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Query: UMLS:C0019693 (HIV)
 170,526 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The HIV-1 Tat protein regulates transcription elongation through binding to the viral TAR RNA stem-loop structure. We have isolated a novel 87 kDa cyclin C-related protein (cyclin T) that interacts specifically with the transactivation domain of Tat. Cyclin T is a partner for CDK9, an RNAPII transcription elongation factor. Remarkably, the interaction of Tat with cyclin T strongly enhances the affinity and specificity of the Tat:TAR RNA interaction, and confers a requirement for sequences in the loop of TAR that are not recognized by Tat alone. Moreover, overexpression of human cyclin T rescues Tat activity in nonpermissive rodent cells. We propose that Tat directs cyclin T-CDK9 to RNAPII through cooperative binding to TAR RNA.
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PMID:A novel CDK9-associated C-type cyclin interacts directly with HIV-1 Tat and mediates its high-affinity, loop-specific binding to TAR RNA. 949 87

Tat stimulates human immunodeficiency virus type 1 (HIV-1) transcription elongation through recognition of the transactivation response (TAR) RNA stem-loop structure at the 5' end of nascent viral transcripts. Recently, a human transcription elongation factor P-TEFb, consisting of CDK9 kinase, cyclin T and other associated factors, has been shown to interact with Tat to restore Tat activation in HeLa nuclear extract depleted of P-TEFb. Here, we report the purification of a P-TEFb complex fraction containing epitope-tagged wild-type CDK9 or kinase-inactive CDK9 and five tightly associated polypeptides. Only wild-type P-TEFb complex with an active CDK9 kinase was able to hyperphosphorylate the C-terminal domain of RNA polymerase II and mediate Tat transactivation in P-TEFb-depleted HeLa nuclear extract. Tat also stimulated transcription elongation by recruitment of the P-TEFb complex to the HIV-1 promoter through a Tat-TAR interaction. A possible mechanism for P-TEFb to become associated with polymerase elongation complexes and function as a general elongation factor was demonstrated by an interaction of P-TEFb with double-stranded RNA molecules through an 87 kDa subunit. Finally, P-TEFb was found to interact with and phosphorylate Tat-SF1, a Tat cofactor required for Tat transactivation. Our data indicate that the various subunits of the human P-TEFb complex may play distinct roles at multiple stages to mediate Tat activation of HIV-1 transcription elongation.
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PMID:Transcription elongation factor P-TEFb mediates Tat activation of HIV-1 transcription at multiple stages. 964 38

HIV-1 Tat activates transcription through binding to human cyclin T1, a regulatory subunit of the TAK/P-TEFb CTD kinase complex. Here we show that the cyclin domain of hCycT1 is necessary and sufficient to interact with Tat and promote cooperative binding to TAR RNA in vitro, as well as mediate Tat transactivation in vivo. A Tat:TAR recognition motif (TRM) was identified at the carboxy-terminal edge of the cyclin domain, and we show that hCycT1 can interact simultaneously with Tat and CDK9 on TAR RNA in vitro. Alanine-scanning mutagenesis of the hCycT1 TRM identified residues that are critical for the interaction with Tat and others that are required specifically for binding of the complex to TAR RNA. Interestingly, we find that the interaction between Tat and hCycT1 requires zinc as well as essential cysteine residues in both proteins. Cloning and characterization of the murine CycT1 protein revealed that it lacks a critical cysteine residue (C261) and forms a weak, zinc-independent complex with HIV-1 Tat that greatly reduces binding to TAR RNA. A point mutation in mCycT1 (Y261C) restores high-affinity, zinc-dependent binding to Tat and TAR in vitro, and rescues Tat transactivation in vivo. Although overexpression of hCycT1 in NIH3T3 cells strongly enhances transcription from an integrated proviral promoter, we find that this fails to overcome all blocks to productive HIV-1 infection in murine cells.
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PMID: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. 983 4

