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

HIV-1 Tat protein regulates viral gene expression by modulating the activity and association of cellular transcription factors with RNA polymerase II (RNAPII). Possible mechanisms include Tat-associated protein kinase(s) and phosphatase(s) that regulate phosphorylation of the C-terminal domain (CTD) of the large subunit of RNAPII. Hypophosphorylated RNAPII (RNAPIIa) is recruited to promoters during formation of a preinitiation complex, whereas hyperphosphorylated RNAPII (RNAPIIo) is associated with the elongation complex. The role of phosphatases in maintaining the equilibrium between the two phosphorylated states of RNAPII, which is required for sustained transcriptional activation from the HIV-1 LTR, is not clear. In this study, we discuss the properties of a Tat-associated CTD phosphatase fractionated from Jurkat T cells. The Tat-associated protein phosphatase (TAPP) is related to the serine/threonine, type 1, protein phosphatase (PP1) family. TAPP dephosphorylates the hyperphosphorylated form of recombinant CTD specifically on serine 2, and augments Tat-mediated transcriptional transactivation of HIV-1 LTR in an in vitro transcription reaction. TAPP is associated with the transcription complex during the early initiation steps, and its release from the HIV-1 promoter coincides with the Tat-specific activation of CDK9. The results suggest a unique role of the Tat-associated phosphatase which regulates viral transcription by target-specific dephosphorylation of RNAPII during the early stages of elongation.
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PMID:A protein phosphatase from human T cells augments tat transactivation of the human immunodeficiency virus type 1 long-terminal repeat. 1203 13

HIV-1 gene expression is regulated by a viral transactivator protein (Tat) which induces transcriptional elongation of HIV-1 long tandem repeat (LTR). This induction requires hyperphosphorylation of the C-terminal domain (CTD) repeats of RNA polymerase II (Pol II). To achieve CTD hyperphosphorylation, Tat stimulates CTD kinases associated with general transcription factors of the promoter complex, specifically TFIIH-associated CDK7 and positive transcription factor b-associated CDK9 (cyclin-dependent kinase 9). Other studies indicate that Tat may bind an additional CTD kinase that regulates the target-specific phosphorylation of RNA Pol II CTD. We previously reported that Tat-associated T-cell-derived kinase (TTK), purified from human primary T-cells, stimulates Tat-dependent transcription of HIV-1 LTR in vivo [Nekhai, Shukla, Fernandez, Kumar and Lamb (2000) Virology 266, 246-256]. In the work presented here, we characterized the components of TTK by biochemical fractionation and the function of TTK in transcription assays in vitro. TTK uniquely co-purified with CDK2 and not with either CDK9 or CDK7. Tat induced the TTK-associated CDK2 kinase to phosphorylate CTD, specifically at Ser-2 residues. The TTK fraction restored Tat-mediated transcription activation of HIV-1 LTR in a HeLa nuclear extract immunodepleted of CDK9, but not in the HeLa nuclear extract double-depleted of CDK9 and CDK7. Direct microinjection of the TTK fraction augmented Tat transactivation of HIV-1 LTR in human primary HS68 fibroblasts. The results argue that TTK-associated CDK2 may function to maintain target-specific phosphorylation of RNA Pol II that is essential for Tat transactivation of HIV-1 promoter. They are also consistent with the observed cell-cycle-specific induction of viral gene transactivation.
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PMID:HIV-1 Tat-associated RNA polymerase C-terminal domain kinase, CDK2, phosphorylates CDK7 and stimulates Tat-mediated transcription. 1204 28

Positive transcription elongation factor-b (P-TEFb) contains CDK9 and cyclin T(1). P-TEFb was affinity purified from a stably transfected cell line that expresses epitope-tagged CDK9, and proteins that appeared to be specifically bound were sequenced. In addition to CDK9, previously identified isoforms of cyclin T (including T(1), T(2A) and T(2B)), HSP90 and CDC37, this analysis identified a novel protein named MCEF. Cloning of its cognate cDNA revealed that MCEF is the newest member of the AF4 family of transcription factors involved in acute lymphoblastic leukemia. MCEF RNA was expressed in all human tissues examined, and antisera directed against recombinant MCEF specifically immunoprecipitated P-TEFb. Ectopic expression of MCEF did not activate HIV-1 replication, and tethering of MCEF to a promoter did not activate transcription.
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PMID:MCEF, the newest member of the AF4 family of transcription factors involved in leukemia, is a positive transcription elongation factor-b-associated protein. 1206 98

