activates=====10580107_155871_5970==>To show that the retarded band observed by EMSA in HIV - tat - treated cells was indeed NF - B , nuclear extracts were incubated with antibodies either to p50 ( NF - B1 ) or to p65 ( RelA ) subunits . ||10799874_155871_3725==>Differential Requirement for p56lck in HIV - tat Versus TNF - Induced Cellular Responses : Effects on NF - { kappa } B , Activator Protein - 1 , c - Jun N - Terminal Kinase , and Apoptosis - - Manna and Aggarwal 164 ( 10 ) : 5156 - - The Journal of Immunology ||10799874_155871_3725==>Differential Requirement for p56 lck in HIV - tat Versus TNF - Induced Cellular Responses : Effects on NF - B , Activator Protein - 1 , c - Jun N - Terminal Kinase , and Apoptosis 1 Sunil K . ||10799874_155871_3725==>HIV - tat protein , like TNF , activates a wide variety of cellular responses , including NF - B , AP - 1 , c - Jun N - terminal kinase ( JNK ) , and apoptosis . ||10799874_155871_3725==>For example , HIV - tat activates the transcription factors NF - B ( 16 , 17 ) and AP - 1 ( 18 ) and associated kinases of the mitogen - activated protein ( MAP ) 3 kinase ( MAPK ) family , including c - Jun N - terminal kinase / stress - activated protein kinase ( JNK / SAPK ) , and MAPK kinase ( MAPKK ) ( 18 ) . ||10799874_155871_3725==>Human immunodeficiency virus - 1 tat protein activates c - Jun N - terminal kinase and activator protein - 1 . ||10982368_155871_5594==> Tat 72aa - mediated MCP - 1 and IL - 8 mRNA induction was susceptible to inhibition by the MEK1 / 2 inhibitor UO126 but was only modestly decreased by the inclusion of the p38 mitogen - activated protein kinase ( MAPK ) inhibitor SB202190 . ||10982368_155871_5594==>Inhibition of Tat 72aa - induced chemokine expression by MEK and p38 MAPK inhibitors . ||10982368_155871_5594==>Using the p38 - specific inhibitor SB202190 ( negative control , SB202474 ) , we demonstrate that increasing concentrations of SB202190 strongly inhibit the induction of IP - 10 mRNA by Tat 72aa ( fig. 6 D ) , with modest inhibition of MCP - 1 ( fig. 6 E ) or IL - 8 ( fig. 6 F ) . ||10982368_155871_5594==>Inhibition of HIV - 1 Tat 72aa - mediated induction of chemokine mRNA by the MEK1 / 2 inhibitor UO126 and the p38 inhibitor SB202190 . ||10982368_155871_5594==>CRT - MG cells were incubated with medium ( ) or stimulated with 50 nM Tat ( T ) for 6 h. Cells were preincubated with the MEK1 / 2 inhibitor UO126 ( A to C ) or the p38 inhibitor SB202190 ( D to F ) for 1 h at the concentrations indicated ( 0.1 to 10 然 ) before stimulation of the cells with 50 nM Tat . ||10982368_155871_5594==>Finding that ERK1 / 2 became phosphorylated after Tat 72aa stimulation ( data not shown ) , UO126 , a very potent and highly specific MEK1 / 2 inhibitor ( 18 ) , was utilized to demonstrate the involvement of the ERK pathway . ||10982368_155871_5594==>In contrast , SB202190 , a specific inhibitor of p38 MAPK activation ( 37 ) , efficiently suppressed IP - 10 mRNA induction by Tat 72aa , while expression of MCP - 1 and IL - 8 mRNAs was affected less . ||10982368_155871_5594==>These findings suggest that Tat activation of MCP - 1 and IL - 8 gene expression is only partially dependent on p38 MAPK activation , as even high concentrations of SB202190 could not completely suppress expression . ||10982368_155871_5594==> Tat - mediated IL - 8 gene regulation is stringently controlled by the ERK1 / 2 pathway , while IP - 10 regulation by Tat 72aa exclusively involves the p38 MAPK pathway . ||10982368_155871_5595==>In accordance with these findings , we have determined that ERK1 / 2 is phosphorylated 20 min after stimulation with 50 nM Tat 72aa ( data not shown ) . ||10982368_155871_5595==>Finding that ERK1 / 2 became phosphorylated after Tat 72aa stimulation ( data not shown ) , UO126 , a very potent and highly specific MEK1 / 2 inhibitor ( 18 ) , was utilized to demonstrate the involvement of the ERK pathway . ||10982368_155871_5595==> Tat - mediated IL - 8 gene regulation is stringently controlled by the ERK1 / 2 pathway , while IP - 10 regulation by Tat 72aa exclusively involves the p38 MAPK pathway . ||10982368_155871_5604==> Tat 72aa - mediated MCP - 1 and IL - 8 mRNA induction was susceptible to inhibition by the MEK1 / 2 inhibitor UO126 but was only modestly decreased by the inclusion of the p38 mitogen - activated protein kinase ( MAPK ) inhibitor SB202190 . ||10982368_155871_5604==>In contrast , Tat - mediated IP - 10 mRNA induction was suppressed by SB202190 but not by the MEK1 / 2 inhibitor UO126 . ||10982368_155871_5604==>Using the highly specific MEK1 / 2 inhibitor UO126 and its negative control UO124 , IL - 8 mRNA induction by Tat was almost completely blocked at a UO126 concentration of 1 然 while UO124 at 10 然 had no inhibitory effect ( fig. 6 C ) . ||10982368_155871_5604==>Inhibition of HIV - 1 Tat 72aa - mediated induction of chemokine mRNA by the MEK1 / 2 inhibitor UO126 and the p38 inhibitor SB202190 . ||10982368_155871_5604==>CRT - MG cells were incubated with medium ( ) or stimulated with 50 nM Tat ( T ) for 6 h. Cells were preincubated with the MEK1 / 2 inhibitor UO126 ( A to C ) or the p38 inhibitor SB202190 ( D to F ) for 1 h at the concentrations indicated ( 0.1 to 10 然 ) before stimulation of the cells with 50 nM Tat . ||10982368_155871_5604==>Finding that ERK1 / 2 became phosphorylated after Tat 72aa stimulation ( data not shown ) , UO126 , a very potent and highly specific MEK1 / 2 inhibitor ( 18 ) , was utilized to demonstrate the involvement of the ERK pathway . ||11160671_155871_5970==>Since Tat interacts with p300 ( 1 , 6 , 7 , 15 ) , it would be of interest to determine if Tat modifies the p300 - CDK2 - cycE - RelA complex , decreasing CDK2 's ability to inhibit the transcriptional activity of RelA . ||12167619_155871_5594==>In addition , we show that extracellular signal - regulated kinase ( ERK ) but not that p38 mitogen - activated protein kinase ( MAPK ) is involved in RSV - tat induced production of NO . ||12167619_155871_5594==>Interestingly , PD98059 , an inhibitor of the ERK pathway , and ERK2 , a dominant - negative mutant of ERK2 , inhibited RSV - tat - induced production of NO through the inhibition of C / EBP but not that of NF - B . ||12167619_155871_5594==>Assay of ERK and p38 MAPK - - U373MG astroglial cells ( 50 - 60 % confluent ) were transfected with different concentrations of either RSV - tat or RSV - CAT . ||12167619_155871_5594==>Role of ERK and p38 MAPK in RSV - tat - induced Production of NO in Human U373MG Astroglial Cells - - In eukaryotic cells , an important group of signaling pathways is the mitogen - activated protein kinase ( MAPK ) signaling cascades ( 38 ) . ||12167619_155871_5594==>Because the activation of MAPK pathways such as ERK and p38 MAPK by lipopolysaccharide and cytokines represents a potential signaling mechanism for NO production during the inflammatory response ( 27 , 39 , 40 ) , we investigated the role of ERK and p38 MAPK in RSV - tat - induced production of NO . ||12167619_155871_5594==>Interestingly , expression of RSV - tat induced the activation of ERK ( fig. 7 ) . ||12167619_155871_5594==>Consistent with the effect of RSV - tat on the induction of NO production and the activation of NF - B and C / EBP , there was an inhibition of ERK activation in cells transfected with higher amount ( 0.5 or 1.0 痢 ) of RSV - tat ( fig. 7 ) . ||12167619_155871_5594==>In contrast , under the same conditions , the activation of p38 MAPK was not detected , suggesting that ERK but not p38 MAPK may play an important role in RSV - tat - induced production of NO in human astroglial cells . ||12167619_155871_5594==>Expression of RSV - tat induces activation of ERK in human U373MG astroglial cells . ||12167619_155871_5594==>Therefore , we investigated the role of ERK and p38 MAPK in RSV - tat - induced production of NO using specific pharmacological inhibitors of MEK - ERK ( PD98059 ) and p38 MAPK ( SB203580 ) . ||12167619_155871_5594==>Consistent with the activation of ERK but not p38 MAPK by RSV - tat , PD98059 but not SB203580 dose - dependently inhibited the production of NO in RSV - tat - transfected cells ( fig. 8 A ) , suggesting that ERK but not p38 MAPK is involved in RSV - tat - induced production of NO . ||12167619_155871_5594==>To further confirm the involvement of ERK but not p38 MAPK in RSV - tat - induced production of NO , we studied the effect of dominant - negative mutants of ERK1 ( ERK1 ) , ERK2 ( ERK2 ) , and p38 ( p38 ) on RSV - tat - induced production of NO . ||12167619_155871_5594==>Interestingly , RSV - tat - induced production of NO was inhibited by ERK2 but not by ERK1 , suggesting that ERK2 but not ERK1 is involved in RSV - tat - induced production of NO ( fig. 8 B ) . ||12167619_155871_5594==>In addition , consistent with the inability of RSV - tat to induce the activation of p38 MAPK and the inability of SB203580 to inhibit RSV - tat - induced production of NO , p38 had no effect on RSV - tat - induced production of NO ( fig. 8 B ) . ||12167619_155871_5594==>Role of ERK and p38 MAPK in RSV - tat - induced NO production in human U373MG astroglial cells . ||12167619_155871_5594==>B , cells were cotransfected with 0.2 痢 of RSV - tat and 0.5 痢 of either an empty vector or ERK1 , ERK2 , or p38 . ||12167619_155871_5594==>Is ERK2 Involved in RSV - tat - induced Activation of NF - B and C / EBP in Human U373MG Astroglial Cells ? - - The results presented in fig. 8 show that RSV - tat induces the production of NO through ERK2 . ||12167619_155871_5594==>Because proinflammatory transcription factors NF - B and C / EBP are also involved in RSV - tat - induced production of NO , we next examined the effect of PD98059 and ERK2 on RSV - tat - induced activation of NF - B and C / EBP . ||12167619_155871_5594==>Because PD98059 inhibits the MEK - ERK pathway , these results suggest that ERK2 is probably involved in RSV - tat - induced activation of C / EBP but not of NF - B . ||12167619_155871_5594==>To further confirm this observation , we tested the effect of ERK2 on RSV - tat - induced activation of NF - B and C / EBP . ||12167619_155871_5594==>Consistent with the effect of PD98059 on RSV - tat - induced activation of NF - B and C / EBP , ERK2 specifically inhibited the activation of C / EBP ( fig. 10 B ) but not of NF - B ( fig. 10 A ) . ||12167619_155871_5594==>These experiments suggest that PD98059 and ERK2 inhibit RSV - tat - induced production of NO by inhibiting the activation of C / EBP but not NF - B . ||12167619_155871_5594==>Effect of ERK2 , the dominant - negative mutant of ERK2 , on RSV - tat - induced activation of NF - B and C / EBP in human U373MG astroglial cells . ||12167619_155871_5594==>Cells were cotransfected with 0.2 痢 of RSV - tat , 0.5 痢 of either an empty vector or ERK2 , and 0.5 痢 of either pBIIX - Luc ( A ) or pC / EBP - Luc ( B ) . ||12167619_155871_5594==>We have found that the expression of RSV - tat induces the activation of ERK only and not p38 MAPK . ||12167619_155871_5594==>Consistently , using pharmacological inhibitors and dominant - negative mutants , we have elucidated that ERK but not p38 MAPK is involved in RSV - tat - induced production of NO . ||12167619_155871_5594==>Interestingly , ERK2 but not ERK1 is involved in RSV - tat - induced production of NO in human astroglial cells . ||12167619_155871_5594==>In addition , RSV - tat - induced ERK2 couples with C / EBP only and not with NF - B . ||12167619_155871_5594==>Taken together , our studies suggest that Tat activates ERK2 , which ultimately couples to the activation of C / EBP but not to NF - B for the induction of iNOS . ||12167619_155871_5595==>To further confirm the involvement of ERK but not p38 MAPK in RSV - tat - induced production of NO , we studied the effect of dominant - negative mutants of ERK1 ( ERK1 ) , ERK2 ( ERK2 ) , and p38 ( p38 ) on RSV - tat - induced production of NO . ||12167619_155871_5595==>Interestingly , RSV - tat - induced production of NO was inhibited by ERK2 but not by ERK1 , suggesting that ERK2 but not ERK1 is involved in RSV - tat - induced production of NO ( fig. 8 B ) . ||12167619_155871_5595==>B , cells were cotransfected with 0.2 痢 of RSV - tat and 0.5 痢 of either an empty vector or ERK1 , ERK2 , or p38 . ||12167619_155871_5595==>Interestingly , ERK2 but not ERK1 is involved in RSV - tat - induced production of NO in human astroglial cells . ||12573582_1026_155807==>ScienceDirect - Virology : HIV - 1 Vpr Activates Cell Cycle Inhibitor p21 / Waf1 / Cip1 : A Potential Mechanism of G2 / M Cell Cycle Arrest ||12573582_1026_155807==>HIV - 1 Vpr Activates Cell Cycle Inhibitor p21 / Waf1 / Cip1 : A Potential Mechanism of G2 / M Cell Cycle Arrest ||12573582_1026_155807==>Here we report the first evidence that Vpr activates the expression and transcription of the cyclin - dependent kinase inhibitor p21 / Waf1 / Cip1 ( hereafter p21 ) , an inhibitor of the G1 and G2 / M phase transitions in T lymphoid and myeloid cells . ||12573582_1026_155807==> Vpr activated p21 protein expression in a dose - dependent manner . ||12573582_1026_155807==> Vpr also caused a three - to eightfold induction of the p21 promoter . ||12573582_1026_155807==>Of note , Vpr activated p21 transcription in endogenous p53 positive cells , but not in p53 - deleted or p53 nonfunctional cells . ||12573582_1026_155807==> Vpr and p53 had an additive effect on p21 transcription . ||12573582_1026_155807==>Mutational analysis indicated that wt Vpr , but not cell cycle inactive Vpr mutants , activated the p21 promoter . ||12573582_1026_155807==>These data demonstrate that HIV - 1 Vpr utilizes the cyclin - dependent kinase inhibitor p21 , in addition to cdc2 , to arrest cells in G2 / M . ||12573582_1026_155807==>Author Keywords : HIV - 1 , Vpr ; cell cycle ; G2 / M phase ; cdk ; p21 ; transcription ; p53 ||9560267_155807_5970==>Densitometric analysis revealed that RelA was 1.3 - fold higher in Vpr transfected cells . ||9560267_155807_5970==>Jurkat cells were cotransfected with 5 痢 of HIV - CAT , 0.2 痢 of RSV - RelA where indicated , 1 痢 of CMV - Vpr where indicated , and increasing amounts of CMV - p300 . ||9560267_155807_5970==>p300 exerted dose - responsive stimulation in combination with Vpr , Rel A , and Vpr plus Rel A , and the Vpr / Rel A / p300 combination was greater than additive ( 100 - fold ) over the effect of p300 plus Vpr ( 36 - fold ) or p300 plus RelA ( 16 - fold ) ( fig. 4 C ) . ||9621077_155871_3791==>Human Immunodeficiency Virus Tat Modulates the Flk - 1 / KDR Receptor , Mitogen - Activated Protein Kinases , and Components of Focal Adhesion in Kaposi 's Sarcoma Cells - - Ganju et al. 72 ( 7 ) : 6131 - - The Journal of Virology ||9621077_155871_3791==>Human Immunodeficiency Virus Tat Modulates the Flk - 1 / KDR Receptor , Mitogen - Activated Protein Kinases , and Components of Focal Adhesion in Kaposi 's Sarcoma Cells Ramesh K . ||9621077_155871_3791==>Recently , HIV - 1 Tat has been shown to act like a cytokine and bind to the Flk - 1 / KDR receptor for the vascular endothelial growth factor A ( VEGF - A ) , which is expressed by KS cells . ||9621077_155871_3791==>Recently , another link between Tat and a cytokine - based model of KS pathogenesis was made by demonstrating that HIV Tat specifically binds with high affinity to the mitogenic Flk - 1 ( but not the Flt - 1 ) receptor , also known as VEGFR - 2 , for VEGF - A ( 3 ) . ||9621077_155871_3791==>In the present study , we observed that Tat treatment activates the Flk - 1 receptor ( VEGFR - 2 ) . ||9621077_155871_3791==>Since HIV Tat has been shown to bind to the Flk - 1 / KDR receptor ( 3 ) and to integrin receptors ( 8 , 60 ) , we analyzed the spectrum of substrates phosphorylated after its stimulation of KS cells . ||9621077_155871_3791==>HIV Tat induces tyrosine phosphorylation and activation of the Flk - 1 / KDR receptor in KS cells . ||9621077_155871_3791==>HIV Tat binds and activates the Flk - 1 / KDR receptor ( VEGFR - 2 ) in vascular endothelial cells ( 3 ) . ||9621077_155871_3791==>We therefore investigated whether HIV Tat activates the Flk - 1 / KDR receptor ( VEGFR - 2 ) in these cells . ||9621077_155871_3791==>The autokinase activity of the Flk - 1 / KDR receptor was also activated upon Tat stimulation ( fig. 2 B ) . ||9621077_155871_3791==>Activation of the Flk - 1 / KDR receptor after HIV Tat treatments . ||9621077_155871_3791==>We observed that HIV Tat treatment of KS cells activated the Flk - 1 / KDR receptor ( VEGFR - 2 ) for VEGF - A . ||9621077_155871_3791==>Recently , Albini et al. ( 3 ) showed that Tat basic peptide can induce tyrosine phosphorylation of the Flk - 1 / KDR receptor ( VEGFR - 2 ) whereas the RGD - containing peptide does not activate the Flk - 1 / KDR receptor . ||9621077_155871_5594==>As shown in fig. 7 , HIV Tat stimulation of KS 38 cells resulted in activation of ERK and JNK kinases as determined by the phosphorylation of MBP and GST - c - Jun , respectively . ||9621077_155871_5594==>( A ) KS cells were stimulated with Tat ( 100 ng / ml ) and immunoprecipitated with ERK - 1 or ERK - 2 antibody and then subjected to an in vitro kinase assay with MBP ( 7 痢 ) as a substrate . ||9621077_155871_5599==>As shown in fig. 7 , HIV Tat stimulation of KS 38 cells resulted in activation of ERK and JNK kinases as determined by the phosphorylation of MBP and GST - c - Jun , respectively . ||9621077_155871_5599==>Activation of MAP kinase and JNK upon Tat stimulation . ||9621077_155871_5599==>c - Src , MAP , and JNK kinases are also activated upon Tat treatment in KS cells . binds=====10545121_155871_904==>As shown in Figure 6B , acetylation by PCAF enhanced the binding of Tat to CDK9 / cycT1 by 3 - fold ( compare lane 2 with lane 3 ) . ||10545121_155871_904==>Garber ME , Wei P , KewalRamani VN , Mayall TP , Herrmann CH , Rice AP , Littman DR and Jones KA ( 1998 ) The interaction between HIV - 1 Tat and human cyclin T1 requires zinc and a critical cysteine residue that is not conserved in the murine CycT1 protein . ||11027346_155871_7852==>Selective CXCR4 antagonism by Tat : Implications for in vivo expansion of coreceptor use by HIV - 1 - - Xiao et al. 97 ( 21 ) : 11466 - - Proceedings of the National Academy of Sciences ||11027346_155871_7852==>Selective CXCR4 antagonism by Tat : Implications for in vivo expansion of coreceptor use by HIV - 1 Hua Xiao * , , Christine Neuveut * , , h. Lee Tiffany * , , Monsef Benkirane , Elizabeth A . ||11027346_155871_7852==>Here , we report that the HIV - 1 Tat protein , which is secreted from virus - infected cells , is a CXCR4 - specific antagonist . ||11027346_155871_7852==>Here we report the first viral factor with this property , the HIV - 1 Tat protein , acting as a non - chemokine CXCR4 - selective antagonist . ||11027346_155871_7852==>HIV - 1 Tat Binds CXCR4 . ||11027346_155871_7852==>Cells expressing either CXCR4 or CCR5 were separately equilibrated with 125 I - labeled ligands ( SDF - 1 , RANTES , or MIP - 1 ) with or without escalating amounts of Tat or 100 nM unlabeled chemokine . ||11027346_155871_7852==> Tat competed efficiently ( as effective as unlabeled SDF - 1 ) for 125 I - SDF - 1a binding to CXCR4 + cells ( fig. 1 a Left ) , but did not compete for binding of either 125 I - RANTES ( fig. 1 a Center ) or 125 I - MIP - 1 to CCR5 ( fig. 1 a Right ) . ||11027346_155871_7852==>Consistent with this , protein affinity chromatography results showed direct binding of glutathione S - transferase ( GST ) - Tat ( ) to CXCR4 without significantly detectable binding to CCR8 , CCR5 , or CCR4 ( fig. 1 b ) . ||11027346_155871_7852==>A similar binding specificity by Tat for CXCR4 protein was also verified in yeast two - hybrid assays ( data not shown ) . ||11027346_155871_7852==> Tat binds directly to CXCR4 and competes for 125 I - SDF - 1 binding to CXCR4 . ||11027346_155871_7852==>( b ) GST - protein affinity chromatography shows direct binding of Tat to CXCR4 . ||11027346_155871_7852==> Tat Antagonizes CXCR4 - Function . ||11027346_155871_7852==>The physical interaction between Tat and CXCR4 ( fig. 1 and data not shown ) directed us to consider functional significance ( i.e. , would extracellular Tat perturb SDF - 1 / CXCR4 signaling ? ) . ||11027346_155871_7852==>Collectively , these results show a functional interaction between Tat and CXCR4 that requires an intact CXC motif . ||11027346_155871_7852==> Tat antagonizes signaling by CXCR4 , but not CCR5 , agonists . ||11027346_155871_7852==>Specificity of Tat for CXCR4 was compared with effects on PBMCs , monocytes , or a CCR5 - expressing HEK293 cell line ( hCCR5 / 293 ; fig. 2 ; panels 2 - 5 ) by CCR5 agonists , RANTES , MIP - 1 , and MIP - 1 . ||11027346_155871_7852==> Tat Inhibits CXCR4 - but Not CCR5 - Tropic Infection of Cells by HIV - 1 . ||11027346_155871_7852==>The above findings suggest that soluble Tat might selectively affect HIV - 1 envelope - CXCR4 interaction . ||11027346_155871_7852==> Tat inhibits CXCR4 - dependent infection of cells by HIV - 1 NL4 - 3 . ||11027346_155871_7852==>Because both Tat and SDF - 1 can bind to CXCR4 ( Figs . ||11027346_155871_7852==>1 and 2 ) , we interpret these results as Tat contributing to further occupy CXCR4 , which might otherwise be vacant despite 800 ng / ml of SDF - 1 . ||11027346_155871_7852==>However , we can not exclude that there could be additionally complex interactions between HIV - 1 envelope / SDF - 1 / Tat with CD4 and CXCR4 . ||11027346_155871_7852==> Tat 's interference with CXCR4 - dependent entry was checked by comparing infection of HOS - CXCR4 cells by X4 - NL4 - 3 with HOS - CCR5 cells by R5 - NLAD8 ( 30 ; fig. 4 b ) . ||11027346_155871_7852==>We also checked Tat 's CXCR4 - specific activity by using the widely accepted MAGI - entry assay ( 31 ) . ||11027346_155871_7852==> Tat inhibited NL4 - 3 entry into HOS - CXCR4 ( lanes 4 and 6 ) but not NLAD8 entry into HOS - CCR5 ( lanes 10 and 12 ) . ||11027346_155871_7852==>RT - normalized virus stocks ( 3 , 000 cpm ) of NL4 - 3 or NLAD8 were used to infect either U373 - MAGI - CXCR4 or U373 - MAGI - CCR5 cells in the presence of MBP ( Tat ) or MBPTat72 ( + Tat ) . ||11027346_155871_7852==>Soluble Tat Selects Against CXCR4 - Tropic Env Residue . ||11027346_155871_7852==>We asked next whether Tat 's selective effect at CXCR4 influences evolution of viral tropism . ||11027346_155871_7852==>By using RT - normalized amounts of virus to infect U373 - MAGI - CXCR4 or U373 - MAGI - CCR5 cells , we found that , after subtracting for background , the R5 proportion of X4 - tropic NL4 - 3 increased from 2 % ( Tat selection ) to 14 % ( + Tat selection ; fig. 5 D ) . ||11027346_155871_7852==>We show that Tat binds CXCR4 ( but not CCR5 ; fig. 1 ) , abolishes SDF - 1 / CXCR4 ( but not - chemokine / CCR5 ) signaling ( fig. 2 ) , inhibits X4 - ( but not R5 - ) mediated viral infection / entry of cells ( Figs . ||11027346_155871_7852==>Currently , we do not understand how selectivity for CXCR4 is achieved by Tat . ||11027346_155871_7852==>Hence , one could view Tat 's interaction with CXCR4 as being contributed in part through its CXC motif and in part through its high density of basic amino acids . ||11027346_155871_7852==>Interestingly , CXCR4 's extracellular surface is extremely acidic whereas CCR5 's surface is more neutral to basic ; these charge properties are also consistent with the Tat / coreceptor specificity described here . ||11027346_155871_7852==>44 ) , it is unclear whether loss of Tat 's CXCR4 antagonism holds the same implication for SHIV / macaques as for HIV - 1 / humans . ||11027346_155871_7852==>Our findings here are somewhat at odds with two recent reports that suggested that Tat up - regulates both CXCR4 and CCR5 ( 45 ) or only CXCR4 ( 46 ) expression . ||11027346_155871_7852==>In our experiments , we consistently have failed to observe either CXCR4 or CCR5 expression being modulated by Tat . ||11027346_155871_7852==>However , we can not exclude that cell surface antagonism of CXCR4 by Tat might lead to compensatory up - regulation of expression . ||11027346_155871_7852==>Indeed , as noted in fig. 3 , several competing effects of Tat likely coexist with optimal suppression of X4 virus requiring a complex interaction of Tat + SDF - 1 at CXCR4 . ||11027346_155871_7852==>Possibly , this emergence is a consequence of progressive degradation of lymphoid architecture by R5 viruses leading to loss of SDF - 1 production ( 16 ) coupled with the contribution to virus replication , at this stage , by Tat 's induction of CXCR4 expression ( 45 , 46 ) . ||11238447_155908_7514==>Key Words : eIF - 5A , CRM1 , nuclear actin , nuclear export , HIV - 1 Rev ||11238447_155908_7514==>A series of studies has shown that the primary target of leucine - rich Rev - like NESs is the export receptor CRM1 / exportin1 and , furthermore , that NES - CRM1 / exportin1 interaction depends on the presence of RanGTP ( Fornerod et al. 1997a ; Fukuda et al. 1997 ; Ossareh - Nazari et al. 1997 ; Stade et al. 1997 ; Askjaer et al. 1998 ) . ||11238447_155908_7514==>Studies with leptomycin B , a specific inhibitor of CRM1 / exportin1 ( Kudo et al. 1998 , Kudo et al. 1999 ) that prevents the formation of stable NES - CRM1 / exportin1 complexes , demonstrated that CRM1 / exportin1 indeed mediates the translocation of all Rev - like NES - containing export cargoes through the NPC ( Fornerod et al. 1997a ; Fukuda et al. 1997 ; Ossareh - Nazari et al. 1997 ; Wolff et al. 1997 ; Engel et al. 1998 ; Freedman and Levine 1998 ; Kudo et al. 1998 ; Toyoshima et al. 1998 ; Wada et al. 1998 ; Stommel et al. 1999 ) . ||11238447_155908_7514==>Antibodies against the following antigens were used : eIF - 5A and Rev ( rabbit antibodies ; Hammerschmid et al. 1994 ) ; CRM1 ( rabbit serum ; Kudo et al. 1997 ) ; actin ( mAb 2G2 ; Gonsior et al. 1999 ) ; phenylalanine饍lycine ( FG ) repeat nucleoporins ( mAb 414 ; Hiss Diagnostics ; Davis and Blobel 1986 ) . ||11238447_155908_7514==>It has been shown that treatment of Xenopus oocytes with the drug leptomycin B , which directly binds to the general export receptor CRM1 / exportin1 and prevents its interaction with leucine - rich NESs ( Kudo et al. 1998 , Kudo et al. 1999 ) , efficiently blocks nucleocytoplasmic translocation of Rev ( Fornerod et al. 1997a ) . ||11238447_155908_7514==>Interestingly , the Rev inhibitory mutant eIF - 5A䤼14 clearly failed to interact with CRM1 / exportin1 ( lane 2 ) . ||11238447_155908_7514==>Moreover , inhibition studies in somatic cells suggested that eIF - 5A acts before or simultaneously with CRM1 / exportin1 in the nuclear export of Rev and Rex ( Elfgang et al. 1999 ) . ||11238447_155908_7514==> CRM1 / exportin1 and associated components ( e.g. , Ran ) then translocate the Rev - NES / eIF - 5A鈪ontaining ribonucleoprotein particle through the pore channel . ||11238447_155908_7514==>When RRE RNA - bound Rev protein was incubated together with CRM1 / exportin1 and RanGTP in RNA gel retardation assays , only an extremely poor level of complex formation was detected ( Askjaer et al. 1998 ) . ||11238447_155908_7514==>Finally , quantification of the Rev - NES𥟟RM1 affinity using an assay that measures the hydrolysis of Ran - bound GTP upon complex formation ( RanGAP assay ) clearly demonstrated that the Rev珹ES displays an extremely low affinity for CRM1 / exportin1 , at a level that is almost indistinguishable from that of an export - deficient mutant NES ( Askjaer et al. 1999 ) . ||12832472_1025_155871==>The formation of a quaternary complex among CDK9 , cyclin T1 , Tat , and TAR RNA determines the recruitment of human P - TEFb to the transcription elongation complex and the efficient synthesis of long productive viral transcripts ( 15 , 18 , 30 , 33 , 44 , 65 ) . ||12832472_1025_155871==>Functional interaction between cyclin T1 / cdk9 and Pur determines the level of TNF promoter activation by Tat in glial cells . ||12832472_1025_155871==>Distinct regions of cyclin T1 are required for binding to CDK9 and for recruitment to the HIV - 1 Tat / TAR complex . ||12832472_1025_155871==>Lentivirus Tat proteins specifically associate with a cellular protein kinase , TAK , that hyperphosphorylates the carboxyl - terminal domain of the large subunit of RNA polymerase II : candidate for a Tat cofactor . ||12832472_1025_155871==>Phosphorylation of the RNA polymerase II carboxyl - terminal domain by CDK9 is directly responsible for human immunodeficiency virus type 1 Tat - activated transcriptional elongation . ||12832472_1025_155871==>Requirement for a kinase - specific chaperone pathway in the production of a Cdk9 / cyclin T1 heterodimer responsible for P - TEFb - mediated Tat stimulation of HIV - 1 transcription . ||12832472_1025_155871==>New insight in cdk9 function : from Tat to MyoD . ||12832472_1025_155871==>A novel CDK9 - associated C - type cyclin interacts directly with HIV - 1 Tat and mediates its high - affinity , loop - specific binding to TAR RNA . ||12832472_1025_155871==> TAK , an HIV Tat - associated kinase , is a member of the cyclin - dependent family of protein kinases and is induced by activation of peripheral blood lymphocytes and differentiation of promonocytic cell lines . ||12832472_1025_155871==> Tat modifies the activity of CDK9 to phosphorylate serine 5 of the RNA polymerase II carboxyl - terminal domain during human immunodeficiency virus type 1 transcription . ||12832472_155871_904==>The interaction between HIV - 1 Tat and human cyclin T1 requires zinc and a critical cysteine residue that is not conserved in the murine CycT1 protein . ||14519844_10015_155030==> Gag p6 - p6 - , Gag pb - p9 - , and Gag pd - PTAP - complemented HIV - 1 was generated as in fig. 4 , but , in this case , luciferase ( control ) - , Tsg101 - , or AIP - 1 / ALIX - specific siRNAs were cotransfected . ||14519844_10015_155030==>The interpretation of these results was slightly complicated by the fact that AIP - 1 / ALIX depletion by using siRNA likely had deleterious effects on cell viability , because a Western blot analysis showed slightly reduced Gag expression at later time points ( fig. 5 C ) . ||14554087_155908_3267==>Missense mutations in the yeast nuclear export factor Crm1p that abrogated Rev NES interaction with the XXFG repeat - containing nucleoporin , Rab / hRIP , had minimal effects on the interaction of GLFG repeat - containing hNup98 . ||14554087_155908_3267==>Author Keywords : HIV - 1 Rev ; Nup98 ; Rab ; REBP ; CRM1 ; Cofactor ; RNA export ||14554087_155908_3267==>The nucleoporins described initially as cofactors for the Rev NES were the XXFG - repeat containing nucleoporin Rab / hRIP and a related yeast nucleoporin , Rip1p ( Bogerd et al. , 1995 , Fritz et al. , 1995 and Stutz et al. , 1995 ) . ||14554087_155908_3267==> Rab / hRIP was also shown to interact with the functionally homologous NES regions ( Hope et al. , 1991 and Mancuso et al. , 1994 ) of the Rex protein of human T cell leukemia virus type 1 ( HTLV - 1 ) and the Rev protein of equine infectious anemia virus ( EIAV ) . ||14554087_155908_3267==>For example , Rab / hRIP failed to interact with the functionally positive NES domains HIV - 1 Rev 59 ? 116 / 76 ? 77s and EIAV Rev 2 ? 66 , whereas hNup98 reacted efficiently . ||14554087_155908_3267==>These differences may account for the particularly strong reactivity of Nup98 ( three - to fivefold greater than Rab / hRIP ) with the Rev NES observed in the yeast two - hybrid assay . ||14554087_155908_3267==>Only - galactosidase activities indicated in a given column are comparable as the reactivity of Rev with Nup98 is three - to fivefold greater than with Rab / hRIP . ||14554087_155908_3267==>We next investigated the requirement of the yeast homolog of the human Rev NES export factor , CRM1 , for interaction of the Rev NES with the kinesin - like nuclear cofactor REBP ( Venkatesh et al. , 2003 ) and the nucleoporin cofactors , Rab / hRIP ( Bogerd et al. , 1995 and Fritz et al. , 1995 ) and Nup98 . ||14554087_155908_3267==>For this purpose , we examined the reactivity of wt and functionally inactive Rev NESs with REBP as well as Rab / hRIP and Nup98 in the Saccharomyces cerevisiae strain W303 ( expressing wt Crm1p ) or in W303 strains carrying viable missense mutations in the Crm1 gene , crm1 - 1 and crm1 - 2 ( Neville et al. , 1997 and Yan et al. , 1998 ) in a two - hybrid protein interaction - based lac Z reporter assay ( Table 2 ) . ||14554087_155908_3267==>Whereas the reactivity of Rab / hRIP with the Rev NES was drastically abrogated in the mutant strains crm1 - 1 and crm1 - 2 by approximately 50 - fold as previously reported ( Neville et al. , 1997 ) , we observed that the reactivity of REBP - y ( the Rev NES - interacting carboxy - terminal 75 aa region of REBP ) and hNup98 was only marginally affected ( ~threefold ) . ||14554087_155908_3267==>Since Rev has been shown to be functional in RRE RNA transport in yeast and in view of the high level of conservation between the s. cerevisiae and human CRM1 proteins ( ~48 % identity and ~57 % similarity ) , these results suggest that Crm1p mutations that strongly affect Rev NES interaction of certain nucleoporins such as the XXFG repeat - containing Rab / hRIP have relatively modest effects on the interaction of other NES cofactors such as REBP and the GLFG repeat - containing Nup98 . ||14554087_155908_3267==>The initial characterization of Rab / hRIP as a nucleoporin cofactor for the leucine - rich nuclear export signals of functionally homologous retroviral Rev proteins in yeast two - hybrid screens of human cDNA expression libraries led to the subsequent identification of a number of nucleoporins of the XXFG , FXFG , and GLFG classes , including Nup98 ( Fritz and Green , 1996 and Stutz et al. , 1996 ) , in random analyses of known FG - repeat containing nuclear pore proteins , as potential targets for Rev NES interaction during nucleocytoplasmic transport . ||14554087_155908_3267==>This may account for the very strong reactivity of hNup98 , three - to fivefold greater than Rab / hRIP , with the Rev NES in yeast protein interaction assays . ||14554087_155908_3267==>Thus , an initial high - affinity interaction with Nup98 may target the Rev exportasome to the inner NPC ; subsequently , a series of variable affinity interactions ( including relatively lower affinity interactions such as with Rab / hRIP ) may serve to propel the export complex through the NPC . ||14554087_155908_3267==>Despite the high degree of homology ( ~50 % ) in the amino acid sequences of the CRM1 proteins of s. cerevisiae and man , we observed that missense crm1 mutations in yeast that strongly abrogated the Rab / hRIP ? Rev NES interaction effected only a marginal reduction in the extent of Rev interaction with Nup98 or the kinesin - like cofactor REBP . ||14554087_155908_3267==>Therefore , the interaction of the GLFG - repeat nucleoporin Nup98 , unlike that of the XXFG - repeat containing Rab / hRIP , with the Rev NES may also involve the coordinate action of other cofactors in the Rev multiprotein export complex . ||14554087_155908_7514==>Author Keywords : HIV - 1 Rev ; Nup98 ; Rab ; REBP ; CRM1 ; Cofactor ; RNA export ||14554087_155908_7514==>Genetic analyses in yeast have demonstrated the requirement of the homolog of the human Rev NES export factor , CRM1 ( Fornerod et al. , 1997b and Stade et al. , 1997 ) , for nucleoporin interaction ( Neville et al. , 1997 ) and in vitro biochemical interaction assays have suggested that CRM1 may mediate RanGTP - dependent interactions of the Rev NES with the FG - repeat regions of nucleoporins ( Askjaer et al. , 1999 , Fornerod et al. , 1997b and Floer and Blobel , 1999 ) . ||14554087_155908_7514==>We next investigated the requirement of the yeast homolog of the human Rev NES export factor , CRM1 , for interaction of the Rev NES with the kinesin - like nuclear cofactor REBP ( Venkatesh et al. , 2003 ) and the nucleoporin cofactors , Rab / hRIP ( Bogerd et al. , 1995 and Fritz et al. , 1995 ) and Nup98 . ||14554087_155908_7514==>For this purpose , we examined the reactivity of wt and functionally inactive Rev NESs with REBP as well as Rab / hRIP and Nup98 in the Saccharomyces cerevisiae strain W303 ( expressing wt Crm1p ) or in W303 strains carrying viable missense mutations in the Crm1 gene , crm1 - 1 and crm1 - 2 ( Neville et al. , 1997 and Yan et al. , 1998 ) in a two - hybrid protein interaction - based lac Z reporter assay ( Table 2 ) . ||14554087_155908_7514==>Whereas the reactivity of Rab / hRIP with the Rev NES was drastically abrogated in the mutant strains crm1 - 1 and crm1 - 2 by approximately 50 - fold as previously reported ( Neville et al. , 1997 ) , we observed that the reactivity of REBP - y ( the Rev NES - interacting carboxy - terminal 75 aa region of REBP ) and hNup98 was only marginally affected ( ~threefold ) . ||14554087_155908_7514==>Since Rev has been shown to be functional in RRE RNA transport in yeast and in view of the high level of conservation between the s. cerevisiae and human CRM1 proteins ( ~48 % identity and ~57 % similarity ) , these results suggest that Crm1p mutations that strongly affect Rev NES interaction of certain nucleoporins such as the XXFG repeat - containing Rab / hRIP have relatively modest effects on the interaction of other NES cofactors such as REBP and the GLFG repeat - containing Nup98 . ||14554087_155908_7514==>However , our recent studies indicate that a Rev 1 ? 116 ( M10 ) ? Nup98 fusion protein is incapable of promoting RRE RNA export under conditions where a Rev 1 ? 