Cyclin T1 has been identified recently as a regulatory subunit of CDK9 and as a component of the transcription elongation factor P-TEFb. Cyclin T1/CDK9 complexes phosphorylate the carboxy terminal domain (CTD) of RNA polymerase II (RNAP II) in vitro. Here we report that the levels of cyclin T1 are dramatically upregulated by two independent signaling pathways triggered respectively by PMA and PHA in primary human peripheral blood lymphocytes (PBLs). Activation of these two pathways in tandem is sufficient for PBLs to enter and progress through the cell cycle. However, the expression of cyclin T1 is not growth and/or cell cycle regulated in other cell types, indicating that regulation of cyclin T1 expression is dependent on tissue-specific signaling pathways. Upregulation of cyclin T1 in stimulated PBLs results in induction of the CTD kinase activity of the cyclin T1/CDK9 complex, which in turn correlates directly with phosphorylation of RNAP II in vivo, linking for the first time activation of the cyclin T1/ CDK9 pair with phosphorylation of RNAP II in vivo. In addition, we report here that endogenous CDK9 and cyclin T1 complexes associate with HIV-1 generated Tat in relevant cells and under physiological conditions (HIV-1 infected T cells). This, together with our results showing that HIV-1 replication in stimulated PBLs correlates with the levels of cyclin T1 protein and associated CTD kinase activity, suggests that the cyclin T1/CDK9 pair is one of the HIV-1 required host cellular cofactors generated during T cell activation.
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PMID:Upregulation of cyclin T1/CDK9 complexes during T cell activation. 987 25

Tat expression is required for efficient human immunodeficiency virus type 1 (HIV-1) reverse transcription. In the present study, we generated a series of 293 cell lines that contained a provirus with a tat gene deletion (Deltatat). Cell lines that contained Deltatat and stably transfected vectors containing either wild-type tat or a number of tat mutants were obtained so that the abilities of these tat genes to stimulate HIV-1 gene expression and reverse transcription could be compared. tat genes with mutations in the amino terminus did not stimulate either viral gene expression or HIV-1 reverse transcription. In contrast, tat mutants in the activation, core, and basic domains of Tat did not stimulate HIV-1 gene expression but markedly stimulated HIV-1 reverse transcription. No differences in the levels of virion genomic RNA or tRNA3Lys were seen in the HIV-1 Deltatat viruses complemented with either mutant or wild-type tat. Finally, overexpression of the Tat-associated kinases CDK7 and CDK9, which are involved in Tat activation of HIV-1 transcription, was not able to complement the reverse transcription defects associated with the lack of a functional tat gene. These results indicate that the mechanism by which tat modulates HIV-1 reverse transcription is distinct from its ability to activate HIV-1 gene expression.
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PMID:Functional domains of Tat required for efficient human immunodeficiency virus type 1 reverse transcription. 997 35

The human immunodeficiency virus type-1 (HIV-1) long terminal repeat (LTR) initiates transcription efficiently but produces only short transcripts in the absence of the trans-activator protein, Tat. To determine whether a cellular enhancer could provide the signals required to recruit an elongation-competent polymerase to the HIV-1 LTR, the B cell-specific immunoglobulin heavy chain gene enhancer (IgHE) was inserted upstream of the LTR. The enhancer increased transcription in the absence of Tat between 6- and 7-fold in transfected B cells, but the full-length transcripts remained at basal levels in HeLa cells, where the enhancer is inactive. RNase-protection studies showed that initiation levels in the presence and absence of the enhancer were constant, but the enhancer significantly increased the elongation capacity of the polymerases. Tat-stimulated elongation is strongly inhibited by the nucleoside analogue 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), which inhibits the Tat-associated kinase, TAK (CDK9). However, polymerases initiating transcription from LTRs carrying the enhancer were able to efficiently elongate in the presence of DRB. Specific repression of TAK by expression in trans of the CDK9 kinase also inhibited Tat-stimulated elongation but did not inhibit enhancer-dependent transcription significantly. Thus, the activation of polymerase processivity by the IgHE involves a unique mechanism which is independent of TAK.
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PMID:Stimulation of Tat-associated kinase-independent transcriptional elongation from the human immunodeficiency virus type-1 long terminal repeat by a cellular enhancer. 1006 3

CDK9 is a cdc2-related kinase protein. Previously named PITALRE, this protein is a serine-threonine kinase involved in many physiological processes. Unlike most of the cdc2-like kinases, its activity is not cell cycle-regulated. CDK9 acts preferentially in processes different from cell-cycle regulation, such as differentiation. Its cyclin partners, cyclins of T family, recently have been isolated. CDK9 immunoprecipitates with several unidentified polypeptides that may regulate its kinase activity. CDK9 has been shown to associate with the HIV-Tat protein, suggesting a possible involvement in AIDS. CDK9 recently was shown to be responsible for the kinase activity associated with the TAK complex and with the P-TEFb complex, suggesting activity also in the transcription process.
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PMID:CDK9 (PITALRE): a multifunctional cdc2-related kinase. 1009 3

