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