116 ( M10 ) ? CRM1 fusion protein mediates efficient nuclear export of such RNAs ( l. Li and L.K . ||14554087_155908_7514==>Despite the high degree of homology ( ~50 % ) in the amino acid sequences of the CRM1 proteins of s. cerevisiae and man , we observed that missense crm1 mutations in yeast that strongly abrogated the Rab / hRIP ? Rev NES interaction effected only a marginal reduction in the extent of Rev interaction with Nup98 or the kinesin - like cofactor REBP . ||14554087_155908_7514==>Explanations for this observation include possibilities that the Rev ? Nup98 interaction is direct , that regions of yeast Crm1p other than those affected by the crm1 mutations participate in Nup98 interaction , or that distinct cofactors mediate the Nup98 ? Rev NES interaction . ||14554087_155908_7514==>It has been argued , based on observations of the in vitro requirements of cofactors for the reactivity of the Rev NES with the FXFG protein Nup42 , that the Rev NES - RanGTP - CRM1 ternary complex facilitates Nup42 interaction ( Floer and Blobel , 1999 ) . ||14554087_155908_7514==>Interestingly , a recent study suggests that the accumulation of Rev - bound RRE RNAs at the NPC is not inhibited by leptomycin B ( Cmarko et al. , 2002 ) , an inhibitor of the Rev NES ? CRM1 interaction ; this may be reflective of the potential for CRM1 - independent association of the Rev - bound RRE RNA cargo with inner NPC components , such as Nup98 , during the initial phase of nuclear export . ||9874563_155807_6908==>As shown in fig. 5 , FLAG - Vpr was coimmunoprecipitated by anti - TFIID ( TBP ) , anti - TFIIB , or anti - GR antibodies in dexamethasone - treated cells , suggesting that Vpr binds to components of the transcription machinery and to the GR , as part of the glucocorticoid - activated transcription initiation complex ( 23 ) . ||9874563_155807_6908==>FLAG - VprL64A was coimmunoprecipitated by anti - TFIIB antibody , suggesting that the TFIIB - binding site of this mutant remains functional , whereas FLAG - Vpr ( 36 - 96 ) was not precipitated by either GR , TFIID , or TFIIB antibodies . ||9874563_155807_6908==>Binding to both factors leads to enhanced incorporation of Vpr into a large transcription complex also including other transcription factors such as TFIID . ||9874563_155807_6908==> Vpr can be precipitated with anti - TFIID ( TBP ) , anti - TFIIB , or anti - hGR antibodies in dexamethasone - treated A204 cells . ||9874563_155807_6908==>Coprecipitation experiments ( fig. 5 ) showed that in the presence of glucocorticoid Vpr became associated not only with the GR , but also with TFIIB and TFIID , consistent with its incorporation into a stable transcription initiation complex , reminiscent of other nuclear receptor coactivators . ||9882380_155348_7374==>Using an in vitro protein - protein binding assay , we reveal a direct interaction between the precursor form of UDG and the viral integrase ( IN ) . ||9882380_155348_7374==>These results demonstrate that the absence of virion - associated UDG is directly related to the absence of the IN domain of the gag - pro - pol precursor . co-localizes with=====11090190_155908_6427==>Identification of a Domain in Human Immunodeficiency Virus Type 1 Rev That Is Required for Functional Activity and Modulates Association with Subnuclear Compartments Containing Splicing Factor SC35 - - D'Agostino et al. 74 ( 24 ) : 11899 - - The Journal of Virology ||11090190_155908_6427==>Identification of a Domain in Human Immunodeficiency Virus Type 1 Rev That Is Required for Functional Activity and Modulates Association with Subnuclear Compartments Containing Splicing Factor SC35 Donna M . ||11090190_155908_6427==>Deletion of the loop resulted in partial accumulation of Rev in SC35 - positive nuclear bodies that resembled nuclear bodies that form in response to inhibition of transcription . ||11090190_155908_6427==>In addition to accumulating in nucleoli , Rev has been demonstrated to partially colocalize with the splicing factor SC35 in nuclear speckles ( 33 ) or in the vicinity of nuclear speckles ( 5 , 20 ) and , when coexpressed with an HIV - 1 RNA target , in the cytoplasm ( 41 ) as well as in subnuclear zones probably corresponding to active sites of transcription and processing ( 6 , 41 ) . ||11090190_155908_6427==>In addition to contributing to the ability of Rev to bind to its RNA target , the loop appears to modulate the protein 's intracellular trafficking and its association with subcellular compartments , as its deletion led to prominent accumulation of Rev in subnuclear domains containing the SC35 splicing factor . ||11090190_155908_6427==>The previous observation that wild - type Rev partially colocalizes with SC35 ( 6 , 33 ) prompted us to examine the distribution of this splicing factor in the context of Rev Loop expression . ||11090190_155908_6427==>Interestingly , in a substantial proportion of the Rev Loop - transfected cells , the SC35 signal was distributed in a small number of regularly shaped nuclear bodies instead of in interchromatin granules and perichromatin fibrils . ||11090190_155908_6427==>The SC35 - positive nuclear bodies were specific to Rev Loop - expressing cells and were not detected in nontransfected cells or in cells transfected with wild - type Rev ( data not shown ) . ||11090190_155908_6427==>In addition , three - dimensional reconstructions of images generated by laser - scanning confocal microscopy showed that while `` normal '' SC35 speckles showed an irregular flake - like morphology , Rev Loop SC35 - positive nuclear bodies showed a very regular spheroid shape ( data not shown ) . ||11090190_155908_6427==> Rev Loop was not detected in bodies resembling normal SC35 - containing nuclear speckles . ||11090190_155908_6427==>These observations indicated that the mutant Rev protein disrupted the normal intracellular distribution of SC35 . ||11090190_155908_6427==>To our knowledge , this is the first description of a Rev mutant that presents this peculiar subnuclear distribution and induces a redistribution of SC35 . ||11090190_155908_6427==>Figure 3 shows the effects of DRB treatment on the distribution of Rev Loop and SC35 in transfected HLtat cells . ||11090190_155908_6427==>Examination of the SC35 pattern showed that treatment with DRB for 3 h resulted in a redistribution of SC35 from interconnected clumps and granules ( fig. 3 A ) into isolated , spheroid nuclear structures that were morphologically indistinguishable from those containing Rev Loop ( fig. 3 B ) . ||11090190_155908_6427==>Effect of DRB on the intracellular accumulation of Rev Loop and SC35 . ||11090190_155908_6427==>Cells treated with DRB for 3 h followed by incubation in the absence of the drug for 1 h showed a normal SC35 staining pattern , with the exception that the cells containing Rev Loop in nuclear bodies also contained SC35 in the same structures ( fig. 3 C ) . ||11090190_155908_6427==>Upon treatment with DRB for 6 h , very few cells expressing Rev Loop were detected , and accumulation of the protein in nuclear bodies was no longer evident ; the SC35 - containing nuclear structures became more numerous and smaller and resembled the pattern previously observed in MDCK cells treated with DRB ( 38 ) . ||11090190_155908_6427==>Effects of heat shock on the distribution of Rev Loop and SC35 . ||11090190_155908_6427==>This prompted us to compare how Rev Loop and SC35 would respond to heat shock . ||11090190_155908_6427==>Cells transfected with Rev Loop were subjected to heat shock at 42蚓 for 10 , 20 , 30 , or 50 min and then analyzed by indirect immunofluorescence using anti - sRev and anti - SC35 antibodies . ||11090190_155908_6427==>Thus , Rev Loop responded to heat shock in a manner more similar to that reported for snRNP antigens than that of SC35 . ||11090190_155908_6427==>As shown in fig. 4 , Rev LoopBL accumulated primarily in the nucleus but was not detected in nuclear bodies ; SC35 exhibited a normal intranuclear distribution in speckles and grains . ||11090190_155908_6427==>To determine whether Rev Loop and SC35 remained in the isolated nuclei after the extraction procedure , we carried out indirect immunofluorescence assays on both intact Rev Loop - transfected cells and extracted nuclei prepared from duplicate transfections . ||11090190_155908_6427==>In contrast , SC35 was readily detected in the extracted nuclei , primarily in the pattern characteristic of nontransfected cells , as well as in nuclear bodies in a few of the Rev Loop - positive nuclei , even though Rev Loop was not detected in these structures ( data not shown ) . ||11090190_155908_6427==>The apparent differential release of SC35 and Rev Loop from the nucleus and nuclear bodies is in line with the results of the heat - shock experiments that showed differential release of the two proteins . ||11090190_155908_6427==> Rev Loop shows a partial distribution in distinct spheroid bodies in the nucleus ; these bodies also contain SC35 and resemble the spheroid nuclear structures observed in cells treated with inhibitors of transcription . ||11090190_155908_6427==>The spheroid SC35 - positive nuclear bodies were observed exclusively in cells expressing Rev Loop , with untransfected cells showing the typical accumulation of SC35 in speckles and grains ( fig. 2 ) . ||11090190_155908_6427==>Interestingly , HLtat cells treated for 3 h with the RNA polymerase II inhibitor DRB exhibited SC35 - containing nuclear bodies that were morphologically indistinguishable from the Rev Loop - containing nuclear bodies ( fig. 3 ) as well as the SC35 - positive nuclear structures observed in previous studies of - amanitin - treated cells . ||11090190_155908_6427==>It is tempting to speculate that the accumulation of SC35 in nuclear bodies in cells expressing Rev Loop reflects inhibition of transcription and / or splicing by the mutant due to inappropriate sequestration of transcription - processing factors , a possibility that is currently being tested . ||11090190_155908_6427==>Upon incubation for 11 h with the drug , Rev Loop was still detected mainly in the nuclei , but nuclear bodies were no longer evident ; staining with anti - SC35 antibody confirmed the presence of nuclear speckles with a normal morphology in leptomycin B - treated cells ( data not shown ) . ||12600646_155459_7431==>Current research suggests an association of Vif with the intermediate filament protein , vimentin , and the tyrosine kinase , Hck , but the significance of these associations remains to be defined . ||12600646_155459_7431==>Given the association of Vif with cytoskeletal proteins such as vimentin , ( Khan et al. , 2001 ) also postulate that intra - virion Vif in association with the NP complex , acts upon entry into target cells as an adaptor to link the complex to a cellular transport pathway facilitating its transport to the nuclear membrane . ||12600646_155459_7431==>Earlier work suggested a connection between the cytoskeletal component vimentin and Vif based on co - localisation of Vif with vimentin in HeLa cells ( Karczewski and Simon ) . ||12600646_155459_7431==>Since there is an association between Vif and Gag during viral assembly , it follows that an interaction of Vif with cytoskeletal components , such as vimentin , is consistent with its proposed function in viral assembly and proper formation of the reverse transcription complex . ||12600646_155459_7431==>However Simon et al. ( 1997 ) were unable to detect co - localisation of Vif and vimentin in non - permissive cells and further studies with Vif expression in Cos - 7 cells shows a dramatic effect on vimentin localisation although Vif and vimentin do not co - localise ( Henzler et al. , 2001 ) . ||12600646_155459_7431==>Thus the significance of the Vif ? vimentin interaction remains unclear . competes with=====10964507_155807_3308==>Indeed Hsp70 stimulated binding of HIV - 1 matrix antigen to GST ? karyopherin fusion protein and rescued nuclear import of a Vpr - defective HIV - 1 strain in vitro . ||10964507_155807_3308==>Binding studies with truncated forms of GST ? karyopherin demonstrated that both Vpr and Hsp70 bind to a region in the amino - terminal part of the karyopherin molecule . ||10964507_155807_3308==> Vpr competed with Hsp70 for binding to karyopherin . ||10964507_155807_3308==>These results suggest the presence of a novel regulatory site on karyopherin which is used by Hsp70 and Vpr to stimulate interaction between the HIV - 1 PIC and karyopherin and thus promote viral nuclear import . ||10964507_155807_3308==>Author Keywords : nuclear import ; Vpr ; Hsp70 ; karyopherin ; matrix antigen ||12832472_10614_155871==>Since Tat also binds to this cyclin T1 N - terminal domain and since the association between 7SK RNA / MAQ1 and P - TEFb competes with the binding of Tat to cyclin T1 , we speculate that the TAR RNA / Tat lentivirus system has evolved to subvert the cellular 7SK RNA / MAQ1 system . ||12832472_10614_155871==> MAQ1 / 7SK RNA and Tat compete for binding to cyclin T1 . ||12832472_10614_155871==>Moreover , MAQ1 and Tat bind the same cyclin T1 mutants in the two - hybrid experiments ( fig. 7B ; see reference 14 ) . ||12832472_10614_155871==>Hence , a possible interference of MAQ1 with P - TEFb / Tat complex formation was investigated with a GST - Tat pull - down assay ( 25 , 61 ) . ||12832472_10614_155871==>Furthermore , neither MAQ1 nor 7SK RNA was retained on GST - Tat beads . ||12832472_10614_155871==>P - TEFb / 7SK RNA / MAQ1 complexes are impaired for Tat binding . ||12832472_10614_155871==>To demonstrate that no posttranslational modification prevented Tat from binding P - TEFb / 7SK RNA / MAQ1 complexes , these were disrupted in vitro by RNase treatment or by an increased salt concentration . ||12832472_10614_155871==>Thus , the presence of 7SK RNA and / or MAQ1 in P - TEFb complexes prevents Tat binding to P - TEFb . ||12832472_10614_155871==>In this study , we identified MAQ1 , a novel cellular protein that belongs to the inactive P - TEFb / 7SK complex and prevents Tat binding to P - TEFb . ||12832472_10614_155871==>The TAR RNA / Tat complex as a viral system that may subvert 7SK RNA / MAQ1 . ||12832472_10614_155871==>The P - TEFb / 7SK RNA / MAQ1 complex evokes the complex that forms among P - TEFb , the HIV Tat protein , and the TAR RNA structure . ||12832472_10614_155871==>( i ) 7SK RNA is essential for MAQ1 binding to P - TEFb , whereas although TAR RNA enhances the interaction between Tat and cyclin T1 ( 63 ) , Tat binding to P - TEFb can occur in the absence of TAR RNA ( 25 , 61 ) . ||12832472_10614_155871==>( ii ) Tat specifically associates with cyclin T1 , whereas MAQ1 and 7SK RNA can associate with P - TEFb containing either cyclin T1 or T2 . ||12832472_10614_155871==>( iv ) P - TEFb bound to Tat may be more active than core P - TEFb ( 61 , 64 ) , whereas P - TEFb is inactive when bound to MAQ1 and 7SK ( 41 , 62 ) . ||12832472_10614_155871==>Despite these differences , Tat and MAQ1 both bind the N terminus of cyclin T1 and 7SK RNA / MAQ1 association prevents Tat binding to P - TEFb in vitro . ||12832472_10614_155871==>Conversely , Tat binding to free P - TEFb may prevent P - TEFb / 7SK RNA / MAQ1 complex formation and the corresponding inhibition of P - TEFb activity . ||12832472_10614_155871==>This competition model does not imply that Tat disrupts the P - TEFb / 7SK RNA / MAQ1 complex in vitro . ||12832472_10614_155871==>However , Tat may trap an active form of P - TEFb , as the P - TEFb / 7SK RNA / MAQ1 complex appears to undergo continuous formation and disruption in vivo . ||12832472_10614_155871==>It is tempting to speculate that the TAR RNA / Tat lentivirus system has evolved to subvert the 7SK RNA / MAQ1 cellular system . ||9111043_155908_5902==>Mutations in the Nuclear Export Signal of Human Ran - binding Protein RanBP1 Block the Rev - mediated Posttranscriptional Regulation of Human Immunodeficiency Virus Type 1 - - Zolotukhin and Felber 272 ( 17 ) : 11356 - - Journal of Biological Chemistry ||9111043_155908_5902==>Mutations in the Nuclear Export Signal of Human Ran - binding Protein RanBP1 Block the Rev - mediated Posttranscriptional Regulation of Human Immunodeficiency Virus Type 1 * ||9111043_155908_5902==>Mutational analysis confirmed that this region is responsible for the cytoplasmic accumulation of RanBP1 and can functionally replace the nuclear export signal of Rev of human immunodeficiency virus type 1 . ||9111043_155908_5902==>We showed that RanBP1 interferes with Rev - mediated expression of human immunodeficiency virus type 1 , whereas the RanBP1 with inactivated nuclear export signal abrogates Rev function . ||9111043_155908_5902==>These findings indicate that Rev and RanBP1 compete for the same nuclear export pathway , whereas Rev - and the CTE - mediated pathways are distinct . ||9111043_155908_5902==>The inhibition of Rev function is independent of the ability of RanBP1 to associate with Ran and therefore , it is not likely a result of interference with Ran function . ||9111043_155908_5902==>These data suggest that RanBP1 interacts with Rev at the putative nuclear receptor and , hence , shares a step in posttranscriptional pathway with Rev . ||9111043_155908_5902==>Here , we show that the NES signals of RanBP1 and Rev functionally cross - interfere , indicating a competition for a common export pathway . ||9111043_155908_5902==>NES ( ) RanBP1 mutants completely abrogate Rev - mediated regulation of HIV - 1 without affecting the regulation mediated by the posttranscriptional control element ( CTE ) of simian retrovirus type 1 . ||9111043_155908_5902==>Taken together , our data suggest that RanBP1 and Rev share a nuclear export pathway , which is distinct from the CTE - mediated pathway . ||9111043_155908_5902==>Comparison of the nuclear export signals of RanBP1 , PKI , and Rev . ||9111043_155908_5902==>To study the function of the identified NES - like sequence , we generated hybrid proteins that have the Rev export signal replaced with the NES - like elements of the human and mouse RanBP1 and , as control , with the previously described NES of PKI . ||9111043_155908_5902==>Comparison to wild type Rev shows that the Rev hybrid proteins containing the NES of human or mouse RanBP1 had about 10 % activity , whereas Rev containing the PKI export signal showed 20 % activity . ||9111043_155908_5902==>Indirect immunofluorescence analysis further revealed that the Rev - RanBP1 hybrid protein localized in the nucleoli and translocated to the cytoplasm upon actinomycin D treatment like Rev ( not shown ) . ||9111043_155908_5902==>Hence , the identified element from RanBP1 can efficiently replace the activation / nuclear export signal of Rev , and the resulting hybrid protein has the characteristic properties of Rev . ||9111043_155908_5902==>The similarity of the nuclear export signals of RanBP1 and Rev suggests that these proteins rely on a common nuclear export pathway . ||9111043_155908_5902==>In contrast , the presence of TD Rev protein M10BL had no effect on RanBP1 localization ( fig. 5 D ) . ||9111043_155908_5902==>In parallel experiments , we showed that the localization of the NES ( ) RanBP1 was not affected by Rev or RevM10BL ( fig. 5 , E and F ) . ||9111043_155908_5902==>Therefore , these findings suggest that Rev and RanBP1 compete for a common NES - specific export pathway . ||9111043_155908_5902==>The cytoplasmic accumulation of RanBP1 can be inhibited by Rev but not by trans - dominant Rev . ||9111043_155908_5902==>Given the finding that Rev interferes with the nuclear export of RanBP1 , we explored whether RanBP1 and its mutants affect Rev function as measured by inhibition of Gag production of HIV - 1 . ||9111043_155908_5902==>We cotransfected the rev molecular clone of HIV - 1 in the presence of Rev expression vector and increasing amounts the wild type and NES ( ) RanBP1 expression plasmids ( fig. 6 ) . ||9111043_155908_5902==>In summary , our results indicate that RanBP1 interferes with the posttranscriptional regulation by Rev , and vice versa , Rev , via its own nuclear export signal , interferes with the nuclear export of RanBP1 . ||9111043_155908_5902==>The strong inhibitory effect of NES ( ) RanBP1 on Rev function has been an unexpected result , since such mutants are not predicted to associate with and , hence , to compete for the putative NES receptors . ||9111043_155908_5902==>The presence of NES ( ) RanBP1 in such a complex may interfere with its transport , resulting in the observed inhibition of Rev activity . ||9111043_155908_5902==>Therefore , we tested whether the presence of RanBP1 or its NES - mutant affect expression of the Rev - independent HIV - 1 molecular clone . ||9111043_155908_5902==>6 C and 6 D , the expression of the Rev - independent HIV - 1 was not affected by RanBP1 or the NES ( - ) RanBP1 . ||9111043_155908_5902==>These data further suggest that RanBP1 and Rev share a specific nuclear export pathway , which is distinct from the CTE - mediated export pathway . ||9111043_155908_5902==>RanBP and NES ( ) RanBP1 specifically inhibit the Rev - mediated posttranscriptional regulation . ||9111043_155908_5902==>The transfection mixtures included 1 痢 of pNL4 - 3 fB supplemented with 0.05 痢 of pBsRev ( Rev - dependent ) or 1 痢 of NL43 Rev ( ) R ( ) .S ( Rev - independent ) and increasing amounts of GFP - RanBP1 or GFP - NES ( ) RanBP1 expression vectors ( plotted on x axis as micrograms in A - E ) . ||9111043_155908_5902==>Saturating amounts of plasmids expressing RBD ( ) RanBP1 or RBD ( ) NES ( ) RanBP1 as well as RanBP1 , NES ( ) RanBP1 , or GFP alone were cotransfected with Rev - regulated ( Rev - dependent ; fig. 7 , bottom ) or CTE - regulated ( Rev - independent ; fig. 7 , top ) molecular clones , and the Gag production was measured with antigen capture assay . ||9111043_155908_5902==>RBD mutation had no significant effect on the inhibitory activity of wild type and NES ( ) RanBP1 , suggesting that the interaction of RanBP1 with Ran does not play a role in its specific effect on Rev function . ||9111043_155908_5902==>In summary , these data indicate that the inhibition of Rev function by RanBP1 is directly mediated through its nuclear export signal and is independent of RanBP1 's interaction with Ran . ||9111043_155908_5902==>Inhibition of Rev - mediated expression of HIV - 1 by RanBP1 and NES ( ) RanBP1 is independent of their ability to associate with Ran . ||9111043_155908_5902==>Human 293 cells were cotransfected with 1 痢 of pNL43 Rev ( ) R ( ) .S ( top panel ) or 1 痢 of pNL4 - 3 fB supplemented with 0.05 痢 of pBsRev ( bottom panel ) in the presence of saturating amounts ( 5 痢 ) of plasmids expressing GFP alone or the GFP - fusion proteins of wild type RanBP1 ( Wt ) , NES ( ) RanBP1 , the RBD ( ) RanBP1 , or the NES ( ) RBD ( ) RanBP1 . ||9111043_155908_5902==>The likely role of RanBP1 in this model is to tether active , nuclear form of Ran to the putative export intermediates that are involved in trafficking of other NES - containing substrates , such as Rev of HIV - 1 . ||9111043_155908_5902==>1 The abbreviations used are : RRE , Rev - responsive element ; HIV - 1 , human immunodeficiency virus type 1 ; SRV - 1 , simian retrovirus type 1 ; CTE , constitutive transport element ; NES , nuclear export signal ; RanBP1 , Ran - binding protein 1 ; RBD , Ran - binding domain ; PKI , protein kinase inhibitor ; GFP , green fluorescent protein ; PBS , phosphate - buffered saline ; TD , transdominant ; PCR , polymerase chain reaction . complexes with=====10393184_1025_155871==>Although the TFIIH kinase has been implicated in CTD phosphorylation and Tat function ( see below ) , other studies have shown that a distinct Tat - associated kinase , TAK , can also hyperphosphorylate the CTD of RNA Pol II ( Herrmann and Rice , 1993 , 1995 ; Yang et al . ||10393184_1025_155871==>The kinase activity of CDK9 ( P - TEFb ) is required for Tat - activity ( Mancebo et al . ||10393184_1025_155871==>( D ) The hSPT5 , Tat - SF1 , CDK9 and nucleolin are physically associated with RNA Pol II in Tat - SF . ||10393184_1025_155871==>, 1993 ) ; PSTAIRE , a cdc2 - like kinase ( Lee and Nurse , 1987 ) ; CKII ; the CDK9 and cyclin T components of P - TEFb ; and human homologues of yeast SPT5 ( also designated Tat - CT1 , Wu - Baer et al . ||10393184_1025_155871==>The P - TEFb components CDK9 and cyclin T showed even broader distributions that did not correlate with Tat stimulatory activity although they also were present in all fractions containing Tat stimulatory activity . ||10393184_1025_155871==>Again , all fractions with Tat stimulatory activity contained these components as well as CDK9 . ||10393184_1025_155871==>Antibodies against the RNA Pol II CTD , the RPB6 subunit of RNA Pol II , CDK9 , hSPT5 , Tat - SF1 and nucleolin generally precipitated significant and roughly comparable relative amounts of the assayed Tat - SF components , as well as excess amounts of the specific antigen ( Figure 4D , lanes 3 , 4 , 6�9 ) . ||10393184_1025_155871==>The main exceptions are the anti - CDK9 antibodies , which did not appear to precipitate an excess of antigen ( Figure 4D , lane 6 ; data not shown ) , and the anti - SPT5 and anti - Tat - SF1 antibodies , which precipitated little to no nucleolin ( lanes 7 and 8 ) . ||10393184_1025_155871==>In a test of this prediction , Western blot analyses of salt - eluted ( 0.3 , 0.7 and 1.0 M KCl ) fractions from affinity columns showed that GST巁at and GST𠼦II ( Figure 5C , lanes 5�7 and 8�10 ) specifically bound Tat - SF components that included RNA Pol II , hSPT5 , XP - E , Tat - SF1 , nucleolin and P - TEFb ( CDK9 and cyclin T ) , whereas GST𤪲P16 specifically retained holoenzyme components that included RNA Pol II , CBP and RHA ( Figure 5C , top ) . ||10393184_1025_155871==>Since hSPT5 and P - TEFb components ( CDK9 and cyclin T ) were also retained by GST𤪲P16 affinity columns , these transcription elongation factors appear not to be Tat - specific cofactors . ||10393184_1025_155871==>Western blot analysis ( Figure 6B ) of PICs isolated from reaction mixtures in which Tat stimulates transcription elongation ( Figure 6A , lanes 4 and 5 ) show the presence of both Tat - SF and RNA Pol II holoenzyme components that include RNA Pol II , Tat - SF1 , nucleolin , P - TEFb ( CDK9 and cyclin T ) , RHA , and SRB / MED proteins ( Figure 6B , lanes 5 and 6 ) . ||10393184_1025_155871==>Consistent with other reports implicating CDK9 as a Tat cofactor , depletion of CDK9 from Tat - SF also impaired Tat function in a reconstituted system that lacks P - TEFb ( data not shown ) . ||10393184_1025_155871==>Gold M , Yang X , Herrmann H and Rice A ( 1998 ) PITALRE , the catalytic subunit of TAK , is required for human immunodeficiency virus Tat transactivation in vivo . ||10393184_1025_155871==>Herrmann C and Rice A ( 1995 ) Lentivirus Tat proteins specifically associate with a cellular protein kinase , TAK , that hyperphosphorylates the carboxy - terminal domain of the large subunit of RNA polymerase II : candidate for a Tat cofactor . ||10393184_1025_155871==>Jones KA ( 1997 ) Taking a new TAK on Tat transactivation . ||10393184_1025_155871==>Wei P , Garber M , Fang S - M , Fisher W and Jones K ( 1998 ) A novel CDK9 - associated C - type cyclin interacts directly with HIV - 1 Tat and mediates its high - affinity , loop - specific binding to TAR RNA . ||10393184_1025_155871==>Yang X , Herrmann C and Rice A ( 1996 ) The human immunodeficiency virus Tat proteins specifically associate with TAK in vivo and require the carboxy - terminal domain of RNA polymerase II for function . ||10393184_1025_155871==>Yang X , Gold MO , Tang DN , Lewis DE , Aguilar - Cordova E , Rice AP and Herrmann CH ( 1997 ) TAK , an HIV - 1 Tat - associated kinase , is a member of the cyclin - dependent family of protein kinases and is induced by activation of peripheral blood lymphocytes and differentiation of promonocytic cell lines . ||10393184_155871_904==>Garber E , Wei P , KewalRamani V , Mayall T , Herrmann C , Rice A , Littman D and Jones K ( 1998 ) The interaction between HIV - 1 Tat and human cyclin T1 requires zinc and a critical cysteine residue that is not conserved in the murine cycT1 protein . ||10454543_1025_155871==>Second , the sensitivity of Tat activation to a spectrum of different drugs mirrors those which inhibit Cdk9 kinase activity in vitro ( 19 ) . ||10454543_1025_155871==>Third , a cellular kinase complex termed TAK ( Tat - activated kinase ) that interacts with the activation domain of Tat and phosphorylates the CTD of Pol II has been identified as P - TEFb ( 12 , 13 , 39 , 45 ) . ||10454543_1025_155871==>Finally , overexpression of a mutant Cdk9 kinase blocks Tat activation of elongation in human cells ( 19 ) . ||10454543_1025_155871==>Repeated depletion of Tat - SF1 , by absorption with specific Tat - SF1 antiserum , reduced the amounts of RAP30 without the depletion of other proteins such as Cdk9 nor RAP74 ( fig. 1 B , lanes 2 , 3 , and 4 ) . ||10454543_1025_155871==>P - TEFb , containing cyclin T and Cdk9 , specifically binds to TAR RNA in the presence of Tat forming a species - specific recognition complex leading to the activation of transcription ( 1 , 33 ) . ||10454543_1025_155871==>Although we did not detect an association of Tat - SF1 with Cdk9 ( probably because of the high - salt washing conditions ) , it is likely that Tat - SF1 would associate with Cdk9 in the presence of Tat . ||10921877_155807_5524==>We provide here biochemical and functional evidence demonstrating that human immunodeficiency virus type 1 ( HIV - 1 ) Vpr mediates G 2 arrest by forming a complex with protein phosphatase 2A ( PP2A ) , an upstream regulator of cdc25 . ||10921877_155807_5524==> Vpr associates with PP2A through a specific interaction with the B55 regulatory subunit . ||10921877_155807_5524==>Interestingly , we found that Vpr association with B55 - containing PP2A targets the enzymatic complex to the nucleus and , importantly , enhances the recruitment and dephosphorylation of the cdc25 substrate . ||10921877_155807_5524==>Our data suggest that Vpr mediates G 2 arrest by enhancing the nuclear import of PP2A and by positively modulating its catalytic activity towards active phosphorylated nuclear cdc25 . ||10964778_1025_155871==>We characterized the protein components of this activity , which include cyclin T1 , CDK9 , Tat - SF1 , and at least three unidentified proteins . ||11704662_1025_155871==>Through its N - terminal basic helix - loop - helix and C - terminal domains , GRIP1 binds to the N - terminal region of Tat and to the host cell protein cyclin T1 , respectively , which is normally complexed with CDK9 as P - TEFb . ||11704662_1025_155871==>The transactivation domain of Tat , localized in the N - terminal region of the molecule , strongly interacts with cyclin T1 , a component of P - TEFb ( p ositive - acting t ranscription e longation f actor - b ) , which also contains CDK9 ( c yclin - d ependent k inase - 9 ) or PIALRE ( 4 , 5 ) . ||11704662_1025_155871==>When we cotransfected Tat , P - TEFb components cyclin T1 and CDK9 , and GRIP1 altogether , we observed strong synergy in increasing dexamethasone - stimulated MMTV LTR activity ( fig. 7 C ) . ||11704662_1025_155871==>HeLa cells were transfected with BS - Tat , pcDNA3 - HA - cyclin T1 / pcDNA3 - HA - CDK9 , and / or pSG5 - GRIP1 - fl , MMTV - LTR - Luc , and pSV40 - - gal . degrades=====12954211_155945_920==>ScienceDirect - Virology : The presence of the casein kinase II phosphorylation sites of Vpu enhances the CD4 + T cell loss caused by the simian ? human immunodeficiency virus SHIVKU - lbMC33 in pig - tailed macaques ||12954211_155945_920==>The presence of the casein kinase II phosphorylation sites of Vpu enhances the CD4 + T cell loss caused by the simian ? human immunodeficiency virus SHIV KU - lbMC33 in pig - tailed macaques ||12954211_155945_920==>To rule out that macaque CC8X had selected for a virus in which there was reversion of the glycine residues at positions 52 and 56 to serine residues and / or compensating mutations occurred in other genes associated with CD4 down - regulation , sequence analysis was performed on amplified vpu sequences isolated from PBMC and from several lymphoid tissues at necropsy . ||12954211_155945_920==>? A Vpu protein with the casein kinase II sites prevents CD4 expression on the cell surface ||12954211_155945_920==> Vpu has been shown to interact with newly synthesized CD4 , re - translocate this molecule across the RER membrane , and target it for destruction via the proteasome pathway ( Fujita et al 1997 ) . ||12954211_155945_920==>Certain highly conserved domains have been shown to be essential to Vpu - mediated CD4 down - regulation . ||12954211_155945_920==>In addition to CD4 down - regulation from the surface , Vpu has also been shown to facilitate virion release from infected cells and this property of Vpu has been associated with the transmembrane domain which has been reported to have an ion channel activity ( Ewart et al 1996 , Schubert et al 1996a and Sansom et al 1998 ) . ||12954211_155945_920==>Our results indicate that the two casein kinase II phosphorylation sites of Vpu contribute to the pathogenicity SHIV KU - 1bMC33 / pig - tailed macaque model of CD4 + T cell loss and provide additional evidence that this protein enhances the pathogenicity of HIV - 1 . ||12954211_155945_920==>Virus - expressed Vpu has been shown to be phosphorylated by casein kinase II and this phosphorylation has been shown to be essential to CD4 down - regulation . ||12954211_155945_920==>A Vpu protein with the casein kinase II sites prevents CD4 expression on the cell surface ||12954211_155945_920==>Using the VpuEGFP reporter , we also analyzed the ability of Vpu S52 , 56G EGFP to prevent CD4 expression on the cell surface . ||12954211_155945_920==>In contrast , transfection of cultures with the pc vpu S52 , 56G EGFP vector still resulted in the expression of CD4 on the cell surface and was similar to cultures transfected with pcEGFP , which expresses only EGFP reporter protein ( Table 1 ) . ||12954211_155945_920==>These results indicate that mutation of the serine residues at positions 52 and 56 to glycine residues abolished the ability of Vpu to prevent CD4 from being expressed on the cell surface . ||12954211_155945_920==>The Vpu S52 , 56G EGFP is does not prevent CD4 from being expressed on the cell surface . ||12954211_155945_920==>HeLa CD4 + cells , which express CD4 , were transfected with pcEGFP , pc vpu EGFP , or pc vpu S52 , 56G EGFP vectors . ||12954211_155945_920==>Results of transfection of HeLa CD4 + cells with vectors expressing EGFP , VpuEGFP , or Vpu S52 , 56G EGFP on cell surface CD4 expression ||12954211_155945_920==>Because of the severe loss of CD4 + T cells in CC8X , one possibility was that there was a selection for mutations in the vpu that resulted in the reversion of the glycine residues at positions 52 and 56 back to serine residues . ||12954211_155945_920==>Another possibility that had to be considered was that because of the mutations engineered in vpu , virus might have been selected for mutations in other viral genes involved in CD4 down - regulation , namely , env and nef , that would have compensated for the lack of a functional Vpu . ||12954211_155945_920==>Studies have shown that three proteins ( Env , Nef , and Vpu ) of HIV - 1 can interact with the CD4 molecule and some investigators have suggested that the CD4 down - regulation function of Vpu is redundant ( Piquet et al. , 1998 ) . ||12954211_155945_920==>While Nef acts preferentially on cell surface CD4 ( Garcia and Miller , 1991 ) , Vpu acts on CD4 in the RER to block the trafficking of newly synthesized CD4 molecules to the cell surface membrane ( Willey et al 1992 ) . ||12954211_155945_920==>Previous studies have shown that the enhancement of virion release and degradation of CD4 can be assigned to different domains of the Vpu protein . ||12954211_155945_920==>While the transmembrane domain of Vpu has been shown to be important for the enhancement of virion release , the cytoplasmic domain of Vpu has been shown to be important in CD4 degradation ( Schubert et al 1996b ) ; ( Paul et al 1998 ) . ||12954211_155945_920==>Regarding Vpu - mediated CD4 degradation , phosphorylation of the two casein kinase II sites in the cytoplasmic domain has been shown to be essential to this function and it happens to be the most conserved sequence in Vpu proteins isolated from different subtypes of HIV - 1 ( Schubert et al 1994 , Paul and Jabbar 1997 and McCormick - Davis et al 2000a ) . ||12954211_155945_920==>Thus , Vpu has a potential role in the pathogenesis of HIV - 1 and SHIV by promoting the degradation of CD4 molecules in these complexes , allowing Env transport to the cell surface for assembly into viral particles . ||12954211_155945_920==>The importance of Vpu in the circulating CD4 + T cell loss caused by SHIV was also demonstrated in a recent study of the entire vpu coding sequence prior to env ( Stephens et al 2002 ) . ||12954211_155945_920==>In this report , we have analyzed the ability of the pathogenic molecular clone SHIV KU - 1bMC33 to cause rapid CD4 + T cell loss and AIDS following site - directed changes that removed the two casein kinase II phosphorylation sites in the cytoplasmic domain of the Vpu protein . ||12954211_155945_920==>We hypothesized that because of the importance of the casein kinase II sites to CD4 degradation by Vpu that the virus would be less pathogenic for macaques . ||12954211_155945_920==>These results indicate that the casein kinase II sites of the Vpu protein are important to the massive CD4 + T cell loss associated with infection with this virus . ||12954211_155945_920==>The obvious question that arose from the results of this study was `` Why did macaque CC8X develop severe CD4 + T cell loss and die at 18 weeks postinoculation ? '' We hypothesized that the virus in this macaque may have selected for mutations in vpu that might have resulted in the reversion of the glycines at positions 52 and 56 to serine residues or compensating amino acid substitutions in other proteins , such as Env . ||12954211_155945_920==>Taken together , the results of this study indicate for the first time that the phosphorylation of Vpu potentiates a dramatic loss in CD4 + T cells following inoculation with pathogenic SHIV KU - 1bMC33 and provide additional evidence that this viral protein is important to the CD4 + T cell loss in HIV - 1 - infected people . ||14561767_155945_920==>The human immunodeficiency virus type 1 Vpu protein acts as an adaptor for the proteasomal degradation of CD4 by recruiting CD4 and - transducin repeat - containing protein ( TrCP ) , the receptor component of the multisubunit SCF - TrCP E3 ubiquitin ligase complex . ||14561767_155945_920==> Vpu increases the release of virus particles from the plasma membrane ( 1 ) through its N - terminal transmembrane domain ( aa 1 - 27 ) , whereas it mediates the degradation of the CD4 receptor in the endoplasmic reticulum ( ER ) ( 2 ) via its cytoplasmic domain ( aa 28 - 81 ) . ||14561767_155945_920==>It was shown recently that Vpu exerts a positive effect on HIV - 1 infectivity by down - modulating CD4 receptor molecules at the surface of HIV - 1 - producing cells ( 3 ) . ||14561767_155945_920==> CD4 degradation requires the phosphorylation of the serine residues at positions 52 and 56 of the Vpu cytoplasmic domain by casein kinase II ( 4 ) and the binding of Vpu to CD4 trapped in the ER via the formation of a complex with the HIV - 1 envelope precursor gp160 ( 5 , 6 ) . ||14561767_155945_920==>We showed previously that Vpu binds to the F box protein - transducin repeat - containing protein ( TrCP ) , the receptor component of the multisubunit SCF - TrCP E3 ubiquitin ligase complex , and connects CD4 to the ubiquitin - proteasome machinery . ||14561767_155945_920==>Besides serving as an adaptor for CD4 degradation , Vpu is a strong competitor that can inhibit the degradation of the TrCP substrates , leading them to accumulate in HIV - 1 - producing cells expressing Vpu . ||14561767_155945_920==>As - catenin accumulates in HIV - 1 - producing cells expressing Vpu , and given that the thymus is one of the major targets of the HIV - 1 infection in AIDS patients , Vpu may be one of the viral factors responsible for the renewal of CD4 + T lymphocytes following their depletion , which is a major consequence of HIV - 1 infection . ||14561767_155945_920==>Such a TrCP sequestration in the cytoplasm probably plays an essential role in the degradation of CD4 mediated by Vpu . ||14561767_155945_920==>Instead of using one of the E3 ubiquitin ligases located in the ER , which are responsible for the ER - associated degradation process ( 44 ) , HIV - 1 has evolved in such a way that , using Vpu , it subverts a mostly nuclear E3 ubiquitin ligase like TrCP to ensure degradation of the CD4 receptor at the ER . ||14561767_155945_920==>Thus , although Vpu is an accessory protein that is not essential for the infection of human CD4 + cells in vitro , it is probably a major pathogenic determinant for HIV infection in vivo . ||7778293_155945_920==>ScienceDirect - Virology : Degradation of CD4 Induced by Human Immunodeficiency Virus Type 1 Vpu Protein : A Predicted Alpha - Helix Structure in the Proximal Cytoplasmic Region of CD4 Contributes to Vpu Sensitivity ||7778293_155945_920==>Degradation of CD4 Induced by Human Immunodeficiency Virus Type 1 Vpu Protein : A Predicted Alpha - Helix Structure in the Proximal Cytoplasmic Region of CD4 Contributes to Vpu Sensitivity ||7778293_155945_920==>The HIV - 1 - encoded Vpu protein induces a rapid and specific degradation of CD4 molecules in the endoplasmic reticulum ( ER ) . ||7778293_155945_920==>In this study , Vpu - induced degradation of CD4 in the ER was investigated by quantitative immunoprecipitation of CD4 following cotransfection of COS - 7 cells with CD4 and Vpu expressors in the presence of brefeldin A , a drug that blocks protein transport from the ER to the Golgi complex , in order to precisely define the sequence ( s ) or structural element ( s ) in the CD4 cytoplasmic domain necessary for Vpu - induced degradation , a panel of deletion and substitution mutants in the cytoplasmic domain of CD4 was generated and analyzed . ||7778293_155945_920==>In agreement with previous reports , our deletion analysis indicates that a region encompassing amino acids 441 to 419 ( KRLLSEKKT ) in the cytoplasmic domain of CD4 was required to confer Vpu sensitivity . ||7778293_155945_920==>However , six specific substitution mutations within this region did not confer CD4 resistance to Vpu , suggesting that neither the amino acid sequence nor the charge of the amino acids in this region was critical to Vpu - induced CD4 degradation . ||7778293_155945_920==>A dileucine motif that is important for internalization of CD4 and Nef - induced CD4 down - regulation was also not required for Vpu - induced CD4 degradation . ||7778293_155945_920==>Interestingly , two substitution mutants ( CD4EMKL and CD4MK407 , 11PP ) located in a more proximal cytoplasmic region of CD4 abolished Vpu - induced CD4 degradation . ||7778293_155945_920==>Computer - assisted analysis of the substitution and deletion mutants conferring CD4 resistance to Vpu - induced degradation indicated that these mutations disrupted a putative alpha - helix formed in the proximal cytoplasmic region of CD4 . ||7778293_155945_920==>Taken together , these studies strongly suggest that a structural element in the proximal cytoplasmic region of CD4 contributes to Vpu sensitivity . ||8659106_155945_920==>ScienceDirect - Virology : The Human Immunodeficiency Virus Type 1 Vpu Protein Tethered to the CD4 Extracellular Domain Is Localized to the Plasma Membrane and Is Biologically Active in the Secretory Pathway of Mammalian Cells : Implications for the Mechanisms of Vpu Function ||8659106_155945_920==>The Human Immunodeficiency Virus Type 1 Vpu Protein Tethered to the CD4 Extracellular Domain Is Localized to the Plasma Membrane and Is Biologically Active in the Secretory Pathway of Mammalian Cells : Implications for the Mechanisms of Vpu Function ||8659106_155945_920==>The HIV - 1 Vpu protein induces the proteolysis of CD4 in the endoplasmic reticulum ( ER ) and enhances the release of virus particles from the plasma membrane . ||8659106_155945_920==>To this end , we generated CD4 / Vpu hybrid proteins and analyzed their biochemical and biological properties in HeLa cells . ||8659106_155945_920==>Importantly , a hybrid protein bearing the CD4 extracellular domain and full - length Vpu induced the degradation of HIV envelope glycoproteins bearing the transmembrane and cytoplasmic domains of CD4 ( Vpu - responsive elements , VRE ) . ||8659106_155945_920==>In addition , a hybrid protein having the extracellular ? transmembrane domains of CD4 and the Vpu cytoplasmic domain was only partially active in inducing the degradation of Vpu - sensitive proteins . ||8659106_155945_920==>Mutational studies have further demonstrated that casein kinase - 2 phosphorylation is critically important in the degradation reaction , but does not regulate membrane trafficking of the CD4 / Vpu hybrid proteins . ||8659106_155945_920==>We also show that the CD4 extracellular domain appended to the Vpu protein is protected from degradation while existing in a complex with Vpu - sensitive ectodomains . ||8659106_155945_920==>Taken together , these studies have revealed that the Vpu protein does not possess sequences that have the ability to sequester CD4 in the intracellular compartments of mammalian cells and that the Vpu protein tethered to the CD4 extracellular domain was biologically active in inducing the degradation of VRE - bearing glycoproteins in the ER . ||8709227_155945_920==> CD4 down - modulation during infection of human T cells with human immunodeficiency virus type 1 involves independent activities of vpu , env , and nef - - Chen et al. 70 ( 9 ) : 6044 - - The Journal of Virology downregulates=====10751368_1231_155871==>There is a trend toward an inhibition of CCR2 and CCR5 after 50 ng / ml or 100 ng / ml of Tat but this is not significant ( n = 3 ; Figure 5B ) . ||10751368_1231_155871==>Densitometric analyses show no significant changes in receptor expression after Tat treatment ; however , there is a trend toward Tat inhibition of CCR2 and CCR5 after 50 and 100 ng / ml treatment of Tat ( n = 3 ) ( B ) . ||10751368_1231_155871==>38 , 45 Tat has also been shown to up - regulate CXCR4 on the surface of resting CD4 + T 33 cells and to mimic the � - chemokines MCP - 1 , MCP - 3 , and eotaxin in their interactions with CCR2 and CCR3 . ||10751368_1234_155871==>There is a trend toward an inhibition of CCR2 and CCR5 after 50 ng / ml or 100 ng / ml of Tat but this is not significant ( n = 3 ; Figure 5B ) . ||10751368_1234_155871==>Densitometric analyses show no significant changes in receptor expression after Tat treatment ; however , there is a trend toward Tat inhibition of CCR2 and CCR5 after 50 and 100 ng / ml treatment of Tat ( n = 3 ) ( B ) . ||11156964_1385_155871==>HIV - 1 Tat protein down - regulates CREB transcription factor expression in PC12 neuronal cells through a phosphatidylinositol 3 - kinase / AKT / cyclic nucleoside phosphodiesterase pathway - - ZAULI et al. 15 ( 2 ) : 483 - - The FASEB Journal ||11156964_1385_155871==>HIV - 1 Tat protein down - regulates CREB transcription factor expression in PC12 neuronal cells through a phosphatidylinositol 3 - kinase / AKT / cyclic nucleoside phosphodiesterase pathway GIORGIO ZAULI * 1 , DANIELA MILANI , PRISCO MIRANDOLA * , , MERI MAZZONI , PAOLA SECCHIERO , SEBASTIANO MISCIA * and SILVANO CAPITANI ||11156964_1385_155871==>The addition of low concentrations ( 0.1�1 nM ) of extracellular HIV - 1 Tat protein to PC12 neuronal cells stimulated a rapid ( peak at 5 min ) elevation of the cAMP intracellular levels , which in turn induced the phosphorylation of CREB transcription factor ( peak at 15 min ) on serine - 133 ( Ser - 133 ) . ||11156964_1385_155871==>In blocking experiments performed with pharmacological inhibitors , Tat decreased the intracellular levels of cAMP and CREB Ser - 133 phosphorylation through a signal transduction pathway involving the sequential activation of phosphatidylinositol 3 - kinase , AKT , and cyclic nucleoside phosphodiesterases . ||11156964_1385_155871==>Moreover , in transient transfection experiments , Tat inhibited transcription of CREB promoter in a manner strictly dependent on the presence of the cAMP - responsive elements ( CRE ) in the CREB promoter . ||11156964_1385_155871==>Consistently , the expression of endogenous CREB protein was significantly reduced