Tat activates transcription from the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) by increasing the processivity of RNA polymerase II. Recently, it has been demonstrated that the cellular kinase CDK9 and its binding partner cyclin T1 are involved in regulating transcriptional elongation and tat-activation. Cyclin T1, CDK9 and Tat bind as a complex to elements in TAR RNA that are required for tat-activation. Here, we used cyclin T1 mutants to define domains in this protein that bind to both CDK9 and Tat and are involved in stimulating tat-activation. The region of cyclin T1 extending from amino acid residues 1 to 263 is necessary for complex formation with Tat bound to TAR RNA and for stimulation of tat-activation in murine cells that are normally poorly responsive to the actions of Tat. In contrast, a smaller region of cyclin T1 was required to bind to CDK9 and stimulate its kinase activity. Recombinant cyclin T1 and CDK9 stimulated both basal and tat-induced in vitro transcriptional elongation from the HIV-1 LTR. The effects of Tat on transcriptional elongation may be mediated by its ability to increase CDK9 phosphorylation of the RNA polymerase II C-terminal domain. These results demonstrate that cyclin T1 interactions with Tat and TAR RNA are critical for activation of HIV-1 gene expression.
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PMID:Cyclin T1 domains involved in complex formation with Tat and TAR RNA are critical for tat-activation. 1032 25

Actinomycin D and alpha-amanitin are commonly used to inhibit transcription. Unexpectedly, however, the transcription of the human immunodeficiency virus (HIV-1) long terminal repeats (LTR) is shown to be activated at the level of elongation, in human and murine cells exposed to these drugs, whereas the Rous sarcoma virus LTR, the human cytomegalovirus immediate early gene (CMV), and the HSP70 promoters are repressed. Activation of the HIV LTR is independent of the NFkappaB and TAR sequences and coincides with an enhanced average phosphorylation of the C-terminal domain (CTD) from the largest subunit of RNA polymerase II. Both the HIV-1 LTR activation and the bulk CTD phosphorylation enhancement are prevented by several CTD kinase inhibitors, including 5, 6-dichloro-1-beta-D-ribofuranosylbenzimidazole. The efficacies of the various compounds to block CTD phosphorylation and transcription in vivo correlate with their capacities to inhibit the CDK9/PITALRE kinase in vitro. Hence, the positive transcription elongation factor, P-TEFb, is likely to contribute to the average CTD phosphorylation in vivo and to the activation of the HIV-1 LTR induced by actinomycin D.
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PMID:The transcriptional inhibitors, actinomycin D and alpha-amanitin, activate the HIV-1 promoter and favor phosphorylation of the RNA polymerase II C-terminal domain. 1034 61

Transcriptional activation of the HIV type 1 (HIV-1) long terminal repeat (LTR) promoter element by the viral Tat protein is an essential step in the HIV-1 life cycle. Tat function is mediated by the TAR RNA target element encoded within the LTR and is known to require the recruitment of a complex consisting of Tat and the cyclin T1 (CycT1) component of positive transcription elongation factor b (P-TEFb) to TAR. Here, we demonstrate that both TAR and Tat become entirely dispensable for activation of the HIV-1 LTR promoter when CycT1/P-TEFb is artificially recruited to a heterologous promoter proximal RNA target. The level of activation observed was indistinguishable from the level induced by Tat and was neither inhibited nor increased when Tat was expressed in trans. Activation by artificially recruited CycT1 depended on the ability to bind the CDK9 component of P-TEFb. In contrast, although binding to both Tat and TAR was essential for the ability of CycT1 to act as a Tat cofactor, these interactions became dispensable when CycT1 was directly recruited to the LTR. Importantly, activation of the LTR both by Tat and by directly recruited CycT1 was found to be at the level of transcription elongation. Together, these data demonstrate that recruitment of CycT1/P-TEFb to the HIV-1 LTR is fully sufficient to activate this promoter element and imply that the sole role of the Tat/TAR axis in viral transcription is to permit the recruitment of CycT1/P-TEFb.
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PMID:Recruitment of cyclin T1/P-TEFb to an HIV type 1 long terminal repeat promoter proximal RNA target is both necessary and sufficient for full activation of transcription. 1039


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