Human immunodeficiency virus, type 1 (HIV-1), Tat protein activates viral gene expression through promoting transcriptional elongation by RNA polymerase II (RNAPII). In this process Tat enhances phosphorylation of the C-terminal domain (CTD) of RNAPII by activating cell cycle-dependent kinases (CDKs) associated with general transcription factors of the promoter complex, specifically CDK7 and CDK9. We reported a Tat-associated T-cell-derived kinase, which contained CDK2. Here, we provide further evidence that CDK2 is involved in Tat-mediated CTD phosphorylation and in HIV-1 transcription in vitro. Tat-mediated CTD phosphorylation by CDK2 required cysteine 22 in the activation domain of Tat and amino acids 42-72 of Tat. CDK2 phosphorylated Tat itself, apparently by forming dynamic contacts with amino acids 15-24 and 36-49 of Tat. Also, amino acids 24-36 and 45-72 of Tat interacted with CTD. CDK2 associated with RNAPII and was found in elongation complexes assembled on HIV-1 long-terminal repeat template. Recombinant CDK2/cyclin E stimulated Tat-dependent HIV-1 transcription in reconstituted transcription assay. Immunodepletion of CDK2/cyclin E in HeLa nuclear extract blocked Tat-dependent transcription. We suggest that CDK2 is part of a transcription complex that is required for Tat-dependent transcription and that interaction of Tat with CTD and a dynamic association of Tat with CDK2/cyclin E stimulated CTD phosphorylation by CDK2.
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PMID:HIV-1 Tat interaction with RNA polymerase II C-terminal domain (CTD) and a dynamic association with CDK2 induce CTD phosphorylation and transcription from HIV-1 promoter. 1211 99

Human immunodeficiency virus type 1 (HIV-1) gene expression is regulated by both cellular transcription factors and Tat. The ability of Tat to stimulate transcriptional elongation is dependent on its binding to TAR RNA in conjunction with cyclin T1 and CDK9. A variety of other cellular factors that bind to the HIV-1 long terminal repeat, including NF-kappaB, SP1, LBP, and LEF, are also important in the control of HIV-1 gene expression. Although these factors have been demonstrated to regulate HIV-1 gene expression by both genetic and biochemical analysis, in most cases a direct in vivo demonstration of their role on HIV-1 replication has not been established. Recently, the efficacy of RNA interference in mammalian cells has been shown utilizing small interfering RNAs (siRNAs) to result in the specific degradation of host mRNAs and decreases the levels of their corresponding proteins. In this study, we addressed whether siRNAs directed against either HIV-1 tat or reverse transcriptase or the NF-kappaB p65 subunit could specifically decrease the levels of these proteins and thus alter HIV-1 replication. Our results demonstrate the specificity of siRNAs for decreasing the expression of these viral and cellular proteins and inhibiting HIV-1 replication. These studies suggest that RNA interference is useful in exploring the biological role of cellular and viral regulatory factors involved in the control of HIV-1 gene expression.
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PMID:RNA interference directed against viral and cellular targets inhibits human immunodeficiency Virus Type 1 replication. 1243 22

P-TEFb, cyclin T1 + CDK9, is needed for the expression of cellular promoters and primate lentiviral long terminal repeats (LTRs). Curiously, cellular and lentiviral promoters differ dramatically in the requirements for positive transcriptional elongation factor (P-TEF) b activity. Lentiviral LTRs, but not cellular promoters, need an RNA-associated P-TEFb/Tat/TAR (trans-activation-responsive) RNA ternary complex. Ternary complex defective murine cycT1 is apparently inactive for lentiviral transcription. Why P-TEFb requires Tat/TAR for LTRs but not for cellular promoters remains unknown. To explore this question, we sought to determine whether DNA targeting of murine and human cyclin T1 can reconstitute a Tat/TAR-independent activity to the HIV-1 LTR. In the absence of Tat and TAR, we found that both HuCycT1 and MuCycT1 can robustly activate the HIV-1 LTR. We further showed that Sp1 is necessary and sufficient for this DNA-targeted activity. Thus, like cellular promoters, HIV-1 LTR can use P-TEFb function without a Tat/TAR RNA complex. This activity could explain recent findings of robust HIV-1 replication in rat cells that cannot form a P-TEFb/Tat/TAR moiety.
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PMID:Tat and trans-activation-responsive (TAR) RNA-independent induction of HIV-1 long terminal repeat by human and murine cyclin T1 requires Sp1. 1245 22

Cyclin T1, together with the kinase CDK9, is a component of the transcription elongation factor P-TEFb which binds the human immunodeficiency virus type 1 (HIV-1) transactivator Tat. P-TEFb facilitates transcription by phosphorylating the carboxy-terminal domain (CTD) of RNA polymerase II. Cyclin T1 is an exceptionally large cyclin and is therefore a candidate for interactions with regulatory proteins. We identified granulin as a cyclin T1-interacting protein that represses expression from the HIV-1 promoter in transfected cells. The granulins, mitogenic growth factors containing repeats of a cysteine-rich motif, were reported previously to interact with Tat. We show that granulin formed stable complexes in vivo and in vitro with cyclin T1 and Tat. Granulin bound to the histidine-rich domain of cyclin T1, which was recently found to bind to the CTD, but not to cyclin T2. Binding of granulin to P-TEFb inhibited the phosphorylation of a CTD peptide. Granulin expression inhibited Tat transactivation, and tethering experiments showed that this effect was due, at least in part, to a direct action on cyclin T1 in the absence of Tat. In addition, granulin was a substrate for CDK9 but not for the other transcription-related kinases CDK7 and CDK8. Thus, granulin is a cellular protein that interacts with cyclin T1 to inhibit transcription.
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PMID:The growth factor granulin interacts with cyclin T1 and modulates P-TEFb-dependent transcription. 1258 88