in PC12 cells by prolonged ( 24�48 h ) treatment with Tat . ||11156964_1385_155871==>This decline in the expression of CREB , which plays an essential role in the survival and function of neuronal cells , anticipated a progressive increase of apoptosis in Tat - treated cells . ||11156964_1385_155871==>, Miscia , s. , Capitani , s. HIV - 1 Tat protein down - regulates CREB transcription factor expression in PC12 neuronal cells through a phosphatidylinositol 3 - kinase / AKT / cyclic nucleoside phosphodiesterase pathway . ||11156964_1385_155871==>Since it has been clearly established that CREB plays a central role in promoting the survival / function of neuronal cells in response to neurotrophic factors ( 22 , 26 , 27 ) , the aim of this study was to investigate whether HIV - 1 Tat protein affects the Ser - 133 phosphorylation levels and expression of CREB in neurons . ||11156964_1385_155871==>Rapid Ser - 133 phosphorylation of CREB induced by Tat protein in PC12 neuronal cells ||11156964_1385_155871==>In the first group of experiments , we investigated whether extracellular Tat modulates the phosphorylation levels of the transcription factor CREB in PC12 neuronal cells . ||11156964_1385_155871==>For this purpose , PC12 cells were serum - starved in order to lower the high levels of endogenous Ser - 133 CREB phosphorylation observed in cells cultured with 10 % HS + 5 % FCS ( not shown ) ; 40 h after serum starvation in D - MEM + 0.1 % FCS , PC12 cells were treated with low concentrations of synthetic Tat protein ( 01�1 nM ) for up to 60 min . ||11156964_1385_155871==>The time course of Tat - mediated CREB phosphorylation in PC12 cells revealed that extracellular Tat maximally increased the levels of Ser - 133 CREB phosphorylation between 15 and 30 min ( fig. 1 and Table 1 ) . ||11156964_1385_155871==>Short - term effect of Tat on CREB Ser - 133 phosphorylation in PC12 neuronal cells . ||11156964_1385_155871==>Levels of Ser - 133 phosphorylated CREB ( P - CREB ) and total CREB were evaluated by Western blot on enriched nuclear fractions obtained from PC12 cells , serum starved for 40 h , and treated for the time indicated ( min ) with Tat ( 1 nM ) or forskolin ( 10 然 ) . ||11156964_1385_155871==>Densitometric analysis of P - CREB protein expression in PC12 cells treated with forskolin or Tat in the absence or presence of various pharmacological inhibitors a ||11156964_1385_155871==>Thus , to ascertain whether PKA was involved in Tat - mediated CREB phosphorylation in PC12 cells , we used the pharmacological compounds H89 , a specific inhibitor for the PKA pathway , and SB203580 , an inhibitor of the p38 MAPK pathway , used as control . ||11156964_1385_155871==>Western blot analysis of PC12 nuclear cell lysates showed that H89 , but not SB203580 , significantly ( P < 0.05 ) inhibited the Tat - mediated Ser - 133 CREB phosphorylation ( fig. 2 and Table 1 ) . ||11156964_1385_155871==>This suggested that activation of the cAMP / PKA pathway was required for the Tat - induced CREB phosphorylation in these cells . ||11156964_1385_155871==>Effect of a PKA pharmacological inhibitor on the short - term Tat - induced CREB Ser - 133 phosphorylation in PC12 cells . ||11156964_1385_155871==>Consistent with a role for the cAMP / PKA pathway in inducing CREB phosphorylation in PC12 cells , Tat stimulated a rapid ( peak at 5 min ) elevation of intracellular cAMP over the basal levels observed in control ( BSA - treated ) cells ( fig. 3 A ) . ||11156964_1385_155871==>At 5�10 min , the combination of Tat plus forskolin showed an additive effect ( P < 0.05 ) on CREB Ser - 133 phosphorylation , as demonstrated by the densitometric analysis of the Western blot bands ( fig. 4 and Table 1 ) . ||11156964_1385_155871==>Effect of combination of Tat and forskolin on short - term CREB Ser - 133 phosphorylation in PC12 cells . ||11156964_1385_155871==>Levels of Ser - 133 phosphorylated CREB ( P - CREB ) and total CREB were evaluated by Western blot on enriched nuclear fractions obtained from PC12 cells treated with BSA ( 0.1 % ) , Tat ( 1 nM ) , forskolin ( 10 然 ) , Tat ( 1 nM ) plus forskolin ( 10 然 ) for the time indicated ( min ) . ||11156964_1385_155871==>We next investigated how extracellular Tat induced the PDE activity responsible for the secondary decrease in the intracellular cAMP levels and in CREB Ser - 133 phosphorylation . ||11156964_1385_155871==>Moreover , pretreatment of PC12 cells with two unrelated pharmacological inhibitors of PI 3 - K ( 10 然 LY294002 and 0.1 然 wortmannin ) before Tat addition significantly increased CREB Ser - 133 phosphorylation in PC12 cells with respect to cells treated with vehicle containing DMSO ( fig. 5 B and Table 1 ) . ||11156964_1385_155871==>B ) Effect of the PI 3 - K / AKT pharmacological inhibitors on the short - term Tat - induced CREB Ser - 133 phosphorylation in PC12 cells . ||11156964_1385_155871==>Inhibition of CREB promoter activity and CREB protein expression after prolonged treatment of PC12 cells with Tat ||11156964_1385_155871==>Thus , in the next group of experiments we investigated whether the ability of extracellular Tat to modulate the levels of intracellular cAMP and CREB Ser - 133 phosphorylation could affect CREB transcription . ||11156964_1385_155871==>This suggested that the Tat - mediated activation of PDE ( fig. 3 A ) was able to down - modulate the CREB promoter activity in PC12 cells . ||11156964_1385_155871==>The transcriptional activity of mut pCREB - CAT was also undetectable in cultures stimulated with forskolin used alone or in combination with Tat , confirming that the CRE sites played a central role in driving CREB transcription ( data not shown ) . ||11156964_1385_155871==>To verify the relevance of Tat - induced down - modulation of CREB promoter , in the next group of experiments we analyzed the total amount of CREB protein in PC12 cells after prolonged Tat treatment ( fig. 7 ) . ||11156964_1385_155871==>For this purpose , cell lysates obtained from whole PC12 cells and nuclei were analyzed for the content of CREB and its levels of phosphorylation on Ser - 133 after 24�48 h from the addition in culture of 1 nM Tat . ||11156964_1385_155871==>The possibility that the loss in CREB protein expression was due to a nonspecific cytotoxicity of Tat was ruled out by an analysis of apoptosis at different time points in Tat - and BSA - treated cells ( Table 3 ) . ||11156964_1385_155871==>Prolonged effect of Tat on CREB Ser - 133 phosphorylation and CREB expression in PC12 neuronal cells . ||11156964_1385_155871==>Levels of Ser - 133 phosphorylated CREB ( P - CREB ) and total CREB were evaluated by Western blot on both whole cell lysates ( cells ) and enriched nuclear fractions ( nuclei ) obtained from PC12 cells , serum starved for 40 h , and treated for the time indicated ( hours ) with Tat ( 1 nM ) . ||11156964_1385_155871==>Densitometric analysis of P - CREB , total CREB and � - tubulin in PC12 cells treated with Tat ( 1 nM ) for up to 48 h a ||11156964_1385_155871==>In fact , after an initial rapid induction of CREB phosphorylation on Ser - 133 , Tat induced a secondary , prolonged down - regulation of CREB expression . ||11156964_1385_155871==>These changes in CREB phosphorylation and expression anticipated and correlated with the bimodal effect of low Tat concentrations on PC12 cell viability : early protection from apoptosis induced by serum withdrawal ( up to 24 h ) , followed by secondary increase of apoptosis ( 72�96 h ) with respect to BSA - treated cells . ||11156964_1385_155871==>Due to its essential role for neuronal cell survival ( 26 , 27 ) and as a mediator of long - term memory ( 50 ) , the Tat - induced down - regulation of CREB expression is expected to have profound detrimental effects on neuronal cell survival and / or function . ||11156964_1385_155871==>Although we can not exclude that the down - regulation of CREB requires internalization and nuclear localization of extracellular Tat protein ( 4 ) , our data strongly suggest that Tat down - modulates CREB gene expression , altering the intracellular levels of cAMP . ||11156964_1385_155871==>Moreover , we have provided a molecular mechanism to explain how low concentrations of extracellular Tat can impair the survival / function of neuronal cells by decreasing the levels of cAMP and the expression of CREB protein . ||11311202_155871_7157==>ScienceDirect - Archives of Oral Biology : Amplification of extracellular matrix and oncogenes in tat - transfected human salivary gland cell lines with expression of laminin , fibronectin , collagens I , III , IV , c - myc and p53 ||11311202_155871_7157==>Amplification of extracellular matrix and oncogenes in tat - transfected human salivary gland cell lines with expression of laminin , fibronectin , collagens I , III , IV , c - myc and p53 ||11311202_155871_7157==>Our preliminary study also demonstrated a decrease in p53 by tat . ||11696595_155945_7185==> Vpu Inhibits the NF - B䤥ependent Expression of the Antiapoptotic Factors Bcl - xL , A1 / Bfl - 1 , and TRAF1 ||11696595_155945_7185==>Cell lysates were analyzed 40 h after infection by immunoblotting to detect expression of Bcl - xL , A1 / Bfl - 1 , TRAF1 , p24 CA , Vpu , or - tubulin . ||11696595_155945_7185==>As for Bcl - xL and A1 / Bfl - 1 , Vpu expression significantly inhibited the TNF - mediated induction of TRAF1 ( fig. 6 B , lane 2 ) . ||11696595_155945_7185==>To assess the physiological relevance of this phenomenon , we analyzed the effect of Vpu on the activation of caspase - 8 , which is regulated by TRAF1 ( 33 ) . ||11696595_155945_7185==>These results confirm that the reduced expression of TRAF1 in Vpu - expressing cells can disturb the equilibrium between pro and antiapoptotic regulators and promote proapoptotic signaling in response to cytokine stimulation . ||11696595_155945_7185==>To validate the results from our inducible CD4U cell lines we examined the effect of Vpu on the expression of antiapoptotic factors such as Bcl - xL , A1 / Bfl - 1 , and TRAF1 in HIV - infected T cells . ||11696595_155945_7185==>In Vpu - expressing cells , the levels of TRAF1 , in response to TNF stimulation , are reduced and no longer sufficient to inhibit the cytokine - induced activation of caspase - 8 ( fig. 7 no . ||12842621_155945_920==>HIV - 1 encodes multiple gene products , Env , Vpu , and Nef , involved in downregulation of CD4 , a major HIV - 1 receptor . ||12842621_155945_920==>We examined the involvement of CD4 downregulation mediated by Vpu and Nef in the modification of virus infectivity . ||12842621_155945_920==>The mutation in vpu increased CD4 incorporation into virions without affecting the Env content in it , inhibiting the attachment step of virions to the CD4 - positive cell surface . ||12842621_155945_920==>Although a single mutation in nef suppresses virus infectivity via a CD4 - independent mechanism , it could augment CD4 incorporation in virions in combination with a vpu mutation . ||12842621_155945_920==>? Vpu - modulated infectivity of virus produced from CD4 - positive cells ||12842621_155945_920==>? A Vpu mutation causes CD4 incorporation into virions ||12842621_155945_920==>In HIV - 1 infections , many reports indicate that the viral regulatory gene products Vpu and Nef also participate in CD4 downregulation , in addition to Env ( Chen et al 1996 , Garcia and Miller 1991 and Strebel et al 1988 ) . ||12842621_155945_920==>Following this finding , it was reported that Vpu shortened the half - life of CD4 molecules , resulting in a reduction of cell - surface CD4 expression in HIV - 1 - infected cells ( Willey et al 1992a ) . ||12842621_155945_920==>Env precursor , gPr160 , is involved in this activity ; Vpu recognizes a gPr160 / CD4 complex formed in the ER membrane ( Bour et al 1995b ) and tags CD4 with ubiquitin ligase , TrCP , directing proteasome - dependent degradation of CD4 ( Margottin et al 1998 and Schubert et al 1998 ) . ||12842621_155945_920==>With vpu and nef mutant viruses , we examined the correlation between their effects on virus infectivity and on modulation of CD4 expression . ||12842621_155945_920==>These results indicated that Vpu affected the total amount of CD4 molecule , and the vpu mutation caused incorporation of CD4 into virions , resulting in impairment of virus infectivity . ||12842621_155945_920==>The amount of Env in virions was not affected by the vpu mutation , suggesting that the reduction of infectivity was due to the inhibition of attachment process caused by Env ? CD4 association on the virion surface . ||12842621_155945_920==> Vpu - modulated infectivity of virus produced from CD4 - positive cells ||12842621_155945_920==>Multiple gene products of HIV - 1 , Vpu , Nef , and Env , act cooperatively in downregulation of CD4 , the major receptor for HIV - 1 infection ( Chen et al 1996 ) . ||12842621_155945_920==>In order to investigate the contribution of CD4 downregulation to virus replication , the viruses harboring frame - shift mutations in vpu ( ? vpu ) , nef ( ? nef ) , and both vpu and nef ( ? vpu / ? nef ) were constructed from an HIV - 1 infectious DNA clone , pNL432 , and their infectivities were examined . ||12842621_155945_920==>Both Vpu and Nef are known to function in modulation of CD4 expression ( Chen et al 1996 ) , suggesting that the phenotype observed with ? vpu / ? nef mutant was a consequence of downregulation of CD4 expression . ||12842621_155945_920==>The infectivity of ? vpu virus obtained from CD4 - negative cells was comparable to that of wildtype . ||12842621_155945_920==>The infectivity of the ? vpu / ? nef virus derived from CD4 - positive cells appeared to be severely impaired . ||12842621_155945_920==>The result of a similar experiment with viruses derived from infected A3.01 cells resembled that of CD4 - positive 293T cells ( fig. 2B ) , indicating that the regulatory effects by vpu and vpu / nef mutations on virus infectivity were dependent upon CD4 expression in producer cells . ||12842621_155945_920==>The CD4 - dependent effect of HIV - 1 ? vpu , ? nef , and ? vpu / ? nef mutants on virion infectivity . ||12842621_155945_920==>Both Vpu and Nef function in the CD4 downregulation , suggesting that the mutations in vpu and / or nef reduce the efficiency of virion release into the culture supernatant . ||12842621_155945_920==>( Bour et al 1999 ) reported that the expression of cell - surface CD4 interfered with Vpu function , resulting in inhibition of virion release . ||12842621_155945_920==>Because 293T cells were used in this report , in which Vpu - dependent enhancement of virion release was inactive , we can not argue that there is an effect of interaction between Vpu and surface CD4 . ||12842621_155945_920==>A Vpu mutation causes CD4 incorporation into virions ||12842621_155945_920==>The reduced infectivity observed with ? vpu and ? vpu / ? nef viruses obtained from CD4 - positive cells might be due to the modification in the composition of viral proteins in the virion . ||12842621_155945_920==>( Lama et al 1999 ) reported that mutations in vpu and / or nef abolished CD4 downregulation , resulting in loss of Env in virion . ||12842621_155945_920==>By using CD4 - expressing 293T cells as the virus producer , although some reduction of Env incorporation in virion was observed with each mutant , there was no significant difference among the mutants , suggesting that it was not responsible for the drastic decrease in infectivity caused by ? vpu and ? vpu / ? nef mutations . ||12842621_155945_920==>These results indicate that the reduction of infectivity found with ? vpu and ? vpu / ? nef viruses from CD4 - positive cells is not due to the consequence of the decreased Env incorporation . ||12842621_155945_920==>Modification of the composition of viral proteins and CD4 by vpu and / or nef mutations . ||12842621_155945_920==>We also examined the effects of vpu and / or nef mutations on the CD4 expression level in producer cells and virions ( fig. 4C ) . ||12842621_155945_920==>With CD4 - expressing 293T cells , cell - associated CD4 was reduced with wildtype and ? nef viruses , but not with ? vpu or ? vpu / ? nef viruses , suggesting that the total amount of CD4 was mainly downregulated by Vpu function . ||12842621_155945_920==> CD4 molecules appeared to be significantly incorporated into ? vpu virions , which were augmented by vpu / nef double mutation . ||12842621_155945_920==> CD4 incorporation into virions by vpu and vpu / nef mutants could be also observed by using A3.01 cells as the producer . ||12842621_155945_920==>These findings suggest that CD4 incorporation into virions contributes to the reduced infectivity of ? vpu and ? vpu / ? nef viruses . ||12842621_155945_920==>On the contrary , in a comparison of attachment efficiencies of the viruses derived from CD4 - positive cells , vpu mutants were significantly reduced in efficiency . ||12842621_155945_920==>suggesting that the regulation of virus infectivity by Vpu function was dependent on CD4 expression in producer cells and that CD4 incorporation into virions had a negative effect on virion infectivity . ||12842621_155945_920==>We found that Vpu function was important for the downregulation of the total amount of CD4 in A3.01 and CD4 - expressing 293T cells and that Nef did not significantly affect CD4 expression ( fig. 4C ) . ||12842621_155945_920==>? vpu , ? nef , and ? vpu / ? nef viruses , however , remarkably lost the effect on surface CD4 expression . ||12842621_155945_920==> Vpu downregulates surface CD4 through the degradation of intracellular CD4 . ||12842621_155945_920==>Because the downregulation of surface CD4 is considered to contribute to the inhibition of superinfection , we examined the inhibitory effects on superinfection of ? vpu , ? nef , and ? vpu / ? nef mutant viruses . ||12842621_155945_920==>The cells infected with a ? vpu / ? nef double mutant virus appeared to allow superinfection to the degree of the control cells ( mock , CD4 - expressing 293T cells ) . ||12842621_155945_920==> CD4 molecules were significantly incorporated when CD4 downregulation was hampered by vpu and / or nef mutations , resulting in reduced attachment efficiency of virions to target cells . ||12842621_155945_920==>Although ( Lama et al 1999 ) reported that vpu and / or nef mutations abolished Env incorporation in exchange for CD4 incorporation in virions , we could not observe the effect on Env incorporation by the mutations . ||12842621_155945_920==> Vpu targets CD4 on the ER for proteasome - dependent degradation , which decreases the total amount of CD4 as shown in fig. 4C ( Bour et al 1995b , Margottin et al 1998 and Schubert et al 1998 ) . ||12842621_155945_920==>Although the effect of Vpu on cell surface CD4 expression was comparable to that of Nef ( fig. 6B ) , the CD4 incorporation into the virion was specific to the ? vpu virus . ||12842621_155945_920==>An early report described that the association between CD4 and gPr160 in the ER attenuated transport of gPr160 to the cytoplasmic membrane and that Vpu rescued Env processing by diminishing CD4 through degradation ( Willey et al 1992b ) , however , our observation was in contrast to that . ||12842621_155945_920==>In CD4 - expressing 293T cells , the vpu mutant did not affect the expression profile of Env in cells or the amount of matured gp120 in the virion , indicating that the association between CD4 and gPr160 did not affect the Env transport to the cell surface , its maturation , or its incorporation into the virion . ||12842621_155945_920==>Although Vpu affects the total amount of CD4 , Nef appears to target the CD4 molecule on the cell surface , which is consistent with previous reports . ||12842621_155945_920==>Although the reduction by the vpu mutation could be due to CD4 incorporation into virions , which by a nef mutation has been reported to be mostly independent of CD4 expression ( Aiken and Trono 1995 and Miller et al 1995 ) . ||12842621_155945_920==> CD4 - dependent regulation of virus infectivity by Nef is also supported by the fact that CD4 incorporation into virions is stimulated by ? vpu / ? nef to a larger extent than by a ? vpu mutation . ||12842621_155945_920==>The regulation of virus infectivity by Nef through CD4 downregulation could be suppressed in the presence of Vpu and may become noticeable in the absence of Vpu . ||12842621_155945_920==>It might be also possible that CD4 - independent enhancement of virus infectivity by Nef is augmented by the ? vpu viruses which are defective for the attachment process . ||12842621_155945_920==>Although it is necessary to further investigate the molecular mechanism of Vpu - and Nef - mediated CD4 regulation and its contribution to virus replication , the results presented in this report raise the possibility that a mutant CD4 molecule resistant to Vpu - and Nef - mediated degradation would be a good candidate for anti - HIV reagents . ||7621073_155871_5243==>Transcription by other Sp1 - dependent promoters , such as MDR1 and the minimal SV40 promoters , is also repressed by Tat , whereas the human - actin promoter is neither activated by Sp1 nor repressed by Tat . enhances=====10400814_155871_5610==>Human Immunodeficiency Virus Type 1 Tat Protein Activates Transcription Factor NF - kappa B through the Cellular Interferon - Inducible , Double - Stranded RNA - Dependent Protein Kinase , PKR - - Demarchi et al. 73 ( 8 ) : 7080 - - The Journal of Virology ||10400814_155871_5610==>Human Immunodeficiency Virus Type 1 Tat Protein Activates Transcription Factor NF - B through the Cellular Interferon - Inducible , Double - Stranded RNA - Dependent Protein Kinase , PKR Francesca Demarchi , 1 Maria Ines Gutierrez , 1 and Mauro Giacca 1 , 2 , * ||10400814_155871_5610==>Here , we show that this activity of Tat requires the function of the cellular interferon - inducible protein kinase PKR . ||10400814_155871_5610==> Tat - mediated NF - B activation and transcriptional induction of the HIV - 1 long terminal repeat were impaired in murine cells in which the PKR gene was knocked out . ||10400814_155871_5610==>Both functions were restored by cotransfection of Tat with the cDNA for PKR . ||10400814_155871_5610==>Expression of a dominant - negative mutant of PKR specifically reduced the levels of Tat transactivation in different human cell types . ||10400814_155871_5610==>Activation of NF - B by Tat required integrity of the basic domain of Tat ; previous studies have indicated that this domain is necessary for specific Tat - PKR interaction . ||10400814_155871_5610==>Given these considerations , we addressed the question of whether the molecular pathway initiated by Tat and leading to the activation of NF - B could be mediated by the activity of PKR . ||10400814_155871_5610==>Mouse NIH 3T3 fibroblasts were transfected with expression plasmids coding for Tat 101 ; for activated Rac , a member of the family of small GTP binding proteins shown to trigger NF - B nuclear translocation ( 37 ) ( the kind gift of Alan Hall ) ; and for PKR , donated by B . ||10400814_155871_5610==>Gel retardation analysis of NF - B complexes induced by Tat in NIH 3T3 and PKR knockout mouse cells . ||10400814_155871_5610==>( B ) Results of transfection of primary mouse PKR 0 / 0 fibroblasts with plasmids expressing Tat 101 ( lane 1 ) , PKR ( lane 2 ) , or both ( lane 3 ) . ||10400814_155871_5610==>The assay was performed with antibodies against the p50 and p65 subunits of NF - B as indicated in the figure and nuclear extracts from PKR 0 / 0 fibroblasts cotransfected with PKR and Tat . ||10400814_155871_5610==>Contrary to what was observed in NIH 3T3 fibroblasts , in Pkr 0 / 0 mouse cells neither Tat nor PKR alone could trigger NF - B activation ( fig. 3 B , lanes 1 and 2 ) , while cotransfection of the two plasmids resulted in a strong synergistic effect ( lane 3 ) , similar to the one obtained by serum addition ( lane 4 ) . ||10400814_155871_5610==>The above results clearly indicate that PKR is involved in Tat - induced activation of NF - B in mouse cells . ||10400814_155871_5610==>For this purpose , we studied the role of Tat and PKR in the activation of transcription from a transfected LTR - CAT cassette in mouse cells lacking a functional PKR as well as in control NIH 3T3 cells . ||10400814_155871_5610==>Under these conditions , Tat considerably enhances the transcription driven by the LTR in NIH 3T3 cells , while PKR does not have any effect ( fig. 4 A ) . ||10400814_155871_5610==>In PKR knockout cells ( fig. 4 B ) the transcriptional effect of Tat is impaired with respect to control cells , but it can be fully restored by cotransfection of a wild - type PKR expression plasmid . ||10400814_155871_5610==> PKR is functionally required for Tat transactivation of the LTR promoter in mouse fibroblasts . ||10400814_155871_5610==>Five micrograms of Tat 101 and / or 5 痢 of PKR expression constructs was cotransfected , as indicated . ||10400814_155871_5610==>Altogether , the data described above demonstrate that the functions of PKR are required for NF - B nuclear translocation and LTR transcription activation by Tat in a murine system . ||10400814_155871_5610==>In all these cell lines , the PKR dominant - negative mutant caused a net decrease in Tat - mediated activation of LTR - driven transcription ( fig. 5 A , B , and C ) . ||10400814_155871_5610==>Dominant - negative PKR impairs LTR activation induced by Tat . ||10400814_155871_5610==>HL3T1 , BF24 , and Jurkat cells ( panels A , B , and C , respectively ) were transfected with 2.5 痢 of pCDNAIII empty vector ( first lanes of each panel ) or with 500 ng of Tat 101 expression vector and 2 痢 of the dominant - negative mutant cDNAs for Ras ( plasmid Ras N17 ) , Rac ( Rac N17 ) , RhoA ( Rho T19N ) , CDC42 ( Cdc42 T17N ) , and PKR ( PKR - M ) as indicated . ||10400814_155871_5610==>The inhibitory effect of the PKR dominant - negative construct on Tat transactivation was mediated in cis by the enhancer region of the LTR . ||10400814_155871_5610==>As shown in fig. 6 , when the vector expressing Tat 101 was cotransfected in HeLa cells together with the PKR dominant - negative construct , transactivation from the wild - type LTR was reduced , while it was unaffected from an LTR promoter bearing the deletion of the NF - B sites ( 30 ) . ||10400814_155871_5610==>An intact LTR enhancer region is required for downregulation of Tat transactivation by dominant - negative PKR . ||10400814_155871_5610==>Altogether the experimental data reported above indicate that PKR is involved in Tat transactivation of the LTR both in rodent and in human cells through the activation of NF - B . ||10400814_155871_5610==>Following different experimental strategies , over the last few years , several laboratories have suggested that a complex interplay exists between Tat , TAR , and PKR , although the results obtained have often been controversial . ||10400814_155871_5610==>Also controversial are the findings that the levels and the activity of PKR are decreased ( 38 ) , increased ( 15 ) , or unaffected ( 34 ) in HIV - 1 - infected and in Tat - expressing cells . ||10400814_155871_5610==>As far as Tat and PKR are concerned , two independent laboratories have recently shown that a physical interaction exists between the two proteins both in vitro and in vivo and that this interaction requires the integrity of the basic domain of Tat ( 12 , 34 ) . ||10400814_155871_5610==>However , it should be pointed out that the same laboratories have indicated , by in vitro studies , that Tat behaves as an inhibitor of PKR activation and as a competitive substrate for phosphorylation , in apparent contradiction with the functional positive role of the PKR - Tat interplay that we are suggesting here . ||10400814_155871_5610==>In this respect , we believe that the conditions for in vitro functional studies of the kinase are likely to be considerably different from those found within the cells , where the relative concentrations of PKR , TAR , Tat , and accessory factors are not easily quantifiable and might vary during the course of infection . ||10400814_155871_5610==>As an alternative explanation , we can not rule out the possibility that the functional requirement of PKR for Tat - mediated NF - B activation , which clearly stems from the set of our experiments , is not directly dependent on the PKR - Tat protein - protein interaction but is mediated by an unidentified intermediate pathway . ||10400814_155871_5610==>The results presented in this work suggest that both Tat 101 , Tat 86 , and Tat 72 ( one exon ; results not shown ) are able to activate NF - B through the PKR pathway , both in rodent and human cells , and that this pathway requires the integrity of the basic domain of the protein . ||10400814_155871_5610==>While it is difficult to rank the relative importance of these mechanisms , we would like to point out that in murine fibroblasts lacking PKR the induction of NF - B nuclear translocation by Tat is almost completely impaired , thus suggesting that , at least in this cell type , the presence of functional PKR is likely to be crucial for this function . ||10400814_155871_5610==>Jeang for the initial suggestion to investigate the role of the Tat - PKR interaction in NF - B induction . ||11956210_155871_2648==>We and others have recently shown that Tat is directly acetylated at lysine 28 , within the activation domain , and lysine 50 , in the TAR RNA binding domain , by Tat - associated histone acetyltransferases p300 , p300 / CBP - associating factor , and hGCN5 . ||11956210_155871_2648==>The other class of Tat co - activators , Tat - associated histone acetyltransferases , are composed of p300 / CBP , p300 / CBP - associating factor ( PCAF ) ( 25 - 27 ) , and hGCN5 ( 28 ) . inactivates=====10921877_155807_983==>The Vpr protein of primate lentiviruses arrests cell cycling at the G 2 / M phase through an inactivation of cyclin B檏34 cdc2 and its upstream regulator cdc25 . ||10921877_155807_995==>The Vpr protein of primate lentiviruses arrests cell cycling at the G 2 / M phase through an inactivation of cyclin B檏34 cdc2 and its upstream regulator cdc25 . ||10921877_155807_995==>We provide here biochemical and functional evidence demonstrating that human immunodeficiency virus type 1 ( HIV - 1 ) Vpr mediates G 2 arrest by forming a complex with protein phosphatase 2A ( PP2A ) , an upstream regulator of cdc25 . ||10921877_155807_995==>Interestingly , we found that Vpr association with B55 - containing PP2A targets the enzymatic complex to the nucleus and , importantly , enhances the recruitment and dephosphorylation of the cdc25 substrate . ||10921877_155807_995==>Our data suggest that Vpr mediates G 2 arrest by enhancing the nuclear import of PP2A and by positively modulating its catalytic activity towards active phosphorylated nuclear cdc25 . ||10958988_155807_983==>In this study , we present direct evidence of genetic suppression of Vpr - induced G2 arrest by cdc2 mutations . ||10958988_155807_983==>Mutations in cdc2 ( cdc2 - 1w and cdc2 - 3w ) reduce the ability of Vpr to induce G2 arrest . ||10958988_155807_983==>A strain with a mutation changing the Tyr15 of Cdc2 to the non - phosphorylated Phe ( Y15F ) eliminated Vpr - induced G2 arrest indicating that Tyr15 of Cdc2 is the sole target for induction of G2 arrest by Vpr . ||10958988_155807_983==>Interestingly , Vpr still induces cell death and morphological changes in the Y15F Cdc2 strain indicating that G2 arrest is not required for morphological changes and cell death . ||10958988_155807_983==> Vpr - induced G2 arrest correlates with hyperphosphorylation of Cdc2 and inactivation of its kinase activity ||10958988_155807_983==> cdc2 Mutations suppress Vpr - induced G2 arrest ||10958988_155807_983==>Preventing phosphorylation of Tyr15 on Cdc2 eliminates Vpr - induced G2 arrest ||10958988_155807_983==>Mutations in Vpr affect cell death similarly in wild - type and Y15F Cdc2 strains ||10958988_155807_983==>As to the mechanism of G2 arrest , there is abundant evidence in human cells that Vpr induces G2 arrest by inhibiting the cyclin B dependent kinase Cdc2 . ||10958988_155807_983==>The activity of the Cdc2 kinase decreases when vpr is expressed in human cells ( He and Re ) , and immunoblot analysis shows that the phosphorylated form of Cdc2 , which migrates slower than the dephosphorylated form on a polyacrylamide gel ( Hayles and Norbury ) increases in human cells when vpr is expressed ( He ; Jowett ; Norbury and Re ) . ||10958988_155807_983==>Expression of the nonphosphorylatable A14T F15Y mutation of Cdc2 overcomes the G2 arrest indicating that Vpr induces G2 arrest in human cells by preventing dephosphorylation of Thr14 and Tyr15 on Cdc2 ( He et al. , 1995 ) . ||10958988_155807_983==> Vpr - induced G2 arrest in fission yeast correlates with hyperphosphorylation of Cdc2 just as it does in human cells ( Zhao et al. , 1996 ) . ||10958988_155807_983==>First , does Vpr induce G2 arrest in fission yeast exclusively through phosphorylation of Tyr15 on Cdc2 ? The observation that a slower migrating form of Cdc2 increases when vpr is expressed in fission yeast is consistent with the idea that phosphorylation of Tyr15 is responsible for the G2 arrest . ||10958988_155807_983==>Immunoblotting for Cdc2 and Vpr has been described previously ( Zhao et al. , 1996 ) . ||10958988_155807_983==> Vpr - induced G2 arrest correlates with hyperphosphorylation of Cdc2 and inactivation of its kinase activity ||10958988_155807_983==>We and others have reported that Vpr induces hyperphosphorylation of the cyclin - dependent kinase Cdc2 in fission yeast and human cells ( He ; Jowett ; Re ; Rogel and Zhao ) . ||10958988_155807_983==>Studies in human cells further showed that the Cdc2 kinase activity is also inhibited during the induction of G2 arrest by Vpr . ||10958988_155807_983==>To determine whether Vpr has a similar inhibitory effect in fission yeast cells , the hyperphosphorylation of Cdc2 was measured in parallel with assays of Cdc2 kinase activity . ||10958988_155807_983==>A gradual decrease in Cdc2 kinase activity was observed starting at 18 h after the switch to minus thiamine media when Vpr was first detected ( fig. 1 A ) . ||10958988_155807_983==>Correlating with this inhibition of Cdc2 kinase by Vpr , a significant increase in the ratio of phosphorylated to dephosphorylated Cdc2 was also detected at these time points ( fig. 1 B ) . ||10958988_155807_983==>G2 arrest induced by Vpr requires Tyr15 phosphorylation of Cdc2 . ||10958988_155807_983==>( B ) Immunoblot analyses of protein extracts prepared after transfer to thiamine - plus ( vpr - repressing ) and - minus ( vpr - expressing ) media with anti - Cdc2 and anti - Vpr . ||10958988_155807_983==> cdc2 Mutations suppress Vpr - induced G2 arrest ||10958988_155807_983==>The vpr gene was expressed in two of the non - conditional cdc2 mutants ( cdc2 - 1w and cdc2 - 3w ) to test whether Vpr induces G2 arrest by increasing the phosphorylation levels of Tyr15 on Cdc2 . ||10958988_155807_983==>The cdc2 - 1w and cdc2 - 3w strains partially suppressed Vpr - induced G2 arrest ( fig. 1 C ) . ||10958988_155807_983==>In contrast to Vpr in wild - type cells where most ( 83.6�3.5 % ) of the G1 cells in vpr - repressing culture shifted to the G2 phase of the cell cycle upon induction of vpr , only 40.5�1.4 % and 55.4�0.8 % ( n =3 ) of the G1 cells in the vpr - repressing cdc2 - 1w and cdc2 - 3w cells shifted to the G2 phase ( fig. 1 C ) . ||10958988_155807_983==>Preventing phosphorylation of Tyr15 on Cdc2 eliminates Vpr - induced G2 arrest ||10958988_155807_983==>The cdc2 - 1w and cdc2 - 3w alleles are partially but not completely resistant to inhibitory phosphorylation of Tyr15 which probably explains why they only partially suppress Vpr - induced G2 arrest . ||10958988_155807_983==>To confirm that phosphorylation of Tyr15 is completely and exclusively responsible for Vpr - induced G2 arrest , we examined the effect of Vpr in a strain with the Y15F Cdc2 mutation . ||10958988_155807_983==>Since Vpr and DNA damage ( Nurse , 1997 ) both induce G2 arrest through phosphorylation of Tyr15 on Cdc2 , Vpr might induce G2 arrest through the DNA damage pathway . ||10958988_155807_983==>Under these same conditions , a wild - type cdc2 strain has an 8.2�0.9 % colony forming ability so Vpr kills Y15F cells at least as efficiently as wild - type cdc2 cells even though Vpr can not induce G2 arrest in the Y15F strain . ||10958988_155807_983==>Under vpr - repressing conditions , Y15F cells show the normal pattern of intense staining of the septa and weaker uniform staining of growing cell walls and have the short , nearly spherical , cells due to the premature mitosis caused by the overactive Y15F Cdc2 kinase ( fig. 3 A , left panel ) ( Gould and Nurse , 1989 ) . ||10958988_155807_983==>There was an unusually heavy streak of the cdc2 + H78R strain on the vpr - on plate which gives the appearance of a small amount of growth , but this was not seen on repetitions of this experiment . ||10958988_155807_983==>Mutations in Vpr affect cell death similarly in wild - type and Y15F Cdc2 strains ||10958988_155807_983==>Point mutations in Vpr can affect the G2 arrest and cell killing activities of Vpr differently when tested in a wild - type Cdc2 yeast strain ( Chen et al. , 1999 ) . ||10958988_155807_983==>Cell cycle analysis by flow cytometry of the Y15F Cdc2 strain carrying the Vpr point mutations showed patterns similar to Cdc2F15Y in fig. 1 C ( data not shown ) indicating that the Vpr mutations did not induce G2 arrest in this strain just as was seen for wild - type Vpr . ||10958988_155807_983==>These four mutant vpr genes were then compared for their ability to kill yeast cells with either a wild - type or a Y15F Cdc2 kinase as a further test of the relationship between G2 arrest and cell killing . ||10958988_155807_983==>Thus , identical patterns of cell killing were seen for the Vpr mutations in the wild - type ( Cdc2 + ) and Y15F Cdc2 strains in this qualitative experiment . ||10958988_155807_983==>In both the wild - type ( Cdc2 + ) and the Y15F Cdc2 strains , the F34I mutation showed reduced cell killing , E25K / N28D showed a slight reduction in cell killing , and the H78R and R88K mutations killed cells nearly as efficiently as the wild - type Vpr ( Chen et al. , 1999 ) . ||10958988_155807_983==>The results presented here are direct evidence that Vpr induces G2 arrest in fission yeast exclusively by increasing the phosphorylation of Tyr15 on Cdc2 . ||10958988_155807_983==>Consistent with the effects of these mutations , Vpr expression causes hyperphosphorylation of Cdc2 and decreased kinase activity ( fig. 1 A ? B ; Zhao et al. , 1996 ) . ||10958988_155807_983==>These results all support the conclusion that Vpr induces G2 arrest in fission yeast solely by increasing the phosphorylation levels of Tyr15 on Cdc2 . ||10958988_155807_983==>Thus , Vpr induces G2 arrest by increasing the levels of inhibitory phosphorylations on Cdc2 both in human and fission yeast cells . ||10958988_155807_983==>G2 arrest induced by Vpr resembles that induced by DNA damage in that both proceed through the phosphorylation of Tyr15 on Cdc2 ( Nurse , 1997 ) . ||10958988_155807_983==>The Y15F Cdc2 strain and Vpr point mutations indicate that G2 arrest is not required for cell killing and morphological changes . ||10958988_155807_983==>In the Y15F Cdc2 strain where there is no induction of G2 arrest , Vpr still induces cell killing and morphological changes . ||10958988_155807_983==>Studies of twelve Vpr point mutations , previously examined in human cells ( Selig et al. , 1997 ) , showed that the mutations often had opposite effects on G2 arrest and cell killing in wild - type Cdc2 fission yeast ( Chen et al. , 1999 ) . ||10958988_155807_983==>Thus , even when G2 arrest was reduced or eliminated both by the H78R or R88K mutation in Vpr and by the Y15F Cdc2 yeast mutation , Vpr still kills the yeast cells . ||10958988_155807_983==>This failure to induce G2 arrest is expected since the mechanism by which Vpr induces G2 arrest , increased Tyr phosphorylation of Cdc28 / Cdc2 , does not lead to G2 arrest in budding yeast . ||10958988_155807_983==>This study demonstrates another similarity in that Vpr induces G2 arrest both in human and fission yeast cells through inhibitory phosphorylation of Cdc2 . ||11531413_155807_983==>Viral protein R ( Vpr ) of human immunodeficiency virus type 1 induces G2 arrest in cells from distantly related eukaryotes including human and fission yeast through inhibitory phosphorylation of tyrosine 15 ( Tyr15 ) on Cdc2 . ||11531413_155807_995==>ScienceDirect - Virology : HIV - 1 Vpr Induces Cell Cycle G2 Arrest in Fission Yeast ( Schizosaccharomyces pombe ) through a Pathway Involving Regulatory and Catalytic Subunits of PP2A and Acting on Both Wee1 and Cdc25 ||11531413_155807_995==>HIV - 1 Vpr Induces Cell Cycle G2 Arrest in Fission Yeast ( Schizosaccharomyces pombe ) through a Pathway Involving Regulatory and Catalytic Subunits of PP2A and Acting on Both Wee1 and Cdc25 ||11531413_155807_995==>Both Wee1 and Cdc25 play a role in Vpr - induced G2 arrest since a wee1 deletion reduces Vpr - induced G2 arrest and a direct in vivo assay shows that Vpr inhibits Cdc25 . ||11531413_155807_995==>Additional support for both Wee1 and Cdc25 playing a role in Vpr - induced G2 arrest comes from a genetic screen , which identified genes whose overexpression affects Vpr - induced G2 arrest . ||11531413_155807_995==>Overexpression of the wos2 gene , an inhibitor of Wee1 , suppresses Vpr - induced G2 arrest while overexpression of rad25 , an inhibitor of Cdc25 , enhances Vpr - induced G2 arrest . ||11531413_155807_995==>These two genes may be part of the uncharacterized pathway for Vpr - induced G2 arrest in which Vpr upregulates PP2A to activate Wee1 and inhibit Cdc25 . ||11878934_155807_983==> Vpr 's ability to arrest cells at the G2 phase of the cell cycle is due to the inactivation of p34 cdc2 cyclin B complex , resulting in hypophosphorylation of substrates involved in cell - cycle progression from G2 to mitosis ( M ) . ||12110603_155807_983==>The Vpr protein of primate lentiviruses arrests cell cycling at the G 2 / M phase through an inactivation of cyclin B檏34 cdc2 and its upstream regulator cdc25 . ||12110603_155807_995==>The Vpr protein of primate lentiviruses arrests cell cycling at the G 2 / M phase through an inactivation of cyclin B檏34 cdc2 and its upstream regulator cdc25 . ||12110603_155807_995==>We provide here biochemical and functional evidence demonstrating that human immunodeficiency virus type 1 ( HIV - 1 ) Vpr mediates G 2 arrest by forming a complex with protein phosphatase 2A ( PP2A ) , an upstream regulator of cdc25 . ||12110603_155807_995==>Interestingly , we found that Vpr association with B55 - containing PP2A targets the enzymatic complex to the nucleus and , importantly , enhances the recruitment and dephosphorylation of the cdc25 substrate . ||12110603_155807_995==>Our data suggest that Vpr mediates G 2 arrest by enhancing the nuclear import of PP2A and by positively