UMLS:C0019693 - FACTA Search

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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

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

Human cyclin T1 (hCycT1), a major subunit of the essential elongation factor P-TEFb, has been proposed to act as a cofactor for human immunodeficiency virus type 1 (HIV-1) Tat. Here, we show that murine cyclin T1 (mCycT1) binds the activation domain of HIV-1 Tat but, unlike hCycT1, cannot mediate Tat function because it cannot be recruited efficiently to TAR. In fact, overexpression of mCycT1, but not hCycT1, specifically inhibits Tat-TAR function in human cells. This discordant phenotype results from a single amino acid difference between hCycT1 and mCycT1, a tyrosine in place of a cysteine at residue 261. These data indicate that the ability of Tat to recruit CycT1/P-TEFb to TAR determines the species restriction of HIV-1 Tat function in murine cells and therefore demonstrate that this recruitment is a critical function of the Tat protein.
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PMID:Recruitment of a protein complex containing Tat and cyclin T1 to TAR governs the species specificity of HIV-1 Tat. 984 10

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 stimulation of HIV-1 transcriptional elongation is species-specific and is believed to require a specific cellular cofactor present in many human and primate cells but not in nonpermissive rodent cells. Human P-TEFb, composed of Cdk9 and cyclin T1, is a general transcription elongation factor that phosphorylates the C-terminal domain of RNA polymerase II. Previous studies have also implicated P-TEFb as a Tat-specific cellular cofactor and, in particular, human cyclin T1 as responsible for the species-specific Tat activation. To obtain functional evidence in support of these hypotheses, we generated and examined the activities of human-rodent "hybrid" P-TEFb complexes. We found that P-TEFb complexes containing human cyclin T1 complexed with either human or rodent Cdk9 supported Tat transactivation and interacted with the Tat activation domain and the HIV-1 TAR RNA element to form TAR loop-dependent ribonucleoprotein complexes. Although a stable complex containing rodent cyclin T1 and human Cdk9 was capable of phosphorylating CTD and mediating basal HIV-1 elongation, it failed to interact with Tat and to mediate Tat transactivation, indicating that the abilities of P-TEFb to support basal elongation and Tat activation can be separated. Together, our data indicated that the specific interaction of human P-TEFb with Tat/TAR, mostly through cyclin T1, is crucial for P-TEFb to mediate a Tat-specific and species-restricted activation of HIV-1 transcription. Amino acid residues unique to human Cdk9 also contributed partially to the formation of the P-TEFb-Tat-TAR complex. Moreover, the cyclin box of cyclin T1 and its immediate flanking region are largely responsible for the specific P-TEFb-Tat interaction.
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PMID:Specific interaction of Tat with the human but not rodent P-TEFb complex mediates the species-specific Tat activation of HIV-1 transcription. 1007 79

Tat stimulates human immunodeficiency virus type 1 (HIV-1) transcriptional elongation by recruitment of the human transcription elongation factor P-TEFb, consisting of Cdk9 and cyclin T1, to the HIV-1 promoter via cooperative binding to the nascent HIV-1 transactivation response RNA element. The Cdk9 kinase activity has been shown to be essential for P-TEFb to hyperphosphorylate the carboxy-terminal domain (CTD) of RNA polymerase II and mediate Tat transactivation. Recent reports have shown that Tat can also interact with the multisubunit transcription factor TFIIH complex and increase the phosphorylation of CTD by the Cdk-activating kinase (CAK) complex associated with the core TFIIH. These observations have led to the proposal that TFIIH and P-TEFb may act sequentially and in a concerted manner to promote phosphorylation of CTD and increase polymerase processivity. Here, we show that under conditions in which a specific and efficient interaction between Tat and P-TEFb is observed, only a weak interaction between Tat and TFIIH that is independent of critical amino acid residues in the Tat transactivation domain can be detected. Furthermore, immunodepletion of CAK under high-salt conditions, which allow CAK to be dissociated from core-TFIIH, has no effect on either basal HIV-1 transcription or Tat activation of polymerase elongation in vitro. Therefore, unlike the P-TEFb kinase activity that is essential for Tat activation of HIV-1 transcriptional elongation, the CAK kinase associated with TFIIH appears to be dispensable for Tat function.
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PMID:Tat activates human immunodeficiency virus type 1 transcriptional elongation independent of TFIIH kinase. 1008 52

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

The human immunodeficiency virus type 1 (HIV-1) Tat protein (hTat) activates transcription initiated at the viral long terminal repeat (LTR) promoter by a unique mechanism requiring recruitment of the human cyclin T1 (hCycT1) cofactor to the viral TAR RNA target element. While activation of equine infectious anemia virus (EIAV) gene expression by the EIAV Tat (eTat) protein appears similar in that the target element is a promoter proximal RNA, eTat shows little sequence homology to hTat, does not activate the HIV-1 LTR, and is not active in human cells that effectively support hTat function. To address whether eTat and hTat utilize similar or distinct mechanisms of action, we have cloned the equine homolog of hCycT1 (eCycT1) and examined whether it is required to mediate eTat function. Here, we report that expression of eCycT1 in human cells fully rescues eTat function and that eCycT1 and eTat form a protein complex that specifically binds to the EIAV, but not the HIV-1, TAR element. While hCycT1 is also shown to interact with eTat, the lack of eTat function in human cells is explained by the failure of the resultant protein complex to bind to EIAV TAR. Critical sequences in eCycT1 required to support eTat function are located very close to the amino terminus, i.e., distal to the HIV-1 Tat-TAR interaction motif previously identified in the hCycT1 protein. Together, these data provide a molecular explanation for the species tropism displayed by eTat and demonstrate that highly divergent lentiviral Tat proteins activate transcription from their cognate LTR promoters by essentially identical mechanisms.
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PMID:Highly divergent lentiviral Tat proteins activate viral gene expression by a common mechanism. 1037 8

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

The human immunodeficiency virus type-1 (HIV-1) Tat protein regulates transcription by stimulating RNA polymerase processivity. Using immobilised templates, we have been able to study the effects of Tat on protein kinase activity during the pre-initiation and elongation stages of HIV-1 transcription. In pre-initiation complexes formed at the HIV-1 LTR, the C-terminal domain (CTD) of RNA polymerase II is rapidly phosphorylated by transcription factor IIH (TFIIH). Addition of Tat does not affect either the rate or the extent of CTD phosphorylation in the pre-initiation complexes. By contrast, Tat is able to stimulate additional CTD phosphorylation in elongation complexes. This reaction creates a novel form of the RNA polymerase that we have called RNA polymerase IIo*. Formation of the RNA polymerase IIo* occurs only after transcription of templates carrying a functional TAR RNA element and is strongly inhibited by low concentrations of 5,6-dichloro-1-beta- D -ribofuranosyl benzimidazole (DRB), a potent inhibitor of CDK9, the protein kinase subunit of the Tat-associated kinase (TAK). Immunoblotting experiments have shown that CDK9 and its associated cyclin, cyclin T1, are present at equivalent levels in both the pre-initiation and elongation complexes. We conclude that activation of the CDK9 kinase, leading to CTD phosphorylation, occurs only in elongation complexes that have transcribed through the Tat-recognition element, TAR RNA.
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PMID:Direct evidence that HIV-1 Tat stimulates RNA polymerase II carboxyl-terminal domain hyperphosphorylation during transcriptional elongation. 1043 93

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