modulating its catalytic activity towards active phosphorylated nuclear cdc25 . incorporates=====11932435_155030_3308==>Of the HIV - 1 genes , gag was found to be sufficient for Hsp70 incorporation , though Hsp70 was roughly equimolar with pol - encoded proteins in virions . ||11932435_155030_3308==>To check whether VLPs formed by HIV - 1 Gag incorporate Hsp70 in the absence of other viral proteins , we PCR amplified and cloned a previously described gag cDNA ( that was modified to be Rev independent ) ( 31 ) into mammalian expression vector pEF / myc / cyto ( Invitrogen ) such that it was in - frame with a myc tag at the carboxyl terminus . ||11932435_155030_3308==>Since MLV does not incorporate Hsp70 , we also cloned MLV gag into the same expression vector as a negative control . ||11932435_155030_3308==>We found that HIV - 1 Gag is sufficient for the incorporation of Hsp70 ( fig. 4C , lane 1 ) . ||11932435_155030_3308==>Even though MLV Gag is well expressed and forms VLPs just as efficiently as HIV - 1 Gag , it does not incorporate Hsp70 ( fig. 4C , lane 2 ) , consistent with the fact that infectious MLV virions do not incorporate Hsp70 ( fig. 3C ) . ||11932435_155030_3308==> Gag is sufficient for Hsp70 incorporation into HIV - 1 virions . ||11932435_155030_3308==>Both the stress - inducible protein Hsp70 and its constitutive form , Hsc70 , interact with various viral proteins and may be involved in the assembly of adenovirus ( 21 ) , enterovirus ( 22 ) , and polyomavirus capsid protein complexes ( 8 ) . ||11932435_155030_3308==>Hence , Hsp70 and Hsc70 could bind to nascent HIV - 1 Gag polyprotein chains and hold them in an assembly - competent conformation during transport to the plasma membrane . ||11932435_155030_3308==>For example , Hsp70 might actively uncoat the viral capsid in a manner similar to its role in the uncoating of clathrin cages ( 6 ) . ||11932435_155030_3329==>Type D retrovirus Gag polyprotein interacts with the cytosolic chaperonin TRiC . ||12670953_10309_155348==>In some cases host proteins , such as the integrase inhibitor 1 ( Ini1 ) factor or the uracil DNA glycosylase ( UNG2 ) enzyme , interact with IN and are specifically incorporated into viral particles ( 21 , 22 ) . ||12670953_10309_155348==>These data indicate that IN promoted UNG2 packaging in the context of the Gag - Pol polyprotein but not in the context of a Vpr弒N fusion . ||12670953_10309_155348==>In trans - complementation assays , we observed that host UNG2 could not be packaged by the Vpr弒N fusion despite being clearly dependent on the presence of IN in the context of the Gag - Pol precursor . ||12670953_10309_155348==>First , the folding of IN processed from the Vpr fusion protein may not accurately reflect the folding of IN processed from the Gag - Pol precursor , leading to differential bindings of UNG2 . ||12670953_10309_155348==>Second , although the IN domain of the Gag - Pol precursor is required for UNG2 packaging , it may not be sufficient . ||12670953_10309_155348==>It may be that a triple interaction between UNG2 and both the reverse transcriptase and IN domains of the Gag - Pol precursor is necessary to allow the efficient packaging of UNG2 . ||12670953_10309_155348==>We have not been able to draw definite conclusions about the importance of UNG2 for HIV - 1 replication , but we have found an interesting mutant of IN that could be rescued by allowing a higher concentration of the very same mutant integrase in the viral particle . ||12670953_10309_155348==>1 The abbreviations used are : HIV - 1 , human immunodeficiency virus , type 1 ; IN , integrase ; IN , IN minus ; CA , capsid ; LTR , long terminal repeat ; UNG2 , uracil DNA glycosylase ; GST , glutathione S - transferase ; TCID 50 , 50 % tissue culture infective dose . ||9882380_155807_7374==>We have previously reported that Vpr associates with the DNA repair enzyme uracil DNA glycosylase ( UDG ) . ||9882380_155807_7374==>In this study , we extended these observations by investigating whether UDG is incorporated into virions and whether this incorporation requires the presence of Vpr . ||9882380_155807_7374==>Our results , with highly purified viruses , show that UDG is efficiently incorporated either into wild - type virions or into Vpr - deficient HIV - 1 virions , indicating that Vpr is not involved in UDG packaging . ||9882380_155807_7374==>We have previously reported , in a study using the yeast two - hybrid system , that Vpr binds to the DNA repair enzyme uracil DNA glycosylase ( UDG ) ( 4 ) . ||9882380_155807_7374==>There exists therefore the possibility that Vpr - associated UDG of primate lentiviruses and dUTPase of nonprimate lentiviruses have similar roles in virus replication . ||9882380_155807_7374==>It is therefore of interest to address whether UDG is incorporated into viral particles and whether the requirement for Vpr is related to such a localization . ||9882380_155807_7374==>Packaging of UDG into viral particles is independent of the presence of Vpr . ||9882380_155807_7374==>To investigate the incorporation of UDG into virions and to test whether Vpr is required in this process , we used wild - type NDK and Vpr mutant NDK virions derived from the productively infected H9 T - cell line . ||9882380_155807_7374==>Unexpectedly , we observed that gradient fractions from Vpr mutant virions also contain UDG ( fig. 1 b , right panel ) . ||9882380_155807_7374==> UDG was also detected in both wild - type and Vpr mutant virions , in two independent experiments , when wild - type AD8 and Vpr mutant AD8 virions derived from productively infected primary macrophages ( 29 ) were analyzed ( data not shown ) . ||9882380_155807_7374==>These results suggest that UDG is indeed a virion - associated protein and that the incorporation of UDG into viral particles may not depend on the presence of packaged Vpr . ||9882380_155807_7374==>Packaging of UDG into viral particles is independent of the presence of Vpr . ||9882380_155807_7374==>Our above results indicating that UDG is packaged within virions through a Vpr - independent mechanism led us to investigate which viral protein is required to incorporate UDG into viral particles . ||9882380_155807_7374==>Individual gradient fractions were then ultracentrifuged and analyzed by Western blotting with anti - p24 , anti - Vpr , and anti - UDG antibody ( fig. 2 ) . ||9882380_155807_7374==>As a control , Pr55 Gag and Vpr overexpressed in cell lysate are shown , as well as endogenous expression of UDG . ||9882380_155807_7374==>We failed to detect the presence of incorporated UDG in gradient fractions containing both Pr55 Gag and Vpr , however . ||9882380_155807_7374==>These results indicate that the gag gene products are probably not important for the incorporation of UDG within virions and confirm that Vpr is not required in this event . ||9882380_155807_7374==>Packaging of UDG into viral particles is independent of the presence of Pr55 Gag and Vpr . ||9882380_155807_7374==>Cell - free supernatant containing virus - like particles was subjected to a linear sucrose density gradient , and individual gradient fractions were collected and analyzed by Western blotting for the presence of Pr55 Gag and Vpr , as well as for the presence of packaged UDG . ||9882380_155807_7374==>As a control , HeLa cell extracts were analyzed by Western blotting for the presence of Pr55 Gag , Vpr , and endogenous UDG . ||9882380_155807_7374==>The cDNA encoding the complete UDG protein ( residues 1 to 304 ) was amplified with appropriate primers by PCR from clone p15 ( 25 ) , kindly provided by G . ||9882380_155807_7374==>GST , GST - Vpr , and GST - UDG ( 52 - 304 ) fusion proteins were prepared as previously described ( 4 ) . ||9882380_155807_7374==>Cell lysate overexpressing UDG was incubated with equivalent amounts of GST , GST - IN , or GST - Vpr , and bound proteins were revealed by Western blotting with an anti - UDG antibody ( fig. 3 b ) . ||9882380_155807_7374==>Binding analysis indicated that the precursor form of UDG preferentially associates with GST - IN , while the two forms of UDG associate with GST - Vpr . ||9882380_155807_7374==>( a ) Cell lysate overexpressing IN was incubated with equivalent amounts of GST , GST - UDG ( 1 - 304 ) , GST - UDG ( 52 - 304 ) , or GST - Vpr fusion proteins affinity purified on GSH - agarose beads . ||9882380_155807_7374==>( b ) Cell lysate overexpressing UDG was incubated with equivalent amounts of GST , GST - IN , or GST - Vpr fusion proteins affinity purified on GSH - agarose beads , and bound proteins were analyzed by Western blotting with rabbit polyclonal anti - UDG antibody . ||9882380_155807_7374==>To demonstrate unequivocally that the IN domain is required for the incorporation of UDG into virions , and that Vpr is not involved in this process , we investigated whether IN mutant viruses or IN - Vpr double mutant viruses are impaired in incorporation of UDG into viral particles . ||9882380_155807_7374==>As expected , the incorporation of UDG into viral particles was observed when wild - type as well as Vpr - defective viruses were analyzed . ||9882380_155807_7374==>Interestingly , viruses defective for the presence of IN but still expressing Vpr in viral particles did not incorporate detectable amounts of UDG . ||9882380_155807_7374==>A similar defect in UDG incorporation into virions was observed when viruses used in the experiments were defective in both IN and Vpr proteins . ||9882380_155807_7374==>Altogether , our data indicate that the IN domain is sufficient for packaging of UDG into virions and that Vpr is not involved in this process . ||9882380_155807_7374==>When virions ( wild type , Vpr defective , IN defective , or Vpr - IN defective ) corresponding to 3 � 10 6 cpm of reverse transcriptase activity were incubated with the uracil - containing substrate , no UDG activity was detected , although the presence of UDG protein is detectable in wild - type and Vpr - defective virions . ||9882380_155807_7374==>We initially reported that UDG has the ability to bind Vpr ( 4 ) . ||9882380_155807_7374==>Although Vpr is incorporated into viral particles , the question arises as to why the intravirion packaging of UDG is not related to the presence of Vpr in virions but to the presence of IN . ||9882380_155807_7374==>It is possible that during assembly and budding , the interaction between Vpr and NCp7 impairs the binding of UDG to Vpr via steric hindrance . ||9882380_155807_7374==>Experiments to test whether UDG , Vpr , and NCp7 can associate as a trimeric complex would resolve this issue . ||9882380_155807_7374==>The other explanation invokes the idea that UDG could bind alternatively Vpr or IN depending on its maturation status . ||9882380_155807_7374==>Consistent with this , our in vivo and in vitro data indicate that IN has the ability to bind the cytoplasmic precursor form of UDG ( this study ) , while Vpr has the ability to bind preferentially the mature form of UDG , as we previously reported with coimmunoprecipitation experiments ( 4 ) . ||9882380_155807_7374==>It is noteworthy that residues 1 to 52 of UDG are important for binding of IN ( this study ) , while residues 222 to 225 have been reported to be important for binding of Vpr ( 5 ) . ||9882380_155807_7374==>We speculate that the pre - mature form of UDG is cleaved after entry in new cells , and then the mature form may interact with Vpr . ||9882380_155807_7374==>The fact that HIV - 1 viruses have evolved by elaborating two viral proteins , IN and Vpr , which both have the ability to bind UDG argues for the importance of these interactions during the viral life cycle . ||9882380_155807_7374==>What then are the respective roles , during the viral life cycle , of the Vpr - UDG and IN - UDG interactions ? We have previously proposed that the Vpr - UDG interaction might play a role similar to that played by the dUTPases of nonprimate lentiviruses , i.e. , the reduction of uracil misincorporation into newly synthesized viral DNA in order to avoid G - to - A substitutions ( 4 ) . ||9882380_155807_7374==>Therefore , one possibility is that the Vpr - UDG and / or IN - UDG interactions participate in the nuclear import of the viral PIC . ||9882380_155807_7374==>It would thus be interesting to determine whether UDG can be found , through its association with Vpr and / or IN , to be an integral part of the PIC . induces=====10393859_155871_4843==>Inhibition of endogenous NO production with the NO synthase ( NOS ) inhibitor L - NMMA causes a significant increase in Tat - induced NF - B activity . ||10393859_155871_4843==>To determine how NO is induced by HIV Tat , reverse transcription polymerase chain reaction was used to demonstrate the induction of NOS - 2 and NOS - 3 mRNA by Tat . ||10393859_155871_4843==>Rather , several Sp - 1 sites adjoining the putative NF - B binding site in the promoter region of NOS - 3 gene are required for the induction of NOS - 3 gene expression by Tat . ||10393859_155871_4843==>The induction of NO by Tat was reduced by L - NMMA , an inhibitor of NOS . ||10393859_155871_4843==> Tat induces NO production that can be blocked by the NOS inhibitor L - NMMA . ||10393859_155871_4843==>15 - 23 In the present study , we observed that L - NMMA , a competitive inhibitor of NOS , enhanced Tat - induced NF - B activity at each time point selected in both the luciferase reporter gene activity assay and the gel shift assay ( Figure 3 , A and B ) . ||10393859_155871_4843==>To further define the role of NO on the inhibition of Tat - induced NF - B activation , we determined the level of NF - B induction by Tat in macrophages from both wild - type and NOS - 2 gene knockout mice . ||10393859_155871_4843==>In NOS - 2 gene knockout macrophages , Tat induced a sustained activation of NF - B , whereas the NO induction by Tat was marginal . ||10393859_155871_4843==>A : Luciferase reporter assay of RAW264.7 cells co - transfected with a 2 x B luciferase reporter and expression vector for Tat in the absence or presence of 200 痠ol / L NOS inhibitor L - NMMA for 6 , 24 , or 48 hours . ||10393859_155871_4843==> Tat induces sustained NF - B activation in NOS - 2 gene knockout macrophages . ||10393859_155871_4843==> Tat Up - Regulated Transcription of NOS - 2 and NOS - 3 Genes ||10393859_155871_4843==>To confirm our observation described above that Tat induced NO production by macrophages , RT - PCR assays were performed to evaluate the induction of NOS - 2 and NOS - 3 mRNA by Tat . ||10393859_155871_4843==>As indicated in Figure 7 , both NOS - 2 and NOS - 3 are induced in the mouse macrophage cell line RAW264.7 cells by Tat transfection . ||10393859_155871_4843==>We have also observed the induction of NOS - 2 and NOS - 3 mRNA by Tat in phorbol myristate acetate ( PMA ) - primed Jurkat T cells and in the THP - 1 human monocyte cell line as well ( data not shown ) . ||10393859_155871_4843==>It has been suggested that Tat induced NOS - 2 mRNA expression through the activation of NF - B , as this nuclear transcription factor plays a crucial role in the transcription of NOS - 2 . ||10393859_155871_4843==> Tat induces NOS - 3 ( eNOS ) and NOS - 2 ( iNOS ) mRNA expression . ||10393859_155871_4843==>Total RNA ( 1 痢 ) isolated from vector ( V ) or Tat ( T ) transfected RAW264.7 cells was used for RT - PCR analysis using specific primers for NOS - 3 or NOS - 2 ( upper panel ) and GAPDH ( lower panel ) as indicated in Materials and Methods . ||10393859_155871_4843==>Nuclear proteins used in the EMSA were extracted from Tat - transfected cells in the absence or presence of NOS inhibitor L - NMMA for 6 or 18 hours . ||10393859_155871_4843==>The results in Figure 10 provide further insight into which of the candidate response elements in the proximal 100 - bp promoter region are functional or nonfunctional in regulating expression of NOS - 3 gene by Tat . ||10393859_155871_4843==>These results demonstrate that the Sp - 1 sites in this proximal region are necessary for the induction of NOS - 3 gene expression by Tat . ||10393859_155871_4843==>The role of the putative NF - B site in the induction of NOS - 3 by Tat , however , appears less important due to the binding of p50 / p50 homodimer , but not p50 / p65 heterodimer , at this site . ||10393859_155871_4843==>CAT activity assay of RAW264.7 cells transfected with a plasmid containing wild - type or mutated NOS - 3 proximal promoter region ( 116 bp ) in the presence of an empty vector ( basal ) or expression vector for Tat . ||10393859_155871_4843==>Our results also show that Tat can induce both NOS - 2 and NOS - 3 mRNA synthesis and hence increase NO production . ||10393859_155871_4843==>To that end , our results provide suggestive evidence that Tat can indeed induce NOS - 2 and NOS - 3 mRNA expression . ||10393859_155871_4843==>These findings are supported by those of Hayman et al who demonstrated the neutralizing effect of a NOS inhibitor on the neurotoxicity of synthetic Tat protein . ||10393859_155871_4843==>It is not surprising therefore that Tat can also promotes NOS - 2 and NOS - 3 mRNA synthesis . ||10393859_155871_4843==>13 , 14 Both the HIV envelope glycoprotein gp120 and the regulatory protein Tat are considered responsible for the induction of NO through the activation of NOS - 2 or NOS - 3 genes . ||10393859_155871_4846==> Tat induces NOS - 3 ( eNOS ) and NOS - 2 ( iNOS ) mRNA expression . ||11160671_1387_155871==>In our experiments , we observed an increase of p65 and CBP , stably binding to the IL - 8 NF - B site in extracts made from Tat - expressing cells at 3 h post - Hu release , coincident with the appearance of IL - 8 mRNA . ||11160671_1387_155871==>It will be of interest to determine if Tat transiently facilitates the association of p65 and CBP during S phase . ||11160671_1387_155871==>Along these lines , Tat has been shown to interact both physically and functionally with p300 and CBP in activation of the HIV long terminal repeat ( 1 , 6 , 15 ) . ||11160671_1387_155871==>Alternatively , Tat may simply enhance the assembly of a stable NF - B complex containing p65 and CBP . ||12167619_155871_4843==>The present study was undertaken to investigate the effect of the HIV - 1 tat gene on the expression of inducible nitric - oxide synthase ( iNOS ) in human U373MG astroglial cells and primary astroglia . ||12167619_155871_4843==>Expression of the tat gene as RSV - tat but not that of the CAT gene as RSV - CAT in U373MG astroglial cells led to the induction of NO production and the expression of iNOS protein and mRNA . ||12167619_155871_4843==>The induction of human iNOS promoter - derived luciferase activity by the expression of RSV - tat suggests that RSV - tat induces the transcription of iNOS . ||12167619_155871_4843==>Activation of NF - B as well as C / EBP by RSV - tat , stimulation of RSV - tat - induced production of NO by the wild type of p65 and C / EBP , and inhibition of RSV - tat - induced production of NO by p65 , a dominant - negative mutant of p65 , and C / EBP , a dominant - negative mutant of C / EBP , suggest that RSV - tat induces iNOS through the activation of NF - B and C / EBP . ||12167619_155871_4843==>This study illustrates a novel role for HIV - 1 tat in inducing the expression of iNOS in human astrocytes that may participate in the pathogenesis of HIV - associated dementia . ||12167619_155871_4843==>We report herein that the HIV - 1 tat gene induces the production of NO and the expression of iNOS through the activation of NF - B and C / EBP in human astrocytes . ||12167619_155871_4843==>Assay of iNOS Promoter - derived Reporter Activity - - Cells plated at 50 - 60 % confluence in 6 - well plates were cotransfected with 0.5 痢 of phiNOS ( 7.2 ) Luc 2 and different amounts of either RSV - tat or RSV - CAT by LipofectAMINE Plus ( Invitrogen ) following the manufacturer 's protocol ( 22 - 24 ) . ||12167619_155871_4843==>Expression of RSV - tat but Not RSV - CAT Induces the Expression of iNOS in Human U373MG Astroglial Cells - - To study the effect of HIV - 1 Tat on the expression of iNOS , we transfected human astroglial cells transiently with HIV - 1 tat gene . ||12167619_155871_4843==>The inhibition of NO production by arginase , an enzyme that degrades the substrate ( L - arginine ) of NOS , and L - NMA , a competitive inhibitor of NOS , but not by D - NMA , a negative control of L - NMA , suggests that RSV - tat induced the production of NO in U373MG astroglial cells through NOS - mediated arginine metabolism ( Table I ) . ||12167619_155871_4843==>To understand the mechanism of NO production , we examined the effect of RSV - tat and RSV - CAT on protein and mRNA levels of iNOS . ||12167619_155871_4843==>However , marked expression of iNOS protein ( fig. 1 B ) and mRNA ( fig. 1 C ) was observed in cells transfected with different doses of RSV - tat . ||12167619_155871_4843==>Similar to the induction of NO production , the expression of iNOS protein and mRNA by RSV - tat was also dose - dependent . ||12167619_155871_4843==>The induction of iNOS protein was maximal in cells transfected with 0.2 痢 ( per well of 6 - well plate ) of RSV - tat ( fig. 1 B ) , whereas the expression of iNOS mRNA was maximal in cells transfected with 0.8 痢 ( 100 - mm dish ) of RSV - tat ( fig. 1 C ) . ||12167619_155871_4843==>Expression of RSV - tat induces the production of NO and the expression of iNOS protein in human U373MG astroglial cells . ||12167619_155871_4843==>Therefore , to understand whether Tat protein is secreted from RSV - tat - transfected astroglial cells and whether Tat protein is in fact responsible for the induction of iNOS , we incubated RSV - tat - transfected cells with anti - Tat antibodies . ||12167619_155871_4843==>Taken together , these observations suggest that the induction of iNOS in RSV - tat - transfected astroglial cells is due to Tat and that Tat protein is secreted from RSV - tat - transfected astroglial cells . ||12167619_155871_4843==>Expression of RSV - tat Induces the Production of NO in Human Primary Astroglia - - Human primary astrocytes have been shown to induce the expression of iNOS in the presence of different proinflammatory cytokines ( 23 , 24 ) . ||12167619_155871_4843==>RSV - tat Induces Human iNOS Promoter - derived Luciferase Activity in Human U373MG Astroglial Cells - - To understand the effect of the tat gene on the transcription of iNOS , U373MG glial cells were cotransfected with phiNOS ( 7.2 ) Luc , a construct containing the human iNOS promoter fused to the luciferase gene , 2 and either RSV - tat or RSV - CAT . ||12167619_155871_4843==>It is evident from fig. 4 that transfection of cells with different amounts of RSV - tat but not RSV - CAT led to the induction of iNOS promoter - derived luciferase activity . ||12167619_155871_4843==>About 3.7 - fold activation of iNOS promoter - derived luciferase activity was observed in cells transfected with 0.2 痢 of RSV - tat ( fig. 4 ) . ||12167619_155871_4843==>Therefore , the observed low induction of 7.2 - kb human iNOS promoter by RSV - tat in human U373MG astroglial cells is probably due to the absence of extra NF - B - and AP - 1 - binding sites . ||12167619_155871_4843==>Expression of RSV - tat induces iNOS promoter - derived luciferase activity in human U373MG astroglial cells . ||12167619_155871_4843==>Cells plated in 6 - well plates were cotransfected with 0.5 痢 of phiNOS ( 7.2 ) Luc ( a construct containing the human iNOS promoter fused to the luciferase gene ) and different amounts of either RSV - CAT or RSV - tat . ||12167619_155871_4843==>Role of NF - B and C / EBP in RSV - tat - mediated Induction of iNOS in U373MG Astroglial Cells - - The presence of NF - B DNA - binding sites in the promoter of iNOS ( 34 - 36 ) and the inhibition of expression of iNOS by the inhibitors of NF - B suggests that NF - B plays an important role in the expression of iNOS ( 22 - 24 , 26 - 28 , 31 , 36 ) . ||12167619_155871_4843==>2 These findings prompted us to ask whether activation of NF - B and C / EBP may be responsible for the induction of iNOS following HIV - 1 tat gene expression in U373MG astroglial cells . ||12167619_155871_4843==>The evidence presented in this manuscript that RSV - tat but not RSV - CAT ( the control plasmid ) induced the production of NO and the expression of iNOS , that anti - Tat antibodies blocked RSV - tat - induced production of NO , and that recombinant Tat protein also induced the production of NO clearly support the conclusion that Tat induces the expression of iNOS in human astrocytes . ||12167619_155871_4843==>In light of these findings , our observations suggest that Tat may also contribute to the expression of iNOS observed in astroglia of HAD brains . ||12167619_155871_4843==>However , Barton et al. ( 49 ) have reported that HIV - 1 Tat inhibits interferon - - induced iNOS activity in murine macrophages . ||12167619_155871_4843==>Therefore , Tat may regulate the expression of iNOS differentially in astrocytes and macrophages . ||12167619_155871_4843==>Our results have shown clearly that the activation of NF - B is important for the expression of iNOS in RSV - tat - transfected astrocytes . ||12167619_155871_4843==>The results presented in this article clearly demonstrate that the activation of C / EBP is also essential for RSV - tat - mediated induction of iNOS in human astrocytes . ||12167619_155871_4843==>Taken together , our studies suggest that Tat activates ERK2 , which ultimately couples to the activation of C / EBP but not to NF - B for the induction of iNOS . ||12167619_155871_4843==>Further studies are under way to delineate the Tat - induced signaling pathway ( s ) that couples with NF - B for the induction of iNOS . ||12167619_155871_4843==>Astroglia are the major glial cell population in the CNS , and therefore , induction of iNOS in astroglia by Tat may be an important source of NO in HAD associated with astrogliosis and neuronal death . inhibits=====11940654_155871_3065==> HDAC1 recruitment and histone hypoacetylation were also seen when Tat activation was inhibited by the overexpression of YY1 . ||11940654_155871_3065==>Conversely , exposure of cells to the histone deacetylase inhibitor trichostatin A or activation of LTR expression by HIV - 1 Tat results in the displacement of HDAC1 from nuc 1 , in association with increased acetylation of histone H4 . ||11940654_155871_3065==>Recruitment of HDAC1 to the LTR nuc 1 can counteract Tat activation and repress LTR expression . ||11940654_155871_3065==>Further , we found that the HIV - 1 activator Tat decreased HDAC1 occupancy , while the LTR host repressor YY1 could overcome this effect , decreasing H4 acetylation at nuc 1 and increasing HDAC1 occupancy . ||11940654_155871_3065==>Transcriptional activation of LTR by Tat correlates with disassociation of HDAC1 from nuc 1 and hyperacetylation of histone H4 at nuc 1 . ||11940654_155871_3065==>We hypothesized that similar counterregulatory changes in HDAC1 occupancy and acetylated histone H4 quantity might occur upon LTR activation by viral factor Tat . ||11940654_155871_3065==>Changes of histone H4 acetylation and HDAC1 occupancy at nuc 1 correlate with Tat activation of LTR . ||11940654_155871_3065==>( C ) ChIP assays showed increased acetylation of histone H4 and decreased HDAC1 occupancy at nuc 1 of LTR upon Tat activation in GFP - positive cells . ||11940654_155871_3065==>Since disassociation of HDAC1 and hyperacetylation of histone H4 at nuc 1 accompany LTR activation by TSA or Tat , repression of LTR expression might correlate with increased HDAC1 occupancy and deacetylation of histone H4 at nuc 1 . ||11940654_155871_3065==>Interaction of YY1 and HDAC1 in vivo is required for repression of Tat by YY1 . ||11940654_155871_3065==>Overexpression of wild - type YY1 inhibits Tat activation and results in increased HDAC1 occupancy and decreased acetylation of histone H4 at nuc 1 ( fig. 6 , lane B compared with lane C ) . ||11940654_155871_3065==>As we have previously described , a glycine / alanine - rich domain mutant of YY1 incapable of HDAC1 interaction was unable to repress LTR activation by Tat ( fig. 6 , lane D ) . ||11940654_155871_3065==>Cell extracts were prepared from GFP - positive cells cotransfected with empty CMV vector , Tat , Tat plus wild - type YY1 , and Tat plus mutant YY1 lacking the HDAC1 interaction domain . ||11940654_155871_3065==>We have shown that expression of the viral activator Tat is associated with histone acetylation at nuc 1 and downregulation of HDAC1 occupancy . ||11940654_155871_3065==>Further , we have identified an inverse relationship between acetylation of histone H4 and HDAC1 occupancy upon activation at LTR by Tat or repression by YY1 . ||11940654_155871_3065==>These observations not only confirm that the transcriptional activation is associated with remodeling of nuc 1 by acetylation but suggest the possibility that histone acetyltransferases or other factors recruited to LTR by Tat may interact with HDAC1 , YY1 , or both and may regulate their functions or interaction . ||11940654_155871_3065==>While both TSA and Tat activation result in the loss of the repression and decreased detection of HDAC1 in ChIP assay , further study is required to define the mechanism ( s ) by which TSA and Tat decrease HDAC1 occupancy . ||11940654_155871_7024==> LSF occupancy did not change at the LTR after Tat activation ( fig. 3D ) . ||11940654_155871_7024==>( D ) Overexpression of Tat did not affect LSF occupancy at nuc 1 . ||11940654_155871_7528==>Counterregulation of Chromatin Deacetylation and Histone Deacetylase Occupancy at the Integrated Promoter of Human Immunodeficiency Virus Type 1 ( HIV - 1 ) by the HIV - 1 Repressor YY1 and HIV - 1 Activator Tat - - He and Margolis 22 ( 9 ) : 2965 - - Molecular and Cellular Biology ||11940654_155871_7528==>Counterregulation of Chromatin Deacetylation and Histone Deacetylase Occupancy at the Integrated Promoter of Human Immunodeficiency Virus Type 1 ( HIV - 1 ) by the HIV - 1 Repressor YY1 and HIV - 1 Activator Tat Guocheng He 1 and David M . ||11940654_155871_7528==>HDAC1 recruitment and histone hypoacetylation were also seen when Tat activation was inhibited by the overexpression of YY1 . ||11940654_155871_7528==>Further , we found that the HIV - 1 activator Tat decreased HDAC1 occupancy , while the LTR host repressor YY1 could overcome this effect , decreasing H4 acetylation at nuc 1 and increasing HDAC1 occupancy . ||11940654_155871_7528==>) , CMV - YY1 ( 46 ) , and CMV - Tat were purified by using an Endofree plasmid kit ( Qiagen , Valencia , Calif . ||11940654_155871_7528==>The overexpression of dnLSF has been shown to relieve YY1 - mediated repression of Tat - activated LTR expression ( 12 , 44 ) . ||11940654_155871_7528==>Interaction of YY1 and HDAC1 in vivo is required for repression of Tat by YY1 . ||11940654_155871_7528==>We compared CAT activity in a HeLa LTR - CAT line cotransfected with GFP alone , GFP plus Tat , GFP plus wild - type YY1 and Tat , and GFP plus mutant YY1 and Tat . ||11940654_155871_7528==>Overexpression of wild - type YY1 inhibits Tat activation and results in increased HDAC1 occupancy and decreased acetylation of histone H4 at nuc 1 ( fig. 6 , lane B compared with lane C ) . ||11940654_155871_7528==>As we have previously described , a glycine / alanine - rich domain mutant of YY1 incapable of HDAC1 interaction was unable to repress LTR activation by Tat ( fig. 6 , lane D ) . ||11940654_155871_7528==>That the acetylation of histone H4 in the presence of Tat and mutant YY1 did not increase ( fig. 6 , lane B compared with lane D ) could be due to incomplete competition of the transfected YY1 mutant with endogenous wild - type YY1 . ||11940654_155871_7528==>Cell extracts were prepared from GFP - positive cells cotransfected with empty CMV vector , Tat , Tat plus wild - type YY1 , and Tat plus mutant YY1 lacking the HDAC1 interaction domain . ||11940654_155871_7528==>Further , we have identified an inverse relationship between acetylation of histone H4 and HDAC1 occupancy upon activation at LTR by Tat or repression by YY1 . ||11940654_155871_7528==>These observations not only confirm that the transcriptional activation is associated with remodeling of nuc 1 by acetylation but suggest the possibility that histone acetyltransferases or other factors recruited to LTR by Tat may interact with HDAC1 , YY1 , or both and may regulate their functions or interaction . ||12154097_10524_155871==>In the case of Tip60 and TAF II 250 , Tat seems to inhibit their HAT activity and consequently modulate the expression of cellular genes ( 5 , 6 ) . ||12154097_10524_155871==> Tip60 , whose HAT activity was previously shown to be inhibited by Tat ( 6 ) , was used as a control . ||12154097_10524_155871==>fig. 1 shows that GST - Tat , but not GST , greatly reduced the HAT activity not only of Tip60 but also of GCN5 and CBP ( Autorad panels ) . ||12154097_10524_155871==> Tat interferes with the HAT activity of Tip60 , GCN5 , and CBP in vitro . ||12154097_10524_155871==>In vitro HAT assays were performed by incubating purified total histones ( 0.33 � M ) and ( 14 C ) acetyl - CoA , with recombinant Tip60 ( left ) , GCN5 ( middle ) , or CBP ( right ) and with 450 n M recombinant purified GST or increasing amounts of GST - Tat : 150 or 300 n M ( + and + + , respectively ) . ||12154097_10524_155871==>The present study strengthens this hypothesis by showing the inhibitory effect of Tat on the histone acetyltransferase activity of three specific HATs : Tip60 , CBP , and GCN5 . ||12154097_155871_8850==>When it interacts with P / CAF , p300 / CBP and GCN5 , an increase in the transactivator potential of Tat on the HIV - 1 LTR has been observed . ||12154097_155871_8850==>Indeed , GCN5 and p300 / CBP can acetylate Tat on its lysines 50 ( Lys 50 ) and 51 ( Lys 51 ) while P / CAF acetylates the viral protein on its lysine 28 ( Lys 28 ) . ||12154097_155871_8850==>The acetylation of Lys 28 of Tat by P / CAF enhances its interaction with the positive transcription elongation factor complex b ( 8 ) . ||12154097_155871_8850==>Moreover , it has recently been shown that the acetylation of Lys 50 of Tat creates a strong binding site for the P / CAF bromodomain ( 12 , 13 ) . ||12154097_155871_8850==>In this case , the bromodomain of P / CAF effectively competes with the TAR RNA and helps the removal of Tat from TAR . ||12154097_155871_8850==>From these experiments we conclude that Tat is capable of inhibiting the activity of CBP , P / CAF , and GCN5 in vitro as well as in vivo . ||12154097_155871_8850==> Tat interferes with the transcriptional activation by CBP , P / CAF , and GCN5 in vivo . ||12154097_155871_8850==>The Repression of Histone Acetylation by Tat Is Not Required for the Tat - dependent Activation of Transcription from HIV - 1 5 ' LTR - - The in vivo Gal4 targeting test was also used to evaluate the role of the Tat C - terminal domain in interfering with the activities of CBP , P / CAF , and GCN5 . ||12154097_155871_8850==>However , for P / CAF and GCN5 , the in vivo results approached those obtained in vitro , since the M1 mutant of Tat was not as efficient as the full - length protein in repressing the activity of these enzymes ( fig. 6 A , compare lanes 9 and 14 with lanes 8 and 13 , respectively ) . ||12154097_155871_8850==>For instance , in the cases of p300 / CBP and P / CAF , E1A not only modulates histone acetylation ( 24 - 28 ) but also , like what was reported here for HIV - 1 Tat , induces a substrate selectivity ( 27 , 29 ) . ||14630801_155871_2833==>Migration of V { delta } 1 and V { delta } 2 T cells in response to CXCR3 and CXCR4 ligands in healthy donors and HIV - 1 - infected patients : competition by HIV - 1 Tat - - Poggi et al. 103 ( 6 ) : 2205 - - Blood ||14630801_155871_2833==>Migration of V 1 and V 2 T cells in response to CXCR3 and CXCR4 ligands in healthy donors and HIV - 1㷽nfected patients : competition by HIV - 1 Tat Alessandro Poggi , Roberta Carosio , Daniela Fenoglio , Sabrina Brenci , Giuseppe Murdaca , Maurizio Setti , Francesco Indiveri , Silvia Scabini , Elisabetta Ferrero , and Maria Raffaella Zocchi ||14630801_155871_2833==>In the present work , we show that ( 1 ) in HIV - 1㷽nfected patients , a population of V 1 T cells coexpressing CXCR4 and CXCR3 is increased in peripheral blood ; ( 2 ) V 1 and V 2 T - cell subsets transmigrate in response to the homeostatic or inflammatory chemokines , IP - 10 / CXCL10 and SDF - 1 / CXCL12 , according to the expression of their specific receptors CXCR3 or CXCR4 ; ( 3 ) IP - 10 / CXCL10� and 6Ckine / CCL21 - induced transmigration are dependent on PI - 3K and CAMKII , activated on the ligation of CXCR3 , whereas SDF - 1 / CXCL12 is dependent only on PI - 3K ; ( 4 ) HIV - 1 Tat , present in the serum of HIV - 1㷽nfected patients , interferes with the chemotactic activity of IP - 10 / CXCL10 , I - 309 / CCL1 , and SDF - 1 / CXCL12 because of the cysteine - rich domain of the protein , which contains CXC and CC chemokine馪ike sequences . ||14630801_155871_2833==>Thus , Tat can downregulate CXCR3 - and CXCR4 - mediated functions . ||14630801_155871_2833==>In the present work , we show that ( 1 ) V 1 T cells are increased in the peripheral blood of HIV - 1㷽nfected patients and express CXCR3 , CXCR4 , or both ; ( 2 ) transendothelial migration driven by CXC and CC chemokines are inhibited by HIV - 1 Tat in the serum of HIV - 1㷽nfected patients ; ( 3 ) transmigration of these cells , obtained from healthy donors , in response to CXCR3 - specific chemokines is dependent on CAMKII in addition to PI - 3K . ||14630801_155871_3627==>In the present work , we show that ( 1 ) in HIV - 1㷽nfected patients , a population of V 1 T cells coexpressing CXCR4 and CXCR3 is increased in peripheral blood ; ( 2 ) V 1 and V 2 T - cell subsets transmigrate in response to the homeostatic or inflammatory chemokines , IP - 10 / CXCL10 and SDF - 1 / CXCL12 , according to the expression of their specific receptors CXCR3 or CXCR4 ; ( 3 ) IP - 10 / CXCL10� and 6Ckine / CCL21 - induced transmigration are dependent on PI - 3K and CAMKII , activated on the ligation of CXCR3 , whereas SDF - 1 / CXCL12 is dependent only on PI - 3K ; ( 4 ) HIV - 1 Tat , present in the serum of HIV - 1㷽nfected patients , interferes with the chemotactic activity of IP - 10 / CXCL10 , I - 309 / CCL1 , and SDF - 1 / CXCL12 because of the cysteine - rich domain of the protein , which contains CXC and CC chemokine馪ike sequences . ||14630801_155871_3627==>Figure 7 shows that transmigration induced by IP - 10 / CXCL10 on V 2 ( Figure 7A , white columns ) or by SDF - 1 / CXCL12 on V 1 T lymphocytes ( Figure 7B , white columns ) was strongly inhibited by wild - type Tat 24 - 51 and by the same peptide that conserves the CXC sequence but is mutated in the CC region ( Tat30 - 31Ala ) ; conversely , no inhibition of the migration induced by the 2 CXC - chemokines was observed when Tat24 - 51 peptide mutated in the CXC sequence ( Tat25 - 27Ala ) was used ( Figure 7A - B ) . ||14630801_155871_6346==>In the present work , we show that ( 1 ) in HIV - 1㷽nfected patients , a population of V 1 T cells coexpressing CXCR4 and CXCR3 is increased in peripheral blood ; ( 2 ) V 1 and V 2 T - cell subsets transmigrate in response to the homeostatic or inflammatory chemokines , IP - 10 / CXCL10 and SDF - 1 / CXCL12 , according to the expression of their specific receptors CXCR3 or CXCR4 ; ( 3 ) IP - 10 / CXCL10� and 6Ckine / CCL21 - induced transmigration are dependent on PI - 3K and CAMKII , activated on the ligation of CXCR3 , whereas SDF - 1 / CXCL12 is dependent only on PI - 3K ; ( 4 ) HIV - 1 Tat , present in the serum of HIV - 1㷽nfected patients , interferes with the chemotactic activity of IP - 10 / CXCL10 , I - 309 / CCL1 , and SDF - 1 / CXCL12 because of the cysteine - rich domain of the protein , which contains CXC and CC chemokine馪ike sequences . ||14630801_155871_6346==>Indeed , the early production of Tat , which is present in the serum and which inhibits the chemokine - driven transmigration of T cells in these patients , may counteract the effects of chemokines released in response to viral infection , such as I - 309 / CCL1 , and may impair the recruitment of cells with antiviral properties . ||14630801_155871_7852==>Migration of V { delta } 1 and V { delta } 2 T cells in response to CXCR3 and CXCR4 ligands in healthy donors and HIV - 1 - infected patients : competition by HIV - 1 Tat - - Poggi et al. 103 ( 6 ) : 2205 - - Blood ||14630801_155871_7852==>Migration of V 1 and V 2 T cells in response to CXCR3 and CXCR4 ligands in healthy donors and HIV - 1㷽nfected patients : competition by HIV - 1 Tat Alessandro Poggi , Roberta Carosio , Daniela Fenoglio , Sabrina Brenci , Giuseppe Murdaca , Maurizio Setti , Francesco Indiveri , Silvia Scabini , Elisabetta Ferrero , and Maria Raffaella Zocchi ||14630801_155871_7852==>In the present work , we show that ( 1 ) in HIV - 1㷽nfected patients , a population of V 1 T cells coexpressing CXCR4 and CXCR3 is increased in peripheral blood ; ( 2 ) V 1 and V 2 T - cell subsets transmigrate in response to the homeostatic or inflammatory chemokines , IP - 10 / CXCL10 and SDF - 1 / CXCL12 , according to the expression of their specific receptors CXCR3 or CXCR4 ; ( 3 ) IP - 10 / CXCL10� and 6Ckine / CCL21 - induced transmigration are dependent on PI - 3K and CAMKII , activated on the ligation of CXCR3 , whereas SDF - 1 / CXCL12 is dependent only on PI - 3K ; ( 4 ) HIV - 1 Tat , present in the serum of HIV - 1㷽nfected patients , interferes with the chemotactic activity of IP - 10 / CXCL10 , I - 309 / CCL1 , and SDF - 1 / CXCL12 because of the cysteine - rich domain of the protein , which contains CXC and CC chemokine馪ike sequences . ||14630801_155871_7852==>HIV - 1 Tat has been reported to function as an antagonist for CXCR4 . ||14630801_155871_7852==>Thus , Tat can downregulate CXCR3 - and CXCR4 - mediated functions . ||14630801_155871_7852==>In the present work , we show that ( 1 ) V 1 T cells are increased in the peripheral blood of HIV - 1㷽nfected patients and express CXCR3 , CXCR4 , or both ; ( 2 ) transendothelial migration driven by CXC and CC chemokines are inhibited by HIV - 1 Tat in the serum of HIV - 1㷽nfected patients ; ( 3 ) transmigration of these cells , obtained from healthy donors , in response to CXCR3 - specific chemokines is dependent on CAMKII in addition to PI - 3K . ||14630801_155871_7852==>Xiao H , Neuveut C , Tiffany HL , et al. Selective CXCR4 antagonism by Tat : implications for in vivo expansion of coreceptor use by HIV - 1 . ||7621073_155871_6667==>Transcription by other Sp1 - dependent promoters , such as MDR1 and the minimal SV40 promoters , is also repressed by Tat , whereas the human - actin promoter is neither activated by Sp1 nor repressed by Tat . ||7621073_155871_6667==>These data demonstrate that Tat differentially affects Sp1 - responsive promoters , depending on promoter architecture . ||8764062_155871_1810==>Interaction of human immunodeficiency virus type 1 Tat with a unique site of TFIID inhibits negative cofactor Dr1 and stabilizes the TFIID - TFIIA complex - - Kashanchi et al. 70 ( 8 ) : 5503 - - The Journal of Virology ||9560267_155807_983==>Similar effects are seen in the absence of Vpr with a kinase - deficient Cdc2 , and overexpression of p300 increases levels of HIV Vpr + replication . ||9560267_155807_983==>The HIV - CAT , RSV - Rel A , CMV - Vpr , CMV - Vpr ( ATG ) , CMV - Cdc2 ( K ) , CMV - p300 , pBS - 3'p300 , WT 12S E1A , p300 12S E1A , and CMV - Vpr mutant expression plasmids have all been described ( 15 , 19 , 20 , 21 , 22 ) . ||9560267_155807_983==> Vpr has been shown to cause growth arrest through its ability to inhibit Cdc2 kinase activity . ||9560267_155807_983==>Cyclin B1嵩dc2 , p300 , and Rel A are associated in the same complex , which is not affected by Vpr overexpression , and effect of kinase - deficient Cdc2 on p300 - dependent HIV transcription . ||9560267_155807_983==>Because Vpr is thought to exert its effect on growth arrest by inhibition of Cdc2 kinase activity and cyclin B1嵩dc2 was found in association with p300 , we next determined whether a kinase - deficient mutant of Cdc2 , Cdc2 ( K ) , could affect HIV transcriptional activation by p300 . ||9560267_155807_983==>Overexpression of Cdc2 ( K ) stimulated HIV - CAT activity , suggesting that direct inhibition of Cdc2 has the same effect on HIV transcription as does indirect Cdc2 inhibition by Vpr . ||9560267_155807_983==>Cotransfection of optimal amounts of Vpr and Cdc2 ( K ) provided no synergistic activation , and this effect was reduced in the absence of the B sites , as seen with Vpr ( data not shown ) , suggesting saturation of an activation pathway common to Vpr and Cdc2 . ||9560267_155807_983==>Thus , the inhibition of p300 - associated Cdc2 activity enhances transcriptional activation by Rel A and p300 , and it is likely that Vpr functions through this mechanism . ||9560267_155807_983==>The mechanism by which Vpr inhibits Cdc2 activity is not completely understood ; however , the Cdc25 protein that activates Cdc2 is inactive in Vpr - expressing cells ( 5 ) , suggesting that proteins which normally control Cdc2 are modulated by Vpr , which in turn affects HIV transcription . ||9560267_155807_983==>Our experiments with the Vpr E25K mutant , which is impaired for nuclear localization but retains the ability to arrest cells , suggest that Vpr regulates Cdc2 - regulatory proteins that reside in the cytoplasm of the cell and further support an indirect role for Vpr in Cdk modulation , which interacts with p300 in the nucleus to affect NF - B transcription . interacts with=====11238447_155908_8021==>Together , our data demonstrate that nuclear export of Rev can be reproduced in a cell - free system provided that the egg extract is prepared without the actin - binding drug cytochalasin B . ||11238447_155908_8021==>In this context it is also important to note that a series of recent studies indeed demonstrated the direct participation of CAN / nup214 , nup153 , and nup98 in the nuclear export of Rev and Rev - mediated viral mRNA export ( Bogerd et al. 1998 ; Ullman et al. 1999 ; Zolotukhin and Felber 1999 ) . ||11238447_155908_8021==>Our findings that ( a ) actin is located at the inner pore fibrils , ( b ) actin interacts in vitro with the export factor eIF - 5A and with the nucleoporin nup62 , and ( c ) Rev and TAP - mediated nuclear export of viral RNAs as well as nuclear export of PKI can be inhibited by introducing either antiactin antibodies or the actin - binding drug latrunculin B into the cell nucleus , indicate a novel and unexpected role for nuclear actin . ||11531413_155807_7465==>ScienceDirect - Virology : HIV - 1 Vpr Induces Cell Cycle G2 Arrest in Fission Yeast ( Schizosaccharomyces pombe ) through a Pathway Involving Regulatory and Catalytic Subunits of PP2A and Acting on Both Wee1 and Cdc25 ||11531413_155807_7465==>HIV - 1 Vpr Induces Cell Cycle G2 Arrest in Fission Yeast ( Schizosaccharomyces pombe ) through a Pathway Involving Regulatory and Catalytic Subunits of PP2A and Acting on Both Wee1 and Cdc25 ||11531413_155807_7465==>Both Wee1 and Cdc25 play a role in Vpr - induced G2 arrest since a wee1 deletion reduces Vpr - induced G2 arrest and a direct in vivo assay shows that Vpr inhibits Cdc25 . ||11531413_155807_7465==>Additional support for both Wee1 and Cdc25 playing a role in Vpr - induced G2 arrest comes from a genetic screen , which identified genes whose overexpression affects Vpr - induced G2 arrest . ||11531413_155807_7465==>Overexpression of the wos2 gene , an inhibitor of Wee1 , suppresses Vpr - induced G2 arrest while overexpression of rad25 , an inhibitor of Cdc25 , enhances Vpr - induced G2 arrest . ||11531413_155807_7465==>These two genes may be part of the uncharacterized pathway for Vpr - induced G2 arrest in which Vpr upregulates PP2A to activate Wee1 and inhibit Cdc25 . ||12642036_155807_6667==>Interestingly , Wang and co - workers ( 29 ) demonstrated that Vpr associated with Sp1 in the context of the G / C box array , but not with either Sp1 or the cis - acting elements alone . ||12719574_155459_5718==>As seen before , there were no obvious differences between wild - type and vif virions in p55 Gag , CA , MA , RT , IN , or gp120 ( 1 , 9 , 17 , 22 , 39 , 44 , 47 , 67 ) . ||12719574_155459_5718==>Of note , contrary to some reports ( 6 , 56 ) but consistent with others ( 9 , 17 , 22 , 39 , 44 , 47 ) , there was no apparent vif - related defect in the processing of the p55 Gag polyprotein . ||12719574_155459_5718==>In particular , the sizes and quantities of the p55 Gag - derived proteins MA , CA , and NC were normal in vif virions . ||12719574_155459_5718==>Importantly , this RP - HPLC analysis provided further evidence that the processing of p55 Gag is normal in vif virions , since the ratios of the p55 Gag derivatives MA , CA , NC , p6 , and p1 were similar in wild - type and vif virions . ||12719574_155459_5718==>Comparison of the fate of the labeled p55 Gag molecules clearly showed that the rate of processing of p55 Gag to CA in both the producer cells ( upper panels ) and virions ( lower panels ) is unaffected by the presence or absence of Vif . ||12719574_155459_5718==>In addition , the kinetics of p55 Gag release from infected cells was very similar for wild - type and vif viruses , suggesting that budding is normal for the vif mutant . ||12719574_155459_5718==>Our finding that cleavage of the p55 Gag polyprotein in HIV - 1 / vif virions occurs with normal kinetics ( fig. 6 ) and generates normal mature Gag proteins ( fig. 3 to 6 ) is in agreement with a number of steady - state analyses ( 9 , 17 , 22 , 39 , 44 , 47 ) and a previous kinetic analysis ( 39 ) but stands opposed to several other reports of aberrant p55 Gag processing ( 6 , 56 ) . ||12719574_155459_5718==>The reason for this discrepancy is not immediately obvious , but the weight of evidence now disfavors the idea that the absence of Vif results in reduced p55 Gag processing . ||12719574_155459_5718==> Vif and the p55 Gag polyprotein of human immunodeficiency virus type 1 are present in colocalizing membrane - free cytoplasmic complexes . ||14554087_155908_4928==>In yeast two - hybrid screens of a human lymph node derived cDNA expression library , we identified the human nucleoporin Nup98 as a highly specific and potent interactor of the Rev NES . ||14554087_155908_4928==>Author Keywords : HIV - 1 Rev ; Nup98 ; Rab ; REBP ; CRM1 ; Cofactor ; RNA export ||14554087_155908_4928==>? Effects of wt and mutant Nup98 protein overexpression on Rev function in 293T and HeLa cells ||14554087_155908_4928==>We report here the results of an extensive series of experiments that identify human Nup98 as a particularly potent interactor of the Rev NES and a physiologically relevant component of Rev - dependent RNA transport . ||14554087_155908_4928==>Three clones encoding the amino - terminal ~500 residues of human Nup98 , which interacted strongly and specifically with the Rev NES , were obtained . ||14554087_155908_4928==>As shown in Table 1 , Nup98 failed to interact with nonspecific baits such as human foamy virus ( HFV ) Bel1 residues 56 ? 227 and HIV - 1 Tat residues 48 ? 101 , but reacted strongly with both Rev 59 ? 116 and full - length Rev ( residues 2 ? 116 ) . ||14554087_155908_4928==>When a panel of Rev NES mutants ( Venkatesh et al. , 1990 , Venkatesh et al. , 2003 and Venkatesh and Chinnadurai , 1990 ) was tested for Nup98 reactivity , functionally defective NES mutants Rev 59 ? 73 , Rev 59 ? 116 / 78 ? 79s , and Rev 59 ? 116 / 81s failed to interact , whereas functionally positive NES mutants Rev 59 ? 98 , Rev 59 ? 116 / 76 ? 77s , and Rev 59 ? 116 / 80s showed evidence of strong reactivity . ||14554087_155908_4928==>In addition , Nup98 also interacted strongly with the functionally equivalent HTLV - 1 Rex NES ( but not with a functionally inactive mutant , ? LSLD , thereof ) and EIAV Rev NES regions . ||14554087_155908_4928==>These differences may account for the particularly strong reactivity of Nup98 ( three - to fivefold greater than Rab / hRIP ) with the Rev NES observed in the yeast two - hybrid assay . ||14554087_155908_4928==>Only - galactosidase activities indicated in a given column are comparable as the reactivity of Rev with Nup98 is three - to fivefold greater than with Rab / hRIP . ||14554087_155908_4928==>We next investigated the requirement of the yeast homolog of the human Rev NES export factor , CRM1 , for interaction of the Rev NES with the kinesin - like nuclear cofactor REBP ( Venkatesh et al. , 2003 ) and the nucleoporin cofactors , Rab / hRIP ( Bogerd et al. , 1995 and Fritz et al. , 1995 ) and Nup98 . ||14554087_155908_4928==>For this purpose , we examined the reactivity of wt and functionally inactive Rev NESs with REBP as well as Rab / hRIP and Nup98 in the Saccharomyces cerevisiae strain W303 ( expressing wt Crm1p ) or in W303 strains carrying viable missense mutations in the Crm1 gene , crm1 - 1 and crm1 - 2 ( Neville et al. , 1997 and Yan et al. , 1998 ) in a two - hybrid protein interaction - based lac Z reporter assay ( Table 2 ) . ||14554087_155908_4928==>Since Rev has been shown to be functional in RRE RNA transport in yeast and in view of the high level of conservation between the s. cerevisiae and human CRM1 proteins ( ~48 % identity and ~57 % similarity ) , these results suggest that Crm1p mutations that strongly affect Rev NES interaction of certain nucleoporins such as the XXFG repeat - containing Rab / hRIP have relatively modest effects on the interaction of other NES cofactors such as REBP and the GLFG repeat - containing Nup98 . ||14554087_155908_4928==>To investigate the role of various Nup98 motifs / domains in the Rev export function , we constructed influenza hemagglutinin ( HA ) - tagged fusion genes ( in the mammalian expression vector pcDNA3 - HA ) , encoding the amino - terminal GLFG repeat - containing or the carboxy - terminal unique regions , inclusive of or without the putative NLS , and tested their properties with respect to subcellular localization and effects on RRE RNA transport . ||14554087_155908_4928==>Effects of wt and mutant Nup98 protein overexpression on Rev function in 293T and HeLa cells ||14554087_155908_4928==>To determine the contribution of various Nup98 domains to HIV RNA transport , we next examined the effects of overexpression of wt and mutant Nup98 proteins on Rev - mediated RRE RNA transport in human cells . ||14554087_155908_4928==>Interestingly , nuclear export of the spliced Rev - encoding mRNA species from pRev - g : wt was unaffected by overexpression of wt Nup98 or its mutants , being comparable to that expressed from the pRev - g : ? 78 ? 79 vector . ||14554087_155908_4928==>Effect of wt and mutant Nup98 expression on Rev function . ||14554087_155908_4928==>These results clearly demonstrate that overexpression of the Rev NES - interacting GLFG - repeat region of Nup98 as well as wt Nup98 in 293T cells ( that support pCDNA3 expression vector replication ) results in pronounced suppression of Rev - dependent unspliced mRNA export . ||14554087_155908_4928==>The initial characterization of Rab / hRIP as a nucleoporin cofactor for the leucine - rich nuclear export signals of functionally homologous retroviral Rev proteins in yeast two - hybrid screens of human cDNA expression libraries led to the subsequent identification of a number of nucleoporins of the XXFG , FXFG , and GLFG classes , including Nup98 ( Fritz and Green , 1996 and Stutz et al. , 1996 ) , in random analyses of known FG - repeat containing nuclear pore proteins , as potential targets for Rev NES interaction during nucleocytoplasmic transport . ||14554087_155908_4928==>Results from our detailed analyses of the Rev NES ? hNup98 interaction offer new insights into the properties of this intriguing nucleoporin and argue persuasively in support of a physiological role for Nup98 in Rev - mediated nucleocytoplasmic transport of RRE - containing HIV RNAs , as previously proposed ( Zolotukhin and Felber , 1999 ) . ||14554087_155908_4928==>Thus , an initial high - affinity interaction with Nup98 may target the Rev exportasome to the inner NPC ; subsequently , a series of variable affinity interactions ( including relatively lower affinity interactions such as with Rab / hRIP ) may serve to propel the export complex through the NPC . ||14554087_155908_4928==>Interestingly , recent studies suggest that Nup98 is a mobile nucleoporin , moving between the nuclear interior and the NPC as well as between the nucleus and the cytoplasm ( Griffis et al. , 2002 ) ; this observation raises the possibility that the Rev ? Nup98 complex alone may display potential , albeit limited , for RRE RNA export . ||14554087_155908_4928==>However , our recent studies indicate that a Rev 1 ? 116 ( M10 ) ? Nup98 fusion protein is incapable of promoting RRE RNA export under conditions where a Rev 1 ? 116 ( M10 ) ? CRM1 fusion protein mediates efficient nuclear export of such RNAs ( l. Li and L.K . ||14554087_155908_4928==>Despite the high degree of homology ( ~50 % ) in the amino acid sequences of the CRM1 proteins of s. cerevisiae and man , we observed that missense crm1 mutations in yeast that strongly abrogated the Rab / hRIP ? Rev NES interaction effected only a marginal reduction in the extent of Rev interaction with Nup98 or the kinesin - like cofactor REBP . ||14554087_155908_4928==>Explanations for this observation include possibilities that the Rev ? Nup98 interaction is direct , that regions of yeast Crm1p other than those affected by the crm1 mutations participate in Nup98 interaction , or that distinct cofactors mediate the Nup98 ? Rev NES interaction . ||14554087_155908_4928==>Therefore , the interaction of the GLFG - repeat nucleoporin Nup98 , unlike that of the XXFG - repeat containing Rab / hRIP , with the Rev NES may also involve the coordinate action of other cofactors in the Rev multiprotein export complex . ||14554087_155908_4928==>Interestingly , a recent study suggests that the accumulation of Rev - bound RRE RNAs at the NPC is not inhibited by leptomycin B ( Cmarko et al. , 2002 ) , an inhibitor of the Rev NES ? CRM1 interaction ; this may be reflective of the potential for CRM1 - independent association of the Rev - bound RRE RNA cargo with inner NPC components , such as Nup98 , during the initial phase of nuclear export . ||14554087_155908_4928==>Since the GLFG region can not localize at the nuclear envelope , this inhibition is probably reflective of intranuclear sequestration of Rev ? RRE RNA complexes at subnuclear sites by inappropriately localized GLFG region ( or wt Nup98 ) protein upon high - level overexpression . ||14554087_155908_4928==>Whether this region plays an important role in Rev transport , other than ensuring appropriate Nup98 localization , remains to be determined . ||14554087_155908_4928==>Interestingly , when exogenous wt Nup98 was expressed at lower levels , as in HeLa cells , significant increase of Rev - dependent RRE RNA export was detectable . ||14554087_155908_4928==>A likely explanation for the discrepancy in the observed effects of exogenously introduced wt Nup98 on Rev - dependent unspliced RNA export in 293T ( which supports expression vector replication ) versus HeLa ( wherein the expression vector can not replicate ) cells is the propensity of highly overexpressed exogenous Nup98 ( in 293T cells ) to localize predominantly in intranuclear , and perhaps inappropriate , structures rather than at the nuclear envelope ; in contrast , lower level overexpression of wt Nup98 , as in HeLa cells , results in primarily nuclear envelope localization of the exogenous protein . ||14554087_155908_4928==>Another intriguing observation is the failure of wt as well as FG - repeat containing mutant Nup98 proteins to suppress spliced ( Rev - expressing ) RNA export in 293T cells in these experiments despite the demonstrated involvement of Nup98 in export of spliced cellular mRNAs through the TAP - p15 pathway ( Reed and Hurt , 2002 ) . ||9054383_155871_2071==>Proteins were resolved on an SDS - polyacrylamide electrophoresis gel , blotted onto a nitrocellulose matrix ( Hybond C , Amersham ) , and analyzed with the appropriate antibodies using standard techniques ( anti - TBP , Promega ; rabbit polyclonal anti - HIV - 2 Tat ; anti - flag ( Kodak ) monoclonal for HIV - 1 Tat ; anti - TFIIH and anti - TFIIE subunits , Santa Cruz Biotechnology ) . ||9054383_155871_2071==>HIV - 1 Tat has previously been shown to associate with several components comprising the HIV - 1 preinitiation complex including SP1 ( 60 ) , TATA - binding protein ( TBP ) and associated factors ( TAFs ) ( 61 ) , the p62 subunit of TFIIH ( 58 ) in addition to RNA polymerase II ( 16 ) . ||9054383_155871_2965==>Proteins were resolved on an SDS - polyacrylamide electrophoresis gel , blotted onto a nitrocellulose matrix ( Hybond C , Amersham ) , and analyzed with the appropriate antibodies using standard techniques ( anti - TBP , Promega ; rabbit polyclonal anti - HIV - 2 Tat ; anti - flag ( Kodak ) monoclonal for HIV - 1 Tat ; anti - TFIIH and anti - TFIIE subunits , Santa Cruz Biotechnology ) . ||9054383_155871_2965==>HIV - 1 Tat has previously been shown to associate with several components comprising the HIV - 1 preinitiation complex including SP1 ( 60 ) , TATA - binding protein ( TBP ) and associated factors ( TAFs ) ( 61 ) , the p62 subunit of TFIIH ( 58 ) in addition to RNA polymerase II ( 16 ) . ||9054383_155871_2966==>Proteins were resolved on an SDS - polyacrylamide electrophoresis gel , blotted onto a nitrocellulose matrix ( Hybond C , Amersham ) , and analyzed with the appropriate antibodies using standard techniques ( anti - TBP , Promega ; rabbit polyclonal anti - HIV - 2 Tat ; anti - flag ( Kodak ) monoclonal for HIV - 1 Tat ; anti - TFIIH and anti - TFIIE subunits , Santa Cruz Biotechnology ) . ||9054383_155871_2966==>HIV - 1 Tat has previously been shown to associate with several components comprising the HIV - 1 preinitiation complex including SP1 ( 60 ) , TATA - binding protein ( TBP ) and associated factors ( TAFs ) ( 61 ) , the p62 subunit of TFIIH ( 58 ) in addition to RNA polymerase II ( 16 ) . ||9054383_155871_2967==>Proteins were resolved on an SDS - polyacrylamide electrophoresis gel , blotted onto a nitrocellulose matrix ( Hybond C , Amersham ) , and analyzed with the appropriate antibodies using standard techniques ( anti - TBP , Promega ; rabbit polyclonal anti - HIV - 2 Tat ; anti - flag ( Kodak ) monoclonal for HIV - 1 Tat ; anti - TFIIH and anti - TFIIE subunits , Santa Cruz Biotechnology ) . ||9054383_155871_2967==>HIV - 1 Tat has previously been shown to associate with several components comprising the HIV - 1 preinitiation complex including SP1 ( 60 ) , TATA - binding protein ( TBP ) and associated factors ( TAFs ) ( 61 ) , the p62 subunit of TFIIH ( 58 ) in addition to RNA polymerase II ( 16 ) . ||9388268_155871_6667==>It has been well established that the Sp1 transcription factor ( 41 ) plays an essential role in the regulation of basal transcription as well as in Tat - mediated transactivation of the HIV - 1 LTR ( 42 - 44 ) . ||9388268_155871_6667==>Moreover in vitro and in vivo data suggest a direct interaction between Sp1 and Tat during transactivation ( 48 , 49 ) . modulates=====11689053_155871_2033==>ScienceDirect - Virology : Enhancement of the p300 HAT Activity by HIV - 1 Tat on Chromatin DNA ||11689053_155871_2033==>Enhancement of the p300 HAT Activity by HIV - 1 Tat on Chromatin DNA ||11689053_155871_2033==>HIV - 1 Tat is able to form a ternary complex with P / CAF and p300 and increase the affinity for CDK9 / P - TEFb CTD kinase complex . ||11689053_155871_2033==>Our previous study demonstrated that Tat binds to p300 / CBP in the minimal HAT domain ( aa 1253 ? 1790 ) and that the interaction results in a change of conformation on p300 / CBP . ||11689053_155871_2033==>Here , we show that the Tat ? p300 interaction increases the HAT activity of p300 on histone H4 that is associated with nucleosomal DNA and not with free histones . ||11689053_155871_2033==>Furthermore , when utilizing an in vitro transcription assay , as well as a Tat mutant virus , we found that ectopic expression of only wild - type Tat in the presence of p300 , and not a lysine 41 Tat mutant , could activate HIV - 1 chromatin DNA , as evidenced by the absence of HIV - 1 virion antigen . ||12134041_155908_7514==>Direct Participation of Sam68 , the 68 - Kilodalton Src - Associated Protein in Mitosis , in the CRM1 - Mediated Rev Nuclear Export Pathway - - Li et al. 76 ( 16 ) : 8374 - - The Journal of Virology ||12134041_155908_7514==>Direct Participation of Sam68 , the 68 - Kilodalton Src - Associated Protein in Mitosis , in the CRM1 - Mediated Rev Nuclear Export Pathway Jinliang Li , 1 , 2 Ying Liu , 1 , 2 Byung Oh Kim , 1 , 2 and Johnny J . ||12134041_155908_7514==>Moreover , digital fluorescence microscopic imaging revealed that down - modulation of Sam68 expression caused exclusive nuclear retention and colocalization of both Rev and CRM1 . ||12134041_155908_7514==>Taken together , these data suggest that adequate Sam68 expression is required for Rev function and , thereby , for HIV - 1 gene expression and viral replication , and they support the notion that Sam68 is directly involved in the CRM1 - mediated Rev nuclear export pathway . ||12134041_155908_7514==>Several cellular factors have been identified to be directly involved in the Rev - mediated nuclear export pathway , such as CRM1 ( 16 , 19 ) , eukaryotic initiation factor 5A ( eIF - 5A ) ( 64 ) , and Rip / Rab ( 7 , 18 ) . ||12134041_155908_7514==>Recent studies have shown that these repeated phenylalanine - glycine ( FG ) - containing nucleoporins bind to the Rev NES indirectly via CRM1 ( 49 ) . ||12134041_155908_7514==>Our results demonstrate that down - modulation of Sam68 expression markedly inhibited HIV - 1 gene expression , replication , and Rev function and that the inhibition was due to a block of CRM1 - mediated Rev nuclear export . ||12134041_155908_7514==>These data together suggest that Sam68 plays a direct role in CRM1 - mediated Rev export from the nucleus to the cytoplasm . ||12134041_155908_7514==>Sam68 expression - induced Rev nuclear export is CRM1 dependent . ||12134041_155908_7514==>Extensive studies have shown that Rev nuclear export is mediated by CRM1 ( 2 , 16 , 19 , 32 , 49 , 53 ) . ||12134041_155908_7514==>Thus , we decided to determine the relationship between Sam68 expression - induced Rev nuclear export and CRM1 - mediated Rev nuclear export . ||12134041_155908_7514==>As expected , Rev was localized in the nucleus ( fig. A ) , while CRM1 was mainly localized at the nuclear membrane , with some in the nucleus and in the cytoplasm ( fig. B ) . ||12134041_155908_7514==>When the transfected cells were treated with ATD , both Rev and CRM1 were considerably shifted into the cytoplasm ( fig. D and E ) . ||12134041_155908_7514==>We then took advantage of an CRM1 - specific inhibitor , leptomycin B ( LMB ) , which is known to prevent CRM1 from binding to Rev and thus to disrupt Rev nuclear export ( 2 , 16 , 19 , 32 , 50 , 75 ) , to determine whether LMB treatment would inhibit Sam68 expression - induced Rev nuclear export . ||12134041_155908_7514==>These results together demonstrated that Sam68 expression - induced Rev nuclear export was CRM1 dependent , suggesting that Sam68 may function as a previously unrecognized cofactor in the CRM1 - mediated Rev nuclear export pathway . ||12134041_155908_7514==>Direct involvement of Sam68 in CRM1 - mediated Rev nuclear export . ||12134041_155908_7514==>To further characterize the roles of Sam68 in the CRM1 - mediated Rev nuclear export pathway , we first determined whether CRM1 bound directly to Sam68 . ||12134041_155908_7514==>Although the NES of Rev has been demonstrated to be directly involved in the interaction of Rev with both Sam68 ( 35 ) and CRM1 ( 16 , 19 , 49 ) , we did not detect any adverse effect of Sam68 expression on CRM1 binding to Rev , or vice versa ( data not shown ) . ||12134041_155908_7514==>These data suggest that Rev binding to Sam68 and to CRM1 may occur independently or simultaneously . ||12134041_155908_7514==>To determine the functional relationship between Sam68 and CRM1 in the Rev nuclear export pathway , we investigated the effect of Sam68 down - modulation on the CRM1 - Rev interaction . ||12134041_155908_7514==>In agreement with the findings of previous studies ( 60 , 79 ) , the results showed that coexpression of CRM1 and Rev relocalized each to the nuclear membrane and led to Rev nuclear export ( fig. 6A and B ) . ||12134041_155908_7514==>As expected , LMB treatment disrupted the CRM1 - Rev interaction , resulting in no Rev nuclear export ( fig. 6D ) and little colocalization of Rev and Sam68 , as they were localized in distinct nuclear compartments ( fig. 6D and E ) . ||12134041_155908_7514==>In contrast , down - modulation of Sam68 expression by expression of the anti - Sam68 antisense RNA expression vector As - Sam68a resulted in little or no Rev nuclear export ( fig. 6G ) and exclusively nuclear localization of both Rev and CRM1 ( fig. 6G and H ) . ||12134041_155908_7514==>Taken together , these results demonstrated that down - modulation of Sam68 expression blocked the CRM1 - mediated Rev nuclear export pathway , suggesting that Sam68 expression - induced Rev nuclear export and CRM1 - mediated Rev nuclear export were mutually dependent . ||12134041_155908_7514==>In addition , these results also raised the possibility that Sam68 may serve to transport the export complex containing CRM1 and Rev from the nucleus to an area in close proximity to the nuclear membrane ( the NPC ) . ||12134041_155908_7514==>Inhibition of CRM1 - mediated Rev nuclear export by Sam68 down - modulation . ||12134041_155908_7514==>It has been well documented that Rev nuclear export is directly mediated by CRM1 ( 2 , 16 , 19 , 32 , 49 , 53 ) . ||12134041_155908_7514==>Therefore , we decided to further elucidate the relationship between Sam68 expression - induced Rev nuclear export and CRM1 - mediated Rev nuclear export . ||12134041_155908_7514==>In addition , our results showed that Sam68 expression - induced Rev nuclear export was CRM1 mediated and that down - modulation of Sam68 expression caused the complex containing CRM1 and Rev to be retained in the nucleus and thus blocked Rev nuclear export . ||12134041_155908_7514==>Taken together , these data suggest that adequate Sam68 expression is required for Rev function by directly regulating the CRM1 - mediated Rev nuclear export pathway . ||12134041_155908_7514==>The NES of Rev has also been found to interact with CRM1 ( 16 , 19 , 49 ) . ||12134041_155908_7514==>Thus , it is conceivable that Sam68 would compete with CRM1 for Rev binding . ||12134041_155908_7514==>However , we were unable to detect any inhibitory effect of Sam68 expression on the binding of CRM1 to Rev or any inhibitory effect of CRM1 expression on the binding of Sam68 to Rev , suggesting that Rev binding to Sam68 and to CRM1 may occur independently or simultaneously . ||12134041_155908_7514==>Subsequent interaction of CRM1 with several nucleoporins , including CAN / Nup214 and Nup98 , has been shown to recruit the complex containing Rev , RanGTP , and RRE - containing RNAs from the nucleus to the nuclear pore ( 17 , 43 , 79 ) . ||12134041_155908_7514==>Nevertheless , we currently have no information concerning the molecular events of Sam68 involvement with the complex of CRM1 , Rev , RanGTP , and RRE - containing mRNAs . ||12134041_155908_7514==>Thus , further studies to understand the precise roles of Sam68 in the CRM1 - mediated Rev nuclear export pathway are warranted . ||12134041_155908_7514==>In summary , our results obtained from the present study , i.e. , the inhibition of HIV - 1 gene expression and viral replication , Rev - dependent gene expression , and CRM1 - mediated Rev nuclear export by Sam68 down - modulation , along with our previous findings that Sam68 expression is able to alleviate a Rev function block in astrocytes through direct interaction and enhanced Rev nuclear export ( 35 ) , support a model ( fig. 7 ) in which Sam68 binds to the complex of Rev , RRE - containing RNAs , CRM1 , and RanGTP , and facilitates transport of the complex to the NPC , within close proximity to the cytoplasmic side of the nuclear membrane , where the complex is docked by direct binding of CRM1 to nucleoporins , followed by the release of CRM1 , Rev , and RRE - containing RNAs into the cytoplasm . ||12134041_155908_7514==>Proposed model for Sam68 function in CRM1 - mediated Rev nuclear export . ||12134041_155908_7514==>Formation of a quadruple complex consisting of Rev , RRE - containing RNA , CRM1 , and RanGTP occurs in the nucleus , possibly independently of Sam68 . ||12134041_155908_7514==>Sam68 then associates with the complex via direct binding to Rev and transports the complex to the NPC , followed by docking onto NPC through CRM1 interaction with nucleoporins . ||12134041_155908_7514==>Translocation of Rev , CRM1 , and RRE - containing RNAs into the cytoplasm leads to the release of Sam68 into the nucleus . ||12134041_155908_7514==>The specificity of the CRM1 - Rev nuclear export signal interaction is mediated by RanGTP . ||9400615_155871_5524==>ScienceDirect - Virology : Increasing the Ratio of PP2A Core Enzyme to Holoenzyme Inhibits Tat - Stimulated HIV - 1 Transcription and Virus Production * 1 ||9400615_155871_5524==>Increasing the Ratio of PP2A Core Enzyme to Holoenzyme Inhibits Tat - Stimulated HIV - 1 Transcription and Virus Production * 1 ||9636359_155030_5478==>We studied chaperone effects on Gag precursor processing using cyclosporin A ( CsA ) to bind CypA and prevent its interaction with p55 Gag . ||9636359_155030_5478==>CsA has a direct effect on HIV - 1 Gag processing that implicates CypA as having an important role in the maturation of HIV - 1 particles . phosphorylates=====8806671_155908_983==>Protein kinase CK1 can not replace CK2 as phosphorylating agent and cdc2 only slowly phosphorylates Rev at one of the two sites affected by MAP kinase . ||9325171_155871_6464==>In addition , in neuronal cells , but not in astrocytes , Tat increased the phosphotyrosine content of Shc , a protein involved in signal transduction downstream of receptor tyrosine kinase activation . ||9621077_155871_2185==>We also report that HIV - 1 Tat treatment enhanced the phosphorylation and association of proteins found in focal adhesions , such as the related adhesion focal tyrosine kinase RAFTK , paxillin , and p130 cas . ||9621077_155871_2185==>We observed that stimulation of KS cells with basic as well as RGD sequence - containing Tat peptides resulted in enhanced phosphorylation of RAFTK and activation of MAP kinase . ||9621077_155871_2185==> RAFTK is tyrosine phosphorylated upon HIV Tat stimulation . ||9621077_155871_2185==>We therefore investigated whether HIV Tat phosphorylates RAFTK . ||9621077_155871_2185==>HIV Tat treatment of KS cells resulted in rapid tyrosine phosphorylation of RAFTK ( fig. 3 , top ) . ||9621077_155871_2185==>Tyrosine phosphorylation of RAFTK by HIV Tat stimulation . ||9621077_155871_2185==>HIV Tat stimulates tyrosine phosphorylation of paxillin and its association with RAFTK . ||9621077_155871_2185==>Furthermore , we observed that paxillin was associated with RAFTK and that this association was enhanced upon HIV Tat treatment ( fig. 4 B ) . ||9621077_155871_2185==>We examined whether Tat treatment , which activated RAFTK , was also able to activate various members of the MAP kinase pathway . ||9621077_155871_2185==> Tat basic and RGD peptides activate RAFTK and the MAP kinase pathway . ||9621077_155871_2185==>Having observed that full - length Tat stimulation resulted in phosphorylation of RAFTK and activation of the MAP kinase pathway , we studied whether treatment with basic or RGD - containing Tat peptides also resulted in activation of these kinases . ||9621077_155871_2185==>Phosphorylation of RAFTK and activation of MAP kinase by basic and RGD domain - containing peptides of Tat . ||9621077_155871_2185==>Since HIV Tat has been shown to affect cell migration , which involves alteration in cytoskeletal elements , we analyzed the phosphorylation of RAFTK , paxillin , and p130 cas , components of focal adhesions . ||9621077_155871_2185==>Our data revealed that Tat induces a rapid tyrosine phosphorylation of RAFTK , which reaches a maximum around 2 min and declines thereafter . ||9621077_155871_2185==>We also observed that there was an enhanced association of paxillin with RAFTK upon Tat stimulation . ||9621077_155871_2185==>Both the basic and RGD Tat peptides resulted in the activation of KS cells , with phosphorylation of RAFTK and activation of MAP kinase . ||9621077_155871_2185==>We add to this report data on Tat stimulation and subsequent activation of the Flk - 1 receptor and phosphorylation of the recently identified focal adhesion components RAFTK and p130 cas . ||9621077_155871_5829==>We also report that HIV - 1 Tat treatment enhanced the phosphorylation and association of proteins found in focal adhesions , such as the related adhesion focal tyrosine kinase RAFTK , paxillin , and p130 cas . ||9621077_155871_5829==>HIV Tat stimulates tyrosine phosphorylation of paxillin and its association with RAFTK . ||9621077_155871_5829==>Thus , we sought to investigate whether HIV Tat treatment of KS 38 cells resulted in changes in the phosphorylation state of paxillin and its association with other proteins . ||9621077_155871_5829==>As shown in fig. 4 A ( top ) , HIV Tat stimulation resulted in enhanced tyrosine phosphorylation of paxillin . ||9621077_155871_5829==>Furthermore , we observed that paxillin was associated with RAFTK and that this association was enhanced upon HIV Tat treatment ( fig. 4 B ) . ||9621077_155871_5829==>KS cells were stimulated with HIV Tat ( 100 ng / ml ) for various times , and stimulated or unstimulated cell lysates were immunoprecipitated ( IP ) with anti - paxillin antibody . ||9621077_155871_5829==>Since HIV Tat has been shown to affect cell migration , which involves alteration in cytoskeletal elements , we analyzed the phosphorylation of RAFTK , paxillin , and p130 cas , components of focal adhesions . ||9621077_155871_5829==>We also observed that there was an enhanced association of paxillin with RAFTK upon Tat stimulation . ||9621077_155871_9564==>We also report that HIV - 1 Tat treatment enhanced the phosphorylation and association of proteins found in focal adhesions , such as the related adhesion focal tyrosine kinase RAFTK , paxillin , and p130 cas . ||9621077_155871_9564==>p130 cas is tyrosine phosphorylated upon HIV Tat treatment . ||9621077_155871_9564==>We observed that HIV Tat treatment also resulted in enhanced tyrosine phosphorylation of p130 cas ( fig. 5 , top ) . ||9621077_155871_9564==>HIV Tat treatment of KS cells stimulates tyrosine phosphorylation of p130 cas . ||9621077_155871_9564==>Unstimulated or HIV Tat - stimulated KS cell lysates were immunoprecipitated ( IP ) with anti - p130 cas . ||9621077_155871_9564==>Since HIV Tat has been shown to affect cell migration , which involves alteration in cytoskeletal elements , we analyzed the phosphorylation of RAFTK , paxillin , and p130 cas , components of focal adhesions . ||9621077_155871_9564==>We add to this report data on Tat stimulation and subsequent activation of the Flk - 1 receptor and phosphorylation of the recently identified focal adhesion components RAFTK and p130 cas . ||9792705_155459_5594==>In this report , we show that HIV - 1 Vif is phosphorylated on Thr 96 and Ser 165 by p44 / 42 mitogen - activated protein kinase ( also known as ERK1 and ERK2 , hereafter referred to as MAPK ) both in vitro and in vivo . ||9792705_155459_5594==>Recombinant Vif was phosphorylated by recombinant p42 MAPK or by MAPK immunoprecipitated from PMA - stimulated HeLa cells with anti - ERK1 and anti - ERK2 as described above in the presence of ( - 32 P ) ATP , or Vif was expressed in and isolated from 293T cells transfected with the pMEK - R4F using a vaccinia virus expression system as described above and metabolically labeled with ( 32 P ) orthophosphate . ||9792705_155459_5594==>Samples with equivalent amounts of protein ( 5 - 10 痢 ) were resolved by 15 % SDS - PAGE and transferred to PVDF membrane for immunoblotting with rabbit anti - ERK1 or anti - ERK1 and anti - ERK2 ( 1 : 1500 dilution of each ) , rabbit anti - phosphorylated MAPK ( 1 : 1000 dilution ) , or rabbit anti - Vif ( 1 : 2000 dilution ) ( 15 ) using the ECL system ( Amersham Pharmacia Biotech ) as described ( 15 ) . ||9792705_155459_5594==>MAPK immunocomplexes were isolated from the cell lysates using anti - ERK1 and anti - ERK2 and incubated with Vif ( A ) or MBP ( B ) in kinase buffer with ( - 32 P ) ATP . ||9792705_155459_5595==>In this report , we show that HIV - 1 Vif is phosphorylated on Thr 96 and Ser 165 by p44 / 42 mitogen - activated protein kinase ( also known as ERK1 and ERK2 , hereafter referred to as MAPK ) both in vitro and in vivo . ||9792705_155459_5595==>Recombinant Vif was phosphorylated by recombinant p42 MAPK or by MAPK immunoprecipitated from PMA - stimulated HeLa cells with anti - ERK1 and anti - ERK2 as described above in the presence of ( - 32 P ) ATP , or Vif was expressed in and isolated from 293T cells transfected with the pMEK - R4F using a vaccinia virus expression system as described above and metabolically labeled with ( 32 P ) orthophosphate . ||9792705_155459_5595==>Samples with equivalent amounts of protein ( 5 - 10 痢 ) were resolved by 15 % SDS - PAGE and transferred to PVDF membrane for immunoblotting with rabbit anti - ERK1 or anti - ERK1 and anti - ERK2 ( 1 : 1500 dilution of each ) , rabbit anti - phosphorylated MAPK ( 1 : 1000 dilution ) , or rabbit anti - Vif ( 1 : 2000 dilution ) ( 15 ) using the ECL system ( Amersham Pharmacia Biotech ) as described ( 15 ) . ||9792705_155459_5595==>MAPK immunocomplexes were isolated from the cell lysates using anti - ERK1 and anti - ERK2 and incubated with Vif ( A ) or MBP ( B ) in kinase buffer with ( - 32 P ) ATP . recruits=====11080476_155871_2959==>Interestingly , wild - type peptide 41 ? 54 , and not other Tat peptides , changes the conformation of the CBP / p300 such that it can acquire and bind better to basal factors such as TBP and TFIIB , indicating that Tat may influence the transcription machinery by helping CBP / p300 to recruit new partners into the transcription machinery . ||11430827_155348_6598==>The HIV - 1 integrase can interact with the protein INI1 ( integrase interactor 1 ) , also called human SNF5 ( hSNF5 ) or BAF47 ( Kalpana et al. 1994 and Wang et al. 1996 ) , which is an essential constituent of the human SWI / SNF chromatin remodeling machine . ||11430827_155348_6598==>In order to define the minimal set of HIV - 1 components necessary to trigger the subcellular redistribution of PML and INI1 , VSV G - pseudotyped HIV - 1 - based particles were produced from a `` multiply attenuated '' HIV vector packaging system , based on the sole expression of gag , pol , and rev ( Dull et al. , 1998 ) . ||11430827_155348_6598==>The cytoplasmic colocalization of the incoming viral genome and INI1 was also observed with Gag - Pol - based HIV vector particles ( Figure 7 , panel e ) . ||11430827_155348_6598==>The binding of HIV - 1 integrase to INI1 makes it likely that this virus has evolved not only to overcome , but also to subvert the antiviral activity revealed by our experiments . ||14657027_1387_155871==>In particular , we and others have shown that Tat associates with : ( i ) the transcriptional co - activators p300 and the highly homologous cAMP - responsive binding protein ( CREB ) binding protein ( CBP ) ; ( ii ) the p300 / CBP - associated factor ( P / CAF ) ; and ( iii ) the general control non - derepressible - 5 ( GCN5 ) protein ( Benkirane et al . ||14657027_1387_155871==>Immunoprecipitation of crosslinked chromatin with antibodies against transcription factors USF ( A ) , p50 ( B ) and p65 ( C ) after TPA or Tat treatment in HL3T1 cells for 5 h. The time course of the binding of histone acetyltransferases GCN5 , CBP and P / CAF to the viral regions nuc - 0 , PPR and nuc - 1 was analyzed by quantitative ChIP after TPA ( D ) or Tat ( E ) treatment . ||14657027_1387_155871==>After treatment with recombinant Tat , histone acetylation and transcriptional activation at 3 and 5 h were paralleled by a marked recruitment of both CBP and P / CAF to both nuc - 0 and PPR ( Figure 8E ) . ||14657027_1387_155871==>In this study we have observed that Tat activation of HL3T1 cells is associated with histone H3 and H4 acetylation at Nuc - 0 , and with promoter occupancy by CBP and P / CAF . ||14657027_1387_155871==>It is also interesting to note that , upon Tat induction , we were able to detect the presence of two HATs , CBP and P / CAF . ||14657027_1387_155871==>Transcription , as measured by mRNA levels , followed the increase in H3 acetylation in both U1 and HL3T1 cells in response to Tat , reflecting the constant presence of CBP at the promoter . ||14657027_155871_2648==>In particular , we and others have shown that Tat associates with : ( i ) the transcriptional co - activators p300 and the highly homologous cAMP - responsive binding protein ( CREB ) binding protein ( CBP ) ; ( ii ) the p300 / CBP - associated factor ( P / CAF ) ; and ( iii ) the general control non - derepressible - 5 ( GCN5 ) protein ( Benkirane et al . ||14657027_155871_2648==>Immunoprecipitation of crosslinked chromatin with antibodies against transcription factors USF ( A ) , p50 ( B ) and p65 ( C ) after TPA or Tat treatment in HL3T1 cells for 5 h. The time course of the binding of histone acetyltransferases GCN5 , CBP and P / CAF to the viral regions nuc - 0 , PPR and nuc - 1 was analyzed by quantitative ChIP after TPA ( D ) or Tat ( E ) treatment . ||14657027_155871_2648==>Col E , Caron C , Seigneurin - Berny D , Gracia J , Favier A and Khochbin S ( 2001 ) The histone acetyltransferase , hGCN5 , interacts with and acetylates the HIV transactivator , Tat . ||14657027_155871_8850==>Strikingly , P / CAF was found associated with the promoter only in response to Tat . ||14657027_155871_8850==>In particular , we and others have shown that Tat associates with : ( i ) the transcriptional co - activators p300 and the highly homologous cAMP - responsive binding protein ( CREB ) binding protein ( CBP ) ; ( ii ) the p300 / CBP - associated factor ( P / CAF ) ; and ( iii ) the general control non - derepressible - 5 ( GCN5 ) protein ( Benkirane et al . ||14657027_155871_8850==>Immunoprecipitation of crosslinked chromatin with antibodies against transcription factors USF ( A ) , p50 ( B ) and p65 ( C ) after TPA or Tat treatment in HL3T1 cells for 5 h. The time course of the binding of histone acetyltransferases GCN5 , CBP and P / CAF to the viral regions nuc - 0 , PPR and nuc - 1 was analyzed by quantitative ChIP after TPA ( D ) or Tat ( E ) treatment . ||14657027_155871_8850==>After treatment with recombinant Tat , histone acetylation and transcriptional activation at 3 and 5 h were paralleled by a marked recruitment of both CBP and P / CAF to both nuc - 0 and PPR ( Figure 8E ) . ||14657027_155871_8850==>In this respect , it should be considered that U1 cells express a Tat protein that is mutated in its N - terminal activation domain , but maintains an intact P / CAF - interacting domain ( Bres et al . ||14657027_155871_8850==>Therefore , it is possible to conceive that this mutated form of Tat still recruits P / CAF at the viral promoter . ||14657027_155871_8850==>In this study we have observed that Tat activation of HL3T1 cells is associated with histone H3 and H4 acetylation at Nuc - 0 , and with promoter occupancy by CBP and P / CAF . ||14657027_155871_8850==>It is also interesting to note that , upon Tat induction , we were able to detect the presence of two HATs , CBP and P / CAF . ||14657027_155871_8850==>Thus , conceivably , Tat appears able to transactivate the HIV - 1 LTR to the highest levels , also by relieving an HDAC1 block and inducing P / CAF . ||14657027_155871_8850==>p300 and P / CAF are coactivators for HIV - 1 Tat . ||14657027_155871_8850==>Bres V , Tagami H , Peloponese JM , Loret E , Jeang KT , Nakatani Y , Emiliani S , Benkirane M and Kiernan RE ( 2002 ) Differential acetylation of Tat coordinates its interaction with the co - activators cyclin T1 and PCAF . ||14657027_155871_8850==>Mujtaba S , He Y , Zeng L , Farooq A , Carlson JE , Ott M , Verdin E and Zhou MM ( 2002 ) Structural basis of lysine - acetylated HIV - 1 Tat recognition by PCAF bromodomain . regulates=====10617616_11140_155871==>Our data suggest a previously unrecognized chaperone - dependent pathway involving the sequential actions of Hsp70 and Hsp90 / Cdc37 in the stabilization / folding of Cdk9 as well as the assembly of an active Cdk9 / cyclin T1 complex responsible for P - TEFb - mediated Tat transactivation . ||10617616_155871_3308==>Our data suggest a previously unrecognized chaperone - dependent pathway involving the sequential actions of Hsp70 and Hsp90 / Cdc37 in the stabilization / folding of Cdk9 as well as the assembly of an active Cdk9 / cyclin T1 complex responsible for P - TEFb - mediated Tat transactivation . ||10617616_155871_3320==>Our data suggest a previously unrecognized chaperone - dependent pathway involving the sequential actions of Hsp70 and Hsp90 / Cdc37 in the stabilization / folding of Cdk9 as well as the assembly of an active Cdk9 / cyclin T1 complex responsible for P - TEFb - mediated Tat transactivation . ||11959860_155871_9733==>HIV - 1 Tat Protein - mediated Transactivation of the HIV - 1 Long Terminal Repeat Promoter Is Potentiated by a Novel Nuclear Tat - interacting Protein of 110 kDa , Tip110 - - Liu et al. 277 ( 26 ) : 23854 - - Journal of Biological Chemistry ||11959860_155871_9733==>HIV - 1 Tat Protein - mediated Transactivation of the HIV - 1 Long Terminal Repeat Promoter Is Potentiated by a Novel Nuclear T at - i nteracting P rotein of 110 kDa , Tip110 * ||11959860_155871_9733==>During our attempts to determine the molecular mechanisms of Tat interaction with brain cells , we isolated a cDNA clone that encodes a novel T at - i nteracting p rotein of 110 kDa or Tip110 from a human fetal brain cDNA library . ||11959860_155871_9733==>In vivo binding of Tip110 with Tat was confirmed by immunoprecipitation and Western blotting , in combination with mutagenesis . ||11959860_155871_9733==>The yeast three - hybrid RNA - protein interaction assay indicated no direct interaction of Tip110 with Tat transactivating response element RNA . ||11959860_155871_9733==>Nevertheless , Tip110 strongly synergized with Tat on Tat - mediated chloramphenicol acetyltransferase reporter gene expression and HIV - 1 virus production , whereas down - modulation of constitutive Tip110 expression inhibited HIV - 1 virus production . ||11959860_155871_9733==>Moreover , digital fluorescence microscopic imaging revealed that Tip110 was expressed exclusively in the nucleus , and within a nuclear speckle structure that has recently been described for human cyclin T and CDK9 , two critical components for Tat transactivation function on HIV - 1 long terminal repeat promoter . ||11959860_155871_9733==>Taken together , these data demonstrate that Tip110 regulates Tat transactivation activity through direct interaction , and suggest that Tip110 is an important cellular factor for HIV - 1 gene expression and viral replication . ||11959860_155871_9733==>During our attempts to characterize the roles of Tat protein in HIV - 1 - induced neuropathogenesis , we isolated a cDNA clone that encodes a novel nuclear T at - i nteracting p rotein of 110 kDa , or Tip110 . ||11959860_155871_9733==>We show that Tip110 directly transactivates the HIV - 1 LTR promoter , but also interacts and synergizes with Tat to argument Tat - mediated transactivation activity . ||11959860_155871_9733==>Interaction between Tat and Tip110 was tested using the yeast two - hybrid assay . ||11959860_155871_9733==>Isolation of the Full - length and 11 - bp Deletion - containing Tip110 cDNAs - - During our attempts to identify the endocytic receptor for Tat on neurons , we used Tat as a bait protein and performed the yeast two - hybrid cDNA screening of a human fetal brain cDNA library . ||11959860_155871_9733==>Thus , we re - named this gene as Tat - interacting protein of 110 kDa , or Tip110 . ||11959860_155871_9733==> Tat Interaction with the Full - length Tip110 - - As described above , we initially isolated Tip110 as a partial cDNA clone , which only contained the N - terminal region of Tip110 . ||11959860_155871_9733==>Thus , we first tested whether the full - length Tip110 would bind to Tat . ||11959860_155871_9733==>We transformed the yeast strain L40 with pAD - Tip110 and pBD - Tat plasmids , in which Tip110 and Tat were fused with Gal4 activation domain ( pACT2 , CLONTECH ) and LexA DNA binding domain ( pBTM116 , a gift from Dr . ||11959860_155871_9733==>Co - transformation of the full - length Tip110 ( AD - Tip110 ) with BD - Tat exhibited a blue color on the filter membrane , whereas no color was developed on the membrane when co - transformed with BTM116 ( fig. 2 ) . ||11959860_155871_9733==>These results confirmed that , like Clone 60 , the full - length Tip110 indeed bound to Tat . ||11959860_155871_9733==>Interaction of Tip110 with Tat . ||11959860_155871_9733==>We then investigated whether Tip110 would bind to Tat in vivo . ||11959860_155871_9733==>Western blot analysis showed that both Tip110 ( fig. 3 , panel WB : His ) and Tat ( fig. 3 , panel WB : Myc ) were expressed in 293T cells at the expected molecular weights . ||11959860_155871_9733==>We then performed immunoprecipitation of cell lysates for Tip110 followed by Western blotting for Tat . ||11959860_155871_9733==>The results showed that Tat was detected in the immunoprecipitation complex of Tip110 only when Tat and Tip110 were co - expressed ( fig. 3 , panel IP : His / WB : Myc ) . ||11959860_155871_9733==>Similar results were obtained by immunoprecipitation for Tat followed by Western blotting for Tip110 ( data not shown ) . ||11959860_155871_9733==>In corroboration with the previous results from the yeast two - hybrid assay , these results demonstrated that Tip110 can form a complex with Tat in vivo . ||11959860_155871_9733==>In vivo binding of Tip110 to Tat . ||11959860_155871_9733==>Specific Binding of Tip110 to Tat - - To further determine the specificity of Tip110 interaction with Tat and also to identify which domain ( s ) of Tip110 are directly involved in Tip110 - Tat interaction , we constructed a series of Tip110 mutants that were deleted for the N - terminal HAT - rich domain , RRM domains , and / or NLS domain ( fig. 4 a ) . ||11959860_155871_9733==>We transfected 293T cells with Tat expression plasmid pTat.Myc , along with each of Tip110 mutants , and determined the complex formation between Tip110 and Tat , as described above . ||11959860_155871_9733==> Tip110 mutants ( fig. 4 b , panel WB : His ) as well as Tat protein ( fig. 2 b , panel WB : Myc ) were expressed in 293T cells at the expected molecular weights . ||11959860_155871_9733==>Transfection of the Tip110 mutants into 293T cells showed that deletion of the NLS domain ( NLS ) , RRM domain ( RRM ) , and both NLS and RRM domains ( CT ) had no effects on Tip110 - Tat complex formation ( fig. 2 b , panel IP : Myc / WB : His ) . ||11959860_155871_9733==>However , deletion of the HAT - rich domain ( NT ) abolished Tip110 binding to Tat . ||11959860_155871_9733==>Direct involvement of the N - terminal HAT - rich domain of Tip110 in Tat binding . ||11959860_155871_9733==>'' b , binding of Tip110 mutants with Tat . ||11959860_155871_9733==>Expression of Tip110 mutant proteins ( WB : His ) or Tat ( WB : Myc ) was determined by Western blot , whereas the binding of Tip110 mutants to Tat was analyzed by immunoprecipitation for Tat followed by Western blot for Tip110 and mutants ( IP : Myc / WB : His ) . ||11959860_155871_9733==>Similarly , we determined the domain ( s ) of Tat that are directly involved in interaction with Tip110 . ||11959860_155871_9733==>We took advantage of a series of Tat deletion mutants in the context of pBTM116 that we previously constructed ( 33 ) and determined Tip110 interaction with each of Tat deletion mutants using the yeast two - hybrid assay . ||11959860_155871_9733==>We transfected the yeast strain L40 with pAD - Tip110 and each of the Tat deletion mutants , as indicated , and plated out the transformants on the SD plates lacking tryptophan and leucine . ||11959860_155871_9733==>The binding between Tip110 and Tat deletion mutants was determined for the - gal gene expression by the filter - gal assay . ||11959860_155871_9733==>These results were further confirmed by a GST `` pull - down '' assay using recombinant GST - Tat and Tip110 proteins and the mutants ( data not shown ) . ||11959860_155871_9733==>Based on these results , we concluded that the N - terminal HAT - rich domain of Tip110 and the core domain of Tat is directly involved in interaction between Tip110 and Tat . ||11959860_155871_9733==>Direct involvement of the Tat core domain ( aa 37 - 48 ) in Tip110 binding . ||11959860_155871_9733==>b , interaction of Tat mutants with Tip110 by the yeast two - hybrid assay . ||11959860_155871_9733==>The L40 yeast strain was transformed with 0.1 痢 of AD - Tip110 and 0.1 痢 each of the Tat mutants in the context of the pBTM116 backbone ( 33 ) . ||11959860_155871_9733==>No Direct Interaction between Tip110 and TAR - - Interaction of Tat with its cognate RNA TAR is prerequisite for Tat transactivation function . ||11959860_155871_9733==>Thus , we transfected the yeast strain L40 - coat with hybrid RNA plasmid ( MS2 , MS2 - TAR , MS2 - RAT containing TAR in a reverse orientation , or MS2 - IRP ) and hybrid protein 2 plasmid ( AD containing the pACT2 backbone , AD - IRP , AD - Tat , and / or AD - Tip110 ) , as indicated in Table I . ||11959860_155871_9733==>No direct interaction between Tip110 and Tat TAR ||11959860_155871_9733==>Next , we determined whether Tip110 expression would have any effects on Tat - TAR interaction using the yeast three - hybrid assay . ||11959860_155871_9733==>We transfected the yeast strain L40 - coat with the MS2 - TAR hybrid RNA plasmid , AD - Tat plasmid , as well as AD - Tip110 plasmid . ||11959860_155871_9733==>The results showed that Tip110 expression exhibited little or no change on the - gal gene expression resulting from Tat - TAR interaction , as the - gal expression in the transformation of AD - Tat and MS2 - TAR was 42.6 � 4.2 units when no pAD - Tip110 was added , 39.4 � 3.3 units when 0.1 痢 of pAD - Tip110 was added , and 41.8 � 4.4 when 10 - fold more pAD - Tip110 ( 1 痢 ) was added ( Table I , lower rows ) . ||11959860_155871_9733==>Similar results were obtained using gel mobility retardation assay with Tip110 and Tat recombinant proteins and 32 P - radiolabeled TAR probe ( data not shown ) . ||11959860_155871_9733==>These results further demonstrated that Tip110 did not bind to TAR , and suggest that Tip110 binding to Tat may occur independently of Tat - TAR interaction . ||11959860_155871_9733==>Potentiation of Tat - mediated Gene Expression by Tip110 - - Because the main function of HIV - 1 Tat protein is to transactivate the HIV - 1 LTR promoter in HIV - 1 gene expression , we examined whether Tip110 interaction with Tat would affect Tat transactivation activity on the HIV - 1 LTR promoter . ||11959860_155871_9733==>Subsequently , we determined the effects of Tip110 on HIV - 1 LTR promoter activity in the presence of Tat . ||11959860_155871_9733==>Nevertheless , the synergistic effects were almost completely abolished by deletion of TAR , the transactivating response element for the Tat protein , from the HIV - 1 LTR promoter ( fig. 6 b , dotted bar ) , suggesting that Tip110 effects on Tat transactivation function were TAR - dependent . ||11959860_155871_9733==>Synergistic effects of Tip110 with Tat on the HIV - 1 LTR promoter . ||11959860_155871_9733==>b , TAR - dependent synergistic effects of Tip110 and Tat on the HIV - 1 LTR promoter . ||11959860_155871_9733==>c , direct binding of Top110 to Tat is required for the synergistic effects of Tip110 and Tat on the HIV - 1 LTR promoter . ||11959860_155871_9733==>Earlier results showed that deletion of the N terminus of Tip110 ( NT ) eliminated Tip110 binding to Tat ( fig. 4 ) . ||11959860_155871_9733==>Thus , to ascertain the relationship between Tip110 binding to Tat and Tip110 effects on Tat transactivation function , we also transfected 293T cells with pLTR - CAT reporter plasmid , 1 ng of pTat.Myc , and 1 ng of each Tip110 mutant ( fig. 4 a ) , and determined the CAT gene expression . ||11959860_155871_9733==>Compared with the wild type ( WT ) , co - expression of Tat with each of Tip110 mutants RRM , CT , and NLS , all of which bound to Tat , did not significantly change Tip110 synergistic effects with Tat on CAT gene expression ( fig. 6 c ) . ||11959860_155871_9733==>In contrast , Tip110 mutant NT mutant , which did not bind to Tat , transactivated CAT gene expression by only 3.9 � 0.8 - fold ( fig. 6 c ) , which was similar to that by 1 ng of pTat.Myc alone ( 3.3 � 0.5 - fold ( fig. 6 a ) or 2.7 � 0.4 - fold ( fig. 6 b ) ) . ||11959860_155871_9733==>These results demonstrated that direct binding of Tip110 to Tat was required for the synergistic effects of Tip110 with Tat on the HIV - 1 LTR promoter . ||11959860_155871_9733==>These data further support the notion that Tip110 can transactivate the HIV - 1 LTR in cooperation with Tat through direct interaction . ||11959860_155871_9733==>To determine the significance of Tip110 protein in Tat - mediated HIV - 1 gene expression , we down - modulated constitutive Tip110 expression and determined its effects on HIV - 1 gene expression . ||11959860_155871_9733==>Taken together , these results suggest that Tip110 plays an important role in HIV - 1 gene expression through interaction with Tat . ||11959860_155871_9733==>Combined with our findings that Tip110 alters Tat - mediated gene expression , these results confirmed that Tip110 is a nuclear protein and that the NLS is located within aa 600 and 670 of Tip110 . ||11959860_155871_9733==>Our studies demonstrate that KIAA0156 , or Tip110 as we termed it , exhibits strong synergistic effects with Tat through direct interaction . ||11959860_155871_9733==>We confirmed in vivo interaction between the full - length Tip110 and Tat using the yeast two - hybrid assay ( fig. 2 ) and Western blot combined with immunoprecipitation ( fig. 3 ) . ||11959860_155871_9733==>Consistent with the role of TPR , our studies identified the N - terminal HAT - rich domain of Tip110 that was responsible for interaction of Tip110 and Tat ( fig. 4 ) . ||11959860_155871_9733==>Like many Tat - interacting proteins / co - factors , Tip110 was found to interact with the core domain of Tat ( fig. 5 ) , which is essential for Tat transactivation function . ||11959860_155871_9733==>Although Tip110 contained two RNA recognition motifs ( fig. 1 ) and has been shown to bind to RNA ( 49 ) , Tip110 did not exhibit the TAR binding activity , or any effects on Tat - TAR interaction , determined by the yeast three - hybrid RNA - protein interaction assay ( Table I ) and the gel mobility retardation assay ( data not shown ) . ||11959860_155871_9733==>Importantly , co - expression of Tip110 and Tat greatly potentiated Tat transactivation function on the LTR - driven reporter gene assay ( fig. 6 b ) . ||11959860_155871_9733==>Furthermore , the synergistic effects were also dependent on the Tat binding activity , as deletion of the Tat - binding HAT - rich domain of Tip110 ( Tip110 NT ) resulted in no changes on Tat - mediated reporter gene expression ( fig. 6 c ) . ||11959860_155871_9733==>These data together demonstrate that Tip110 is capable of targeting TAR through interaction with Tat , as well as interacting with other regulatory regions or elements other than TAR within the HIV - 1 LTR promoter . ||11959860_155871_9733==>The enhancement effects were correlated with Tat binding activity of Tip110 , as Tip110 NT mutant exhibited no effects on HIV - 1 gene expression ( fig. 7 a ) . ||11959860_155871_9733==>These results support an essential role of Tip110 in the process of Tat - mediated HIV - 1 gene expression . ||11959860_155871_9733==>Our studies did not rule out the possibility that Tip110 binding to Tat may play a role in the splicing process of HIV - 1 viral mRNA transcripts and / or other Tat - mediated processes , such as HIV - 1 reverse transcription ( 57 ) . ||11959860_155871_9733==>Therefore , additional studies are needed to define the molecular mechanisms of Tip110 function in Tat - mediated HIV - 1 gene expression . ||11959860_155871_9733==>The abbreviations used are : HIV , human immunodeficiency virus ; Tip110 , Tat - interacting protein of 110 kDa ; TAR , Tat transactivating response element ; P - TEFb , positive transcription elongation factor b ; LTR , long terminal repeat ; CAT , chloramphenicol acetyltransferase ; HAT , half - a - tetratricopeptide repeat ; TPR , tetratricopeptide repeat ; RRM , RNA recognition motif ; NLS , nuclear localization signal ; SD , synthetic dropout medium ; LRP , low density lipoprotein receptor - related protein ; AD , Gal4 activation domain - containing yeast vector ; BD , LexA DNA binding domain - containing yeast vector ; - gal , - galactosidase ; RTase , reverse transcriptase ; GFP , green fluorescence protein ; DAPI , 4 , 6 - diamidino - 2 - phenylindole ; aa , amino acid ( s ) ; GST , glutathione S - transferase ; ORF , open reading frame . ||12368300_1025_155871==>The human immunodeficiency virus type 1 ( HIV - 1 ) Tat protein is essential for viral replication and stimulates transcription of the integrated provirus by recruiting the kinase complex TAK / P - TEFb , composed of cyclin T1 ( CycT1 ) and Cdk9 , to the viral TAR RNA element . ||12368300_1025_155871==>The transactivation function of Tat is mediated by a cellular protein kinase complex termed TAK / P - TEFb ( Tat - associated kinase / positive transcription elongation factor b ) , which binds along with Tat to TAR RNA , a 59 - nucleotide stem - loop structure located at the 5 ' ends of all nascent HIV - 1 transcripts . ||12368300_1025_155871==>The CycT1 subunit of the TAK / P - TEFb complex interacts directly with the transactivation domain of Tat to form a zinc - dependent complex that is important for the species - specific activation of HIV - 1 transcription ( 3 , 8 , 11 ) . ||12368300_1025_155871==>There are multiple P - TEFb complexes in human cells that contain Cdk9 but that differ according to their cyclin regulatory subunits , CycT1 , - T2a , - T2b , and - K ; only CycT1 is capable of interaction with the Tat protein . ||12368300_1025_155871==>Because TAK / P - TEFb is essential for Tat function , under some circumstances it may be one of the limiting factors for HIV transcription and replication in these cell types . ||12368300_1025_155871==>In agreement with this transient induction of the CycT1 protein , TAK / P - TEFb kinase function and Tat transactivation of the HIV - 1 LTR are also transiently induced during MDM differentiation . ||12368300_1025_155871==>Arrows , phosphorylated GST - Tat - 2 , Cdk9 , and hyper - and hypophosphorylated CTD ( CTDo and CTDa , respectively ) . ||12368300_1025_155871==>Additionally , TAK / P - TEFb phosphorylates the GST - Tat - 2 protein , resulting in two forms that migrate differently in SDS - polyacrylamide gels . ||12368300_1025_155871==> CDK9 autophosphorylation regulates high - affinity binding of the human immunodeficiency virus type 1 Tat - P - TEFb complex to TAR RNA . ||12368300_1025_155871==> Tat - associated kinase , TAK , activity is regulated by distinct mechanisms in peripheral blood lymphocytes and promonocytic cell lines . ||12368300_1025_155871==>Lentivirus Tat proteins specifically associate with a cellular protein kinase , TAK , that hyperphosphorylates the carboxyl - terminal domain of the large subunit of RNA polymerase II : candidate for a Tat cofactor . ||12368300_1025_155871==>A novel CDK9 - associated C - type cyclin interacts directly with HIV - 1 Tat and mediates its high - affinity , loop - specific binding to TAR RNA . ||12368300_1025_155871==> TAK , an HIV Tat - associated kinase , is a member of the cyclin - dependent family of protein kinases and is induced by activation of peripheral blood lymphocytes and differentiation of promonocytic cell lines . ||12368300_1025_155871==>The human immunodeficiency virus Tat proteins specifically associate with TAK in vivo and require the carboxyl - terminal domain of RNA polymerase II for function . ||12368300_155871_904==>The human immunodeficiency virus type 1 ( HIV - 1 ) Tat protein is essential for viral replication and stimulates transcription of the integrated provirus by recruiting the kinase complex TAK / P - TEFb , composed of cyclin T1 ( CycT1 ) and Cdk9 , to the viral TAR RNA element . ||12368300_155871_904==>In transient transfection assays , the ability of Tat to transactivate the HIV long terminal repeat ( LTR ) in the late differentiated macrophages was greatly diminished relative to its ability to transactivate the HIV LTR in early differentiated cells , strongly suggesting that CycT1 is limiting for Tat function in late differentiated macrophages . ||12368300_155871_904==>The CycT1 subunit of the TAK / P - TEFb complex interacts directly with the transactivation domain of Tat to form a zinc - dependent complex that is important for the species - specific activation of HIV - 1 transcription ( 3 , 8 , 11 ) . ||12368300_155871_904==>There are multiple P - TEFb complexes in human cells that contain Cdk9 but that differ according to their cyclin regulatory subunits , CycT1 , - T2a , - T2b , and - K ; only CycT1 is capable of interaction with the Tat protein . ||12368300_155871_904==>In agreement with this transient induction of the CycT1 protein , TAK / P - TEFb kinase function and Tat transactivation of the HIV - 1 LTR are also transiently induced during MDM differentiation . ||12368300_155871_904==>This observation suggests that the transient induction of CycT1 during MDM differentiation regulates HIV Tat function , and this is likely to be important for HIV - 1 replication levels in macrophages . ||12368300_155871_904==>Levels of Tat transactivation of HIV - 1 LTR in MDMs correlates with the transient expression of CycT1 . ||12368300_155871_904==>The unexpected decline of CycT1 protein expression late in MDM differentiation predicts that the HIV Tat transactivation function will be low or absent at these late time points . ||12368300_155871_904==>For each of the four MDM preparations , Tat transactivation activity was low to absent at late time points when CycT1 protein levels were very low . ||12368300_155871_904==>This result suggests that the level of CycT1 expression during MDM differentiation regulates Tat transactivation of the HIV - 1 LTR , and this is likely to be crucial for HIV - 1 replication levels in MDMs . ||12368300_155871_904==> Tat transactivation activity for the HIV - 1 LTR is high when CycT1 expression is at a peak level during early macrophage differentiation , and it decreases to low or undetectable levels at late time points when CycT1 protein levels decline . ||12368300_155871_904==>This result strongly suggests that the CycT1 protein is limiting for Tat function and therefore also for replication of HIV - 1 in macrophages . ||12368300_155871_904==>We attempted plasmid cotransfections to address whether CycT1 alone is limiting for Tat function late in MDM differentiation . ||12368300_155871_904==>However , no significant enhancement of Tat activity for the HIV - 1 LTR could be observed in MDMs transfected with a CycT1 expression plasmid at late times when CycT1 expression was low ( data not shown ) . ||12368300_155871_904==>The inability to restore Tat function by using the CycT1 expression plasmid may be due to low transfection efficiencies and / or rapid degradation of exogenously expressed CycT1 . ||12368300_155871_904==>The finding that CycT1 is induced only transiently during macrophage differentiation has implications for lentiviruses in addition to HIV - 1 , notably bovine immunodeficiency virus and equine infectious viruses ( EIAV ) , both of which also use a viral Tat protein / TAR RNA mechanism , and therefore CycT1 , to activate transcription directed by the viral LTR ( 1 , 2 , 39 ) . ||12368300_155871_904==>The transient induction of CycT1 appears to regulate Tat transactivation function in these cells , and this may play a critical role in the establishment of latency and the subsequent reactivation of HIV - 1 in infected macrophages . ||12368300_155871_904==>The interaction between HIV - 1 Tat and human cyclin T1 requires zinc and a critical cysteine residue that is not conserved in the murine CycT1 protein . ||12573582_155807_7157==>Of note , Vpr activated p21 transcription in endogenous p53 positive cells , but not in p53 - deleted or p53 nonfunctional cells . ||12573582_155807_7157==> Vpr and p53 had an additive effect on p21 transcription . ||12573582_155807_7157==>Author Keywords : HIV - 1 , Vpr ; cell cycle ; G2 / M phase ; cdk ; p21 ; transcription ; p53 requires=====11070027_142_155348==>It remains unresolved to what extent the retroviral enzymes reverse transcriptase ( gap filling ) and integrase ( DNA splicing ( 11 ) ) or the eukaryotic host proteins , such as DNA - PK and PARP , contribute to the DNA repair of the single - stranded DNA gaps that remain after retrovirus integration . ||11248084_142_155348==>As retroviral infection requires integrase - catalyzed DNA strand breaks , we examined infection of pseudotyped HIV type I in fibroblasts from mice with a targeted deletion of PARP - 1 . ||11248084_142_155348==> PARP - 1 , poly ( ADP - ribose ) polymerase - 1 ; PARP - 1 + / + , PARP - 1 wild type ; PARP - 1 / , genetic deficiency of PARP - 1 ; NAD + , - nicotinamide adenine dinucleotide ; HIV - 1 , HIV type I ; IN , integrase ; LTR , long terminal repeat ; MEFs , mouse embryonic fibroblasts ; FACS , fluorescence - activated cell sorter ; EGFP , enhanced green fluorescent protein ; HIV - EGFP E , envelope protein of HIV - 1 replaced with EGFP ; GAPDH , glyceraldehyde - 3 - phosphate dehydrogenase . ||7666561_155871_6667==> Sp1 transcription factor is required for in vitro basal and Tat - activated transcription from the human immunodeficiency virus type 1 long terminal repeat - - Sune and Garcia - Blanco 69 ( 10 ) : 6572 - - The Journal of Virology ||9334325_1025_155871==>In addition , we found that PITALRE associated with the activation domain of HIV - 1 Tat , indicating that P - TEFb is a Tat - associated kinase ( TAK ) . ||9334325_1025_155871==>Like P - TEFb , this Tat - associated kinase ( TAK ) is sensitive to DRB ( Yang et al. 1996 ) . ||9334325_1025_155871==>It has been suggested that Tat might function by recruiting TAK to phosphorylate the CTD ( Yang et al. 1996 ) . ||9334325_1025_155871==> Tat transactivation is sensitive to DRB ( Braddock et al. 1991 ; Marciniak and Sharp 1991 ) and requires the CTD ( Chun and Jeang 1996 ; Parada and Roeder 1996 ; Yang et al. 1996 ) , and Tat associates with a DRB - sensitive CTD kinase ( TAK ) ( Herrmann and Rice 1993 , 1995 ; Chun and Jeang 1996 ; Yang et al. 1996 ) . ||9334325_1025_155871==>GST - Tat fusions containing mutations in the activation domain that abolish Tat transactivation ( Rice and Carlotti 1990 ; Herrmann and Rice 1995 ) were not able to pull down TAK ( fig. 5 A ) . ||9334325_1025_155871==>The proteins associated with the Tat constructs were probed with anti - PITALRE antibody by Western blot analysis ( fig. 5 B ) . ||9334325_1025_155871==> PITALRE was detected only when the constructs contained an intact Tat transactivation domain . ||9334325_1025_155871==> PITALRE associates with the activation domain of HIV - 1 Tat . ||9334325_1025_155871==>Others have shown that TFIIH can associate with Tat ( Parada and Roeder 1996 ; Garcia - Martinez et al. 1997b ) , but under the conditions used , we did not detect any p62 subunit of TFIIH bound to Tat nor did we detect a reduction in the amount of the subunit in the PITALRE - depleted extract . ||9334325_1025_155871==>To further confirm that human P - TEFb can associate with Tat , TAK was partially purified from HeLa cells by sequential chromatography on DEAE , heparin , and SP resins . ||9334325_1025_155871==>To investigate the function of P - TEFb in Tat transactivation directly , we compared the effect of Tat on initiation and elongation in whole or PITALRE - depleted extracts ( fig. 7 ) . ||9334325_1025_155871==>When PITALRE - depleted extract was used , the majority of the stimulatory effect of Tat was abolished . ||9334325_1025_155871==>This suggests that other required factors were removed by the depletion of PITALRE or that Drosophila P - TEFb lacks appropriate domains required for interaction with Tat or other cofactors . ||9334325_1025_155871==>Addition of the high salt - washed PITALRE immunoprecipitate had no effect on runoff in the presence or absence of Tat ( data not shown ) , indicating that immobilization had a negative impact on the function of P - TEFb . ||9334325_1025_155871==>The interaction of TAK with Tat survives exhaustive washing under stringent conditions and has been observed in vitro ( Herrmann and Rice 1993 , 1995 ; Chun and Jeang 1996 ) and in vivo ( Yang et al. 1996 ) . ||9334325_1025_155871==>Furthermore , TAK ( P - TEFb ) binds to the Tat proteins of HIV - 2 and equine infectious anemia virus ( Herrmann and Rice 1993 , 1995 ) , which both appear to function through the same cofactor as HIV - 1 Tat ( Carroll et al. 1992 ; Madore and Cullen 1993 ) . ||9334325_1025_155871==>Preparation of Tat fusion proteins and the TAK pull - down assay were conducted as described by Herrmann and Rice ( 1993 , 1995 ) with modifications . ||9334325_1025_155871==>Nuclear extract , cytoplasmic extract , or fractions containing partially purified TAK were incubated with glutathione - Sepharose beads containing GST - Tat fusion proteins for 1 hr at 4蚓 with gentle rocking . ||9334326_1025_155871==>Loss of Tat transactivation in extracts depleted of the kinase subunit of human P - TEFb , PITALRE , was reversed by addition of partially purified human P - TEFb . ||9334326_1025_155871==>Transfection experiments with wild - type or kinase knockout PITALRE demonstrated that P - TEFb is required for Tat function . ||9334326_1025_155871==>In an accompanying paper ( Zhu et al. , this issue ) we report that PITALRE , a member of the CDC2 family of protein kinases , is the catalytic subunit of human P - TEFb and associates with the activation domain of Tat . ||9334326_1025_155871==>To assess more directly the requirement of P - TEFb in Tat activation , we immunodepleted nuclear extracts with either antibodies to PITALRE or a control antibody and measured the ability of the treated extracts to support Tat transactivation in vitro . ||9334326_1025_155871==>In contrast , the extract depleted with antibodies against PITALRE did not restore transactivation by Tat , suggesting that human P - TEFb is required for Tat transactivation in vitro ( fig. 5 C , lanes 9 - 12 ) . ||9334326_1025_155871==>( C ) HeLa cell - purified P - TEFb restores Tat transactivation to in vitro transcription reactions reconstituted with nuclear extracts that had been immunodepleted with antibodies against PITALRE . ||9334326_1025_155871==>Western blot analyses indicated that immunodepletion with PITALRE antibodies did not remove either components of the basal transcriptional apparatus ( data not shown ) or Cdk7 , another CTD kinase that has been implicated in Tat activation , whereas greater than 95 % of PITALRE was removed by the immunodepletion experiment ( fig. 5 D ) . ||9334326_1025_155871==>Addition of the HeLa - purified P - TEFb fraction to transcription reactions that contained anti - PITALRE - depleted nuclear extracts restored both Tat - responsiveness and DRB sensitivity ( fig. 5 C , lanes 13 - 16 ) . ||9334326_1025_155871==>A PITALRE kinase mutant has a dominant - negative effect on Tat transactivation ||9334326_1025_155871==>We then analyzed the effect of ectopic expression of either the wild - type or mutated PITALRE on Tat - independent and Tat - dependent transcriptional activation in the transfected cells . ||9334326_1025_155871==>Neither the wild - type nor the mutant PITALRE derivative affected Tat - independent transcription from the HIV LTR ( fig. 6 A , B ) . ||9334326_1025_155871==>Interestingly , expression of the wild - type PITALRE , but not the kinase mutant consistently enhanced Tat - dependent transcription in both cell types ( fig. 6 A , B ) , suggesting that recruitment of P - TEFb kinase to the HIV LTR is dependent on Tat . ||9334326_1025_155871==>More importantly , Tat - dependent luciferase activity was inhibited on overexpression of mutant PITALRE in Jurkat and HeLa cells ( fig. 6 A , B ) . ||9334326_1025_155871==>Inhibition of Tat - dependent luciferase activity by mutant PITALRE was observed only under conditions that supported robust Tat transactivation . ||9334326_1025_155871==>Jurkat cells supported the strongest Tat - dependent activation ( > 2500 - fold ) and the most dramatic inhibition of Tat - mediated transcription by the mutant PITALRE kinase ( fig. 6 A ) . ||9334326_1025_155871==>HeLa cells supported intermediate levels of Tat transactivation ( an average of 50 - fold ) , which were inhibited consistently ~40 % by the mutant PITALRE kinase . ||9334326_1025_155871==>( A ) Inhibition of Tat - dependent transactivation by overexpression of mutated PITALRE in Jurkat cells . ||9334326_1025_155871==>All transfections were performed with a wild - type LTR - luciferase reporter with ( + ) or without ( ) a Tat expression vector and without ( - ) or with wild - type ( wt ) or mutated ( mt ) PITALRE expression vectors as indicated . ||9334326_1025_155871==>( B ) Stimulation of Tat - dependent activation in HeLa cells by wild - type , but not mutated PITALRE . ||9334326_1025_155871==>Therefore , the loss of Tat transactivation on treatment of the extract with antibodies to PITALRE implies that cofactors required for Tat function were removed by the immunodepletion process . ||9334326_1025_155871==>Direct evidence of a role for P - TEFb in Tat activation in vivo is provided by transient transfection experiments in Jurkat and HeLa cells with either the wild - type PITALRE subunit or one containing a null mutation ( fig. 6 ) . ||9334326_1025_155871==> Tat - independent transcription from the HIV LTR was equivalent whether or not the PITALRE gene carried the null mutation . ||9334326_1025_155871==>In the presence of the wild - type , but not the mutated PITALRE gene , Tat - activated transcription was stimulated consistently in both cell types , indicating that transcriptional enhancement is dependent on the enzymatic activity of the wild - type gene . ||9334326_1025_155871==>Expression vectors were synthesized with pCDNA 3 ( Promega ) as the parent vector and used to drive the expression of Tat , wild - type PITALRE , and mutated PITALRE . ||9557739_1025_155871==> PITALRE , the Catalytic Subunit of TAK , Is Required for Human Immunodeficiency Virus Tat Transactivation In Vivo - - Gold et al. 72 ( 5 ) : 4448 - - The Journal of Virology ||9557739_1025_155871==> PITALRE , the Catalytic Subunit of TAK , Is Required for Human Immunodeficiency Virus Tat Transactivation In Vivo Moses O . ||9557739_1025_155871==>The human cdc2 - related kinase PITALRE is the catalytic component of TAK , the Tat - associated kinase . ||9557739_1025_155871==>Previously , we have proposed that TAK is a cellular factor that mediates Tat transactivation function . ||9557739_1025_155871==>Here we demonstrate that transient overexpression of PITALRE specifically squelches Tat - 1 activation of both a transfected and an integrated human immunodeficiency virus type 1 ( HIV - 1 ) long terminal repeat ( LTR ) , suggesting that PITALRE mediates Tat function as a multiprotein complex . ||9557739_1025_155871==>A catalytic mutant of PITALRE , D167N , was found to be more efficient than wild - type PITALRE in squelching Tat transactivation . ||9557739_1025_155871==>Additionally , we show that artificial targeting of PITALRE to a nascent RNA element , in the absence of Tat , activated HIV - 1 LTR expression . ||9557739_1025_155871==>These results indicate that a PITALRE - containing complex mediates transactivation by Tat and suggest that Tat proteins function by localizing such a PITALRE - containing complex to the site of the transcribing provirus . ||9557739_1025_155871==>Based on biochemical observations , a number of cofactors that could constitute the cellular interface to Tat function have been proposed , including cellular general transcription factor TFIIH ( 11 , 31 ) , Tat - SF1 ( 46 ) , and Tat - associated kinase ( TAK ) ( 14 , 15 , 42 ) . ||9557739_1025_155871==>To determine the role of TAK in Tat - mediated viral transcription , in this study , wild - type PITALRE or catalytically inactive mutant PITALRE was transiently overexpressed and the resultant effect on transactivation of the HIV - 1 long terminal repeat ( LTR ) by Tat - 1 was observed . ||9557739_1025_155871==>These studies have provided evidence that TAK mediates Tat 's transactivation function in vivo . ||9557739_1025_155871==>Transiently expressed wild - type PITALRE and catalytic mutant D167N specifically associate with Tat in vitro . ||9557739_1025_155871==>Transiently expressed wild - type and catalytic mutant PITALRE proteins specifically associate with Tat in vitro . ||9557739_1025_155871==>To determine whether the transiently expressed PITALRE proteins were capable of specific association with Tat proteins , in vitro glutathione S - transferase ( GST ) `` pull - down '' assays were performed with wild - type HIV - 2 Tat ( Tat - 2 ) and the transactivation - defective C59A Tat - 2 mutant protein . ||9557739_1025_155871==>An immunoblot performed with anti - PITALRE antibodies indicated that endogenous HeLa PITALRE and both recombinant PITALRE and D167N associated with the GST fusion to wild - type but not mutant Tat - 2 ( fig. 1 B ) . ||9557739_1025_155871==>We conclude from these experiments that the D167N mutant lacks a detectable catalytic function and , like wild - type PITALRE , is able to specifically associate in a TAK complex with Tat - 2 . ||9557739_1025_155871==> PITALRE association with Tat proteins could require that PITALRE first assemble into a complex with other cellular factors . ||9557739_1025_155871==>Using baculovirus - expressed PITALRE and Escherichia coli - expressed PITALRE , we have been unable to observe a specific association between PITALRE and Tat proteins in vitro ( unpublished observations ) . ||9557739_1025_155871==>The ability of recombinant PITALRE isolated from cells to associate with Tat - 2 in vitro , as shown in fig. 1 B , may require an additional human cellular component . ||9557739_1025_155871==> PITALRE overexpression in HeLa cells inhibits transactivation by Tat - 1 . ||9557739_1025_155871==>To examine the effects of overexpression of wild - type and catalytic mutant PITALRE proteins on Tat function in vivo , HeLa cells were cotransfected with an HIV - 1 LTR luciferase reporter plasmid , a Tat - 1 expression plasmid , and a plasmid expressing PITALRE or D167N ( fig. 2 A ) . ||9557739_1025_155871==> PITALRE overexpression resulted in 2.7 - fold inhibition of Tat transactivation function , and overexpression of D167N resulted in 5.8 - fold inhibition of transactivation . ||9557739_1025_155871==>Effect of PITALRE overexpression on transactivation by HIV - 1 Tat and HTLV - 1 Tax . ||9557739_1025_155871==>( A ) PITALRE overexpression inhibits transactivation by Tat - 1 . ||9557739_1025_155871==>HeLa cells ( 50 % confluent in 6 - cm - diameter dishes ) were transfected with 15 ng of an HIV - 1 LTR luciferase reporter plasmid ( 1 ) , 1 , 500 ng of a simian virus 40 early promoter - galactosidase ( - gal ) reporter plasmid ( pCH110 ; Pharmacia ) , 25 ng of CMV2 - FLAG - Tat - 1 , and 500 ng of the CMV2 - FLAG parental vector ( Kodak ) or a FLAG - PITALRE plasmid , as indicated . ||9557739_1025_155871==>In the experiment presented in fig. 2 , the Tat , Tax , and PITALRE proteins were expressed from the same cytomegalovirus ( CMV ) promoter ; therefore , it is highly unlikely that the levels of inhibition of Tat transactivation by the wild - type and D167N PITALRE proteins can be explained by reduced Tat protein levels . ||9557739_1025_155871==>These results indicate that both wild - type PITALRE and D167N , upon overexpression in HeLa cells , can specifically squelch Tat - mediated transactivation . ||9557739_1025_155871==>The ability of wild - type overexpressed PITALRE to squelch Tat function suggests that TAK function requires one or more subunits in addition to PITALRE , such that overexpression of one subunit disrupts the stoichiometry of the functional complex . ||9557739_1025_155871==>The observation that D167N squelches Tat function more efficiently than does wild - type PITALRE suggests that the catalytic function of PITALRE is important for TAK function and is consistent with our previously proposed model that the function of TAK during HIV transcription is to phosphorylate the CTD of RNAPII ( 15 ) . ||9557739_1025_155871==>To determine whether PITALRE overexpression could inhibit Tat activation of an integrated HIV - 1 LTR , we examined Jurkat T - cell line 1G5 , which contains an integrated HIV - 1 LTR with luciferase as the reporter protein ( 1 ) . ||9557739_1025_155871==>1G5 cells were cotransfected by electroporation with a Tat - 1 expression plasmid and a PITALRE or D167N expression plasmid ( fig. 3 ) . ||9557739_1025_155871==>Measurement of Tat - induced luciferase activities at 24 h posttransfection yielded 60.1 luciferase U in the presence of the parental vector , 18.4 U upon wild - type PITALRE overexpression , and 7.8 U upon D167N overexpression . ||9557739_1025_155871==>Therefore , in 1G5 cells , PITALRE overexpression inhibited Tat - induced luciferase expression 3.3 - fold , while D167N overexpression inhibited Tat - induced luciferase expression 7.7 - fold . ||9557739_1025_155871==> PITALRE overexpression inhibits Tat activation of an integrated HIV - 1 LTR . ||9557739_1025_155871==>1G5 cells ( 3.0 � 10 6 ) were transfected with 1 痢 of a CMV2 - FLAG - Tat - 1 and 10 痢 of a CMV2 - FLAG or FLAG - PITALRE plasmid by electroporation ( Gene Pulser ; Bio - Rad ) at 250 V and 960 湩 in 400 痞 of serum - free RPMI medium . ||9557739_1025_155871==>Both wild - type PITALRE and D167N squelch Tat - mediated transactivation , and D167N squelches more efficiently than wild - type PITALRE . ||9557739_1025_155871==>These results obtained with 1G5 cells indicate , additionally , that PITALRE overexpression can squelch Tat - 1 - mediated transactivation of an integrated HIV - 1 LTR . ||9557739_1025_155871==>Our observation that Tat associates with TAK led us to suggest the model for the mechanism of action of Tat shown in fig. 4 A . ||9557739_1025_155871==>This model proposes that by binding to TAR RNA , Tat is able to functionally recruit TAK to the RNAPII complex , leading to hyperphosphorylation of the CTD and a resultant activation of transcription ( 15 ) . ||9557739_1025_155871==>This model proposes that Tat recruits a cellular protein kinase , TAK , to the RNAPII holoenzyme ( 15 ) . ||9557739_1025_155871==>In an additional experiment , we compared activation of the reporter plasmid by PITALRE - Rev and Tat - Rev fusion proteins ( fig. 5 B ) . ||9557739_1025_155871==>The Tat - Rev protein was considerably more active , resulting in 37 - fold activation versus 4.8 - fold activation by the PITALRE - Rev protein . ||9557739_1025_155871==>The observation that PITALRE targeted to a nascent RNA element , in the absence of Tat , can activate HIV - 1 LTR - driven gene expression is consistent with the suggestion that PITALRE can act as a transcription activator . ||9557739_1025_155871==>The relatively low levels of activation by PITALRE - Rev may indicate that other factors , such as its cyclin partner or additional TAK components , are needed to reconstitute the total functional entity that is interfaced by Tat . ||9557739_1025_155871==>While this report was in preparation , we became aware of two recent studies describing the involvement of PITALRE in Tat transactivation ( 25 , 47 ) . ||9557739_1025_155871==>Using an vitro transcription assay , both studies demonstrated that PITALRE is required for Tat transactivation in vitro . ||9557739_1025_155871==>Additionally , one study also investigated the effect of transient overexpression of PITALRE on Tat transactivation in vivo ( 25 ) ; however , those data , in contrast to the results presented here , indicate activation of Tat function by overexpression of wild - type PITALRE . ||9557739_1025_155871==>However , our finding that overexpression of wild - type PITALRE can inhibit Tat function is consistent with the proposal that PITALRE functions in vivo as a multiprotein complex . ||9557739_1025_155871==>In agreement with our results , in this recent study it was also observed that a catalytic mutant of PITALRE does inhibit Tat transactivation ( 25 ) . ||9557739_1025_155871==>Taken together , recent work by several laboratories has demonstrated that PITALRE does , indeed , mediate Tat transactivation . stabilizes=====14561767_155945_468==>We showed that the expression of a Vpu - green fluorescent fusion protein prevented the proteosomal degradation of TrCP substrates such as - catenin , I B , and ATF4 , which are normally directly targeted to the proteasome for degradation . ||14561767_155945_468==>The overexpression of ATF4 also provoked accumulation of - catenin , but to a lower level than that resulting from the expression of Vpu . ||14561767_155945_468==>The signal for the recognition of all these cellular ligands by TrCP is the phosphorylation of one or two serine residues present in a conserved motif , DSG XX S for Vpu , - catenin , and I B and DSG XXX S for ATF4 , p105 , and p100 . ||14561767_155945_468==>HeLa cells were transiently transfected by electroporation when using plasmids encoding HA - ATF4 , Vpu - GFP , Vpuc - GFP , or TrCP - FLAG or by the calcium method when using pNL4 - 3 or its variants . ||14561767_155945_468==>Western Blotting Analysis 𣶷amples containing 8 x 10 6 HeLa cells were transiently transfected by electroporation with Vpuc - GFP , Vpuc2.6 - GFP , Vpu - GFP , Vpu2.6 - GFP , or HA - ATF4 . ||14561767_155945_468==>Expression of Vpu Results in Accumulation of ATF4 𦝁o examine whether ATF4 , a newly identified substrate of TrCP , accumulated in the presence of Vpu like - catenin and I B , we analyzed the effect of Vpuc - GFP on cells co - transfected with HA - ATF4 expression vector . ||14561767_155945_468==>A , expression of Vpu - GFP in HeLa cells results in accumulation of transfected HA - ATF4 . ||14561767_155945_468==>B , expression of Vpu - GFP has a stronger effect on the accumulation of endogenous - catenin than does the expression of HA - ATF4 . ||14561767_155945_468==>In conclusion , these data suggest that Vpu is a more potent competitive inhibitor of TrCP for the inhibition of - catenin degradation than the overproduced ATF4 . ||14561767_155945_468==>In fact , - catenin , I B , and ATF4 accumulate , and the transcriptional activity of - catenin is up - regulated in the presence of Vpu . ||14561767_155945_468==>The accumulation of ATF4 as a result of Vpu production might disrupt the metabolism of T cells infected by HIV - 1 . ||8764062_155871_2958==>Interaction of human immunodeficiency virus type 1 Tat with a unique site of TFIID inhibits negative cofactor Dr1 and stabilizes the TFIID - TFIIA complex - - Kashanchi et al. 70 ( 8 ) : 5503 - - The Journal of Virology stimulates=====10393184_155871_5435==>This study ( Figure 4D , right ) showed that antibodies directed against the CTD and a small RNA Pol II subunit ( RPB6 ) specifically co - immunoprecipitated RNA Pol II , hSPT5 , Tat - SF1 and ( to a lesser extent ) nucleolin ( lanes 3�4 ) , whereas control anti - TFIIB and anti - RPC82 antibodies did not ( lanes 2 and 5 ) . ||10393184_155871_5435==>Antibodies against the RNA Pol II CTD , the RPB6 subunit of RNA Pol II , CDK9 , hSPT5 , Tat - SF1 and nucleolin generally precipitated significant and roughly comparable relative amounts of the assayed Tat - SF components , as well as excess amounts of the specific antigen ( Figure 4D , lanes 3 , 4 , 6�9 ) . ||10393184_155871_6829==>Keywords : nucleolin , P - TEFb , SPT5 , Tat - activated HIV - 1 transcription , Tat - SF ||10393184_155871_6829==>, 1993 ) ; PSTAIRE , a cdc2 - like kinase ( Lee and Nurse , 1987 ) ; CKII ; the CDK9 and cyclin T components of P - TEFb ; and human homologues of yeast SPT5 ( also designated Tat - CT1 , Wu - Baer et al . ||10393184_155871_6829==>The main exceptions are the anti - CDK9 antibodies , which did not appear to precipitate an excess of antigen ( Figure 4D , lane 6 ; data not shown ) , and the anti - SPT5 and anti - Tat - SF1 antibodies , which precipitated little to no nucleolin ( lanes 7 and 8 ) . ||10393184_155871_6829==>Wu - Baer F , Lane W and Gaynor R ( 1998 ) Role of the human homolog of the yeast transcription factor SPT5 in HIV - 1 Tat - activation . ||10438593_1022_155871==>Several groups have suggested that in addition to activating CDK9 , Tat might regulate the phosphorylation of the RNA polymerase II carboxyl - terminal domain in pre - initiation complexes by activating CDK7 , the kinase associated with TFIIH ( Cujec et al 1997 , Garcia - Martinez et al 1997 and Parada and Roeder 1996 ) . ||10438593_1022_155871==>Step 2 : the CDK7 kinase present in TFIIH phosphorylates the CTD of RNA polymerase , in the absence of Tat . ||10438593_1022_155871==>It is important to note that because we are able to analyse kinase activity using active pre - initiation complexes assembled in the presence of template DNA , our experiments are less prone to artefacts , such as the binding of Tat to a conserved sequence in the kinase domain of CDK7 ( TFIIH ) that is inaccessible in the pre - initiation complex , than experiments performed using purified components . ||10438593_1022_155871==>These results are consistent with a recent report by ( Chen and Zhou 1999 ) , who observed that Tat can still stimulate transcription elongation after the removal of CDK7 from TFIIH by treatment with high salt and immunodepletion . ||10438593_155871_2071==>Abbreviations : CDK , cyclin - dependent kinase ; CTD , carboxyl - terminal domain ; DNA - PK , DNA - dependent protein kinase ; DRB , 5 , 6 - dichloro - 1 - - - ribofuranosyl benzimidazole ; H8 , N - ( 2 - ( methylamino ) ethyl ) - 5 - isoquinoline sulphonamide ; HIV - 1 , human immunodeficiency virus type - 1 ; TAK , Tat - associated kinase ; TAR , trans - activation response element ; TFIIH , transcription factor IIH ||10438593_155871_2071==>The results demonstrate that Tat can stimulate TAK activity in elongation complexes but does not affect the rate of phosphorylation by TFIIH of the CTD of RNA polymerase present in pre - initiation complexes . ||10438593_155871_2071==>Several groups have suggested that in addition to activating CDK9 , Tat might regulate the phosphorylation of the RNA polymerase II carboxyl - terminal domain in pre - initiation complexes by activating CDK7 , the kinase associated with TFIIH ( Cujec et al 1997 , Garcia - Martinez et al 1997 and Parada and Roeder 1996 ) . ||10438593_155871_2071==>In contrast to previous reports using purified TFIIH ( Cujec et al 1997 , Garcia - Martinez et al 1997 and Parada and Roeder 1996 ) , we have found that TFIIH - dependent phosphorylation of the RNA polymerase II CTD in the assembled pre - initiation complex is not stimulated by Tat ( Figure 2 and Figure 3 ) . ||10438593_155871_2071==>Step 2 : the CDK7 kinase present in TFIIH phosphorylates the CTD of RNA polymerase , in the absence of Tat . ||10438593_155871_2071==>In contrast to previous reports suggesting that Tat can stimulate phosphorylation of RNA polymerase CTD by TFIIH ( Garcia - Martinez et al 1997 and Parada and Roeder 1996 ) , we have been unable to detect any increase in CTD phosphorylation at the pre - initiation complex after addition of Tat . ||10438593_155871_2071==>It is important to note that because we are able to analyse kinase activity using active pre - initiation complexes assembled in the presence of template DNA , our experiments are less prone to artefacts , such as the binding of Tat to a conserved sequence in the kinase domain of CDK7 ( TFIIH ) that is inaccessible in the pre - initiation complex , than experiments performed using purified components . ||10438593_155871_2071==>These results are consistent with a recent report by ( Chen and Zhou 1999 ) , who observed that Tat can still stimulate transcription elongation after the removal of CDK7 from TFIIH by treatment with high salt and immunodepletion . ||10438593_155871_2965==>Abbreviations : CDK , cyclin - dependent kinase ; CTD , carboxyl - terminal domain ; DNA - PK , DNA - dependent protein kinase ; DRB , 5 , 6 - dichloro - 1 - - - ribofuranosyl benzimidazole ; H8 , N - ( 2 - ( methylamino ) ethyl ) - 5 - isoquinoline sulphonamide ; HIV - 1 , human immunodeficiency virus type - 1 ; TAK , Tat - associated kinase ; TAR , trans - activation response element ; TFIIH , transcription factor IIH ||10438593_155871_2965==>The results demonstrate that Tat can stimulate TAK activity in elongation complexes but does not affect the rate of phosphorylation by TFIIH of the CTD of RNA polymerase present in pre - initiation complexes . ||10438593_155871_2965==>Several groups have suggested that in addition to activating CDK9 , Tat might regulate the phosphorylation of the RNA polymerase II carboxyl - terminal domain in pre - initiation complexes by activating CDK7 , the kinase associated with TFIIH ( Cujec et al 1997 , Garcia - Martinez et al 1997 and Parada and Roeder 1996 ) . ||10438593_155871_2965==>In contrast to previous reports using purified TFIIH ( Cujec et al 1997 , Garcia - Martinez et al 1997 and Parada and Roeder 1996 ) , we have found that TFIIH - dependent phosphorylation of the RNA polymerase II CTD in the assembled pre - initiation complex is not stimulated by Tat ( Figure 2 and Figure 3 ) . ||10438593_155871_2965==>Step 2 : the CDK7 kinase present in TFIIH phosphorylates the CTD of RNA polymerase , in the absence of Tat . ||10438593_155871_2965==>In contrast to previous reports suggesting that Tat can stimulate phosphorylation of RNA polymerase CTD by TFIIH ( Garcia - Martinez et al 1997 and Parada and Roeder 1996 ) , we have been unable to detect any increase in CTD phosphorylation at the pre - initiation complex after addition of Tat . ||10438593_155871_2965==>It is important to note that because we are able to analyse kinase activity using active pre - initiation complexes assembled in the presence of template DNA , our experiments are less prone to artefacts , such as the binding of Tat to a conserved sequence in the kinase domain of CDK7 ( TFIIH ) that is inaccessible in the pre - initiation complex , than experiments performed using purified components . ||10438593_155871_2965==>These results are consistent with a recent report by ( Chen and Zhou 1999 ) , who observed that Tat can still stimulate transcription elongation after the removal of CDK7 from TFIIH by treatment with high salt and immunodepletion . ||10438593_155871_2966==>Abbreviations : CDK , cyclin - dependent kinase ; CTD , carboxyl - terminal domain ; DNA - PK , DNA - dependent protein kinase ; DRB , 5 , 6 - dichloro - 1 - - - ribofuranosyl benzimidazole ; H8 , N - ( 2 - ( methylamino ) ethyl ) - 5 - isoquinoline sulphonamide ; HIV - 1 , human immunodeficiency virus type - 1 ; TAK , Tat - associated kinase ; TAR , trans - activation response element ; TFIIH , transcription factor IIH ||10438593_155871_2966==>The results demonstrate that Tat can stimulate TAK activity in elongation complexes but does not affect the rate of phosphorylation by TFIIH of the CTD of RNA polymerase present in pre - initiation complexes . ||10438593_155871_2966==>Several groups have suggested that in addition to activating CDK9 , Tat might regulate the phosphorylation of the RNA polymerase II carboxyl - terminal domain in pre - initiation complexes by activating CDK7 , the kinase associated with TFIIH ( Cujec et al 1997 , Garcia - Martinez et al 1997 and Parada and Roeder 1996 ) . ||10438593_155871_2966==>In contrast to previous reports using purified TFIIH ( Cujec et al 1997 , Garcia - Martinez et al 1997 and Parada and Roeder 1996 ) , we have found that TFIIH - dependent phosphorylation of the RNA polymerase II CTD in the assembled pre - initiation complex is not stimulated by Tat ( Figure 2 and Figure 3 ) . ||10438593_155871_2966==>Step 2 : the CDK7 kinase present in TFIIH phosphorylates the CTD of RNA polymerase , in the absence of Tat . ||10438593_155871_2966==>In contrast to previous reports suggesting that Tat can stimulate phosphorylation of RNA polymerase CTD by TFIIH ( Garcia - Martinez et al 1997 and Parada and Roeder 1996 ) , we have been unable to detect any increase in CTD phosphorylation at the pre - initiation complex after addition of Tat . ||10438593_155871_2966==>It is important to note that because we are able to analyse kinase activity using active pre - initiation complexes assembled in the presence of template DNA , our experiments are less prone to artefacts , such as the binding of Tat to a conserved sequence in the kinase domain of CDK7 ( TFIIH ) that is inaccessible in the pre - initiation complex , than experiments performed using purified components . ||10438593_155871_2966==>These results are consistent with a recent report by ( Chen and Zhou 1999 ) , who observed that Tat can still stimulate transcription elongation after the removal of CDK7 from TFIIH by treatment with high salt and immunodepletion . ||10438593_155871_2968==>Abbreviations : CDK , cyclin - dependent kinase ; CTD , carboxyl - terminal domain ; DNA - PK , DNA - dependent protein kinase ; DRB , 5 , 6 - dichloro - 1 - - - ribofuranosyl benzimidazole ; H8 , N - ( 2 - ( methylamino ) ethyl ) - 5 - isoquinoline sulphonamide ; HIV - 1 , human immunodeficiency virus type - 1 ; TAK , Tat - associated kinase ; TAR , trans - activation response element ; TFIIH , transcription factor IIH ||10438593_155871_2968==>The results demonstrate that Tat can stimulate TAK activity in elongation complexes but does not affect the rate of phosphorylation by TFIIH of the CTD of RNA polymerase present in pre - initiation complexes . ||10438593_155871_2968==>Several groups have suggested that in addition to activating CDK9 , Tat might regulate the phosphorylation of the RNA polymerase II carboxyl - terminal domain in pre - initiation complexes by activating CDK7 , the kinase associated with TFIIH ( Cujec et al 1997 , Garcia - Martinez et al 1997 and Parada and Roeder 1996 ) . ||10438593_155871_2968==>In contrast to previous reports using purified TFIIH ( Cujec et al 1997 , Garcia - Martinez et al 1997 and Parada and Roeder 1996 ) , we have found that TFIIH - dependent phosphorylation of the RNA polymerase II CTD in the assembled pre - initiation complex is not stimulated by Tat ( Figure 2 and Figure 3 ) . ||10438593_155871_2968==>Step 2 : the CDK7 kinase present in TFIIH phosphorylates the CTD of RNA polymerase , in the absence of Tat . ||10438593_155871_2968==>In contrast to previous reports suggesting that Tat can stimulate phosphorylation of RNA polymerase CTD by TFIIH ( Garcia - Martinez et al 1997 and Parada and Roeder 1996 ) , we have been unable to detect any increase in CTD phosphorylation at the pre - initiation complex after addition of Tat . ||10438593_155871_2968==>It is important to note that because we are able to analyse kinase activity using active pre - initiation complexes assembled in the presence of template DNA , our experiments are less prone to artefacts , such as the binding of Tat to a conserved sequence in the kinase domain of CDK7 ( TFIIH ) that is inaccessible in the pre - initiation complex , than experiments performed using purified components . ||10438593_155871_2968==>These results are consistent with a recent report by ( Chen and Zhou 1999 ) , who observed that Tat can still stimulate transcription elongation after the removal of CDK7 from TFIIH by treatment with high salt and immunodepletion . ||10454543_155871_6829==> Tat - SF1 Protein Associates with RAP30 and Human SPT5 Proteins - - Kim et al. 19 ( 9 ) : 5960 - - Molecular and Cellular Biology ||10454543_155871_6829==> Tat - SF1 Protein Associates with RAP30 and Human SPT5 Proteins Jae B . ||10454543_155871_6829==>The cellular proteins Tat - SF1 and human SPT5 ( hSPT5 ) are required for Tat activation as shown by immunodepletion with specific sera and complementation with recombinant proteins . ||10454543_155871_6829==>A recent study has suggested that the human homolog of the yeast transcription factor SPT5 is also involved in Tat - activated elongation of transcription ( 35 ) . ||10454543_155871_6829==>This study shows that Tat - SF1 efficiently associates with the RAP30 subunit of TFIIF and inefficiently associates with Pol II and human SPT5 ( hSPT5 ) . ||10454543_155871_6829==>Interestingly , Pol II ( largest subunit : Pol II - LS ) and human SPT5 ( hSPT5 ) were also found to be associated with Tat - SF1 ( fig. 2 A ) . ||10454543_155871_6829==>When compared with the interaction between Tat - SF1 and RAP30 , the association between Tat - SF1 and hSPT5 is inefficient , since less than 5 % of the input SPT5 protein was immunoprecipitated with - Tat - SF1 sera . ||10454543_155871_6829==>Recombinant Tat - SF1 and SPT5 proteins complement Tat activation of transcription . ||10617616_155871_904==>A , recombinant Cdk9 / cyclin T1 dimer ( rCdk9 / CycT1 ) purified from baculovirus - infected insect cells was compared with affinity - purified Cdk9 - HA fraction ( - HA IP ) in Tat - specific transcription assays . ||10617616_155871_904==>Addition of 0.5 痞 of the recombinant Cdk9 / cyclin T1 dimer ( rCdk9 / CycT1 ) to the depleted extract also resulted in a major increase in basal HIV - 1 elongation and , more importantly , mediated Tat transactivation , although to a lesser extent than the Cdk9 - HA IP ( fig. 3 A , lanes 3 and 4 showed 18 - fold and lanes 5 and 6 showed 6.7 - fold Tat activation when normalized to internal controls ) . ||10617616_155871_904==>In a separate experiment ( fig. 3 A , lanes 13 - 18 ) , rCdk9 / CycT1 and the Cdk9 - HA IP supported a comparable fold of Tat activation ( lanes 15 and 16 showed 5.5 - fold , and lanes 17 and 18 showed 6.4 - fold Tat activation when normalized to internal controls ) . ||10617616_155871_904==>Results from several independent experiments indicated that on average the fold of Tat activation mediated by rCdk9 / CycT1 was about 50 % of that mediated by the purified Cdk9 - HA fraction . ||10617616_155871_904==>However , disregarding the relative effectiveness of the two P - TEFb preparations , the observation that rCdk9 / CycT1 can indeed support Tat transactivation strongly argues that in addition to the general elongation activity of P - TEFb , the specific ability of P - TEFb to mediate Tat activation resides within the Cdk9 / cyclin T1 heterodimer . ||10617616_155871_904==>In agreement with this model , increasing the concentration of rCdk9 / CycT1 in transcription reactions was found to suppress the degree of Tat activation by increasing the efficiency of elongation independent of Tat ( fig. 3 A , lanes 7 - 12 ) . ||10617616_155871_904==>It is likely that high concentrations of rCdk9 / CycT1 facilitated the association of the dimer with polymerase elongation complexes and thereby bypassed the requirement of Tat for efficient elongation . ||10617616_155871_904==>Importantly , this Cdk9 / HA - CycT1 complex has been shown to be an active P - TEFb for mediating Tat transactivation ( 5 ) . ||10866664_1025_155871==> Tat Modifies the Activity of CDK9 To Phosphorylate Serine 5 of the RNA Polymerase II Carboxyl - Terminal Domain during Human Immunodeficiency Virus Type 1 Transcription - - Zhou et al. 20 ( 14 ) : 5077 - - Molecular and Cellular Biology ||10866664_1025_155871==> Tat Modifies the Activity of CDK9 To Phosphorylate Serine 5 of the RNA Polymerase II Carboxyl - Terminal Domain during Human Immunodeficiency Virus Type 1 Transcription Meisheng Zhou , Matthew A . ||10866664_1025_155871==>Remarkably , in the presence of Tat , the substrate specificity of CDK9 is altered , such that the kinase phosphorylates both serine 2 and serine 5 . ||10866664_1025_155871==> Tat - induced CTD phosphorylation by CDK9 is strongly inhibited by low concentrations of 5 , 6 - dichloro - 1 - - D - ribofuranosylbenzimidazole , an inhibitor of transcription elongation by RNAP II . ||10866664_1025_155871==>These studies suggest that the ability of Tat to increase transcriptional elongation may be due to its ability to modify the substrate specificity of the CDK9 complex . ||10866664_1025_155871==>The second CTD kinase , TAK ( Tat - associated kinase ) , was first reported by Herrmann and Rice ( 22 ) . ||10866664_1025_155871==>Importantly , immunodepletion of CDK9 from HeLa nuclear extract eliminated basal transcription elongation and Tat transactivation ( 39 , 84 , 85 ) . ||10866664_1025_155871==>Thus , the ability of Tat to recruit cyclin T1 - CDK9 to TAR not only stimulates HIV - 1 transcriptional elongation but also governs the species specificity of HIV - 1 Tat transactivation . ||10866664_1025_155871==>Remarkably , Tat modifies the substrate specificity of PIC CDK9 , allowing CDK9 to phosphorylate serine 2 and serine 5 . ||10866664_1025_155871==> CDK9 ( a subunit of P - TEFb ) , CDK7 , cyclin H , Mat1 , and p62 ( subunits of TFIIH ) , CDK8 , and Tat were detected with specific antibodies as indicated above . ||10866664_1025_155871==>Figure 2 shows the Western blot analysis for P - TEFb ( CDK9 ) , TFIIH ( CDK7 , cyclin H , Mat1 , and p62 subunits ) , CDK8 , and Tat . ||10866664_1025_155871==>In contrast to the results obtained with CDK7 and CDK9 , we find that CDK8 is not present in the HIV - 1 PIC in the absence or presence of Tat ( fig. 2 C , lanes 1 and 3 ) . ||10866664_1025_155871==>Western blot analysis of the purified HIV - 1 PICs was then done with anti - CDK9 , anti - CDK7 , anti - cyclin H , anti - Mat1 , anti - p62 , anti - CDK8 , and anti - Tat antibodies . ||10866664_1025_155871==>Basal and Tat - activated transcription differ in their requirements for CDK7 and CDK9 . ||10866664_1025_155871==>Biotinylated HIV - 1 LTR templates were incubated with mock - depleted , CDK8 - depleted , CDK7 - depleted , CDK9 - depleted , and CDK7 - and CDK9 - depleted extracts in the absence ( lanes 1 to 5 ) or presence ( lanes 6 to 10 ) of Tat , and PICs were then purified with streptavidin - coated magnetic beads . ||10866664_1025_155871==>Biotinylated HIV - 1 LTR templates were incubated with the mock - depleted , CDK8 - depleted , CDK7 - depleted , CDK9 - depleted , and CDK7 - and CDK9 - depleted extracts in the absence or presence of Tat , and PICs were purified with streptavidin - coated magnetic beads . ||10866664_1025_155871==>In the absence of Tat , the depletion of CDK7 or CDK9 decreased HIV - 1 transcription approximately 3 - and 10 - fold , respectively ( lanes 1 to 4 ) . ||10866664_1025_155871==>In contrast , CDK9 depletion decreased Tat transactivation by more than 16 - fold to essentially background levels ( lanes 6 and 9 ) . ||10866664_1025_155871==>When both CDK7 and CDK9 were depleted from the extract , the basal transcription and Tat transactivation were eliminated ( lanes 5 and 10 ) . ||10866664_1025_155871==>Biotinylated HIV - 1 LTR templates were incubated with mock - depleted , CDK7 - depleted , CDK9 - depleted , or CDK7 - and CDK9 - depleted extracts in the absence ( lanes 1 to 4 ) or presence ( lanes 5 to 8 ) of Tat , and PICs were then purified with streptavidin - coated magnetic beads . ||10866664_1025_155871==>Biotinylated HIV - 1 LTR templates were incubated with mock - depleted , CDK7 - depleted , CDK9 - depleted , or CDK7 - and CDK9 - depleted extracts in the absence ( lanes 1 to 4 ) or presence ( lanes 5 to 8 ) of Tat . ||10866664_1025_155871==>The analysis of kinase activity in HIV - 1 PICs formed in the presence of Tat provided evidence that the substrate specificity of CDK9 is altered in the Tat complexes . ||10866664_1025_155871==>Since the serine 5 kinase activity was lost when CDK9 was also depleted from the extract ( fig. 5 , bottom , lane 8 ) , this result suggests that CDK9 phosphorylates serine 5 in the HIV - 1 Tat transcription complex . ||10866664_1025_155871==>In the absence of Tat , CDK9 phosphorylates only serine 2 . ||10866664_1025_155871==>It should be stressed that these studies , which clearly show the specificity of CDK9 kinase activity in the absence or presence of Tat , are done in the context of the transcription complex and the authentic substrate , the CTD of RNAP II . ||10866664_1025_155871==>CTD phosphorylation by Tat - modified CDK9 is sensitive to DRB . ||10866664_1025_155871==>The results of this experiment demonstrate that serine 2 and serine 5 phosphorylation by Tat - induced CDK9 is sensitive to low concentrations of DRB . ||10866664_1025_155871==>CTD phosphorylation by Tat - modified CDK9 is sensitive to DRB . ||10866664_1025_155871==>Since CDK7 and Tat - induced CDK9 phosphorylate serine 5 , the ability of Tat to modify the substrate specificity of the CDK9 complex would most likely become important when CDK7 was released from the transcription complex . ||10866664_1025_155871==>The amount of CDK9 associated with the complex is not modified by the presence or absence of Tat . ||10866664_1025_155871==>The above - described results suggest that Tat - induced phosphorylation of serine 5 by CDK9 might be important after transcription has reached the + 36 position , at which time CDK7 has been released from the complex . ||10866664_1025_155871==> Tat directly modifies the substrate specificity of CDK9 in the recombinant P - TEFb complex . ||10866664_1025_155871==>The above - described experiments suggest that Tat modifies the substrate specificity of the CDK9 - containing P - TEFb complex . ||10866664_1025_155871==>The results presented in fig. 9 B demonstrate several important points about the functional interaction between Tat and CDK9 . ||10866664_1025_155871==>First , in the absence of Tat , CDK9 phosphorylates the CTD at serine 2 but not at serine 5 ( fig. 9 B , lane 1 ) . ||10866664_1025_155871==>The addition of a Tat mutant containing an amino acid substitution at cysteine 22 failed to increase CDK9 activity ( fig. 9 B , top , compare lane 1 with lanes 2 to 5 ) . ||10866664_1025_155871==>Remarkably , in the presence of Tat , CDK9 was also able to phosphorylate serine 5 ( bottom , compare lane 1 with lanes 6 to 9 ) . ||10866664_1025_155871==>In results similar to those presented in fig. 6 , the addition of low concentrations of DRB to the kinase reactions inhibited serine 2 and serine 5 phosphorylation by Tat - induced CDK9 ( fig. 9 C ) . ||10866664_1025_155871==>These results provide direct evidence that Tat modifies the substrate specificity of the CDK9 enzyme and that the induction of kinase activity observed in the presence of Tat is primarily due to phosphorylation at serine 5 . ||10866664_1025_155871==>Ramanathan et al. have recently reported that serine 5 is essential for phosphorylation by the Tat - TAK complex ( Tat - P - TEFb complex ) ( 55 ) . ||10866664_1025_155871==>Mutation of serines 2 and 7 did not affect the activity of the Tat - TAK complex . ||10866664_1025_155871==>Recent studies strongly imply that the Tat cofactor is a cellular protein kinase termed TAK which interacts with the activation domain of Tat and phosphorylates CTD ( 21 , 22 , 79 ) . ||10866664_1025_155871==>The results presented in this study further suggest that the ability of Tat to alter the substrate specificity of CDK9 would allow the continued hyperphosphorylation of the RNAP II CTD at serine 2 and serine 5 in the Tat transcription elongation complex . ||10866664_1025_155871==>The results presented in the latter study demonstrate that Tat , in fact , does modify the activity of the P - TEFb - associated CDK9 kinase , altering the specificity of CTD phosphorylation to allow CDK9 to phosphorylate serine 5 . ||12444143_155807_8685==>HIV - 1 Protein Vpr Suppresses IL - 12 Production from Human Monocytes by Enhancing Glucocorticoid Action : Potential Implications of Vpr Coactivator Activity for the Innate and Cellular Immunity Deficits Observed in HIV - 1 Infection Marco Mirani * , Ilia Elenkov , Simona Volpi , Naoki Hiroi * , George P . suppresses=====12167863_155459_60489==>Here , we describe a unique cellular gene , CEM15 , whose transient or stable expression in cells that do not normally express CEM15 recreates this phenotype , but whose antiviral action is overcome by the presence of Vif . ||12167863_155459_60489==>Because the Vif : CEM15 regulatory circuit is critical for HIV - 1 replication , perturbing the circuit may be a promising target for future HIV / AIDS therapies . ||12167863_155459_60489==>Varying doses of pCEM15 : HA ( a vector that encodes an epitope - tagged version of the predicted 384 - amino - acid open reading frame of CEM15 ) were co - transfected with either wild - type or vif versions of an X4 provirus expression vector and an internal control luciferase - expressing plasmid . ||12167863_155459_60489==>Figure 3 Expression of CEM15 in permissive cells inhibits the infectivity of HIV - 1 / vif . ||12167863_155459_60489==>In contrast to the wild - type virus , which was largely unaffected by CEM15 expression , the vif virus displayed minimal levels of infection at all doses of CEM15 tested ( for example , 97 % reduction in infectivity with a sixfold excess of vif provirus over pCEM15 : HA , relative to provirus alone ) . ||12167863_155459_60489==>Whole - cell lysates from one set of vif transfections were also examined by immunoblotting to confirm the titration curve for CEM15 : HA expression ( fig. 3c ) , and for luciferase expression to establish that CEM15 does not affect cell physiology or gene expression in a pleiotropic manner ( fig. 3d ) . ||12167863_155459_60489==>The effects of CEM15 on virus infectivity were essentially the same as for the X4 viruses , thus demonstrating that the inhibition of vif infection by CEM15 is not strain specific . ||12167863_155459_60489==>Taken together , these data reveal that CEM15 shows all the features expected for a gene that establishes the non - permissive phenotype ; that is , its expression in permissive cells inhibits vif virus production qualitatively ( inhibition of infectivity ) , but not quantitatively . ||12167863_155459_60489==>In contrast , the CEM15 - expressing cells allowed robust replication by the wild - type virus , but low or no detectable vif virus growth ( for example , for the challenges with an intermediate inoculum ( fig. 4b ) , there was a roughly 300 - fold reduction in output of vif virus relative to wild - type virus at day 10 ) . ||12167863_155459_60489==>Although we suspect that a lack of adequate CEM15 expression in a subset of the cells accounts for the residual levels of HIV - 1 / vif replication in cultures challenged with higher doses , the possible contribution of other genes to the complete non - permissive phenotype can not be ruled out 11 . ||12167863_155459_60489==>Figure 4 Stable expression of CEM15 in CEM - SS cells selectively inhibits HIV - 1 / vif replication . ||12167863_155459_60489==>To determine whether CEM15 influences Vif incorporation , and whether CEM15 itself can be packaged , wild - type and vif virions produced in 293T cells in the presence or absence of CEM15 : HA were purified and analysed by immunoblotting ( fig. 5 ) . ||12167863_155459_60489==>On the basis of this transfection experiment , Vif packaging is not affected by CEM15 ( lanes 1 and 3 ) , and CEM15 can be incorporated into virions irrespective of Vif expression ( lanes 3 and 4 ) . ||12167863_155459_60489==>Whether the residues in CEM15 that correspond to those required for deaminase function also operate in rendering vif viruses non - infectious in non - permissive cells remains to be determined . ||12167863_155459_60489==> CEM15 is an efficient and specific inhibitor of HIV - 1 / vif infectivity . ||12167863_155459_60489==>Because CEM15 is selectively expressed in non - permissive cells ( fig. 2 ) , and its anti - HIV phenotype is readily overcome by the levels of Vif expressed during normal HIV - 1 replication ( fig. 4 ) , we assert that the CEM15 protein is the human cell target for Vif function . ||12167863_155459_60489==>Because apobec - 1 binds to and edits mRNA 18 , and because Vif has been reported not only to bind viral genomic RNA but also to depend on this interaction for normal incorporation into virions 20 - 22 , CEM15 may affect vif virions through their RNA components . ||12167863_155459_60489==>On the basis of these findings , we propose that CEM15 comprises a significant part ( or all ) of a form of innate antiviral resistance that acts during the late stages of the viral life cycle , and suggest that relieving its suppression by Vif may lend a fresh perspective to the area of HIV / AIDS therapies . ||12167863_155459_60489==>Immunoblotting and luciferase assays CEM15 : HA , the heterogeneous nuclear ( hn ) RNP C1 / 2 proteins , p24 Gag and Vif were detected in whole - cell lysates ( transfected or transduced cultures ) or lysates of purified virions using the 16B12 ( Covance ) , 4F4 , 24 - 3 or 319 monoclonal antibodies 30 , respectively , for primary hybridization and enhanced chemiluminescence . ||12600646_155459_3055==>Current research suggests an association of Vif with the intermediate filament protein , vimentin , and the tyrosine kinase , Hck , but the significance of these associations remains to be defined . ||12600646_155459_3055==>Using a GST pull down assay researchers found that GST - Vif specifically interacts with the tyrosine kinase , Hck ( Hassaine et al. , 2001 ) . ||12600646_155459_3055==>Overexpression of Hck was found to have an inhibitory effect on HIV - 1 replication in the absence of Vif . ||12600646_155459_3055==>While Hck is expressed at lower levels in H9 cells it is not detectable in CD4 + T - cells , thus the significance of the Vif / Hck interaction in non - permissive cells remains to be elucidated and further work will be required to fully understand the biological significance of this interaction . ||12719574_155459_60489==>Evidence suggests that Vif acts late in the viral life cycle during assembly , budding , and / or maturation to counteract the antiviral activity of the CEM15 protein and possibly other antiviral factors . ||12719574_155459_60489==>Recently , we have used gene transfer experiments to show that the CEM15 protein ( also termed APOBEC3G ( 37 ) ) is at least partially responsible for this antiviral activity ( 55 ) ; whether Vif interacts directly with this protein is unknown . ||12719574_155459_60489==>We have recently reported that an epitope - tagged version of CEM15 , the T - cell - expressed antiviral factor that is counteracted by Vif , is incorporated in HIV - 1 particles when overexpressed in virus - producing cells ( 55 ) . ||12719574_155459_60489==>Once antibodies are developed to endogenous CEM15 , it will be important to determine whether this protein is differentially encapsidated into wild - type and vif virions under physiological conditions . ||12719574_155459_60489==>Since CEM15 shares significant sequence similarity with cytidine deaminases ( 55 ) , it is plausible that viral ( or even cellular ) RNAs ( messenger , genomic , or ) or DNAs are subjected to sequence modification in nonpermissive cells in the absence of Vif . ||12719574_155459_60489==>Nevertheless , in view of the potential deaminase activity of CEM15 , an intriguing possibility is that Vif protects against CEM15 - mediated RNA modifications via its ability to associate with RNA ( 17 , 39 , 68 ) . ||12719574_155459_60489==>In any case , it can be expected that a molecular description of CEM15 function will suggest additional aspects of vif virion structure , composition , and activity that should be assayed . ||12830140_155459_60489==> APOBEC3G was identified in the non - permissive human T cell line ( CEM ) as a gene whose product does not allow propagation of virion infectivity factor ( Vif ) 䤥eficient HIV 5 . ||12830140_155459_60489==>Accordingly , expression of APOBEC3G in cell lines that normally support growth of Vif - deficient virus ( for example , CEM - SS or 293T 5 ) renders them resistant to its growth . ||12830140_155459_60489==>HIV Vif can counteract APOBEC3G - mediated immunity , but how this occurs is not yet known . ||12830140_155459_60489==>The APOBEC3G - dependent minus - strand dC dT transition mutations accumulated to a great extent only in the absence of the HIV Vif protein 1 - 4 . ||12830140_155459_60489==>Expression of HIV Vif in virus - producing cells was able to reverse most of the antiviral effects attributable to APOBEC3G . ||12830140_155459_60489==>How Vif inhibits APOBEC3G and thereby facilitates viral infectivity is unknown , although ( and perhaps not unexpectedly ) a balance between the activities of these two proteins is probably an important factor in determining overall infectivity . ||12830140_155459_60489==>In support of this , readily detectable dC dT transition mutations were still apparent in wild - type ( Vif + ) HIV grown on APOBEC3G - expressing cells 2 , and transient Vif expression was able to fully restore infectivity of murine leukemia virus produced on a stable APOBEC3G - expressing cell line but only partially restored it on a sister clone expressing threefold more APOBEC3G 3 . ||12830140_155459_60489==>Indeed , such severance could explain the observation that Vif - deficient HIV grown in APOBEC3G cells can synthesize but not accumulate normal amounts of DNA replication intermediates after infection of susceptible target cells 27 . ||12830140_155459_60489==>Clearly , just as these advances have answered many questions , many new ones have arisen ( How is APOBEC3G targeted to the viral DNA ? ) and some older ones have been re - emphasized ( How does Vif work to counteract APOBEC3G ? ) . ||12970355_155459_60489==> Vif functions to counteract an anti - HIV - 1 cellular factor in non - permissive cells , CEM15 / Apobec - 3G , which shares a cytidine deaminase motif . ||12970355_155459_60489==>However , precise mechanisms how exactly the enzymatic activity of CEM15 / Apobec - 3G regulates the virion infectivity and how Vif protein overcomes the function of this protein remain unclear . ||12970355_155459_60489==>We transfected pNL43 - Luc ( WT ) or pNL43 / vif - Luc ( vif ) together with pcDNA4 / His - CEM15 ( A ) or pDON / EGFP - CEM15 ( B ) ( an increasing amount of CEM15 / Apobec - 3G with the empty parental vector making up the balance ) into HEK293T cells . ||12970355_155459_60489==>Infectivity Assay� Luciferase reporter viruses with or without Vif were prepared in HEK293T cells by cotransfection of pNL43 - Luc or pNL43 / vif - Luc together with a mock - vector or expression vectors for CEM15 / Apobec - 3G or its mutants by the calcium phosphate method . ||12970355_155459_60489==>Expression of CEM15 / Apobec - 3G Is Sufficient for Non - permissive Phenotype� First , we transfected pcDNA4 / His - CEM15 or pDON / EGFP - CEM15 together with pNL43 / Luc ( WT ) or pNL43 / vif - Luc ( vif ) into HEK293T cells , prepared viruses , and tested the infectivity . ||12970355_155459_60489==>As shown in fig. 1 , transient expression of His - CEM15 or EGFP - CEM15 clearly suppressed the infectivity of vif virions in a dose - dependent manner as reported previously ( 6 ) . ||12970355_155459_60489==>This indicated that His - and EGFP - tagged protein could suppress the infectivity of vif virions and that expression of CEM15 / apobec - 3G was sufficient to give non - permissive phenotype to HEK293T cells . ||12970355_155459_60489==>The DNA Editing Activities of E67Q and E259Q Mutants Were Both Retained but Impaired to the Same Extent� To address the above issue , we next examined the occurrence of hypermutation in the viral DNA induced by expression of these mutants , since recent reports showed that expression of CEM15 / Apobec - 3G introduced G to A hypermutation in the viral DNA in the absence of Vif protein ( 15 � 17 ) . ||12970355_155459_60489==>A , vif virions with CEM15 / Apobec - 3G . ||12970355_155459_60489==>We have also shown that CEM15 / Apobec - 3G induced G to A mutations in the viral DNA of wild type HIV - 1 ( with Vif ) . ||12970355_155459_60489==>These data suggest that CEM15 / Apobec - 3G acts on wild type virus to a lesser extent , even in the presence of Vif protein . ||12970355_155459_60489==>However , it still remains unclear how HIV - 1 Vif protein counteracts and inhibits the function of CEM15 / Apobec - 3G to make infectious viruses from non - permissive cells . synergizes with=====10438928_155871_2247==>However , bFGF , but not VEGF , synergizes with Tat in vivo and induces endothelial cells to migrate , to adhere , and to grow in response to Tat in vitro . ||10438928_155871_2247==> Tat angiogenic effects correlate with the expression of the v � 3 integrin that is induced by bFGF and binds the arginine - glycine - aspartic acid ( RGD ) region of Tat . ||10438928_155871_2247==>Finally , KS lesion formation induced by bFGF and Tat in nude mice is blocked by antagonists of RGD - binding integrins . ||10438928_155871_2247==>In fact , Tat promotes the development of angioproliferative KS - like lesions in mice only when injected with suboptimal ( nonlesion forming ) amounts of basic fibroblast growth factor ( bFGF ) ( 8 ) , an angiogenic molecule highly expressed by KS cells both in vitro and in primary lesions ( 8 , 25 , 26 , 27 ) . ||10438928_155871_2247==>In this study , we show that the in vivo KS - promoting effect of Tat is triggered by IC expressed in KS lesions and that it requires both the presence of bFGF , but not of vascular endothelial growth factor ( VEGF ) , and the binding of the RGD region of Tat to RDG - binding integrin receptors such as v � 3 . ||10438928_155871_2247==>IC induce expression of bFGF and VEGF ; however , only bFGF synergizes with Tat to induce KS - like lesions ||10438928_155871_2247==>Injection of combined IC at concentrations exerting angiogenic synergy with Tat induced high levels of expression of both bFGF and VEGF ( fig. 1 ) . ||10438928_155871_2247==>In contrast , lower IC concentrations , not sufficient to synergize with Tat in promoting angiogenesis ( Table I ) , were not capable of inducing a significant bFGF and / or VEGF expression ( data not shown ) . ||10438928_155871_2247==>These findings strongly suggested that IC may synergize with Tat in vivo by promoting bFGF and VEGF expression . ||10438928_155871_2247==>To investigate whether these two angiogenic factors were both involved in the in vivo synergistic effects of Tat and IC , mice were inoculated with suboptimal ( nonlesion forming ) amounts of bFGF or VEGF alone or combined with Tat . ||10438928_155871_2247==>In the presence of suboptimal amounts of bFGF , Tat increased KS - like lesion formation from 7 to 60 % of the inoculated mice ; on the contrary , no angiogenic synergy was observed when Tat and suboptimal amounts of VEGF were simultaneously injected in nude mice ( Table II ) . ||10438928_155871_2247==>Thus , although IC induce expression of both bFGF and VEGF , Tat synergizes with bFGF , but not with VEGF to promote KS - like lesion formation in mice . ||10438928_155871_2247==> bFGF , but not VEGF , synergizes with Tat in inducing the development of angioproliferative KS - like lesions in nude mice 1 ||10438928_155871_2247==> bFGF but not VEGF induces endothelial cells to become responsive to the growth , migration , and adhesion effects of Tat ||10438928_155871_2247==>Because IC induce both bFGF and VEGF expression , but bFGF and not VEGF cooperates with Tat in inducing angiogenic lesions in mice , experiments were addressed to verify whether bFGF or VEGF were capable of inducing EC to become responsive to the in vitro angiogenic effects of Tat , as found previously with IC . ||10438928_155871_2247==>The culture of HUVEC with bFGF induced these cells to adhere onto immobilized Tat , whereas exposure to VEGF had no effects ( fig. 2 ) . ||10438928_155871_2247==>Similarly , HUVEC cultured with bFGF , but not after culture with VEGF , migrated and proliferated in response to soluble Tat ( fig. 2 ) . ||10438928_155871_2247==>Thus , bFGF , but not VEGF , synergizes with Tat in vivo because only bFGF induces EC responsiveness to the adhesion , migration , and growth effects of Tat . ||10438928_155871_2247==>Induction of EC responsiveness to the migration , adhesion , and growth effects of Tat by bFGF , but not by VEGF . ||10438928_155871_2247==>Because only IC or bFGF , but not VEGF , synergized with Tat to promote lesions in mice and to induce EC growth , migration , and adhesion to Tat , immunohistochemical stainings were performed to analyze the type of integrins expressed in mice upon injection of bFGF , VEGF , or IC . ||10438928_155871_2247==>Thus , � 3 expression induced by IC or bFGF correlated with the angiogenic synergy by Tat ( Tables I , II , and III ) . ||10438928_155871_2247==>This suggested that the binding of Tat RGD region to v � 3 is required for the synergistic effect of combined bFGF and Tat . ||10438928_155871_2247==>To evaluate whether the in vivo angiogenic effect of combined Tat and bFGF requires the engagement of RGD - binding integrins , in vivo experiments were performed with integrin antagonists . ||10438928_155871_2247==>As shown in fig. 4 , competitors of RGD - binding integrins such as RGD peptides ( 31 , 38 ) blocked the development of KS - like lesions induced by the injection of combined Tat and bFGF in mice . ||10438928_155871_2247==>Thus , the engagement of RGD - binding integrins by Tat is required to observe the synergistic angiogenic KS - promoting effect of bFGF and Tat . ||10438928_155871_2247==>Block of the angiogenic KS - promoting effect of combined Tat and bFGF by RGD - integrin antagonists . ||10438928_155871_2247==>Shown are x 200 magnification of the hematoxylin - eosin staining of representative tissues from mice injected with bFGF + Tat ( upper left panel ) , protein resuspension buffer ( upper right panel ) , bFGF + Tat + RGD peptides ( lower left panel ) , and bFGF + Tat + RGE peptides ( lower right panel ) . ||10438928_155871_2247==>Injection of combined Tat and bFGF induced in all mice angiogenesis ( average value of 7 ) , spindle cell growth ( average value of 5 ) , and hemorrhagies ( average value of 3 ) . ||10438928_155871_2247==>When bFGF and Tat were injected in the presence of GRGDS peptides , angiogenesis , spindle cell growth , and hemorrhagies were reduced to 39 % , 44 % , and 0 % , respectively . ||10438928_155871_2247==>In contrast , injection of bFGF and Tat with mutated GRGES peptides gave the same values as the positive control ( 100 % per each histological alteration ) . ||10438928_155871_2247==>In fact , injection of combined IL - 1� , TNF - , IFN - , and Tat promotes in nude mice the development of angioproliferative KS - like lesions in which both bFGF and VEGF are highly expressed ( Table I and fig. 1 ) . ||10438928_155871_2247==>Nevertheless , to exert angiogenic synergy with Tat , however , IL - 1� , TNF - , and IFN - have to be injected at concentrations sufficient to induce a significant level of bFGF and / or VEGF expression ( Table I and fig. 1 ) . ||10438928_155871_2247==>In fact , our recent work indicates that Tat releases sequestered , extracellular - bound bFGF into a soluble form by competing for its heparin binding sites ( 42 ) . ||10438928_155871_2247==>As a consequence , Tat enhances the mitogenic effect of bFGF on EC ( 42 ) . ||10438928_155871_2247==>Consistent with these in vitro findings , in this study we have shown that Tat exerts in vivo synergistic angiogenic effects with bFGF , but not with VEGF ( Table II ) . ||10438928_155871_2247==>In agreement with these in vivo findings , the exposure of EC to bFGF induces their adhesion onto immobilized Tat as well as their migration and growth in response to soluble Tat . ||10438928_155871_2247==>These results indicate that bFGF is specifically required for Tat angiogenic effect . ||10438928_155871_2247==>Previous work demonstrated that bFGF and VEGF promote angiogenesis by inducing distinct integrin pathways : VEGF promotes the expression of v � 5 ( 37 ) , an integrin that binds Tat basic sequence ( 29 ) , whereas bFGF induces the expression of the v � 3 integrin ( 37 ) , which binds Tat RGD region ( 12 ) . ||10438928_155871_2247==>Immunohistochemical analyses of the mice tissues indicated that , differently from VEGF - induced � 5 expression , the expression of � 3 , which is induced by IC or bFGF , correlates with Tat angiogenic effects ( Tables I , II , and III , and fig. 3 ) . ||10438928_155871_2247==>These results suggested that the selective angiogenic effect of Tat could be due to the specific interaction of its RGD sequence with the v � 3 integrin , whose expression is triggered by bFGF or IC , but not by VEGF . ||10438928_155871_2247==>In fact , the injection of RGD peptides , which are known inhibitors of specific integrin function ( 31 , 38 ) , but not of mutated control RGE peptides , inhibits the development of angioproliferative lesions induced in nude mice by combined Tat and bFGF ( fig. 4 ) . ||10438928_155871_2247==>IC and bFGF are highly expressed in AIDS - KS lesions ( 8 , 16 , 24 , 27 ) , in which extracellular Tat costains with v � 3 on both EC and KS cells ( 8 ) . ||10438928_155871_7124==>In this study , we show that a combination of the same inflammatory cytokines increased in KS lesions , namely IL - 1� , TNF - , and IFN - , synergizes with Tat to promote in nude mice the development of angioproliferative KS - like lesions that are not observed with each factor alone . ||10438928_155871_7124==>Among them , IL - 1� , TNF - , and IFN - play a major role in inducing EC responsiveness to the in vitro effects of Tat ( 15 , 16 , 17 , 18 ) . ||10438928_155871_7124==>Again , the injection of mice with IL - 1� , TNF - , and IFN - combined together at 0.1 痢 each had no effects , even in the presence of Tat . ||10438928_155871_7124==>Thus , IL - 1� , TNF - , and IFN - are all required to observe the synergy with Tat in inducing macroscopic vascular lesions and histological changes closely resembling early KS in humans . ||10438928_155871_7124==>Table i. Combined IL - 1� , TNF - , and IFN - synergize with Tat in inducing the development of angioproliferative KS - like lesions in nude mice 1 ||10438928_155871_7124==>Previous work indicated that the angiogenic KS - promoting effects of Tat are induced in vitro by combined IC , including IL - 1� , TNF - , and IFN - ( 12 , 13 , 15 , 16 , 17 , 18 ) . ||10438928_155871_7124==>In fact , injection of combined IL - 1� , TNF - , IFN - , and Tat promotes in nude mice the development of angioproliferative KS - like lesions in which both bFGF and VEGF are highly expressed ( Table I and fig. 1 ) . ||10438928_155871_7124==>Nevertheless , to exert angiogenic synergy with Tat , however , IL - 1� , TNF - , and IFN - have to be injected at concentrations sufficient to induce a significant level of bFGF and / or VEGF expression ( Table I and fig. 1 ) . ||10454543_155871_2963==> Tat - SF1 Protein Associates with RAP30 and Human SPT5 Proteins - - Kim et al. 19 ( 9 ) : 5960 - - Molecular and Cellular Biology ||10454543_155871_2963==> Tat - SF1 Protein Associates with RAP30 and Human SPT5 Proteins Jae B . ||10454543_155871_2963==>Surprisingly , the RAP30 protein of the heterodimeric transcription TFIIF factor is associated with Tat - SF1 , while the RAP74 subunit of TFIIF is not coimmunoprecipitated with Tat - SF1 . ||10454543_155871_2963==>These results suggest that Tat - SF1 and hSPT5 are indispensable cellular factors supporting Tat - specific transcription activation and that they may interact with RAP30 in controlling elongation . ||10454543_155871_2963==>This study shows that Tat - SF1 efficiently associates with the RAP30 subunit of TFIIF and inefficiently associates with Pol II and human SPT5 ( hSPT5 ) . ||10454543_155871_2963==>Surprisingly , coimmunoprecipitation experiments show that Tat - SF1 forms a stable complex with the p30 subunit of the general transcription factor TFIIF ( RAP30 ) ( fig. 1 A , lanes 3 and 4 ) . ||10454543_155871_2963==>Immunoprecipitation by antiserum to either Tat - SF1 or RAP30 produced similar amounts of the RAP30 and Tat - SF1 polypeptides , respectively . ||10454543_155871_2963==>The levels of coimmunoprecipitated Tat - SF1 and RAP30 polypeptides were about 20 % of that present in the input nuclear extract ( fig. 1 , lane 1 ) . ||10454543_155871_2963==>In contrast , coimmunoprecipitates of Tat - SF1 contained negligible amounts of the RAP74 protein , which associates with RAP30 to form the heterodimeric TFIIF ( data not shown ) . ||10454543_155871_2963==>Repeated depletion of Tat - SF1 , by absorption with specific Tat - SF1 antiserum , reduced the amounts of RAP30 without the depletion of other proteins such as Cdk9 nor RAP74 ( fig. 1 B , lanes 2 , 3 , and 4 ) . ||10454543_155871_2963==>The interaction between Tat - SF1 and RAP30 appears to be very specific since a similar association with other elongation factors , such as the elongin A , B , and C complex and TFIIS , was not detected ( data not shown ) . ||10454543_155871_2963==>Furthermore , the interaction between Tat - SF1 and RAP30 is probably direct since both polypeptides coimmunoprecipitated with either antiserum when added as purified proteins to a reaction ( data not shown ) . ||10454543_155871_2963==> Tat - SF1 associates with RAP30 of TFIIF . ||10454543_155871_2963==>( A ) Tat - SF1 coimmunoprecipitated with RAP30 . ||10454543_155871_2963==>HeLa total cell extracts ( 250 痢 ) were used for immunoprecipitation with antibodies against preimmune ( PI , lane 2 ) , Tat - SF1 ( lane 3 ) ( 44 ) , and RAP30 of TFIIF ( lane 4 ) ( Santa Cruz Biotechnology ) . ||10454543_155871_2963==>Western blots of the immunoprecipitates were probed with anti - Tat - SF1 ( top ) and anti - RAP30 ( bottom ) antibodies . ||10454543_155871_2963==>( B ) Tat - SF1 - depleted HeLa nuclear extracts contained decreased levels of RAP30 . ||10454543_155871_2963==>After each depletion step , aliquots were taken and Western blotting was performed with antibodies against Tat - SF1 and RAP30 . ||10454543_155871_2963==>When compared with the interaction between Tat - SF1 and RAP30 , the association between Tat - SF1 and hSPT5 is inefficient , since less than 5 % of the input SPT5 protein was immunoprecipitated with - Tat - SF1 sera . ||10454543_155871_2963==>In accordance with fig. 1 and 2 , these eluted fractions contained Pol II - LS , RAP30 , and hSPT5 , as well as Tat - SF1 ( fig. 2 C , lanes 3 , 4 , and 5 ) . ||10454543_155871_2963==>These results confirm that Tat - SF1 forms protein complexes with Pol II , RAP30 , and hSPT5 . ||10454543_155871_2963==>Aliquots of each depleted extract were probed with antibodies against Pol II CTD , hSPT5 , Tat - SF1 , RAP30 , and TBP . ||10454543_155871_2963==>Furthermore , the addition of recombinant TFIIF ( RAP30 and RAP74 ) into Tat - SF1 - depleted nuclear extract increased both Tat - dependent and Tat - independent transcription ( data not shown ) . ||10454543_155871_2963==>Surprisingly , a significant fraction of Tat - SF1 is associated with the RAP30 subunit and not to the other subunit of TFIIF , RAP74 . ||10454543_155871_2963==>This suggests that RAP30 , which is tightly bound to elongating Pol II , may be an important factor in the mechanism of Tat activation . ||10454543_155871_2963==> RAP30 , as part of the elongating complex , probably also associates with Tat - SF1 . ||10454543_155871_2963==>This association appears to compete with the binding of RAP74 to RAP30 since RAP74 did not efficiently coprecipitate with either Tat - SF1 or RAP30 antiserum . ||10454543_155871_2963==>The association of Tat - SF1 with RAP30 might position the former in the elongation complex in close proximity to the Pol II . ||10454543_155871_2963==>Since Tat - SF1 also binds the hSPT5 protein which also associates with Pol II , the mutual interaction of these three proteins ( RAP30 , Tat - SF1 , and hSPT5 ) could greatly stabilize the complex on the Pol II . ||12221105_155871_4782==>HIV Tat also cooperated with the Sp1 and CTF activation domains to enhance transcript elongation and EDI skipping . ||12221105_155871_4782==>Class I activators , such as Sp1 and CTF / NF1 , stimulate initiation ; class IIA activators , of which HIV - 1 Tat is the best example , stimulate predominantly elongation ( 18 , 19 ) ; and class IIB activators , which include VP16 , p53 , and E2F1 , stimulate both initiation and elongation . ||12221105_155871_4782==>HIV - 1 Tat , Sp1 , and CTF Activation Domains Alone Have No Effect on EDI Splicing - - fig. 1 C shows that stimulation of exon skipping by Gal4 - VP16 correlates directly with pol II elongation capacity . ||12221105_155871_4782==> Tat Synergizes with Type I Activators to Enhance EDI Exon Skipping - - Blau et al. ( 17 ) showed that type I activators such as Sp1 , SW6 , and CTF specifically synergize with Tat in transcriptional activation by increasing the efficiency of elongation whereas type IIB activators such as VP16 do not , when present in multiple copies at the promoter . ||12221105_155871_4782==>In contrast , type I activators Gal4 - SW6 , Gal4 - Sp1 , and Gal4 - CTF synergize strongly with Tat in enhancing EDI skipping and reducing EDI inclusion ( lanes 5 , 7 , and 9 ) . ||12221105_155871_4782==>In this report we analyzed the individual and combined effects on alternative splicing of five transcription factors ( VP16 , SW6 , Sp1 , CTF , and Tat ) acting on a single promoter . ||12221105_155871_4782==>In agreement with these observations , Tat synergizes with SW6 , Sp1 , and CTF but not with VP16 in promoting transcriptional processivity in our system and therefore in inhibiting EDI inclusion ( fig. 2 ) . ubiquitinated by=====11087860_155030_4734==>The release defect of a minimal Gag construct could also be corrected through the attachment of a peptide that serves as a physiological docking site for the ubiquitin ligase Nedd4 . ||11991975_10537_155030==>The simplest explanation for this observation is that the affected Gag species harbored a Lys63 - linked diubiquitin chain . ||12610113_155030_4734==>Proteins related to the Nedd4 family of ubiquitin protein ligases interact with the L domain of Rous sarcoma virus and are required for gag budding from cells . upregulates=====11311202_155871_4609==>ScienceDirect - Archives of Oral Biology : Amplification of extracellular matrix and oncogenes in tat - transfected human salivary gland cell lines with expression of laminin , fibronectin , collagens I , III , IV , c - myc and p53 ||11311202_155871_4609==>Amplification of extracellular matrix and oncogenes in tat - transfected human salivary gland cell lines with expression of laminin , fibronectin , collagens I , III , IV , c - myc and p53 ||11311202_155871_4609==> Tat - amplified transcription of the major basement membrane protein laminin , as well as of fibronectin , collagen I and III , and c - myc oncogene was demonstrated . ||11311202_155871_4609==>As tat modulates expression of the oncoproteins c - jun and c - fos and oncogene c - myc , the increased expression of c - myc suggests a role for tat in the development of salivary gland neoplasia or lymphoma development in HIV infection . ||11311202_155871_4609==>The molecular basis of c - myc function in cell replication is not entirely clear but these data from salivary gland cell lines transfected with tat suggest that there is amplification of c - myc by tat and that neoplastic transformation may be enhanced . ||11531413_155807_5524==>ScienceDirect - Virology : HIV - 1 Vpr Induces Cell Cycle G2 Arrest in Fission Yeast ( Schizosaccharomyces pombe ) through a Pathway Involving Regulatory and Catalytic Subunits of PP2A and Acting on Both Wee1 and Cdc25 ||11531413_155807_5524==>HIV - 1 Vpr Induces Cell Cycle G2 Arrest in Fission Yeast ( Schizosaccharomyces pombe ) through a Pathway Involving Regulatory and Catalytic Subunits of PP2A and Acting on Both Wee1 and Cdc25 ||11531413_155807_5524==>It was found that protein phosphatase 2A ( PP2A ) plays an important role in the induction of G2 arrest by Vpr since mutations in genes coding for a regulatory or catalytic subunit of PP2A reduce Vpr - induced G2 arrest . ||11531413_155807_5524==> Vpr was also found to upregulate PP2A , supporting a model in which Vpr activates the PP2A holoenzyme to induce G2 arrest . ||11531413_155807_5524==>These two genes may be part of the uncharacterized pathway for Vpr - induced G2 arrest in which Vpr upregulates PP2A to activate Wee1 and inhibit Cdc25 . ||11909874_155871_355==>HIV Tat Binds Egr Proteins and Enhances Egr - dependent Transactivation of the Fas Ligand Promoter - - Yang et al. 277 ( 22 ) : 19482 - - Journal of Biological Chemistry ||11909874_155871_355==>HIV Tat Binds Egr Proteins and Enhances Egr - dependent Transactivation of the Fas Ligand Promoter * ||11909874_155871_355==>HIV Tat can enhance activation - induced up - regulation of Fas ligand ( FasL ) , which may contribute to T cell apoptosis in human immune deficiency virus ( HIV ) - infected individuals . ||11909874_155871_355==>Given the evidence that FasL - Fas interactions may account for bystander killing of T cells in patients infected with HIV ( 35 ) , one of the more intriguing activities ascribed to Tat is that it synergizes with T cell activating stimuli in the up - regulation of FasL expression ( 36 ) . ||11909874_155871_356==>HIV Tat can enhance activation - induced up - regulation of Fas ligand ( FasL ) , which may contribute to T cell apoptosis in human immune deficiency virus ( HIV ) - infected individuals . ||11909874_155871_356==>We have assessed functional and physical interactions between Tat and the Egr family of transcription factors ( Egr - 1 , - 2 , and - 3 ) , the latter two of which are major participants in activation - induced FasL up - regulation . ||11909874_155871_356==>Here we report that whereas Tat itself has no effect on the FasL promoter , it binds to Egr - 2 and - 3 and synergizes with them to superinduce expression of a FasL promoter - driven reporter . ||11909874_155871_356==>A Tat molecule containing a single amino acid substitution that results in the loss of transactivation activity for the HIV long terminal repeat still binds Egr - 3 but can no longer enhance Egr - mediated transactivation of the FasL promoter . ||11909874_155871_356==>Furthermore , the mutated Tat acts as a dominant negative inhibitor , blocking the superinduction of FasL caused by wild type Tat . ||11909874_155871_356==>Because Tat is present in virus - infected cells and in the serum of HIV - infected individuals , these results suggest that increased expression of FasL in these circumstances may result from the cooperative activities of activation - induced Egrs and Tat . ||11909874_155871_356==>Given the evidence that FasL - Fas interactions may account for bystander killing of T cells in patients infected with HIV ( 35 ) , one of the more intriguing activities ascribed to Tat is that it synergizes with T cell activating stimuli in the up - regulation of FasL expression ( 36 ) . ||11909874_155871_356==>In this study , we asked if the ability of Tat to synergize with activation to superinduce FasL reflects an interaction , direct or indirect , with Egr family transcription factors . ||11909874_155871_356==>Here we report that Tat physically interacts with all Egr family members and synergizes with Egr - 2 and - 3 but not Egr - 1 to increase the expression of a reporter gene driven by the FasL promoter , an activity that depends upon both an intact Egr binding site in the promoter and the transactivation activity of Tat . ||11909874_155871_356==>Furthermore , a transactivation - deficient form of Tat acts as a dominant negative , abrogating the ability of native Tat to co - activate the FasL promoter . ||11909874_155871_356==>These results provide a molecular mechanism for the ability of Tat to synergistically enhance FasL expression , and they suggest a possible means for interfering with this phenomenon . ||11909874_155871_356==> Tat Synergizes with Egr - 2 and Egr - 3 to Induce the FasL Promoter - - Ectopic expression of Egr - 2 or Egr - 3 but not Egr - 1 can induce expression of luciferase reporters driven by the 511 - bp sequence of FasL promoter or the 16 - bp FLRE ( Egr binding site ) in the FasL promoter . ||11909874_155871_356==>To determine whether Tat can synergize with Egrs in transactivating the FasL promoter , HeLa cells were cotransfected with a luciferase reporter construct containing the 511 - bp FasL promoter and vectors expressing Tat with or without Egr - 1 , Egr - 2 , or Egr - 3 ( fig. 1 A ) . ||11909874_155871_356==>When Egr - 2 or Egr - 3 and Tat were coexpressed , however , there was an 8 - to 10 - fold up - regulation of FasL promoter activity . ||11909874_155871_356==>Furthermore , similar to the full - length 511 - bp FasL promoter , whereas Tat by itself had no effect on luciferase activity , it substantially increased reporter activity when coexpressed with either of these Egr family members . ||11909874_155871_356==>HIV Tat enhances transactivation of FasL promoter by Egr - 2 and - 3 . ||11909874_155871_356==>Therefore , we asked if Tat could also synergize with Egr - 1 in FasL induction . ||11909874_155871_356==>Those results demonstrate that HIV Tat synergizes with Egr - 2 and - 3 to activate the FasL promoter and that this requires the binding of the Egrs to the FLRE in the FasL promoter . ||11909874_155871_356==> Tat Binds Egrs - - Because Tat enhancement of FasL promoter transcription depends on the concomitant presence of Egr - 2 or - 3 , we asked whether these molecules interact physically . ||11909874_155871_356==>Because this region is vital for Tat - mediated gene transactivation , these results suggest that direct interaction between the activation domain of Tat and Egrs is responsible for the superinduction of the FasL promoter . ||11909874_155871_356==>Taken together , these data argue that binding of transactivation - competent HIV Tat to the biologically active Egrs ( Egr - 2 and - 3 ) is necessary for synergism between these two transcriptional regulators at induction of the FasL promoter . ||11909874_155871_356==>Given the fact that Tat can be secreted by infected cells and detected in serum from HIV - infected individuals and can cross the plasma membrane of uninfected cells , the observation that exogenous Tat is able to enhance the elevation of FasL mRNA following T cell activation or CD4 cross - linking in vitro suggests that Tat contributes to the up - regulation of FasL in vivo . ||11909874_155871_356==> Tat can enhance NF - B activation via induction of oxidative stress and down - regulation of Mn 2 + - dependent superoxide dismutase expression in T cells ( 61 ) , suggesting that activation of NF - B may mediate the synergistic action of T cell activation and HIV Tat in the up - regulation of FasL . ||11909874_155871_356==>This was supported by the finding that NF - B sites were required for the Tat - mediated increase of transactivation through FasL promoter ( 62 ) . ||11909874_155871_356==>The data presented in this report demonstrate a different mechanism for Tat enhancement of FasL expression : synergism with activation - induced Egr - 2 and - 3 . ||11909874_155871_356==>Therefore , it is conceivable that Nef induces the Egr family proteins in infected cells and together with Tat up - regulates FasL following HIV infection . ||11909874_155871_356==>If so , interfering with the Tat - Egr interaction might reduce FasL expression and the secondary depletion of T cells during HIV infection . ||12539042_155871_3665==>Two of the Tat - induced genes , interferon regulatory factor - 7 ( IRF7 ) and signal transducer and activator of transcription 1 ( STAT1 ) , are transcriptional regulators of IFN - inducible genes 24 , 25 , so it is possible that one or more of these transcription factors is responsible for induction of the other genes . ||12539042_155871_4283==>Genes encoding four different chemokines ( interferon inducible protein - 10 ( IP - 10 ) , human monokine induced by interferon - ( HuMIG ) , monocyte chemoattractant protein - 2 ( MCP - 2 ) and monocyte chemoattractant protein - 3 ( MCP - 3 ) ) were induced in iDC by both Tat expression and HIV - 1 infection ( fig. 2 ) . ||12539042_155871_4283==>The amounts of IP - 10 , HuMIG , MCP - 2 and MCP - 3 in tissue culture supernatants increased after cells were infected with adeno - Tat and three different strains of HIV - 1 ( fig. 3 ) . ||12539042_155871_4283==> HuMIG induction was observed at the RNA and protein levels in both HIV - 1 - infected and adeno - Tat - treated cells . ||12539042_155871_4283==>a , ELISA of IP - 10 , HuMIG , MCP - 2 and MCP - 3 in culture supernatants of iDCs from 4 different donors ( A , B , C and D ) , 30 h after infection with adenovirus expressing LacZ ( ) , Tat ( ) or Nef ( ) ( donor D only ) . ||12539042_155871_4283==>In light of these results , the induction of IP - 10 , HuMIG , MCP - 2 and MCP - 3 in iDC by Tat and HIV - 1 seems especially important . ||12539042_155871_4283==>The observation that HIV - 1 Tat increases levels of the T - cell chemokines IP - 10 and HuMIG in immature dendritic cells has several implications . ||12539042_155871_5610==> Tat also interacts with the protein kinase PKR ( ref . ||12539042_155871_6772==>Two of the Tat - induced genes , interferon regulatory factor - 7 ( IRF7 ) and signal transducer and activator of transcription 1 ( STAT1 ) , are transcriptional regulators of IFN - inducible genes 24 , 25 , so it is possible that one or more of these transcription factors is responsible for induction of the other genes . ||12539042_155871_8743==>Of the IFN - inducible genes affected by Tat , TRAIL ( TNF - related apoptosis - inducing ligand / Apo - 2 ligand ) is particularly interesting , as it has been shown to trigger apoptosis in T cells from HIV - 1 infected individuals , whereas T cells from uninfected controls are completely resistant to TRAIL - induced apoptosis 39 , ||12767990_155871_8743==>It is well known that Tat induces tumor necrosis factor - related apoptosis - induced ligand ( TRAIL ) in peripheral blood mononuclear cells ( PBMC ) , and we show that the majority of TRAIL is produced by the monocyte subset of PBMC . ||12767990_155871_8743==>Human monocytes and U937 monoblastoid cells did not take up soluble HIV Tat - 86 , as T cells did , yet produced more TRAIL than did T cells . ||12767990_155871_8743==> TRAIL secretion was induced by Tat and by a cysteine - rich peptide of Tat but not by sulfhydryl - modified Tat toxoid . ||12767990_155871_8743==>The cytotoxicity of Tat - stimulated monocyte medium could be blocked by a TRAIL - neutralizing antibody . ||12767990_155871_8743==>T cells treated with Tat did not secrete enough TRAIL to mediate cell death in our assay . ||12767990_155871_8743==>The production of TRAIL by Tat - stimulated monocytes provides a mechanism by which HIV infection can destroy uninfected bystander cells . ||12767990_155871_8743==>This study demonstrates an intracellular and extracellular increase in TRAIL once monocytes are exposed to HIV Tat or to a cysteine - rich peptide of Tat . ||12767990_155871_8743==>In contrast to monocytes , T cells secreted relatively little TRAIL upon exposure to Tat . ||12767990_155871_8743==>To detect cell surface TRAIL , U937 cells , primary human monocytes , Jurkat cells , or primary human CD4 + cells ( 2 x 10 6 cells ) were cultured with or without HIV Tat at 37蚓 for 24 h and then incubated with PE - labeled anti - TRAIL ( RIK - 2 ; Pharmingen ) before a final wash and fixation in 1 % paraformaldehyde - PBS for flow cytometry . ||12767990_155871_8743==>HIV Tat increases the expression of TRAIL in U937 cells and human monocytes . ||12767990_155871_8743==>Fresh human monocytes or the promonocytic cell line U937 were stimulated with HIV Tat protein ( 0 to 1 , 000 ng / ml ) at 37蚓 , and lysates were collected after 20 h. Immunoblotting results showed that HIV Tat increases the expression of TRAIL in a dose - dependent manner , with 100 ng / ml being the optimum concentration of HIV Tat ( fig. 2A , part a ) . ||12767990_155871_8743==>At a higher concentration of Tat ( 1 , 000 ng / ml ) , the expression of TRAIL was decreased . ||12767990_155871_8743==>The same results were obtained with U937 cells ; however , no Tat stimulation of TRAIL could be detected in fresh human T cells or in Jurkat cells ( fig. 2A , part b ) . ||12767990_155871_8743==>Chemically modified Tat toxoid , with an average of two cysteines inactivated per molecule , was incapable of inducing TRAIL in monocytic cells ( fig. 2A , part a ) . ||12767990_155871_8743==>HIV Tat increases TRAIL expression in U937 cells and human monocytes , but Tat toxoid does not . ||12767990_155871_8743==>( A ) TRAIL expression increases with increasing doses of Tat protein . ||12767990_155871_8743==> TRAIL production in fresh human T cells or in Jurkat cells was not stimulated by exposure to Tat . ||12767990_155871_8743==>( B ) HIV Tat induces the transcription of TRAIL mRNA in U937 cells and monocytes . ||12767990_155871_8743==>Transcription of control � - actin or TRAIL / APO2L mRNA in U937 cells is shown after 0 , 2 , 6 , 12 , or 24 h of incubation in the presence of HIV Tat ( 100 ng / ml ) . ||12767990_155871_8743==>HIV Tat increases TRAIL mRNA transcription in U937 cells and human monocytes . ||12767990_155871_8743==>The transcription of TRAIL mRNA was rapidly induced within 2 h after the addition of Tat , and expression was sustained throughout overnight culture ( fig. 2B ) in both U937 cells and in freshly isolated human monocytes . ||12767990_155871_8743==>Since Tat is easily oxidized , mRNA expression dropped off after 12 h. In parallel experiments with T cells , TRAIL mRNA was not detectable . ||12767990_155871_8743==>To identify the portion of Tat responsible for inducing TRAIL , we synthesized six overlapping peptides covering different portions of Tat . ||12767990_155871_8743==> Tat 16 - 35 , which contains six of the seven cysteines of Tat , induced TRAIL expression in monocytes , while other peptides did not ( fig. 3 ) . ||12767990_155871_8743==>The cysteine - rich region of Tat is responsible for inducing monocytes to express TRAIL . ||12767990_155871_8743==> Tat induced little cell surface TRAIL on monocytes . ||12767990_155871_8743==>We used flow cytometry to determine whether HIV Tat induced the expression of TRAIL on the surface of human monocytes or U937 cells . ||12767990_155871_8743==> Tat treatment for 20 h only slightly increased the membrane expression of TRAIL in U937 cells and monocytes in comparison to what was seen with controls ( fig. 4A and B ) . ||12767990_155871_8743==> Tat did not affect surface expression of TRAIL on Jurkat cells or on primary CD4 + cells ( fig. 4C and D ) . ||12767990_155871_8743==>HIV Tat only slightly increases membrane - associated TRAIL expression in U937 cells and monocytes . ||12767990_155871_8743==>U937 cells ( A ) , human monocytes ( B ) , Jurkat cells ( C ) , and primary CD4 + cells ( D ) were stimulated with HIV Tat ( 100 ng / ml ) for 20 h , stained with PE - conjugated mouse anti - human TRAIL or mouse IgG1 control monoclonal antibody as described in Materials and Methods , and then subjected to flow cytometry . ||12767990_155871_8743==>Release of soluble TRAIL in the media from U937 cells and human monocytes upon stimulation with HIV Tat . ||12767990_155871_8743==>We tested whether HIV Tat stimulation resulted in the majority of TRAIL being released from monocytes or T cells . ||12767990_155871_8743==>Using a Western blotting assay , we clearly showed that Tat stimulation resulted in the appearance of TRAIL in the media from U937 cell ( fig. 5A ) or human monocyte cultures ( fig. 5B ) . ||12767990_155871_8743==>HIV Tat increases secretion of soluble TRAIL in media of U937 cells and monocytes . ||12767990_155871_8743==>By comparing commercially available TRAIL protein with Tat - induced TRAIL in extracts , we estimated that our Western blots detected as little as 10 ng of TRAIL . ||12767990_155871_8743==>At 20 h after Tat exposure , a little more TRAIL was found intracellularly than was found extracellularly ( per cell ) in the secreted form . ||12767990_155871_8743==>This indicates that soluble TRAIL is released from Tat - stimulated monocytes . ||12767990_155871_8743==>( C ) HIV Tat - stimulated human monocytes release functional soluble TRAIL . ||12767990_155871_8743==>We demonstrated that TRAIL expression by monocytes far exceeds TRAIL expression by T cells after a brief exposure to Tat . ||12767990_155871_8743==>Fifty nanograms ( or 5 nM ) of Tat / ml initiates TRAIL production , which is within range of the 1 ng / ml found in HIV - infected sera ( 59 ) , especially considering that Tat effects are more likely to occur in solid tissues than in circulation . ||12767990_155871_8743==>Exposure to Tat only slightly increases membrane - bound TRAIL expression but significantly increases de novo transcription and secretion of TRAIL . ||12767990_155871_8743==>The mechanism by which Tat induces monocyte TRAIL expression is not known . ||12767990_155871_8743==>Either very small amounts of intracellular Tat are required to induce TRAIL or Tat can induce TRAIL expression via extracellular signal transduction . ||12767990_155871_8743==>Here we show that after stimulation with HIV Tat protein , human monocytes upregulate TRAIL mRNA within 2 h and release the soluble form of TRAIL into the medium . ||12767990_155871_8743==>We report here that TRAIL released from Tat - stimulated monocytes is the major cytotoxic agent responsible for killing T cells , since T cells treated with Tat for the same amount of time did not upregulate any detectable TRAIL mRNA or produce enough TRAIL to be detected by our immunoblotting or cytotoxicity assays . ||12767990_155871_8743==>Using flow cytometry , Zhang et al. ( 64 ) showed that HIV Tat upregulated TRAIL on the surface of monocyte - derived macrophages . ||12767990_155871_8743==>In this study , we employed promonocytic U937 cells and human monocytes , but we failed to find a significant increase in cell surface TRAIL in these cells after Tat stimulation . ||12767990_155871_8743==>To determine which portion of Tat was responsible for inducing TRAIL , we used overlapping synthetic peptides from all regions of Tat . ||12767990_155871_8743==>Only one peptide , Tat 16 - 35 , containing six of the seven cysteines in Tat , was capable of inducing monocyte TRAIL . ||12767990_155871_8743==> TRAIL induction required 50 ng ( about 20 nM ) of peptide / ml , so full - length Tat ( inducing TRAIL at 5 nM ) is at least fourfold more potent than peptide . ||12767990_155871_8743==>Peptide Tat 16 - 35 can induce TRAIL ( this study ) and can activate NF - B but can not mediate transcriptional transactivation ( i. Tikhonov , unpublished results ) . ||12767990_155871_8743==>Others have shown that Jurkat cells stably transfected with HIV - tat are less susceptible to TRAIL - mediated cell death than are untransfected cells ( 20 ) . ||12767990_155871_8743==>In summary , we described the rapid induction and secretion of TRAIL in HIV Tat - stimulated monocytes , and its ability to selectively eliminate uninfected T cells . ||12767990_155871_8743==>Identification of a potential HIV - induced source of bystander - mediated apoptosis in T cells : upregulation of TRAIL in primary human macrophages by HIV - 1 Tat . ||9269771_155871_6401==>We found that the treatment of EC with HIV - tat , like that with TNF , induces the cell surface expression of intercellular adhesion molecule - 1 ( ICAM - 1 ) , vascular cell adhesion molecule - 1 ( VCAM - 1 ) , and endothelial leukocyte adhesion molecule - 1 ( ELAM - 1 ) and that induction of all three molecules was dependent on protein synthesis . ||9269771_155871_6401==>HIV - tat induces the expression of ICAM - 1 , VCAM - 1 , and ELAM - 1 . ||9269771_155871_6401==>To determine the effect of HIV - tat , EC were treated with either buffer ( control ) or HIV - Tat ( 100 ng / mL ) for 6 hours at 37蚓 , detached by a brief exposure to trypsin / EDTA , stained using monoclonal antibody to various adhesion molecules , and examined for the expression of ICAM - 1 , VCAM - 1 , and ELAM - 1 by flow cytometry . ||9269771_155871_6401==>This effect was also time - dependent ( Fig 3 ) , because treatment of cells with 100 ng / mL HIV - tat led to maximum induction of VCAM - 1 and ELAM - 1 in approximately 6 hours , whereas the induction of ICAM - 1 did not reach maximum even after 12 hours . ||9269771_155871_6401==>The quatitation of these results ( Table 1 ) also shows that TNF potentiates the effects of HIV - tat for the induction of ICAM - 1 , VCAM - 1 , and ELAM - 1 . ||9269771_155871_6401==>The results in Fig 5 and Table 1 indicate that TNF potentiates the effects of HIV - tat for the induction of ICAM - 1 , VCAM - 1 , and ELAM - 1 . ||9269771_155871_6401==>To determine that adhesion molecules induced by HIV - tat / TNF are involved in cellular adhesion , after treatment with either HIV - tat ( 100 ng / mL ) or TNF ( 100 ng / mL ) for 6 hours , EC were treated with antibodies against either ICAM - 1 or VCAM - 1 or ELAM - 1 or a combination for 1 hour at room temperature and then examined for adhesion to HL - 60 cells . ||9269771_155871_6401==>In the present report , we examined the effect of HIV - tat protein on the EC surface expression of the adhesion molecules ICAM - 1 , VCAM - 1 , and ELAM - 1 . ||9269771_155871_6401==>Like HIV - tat , TNF interacts with human EC and causes the de novo synthesis of ICAM - 1 , VCAM - 1 , and ELAM - 1 . ||9269771_155871_6401==>24 Our current results indicate that exposure of EC to exogenous HIV - tat induces ICAM - 1 , VCAM - 1 , and ELAM - 1 . ||9269771_155871_6401==>Hofman et al 31 showed an increase in expression of ELAM - 1 by HIV - tat in human umbilical vein EC within 4 hours and its complete disappearance by 24 hours . ||9269771_155871_6401==>It is not due to the lack of activity of HIV - tat protein , because it induced ELAM - 1 in both the studies . ||9269771_155871_6401==>Our report is the first to indicate that , besides ELAM - 1 and VCAM - 1 , HIV - tat also induces ICAM - 1 .