The Tat protein of human immunodeficiency virus type 1 (HIV-1) trans-activates HIV-1 transcription by functionally interacting with a number of cellular proteins, among which the Sp1 transcription factor. We recently demonstrated that Tat does not directly interact with Sp1 either in vitro or in vivo, and we suggested that other protein(s) could indirectly mediate Tat-Sp1 interaction. In keeping, here we showed that addition of HeLa cell nuclear extracts to purified Tat and Sp1 proteins allows the formation of a Tat/Sp1 complex in in vitro binding assays. In an attempt to identify the partner(s) that bridge Tat and Sp1, we developed a yeast multi-protein system, in which cellular proteins recently shown to play a relevant role in Tat function, namely TATA box-binding protein, cyclin T1, CDK9, and cyclin T1/CDK9 complex, were coexpressed, individually or in pair-wise combination, with Tat and Sp1 hybrids. We demonstrated that none of these candidate partners bridges Tat and Sp1. However, our yeast multi-protein system, which allows simple and rapid detection of interactions among up to four proteins, will be most helpful to further dissect the interaction of Tat and Sp1 with other candidate partners that participate in the assembly of transcriptionally active complexes at the HIV-1 LTR.
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PMID:Interaction of Sp1 transcription factor with HIV-1 Tat protein: looking for cellular partners. 1275 6

The HIV type 1 (HIV-1) Tat protein stimulates transcription elongation by recruiting P-TEFb (CDK9/cyclin T1) to the transactivation response (TAR) RNA structure. Tat-induced CDK9 kinase has been shown to phosphorylate Ser-5 of RNA polymerase II (RNAP II) C-terminal domain (CTD). Results presented here demonstrate that Tat-induced Ser-5 phosphorylation of CTD by P-TEFb stimulates the guanylyltransferase activity of human capping enzyme and RNA cap formation. Sequential phosphorylation of CTD by Tat-induced P-TEFb enhances the stimulation of human capping enzyme guanylyltransferase activity and RNA cap formation by transcription factor IIH-mediated CTD phosphorylation. Using an immobilized template assay that permits isolation of transcription complexes, we show that Tat/TAR-dependent phosphorylation of RNAP II CTD stimulates cotranscriptional capping of HIV-1 mRNA. Upon transcriptional induction of latently infected cells, accumulation of capped transcripts occurs along with Ser-5-phosphorylated RNAP II in the promoter proximal region of the HIV-1 genome. Therefore, these observations suggest that Tat/TAR-dependent phosphorylation of RNAP II CTD is crucial not only in promoting transcription elongation but also in stimulating nascent viral RNA capping.
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PMID:The Tat/TAR-dependent phosphorylation of RNA polymerase II C-terminal domain stimulates cotranscriptional capping of HIV-1 mRNA. 1456 24

Tat-mediated trans-activation of the HIV-1 long terminal repeat (LTR) occurs through the phosphorylation of the carboxy-terminal domain of the RNA polymerase II. The kinase complex, pTEFb, composed of cyclin T1 (CycT1) and CDK9, mediates this process. The trans-activation response (TAR) RNA-binding protein 2 (TRBP2) increases HIV-1 LTR expression through TAR and protein kinase R (PKR) binding, but not through interactions with the Tat-CycT1-CDK9 complex. TRBP2 and the Tat-CycT1-CDK9 complex have overlapping binding sites on TAR RNA. TRBP2 and CycT1 increased Tat trans-activation in NIH 3T3 cells with additive effects. Upon transfection of HIV-1 pLAI, pNL4-3, pMAL, and pAD molecular clones, reverse transcriptase (RT) activity and p24 concentration were decreased 200- to 900-fold in NIH 3T3 cells compared with HeLa cells in both cells and supernatants. In murine cells, cotransfection of the HIV clones with CycT1 or TRBP2 increased modestly the expression of RT activity in cell extracts. The analysis of Gag expression in murine cells transfected with CycT1 compared with human cells showed a 20-fold decrease in expression and a strong processing defect. The expression of both CycT1 and TRBP2 had a more than additive activity on RT function in cell extracts and on viral particle production in supernatant of murine cells. These results suggest an activity of CycT1 and TRBP2 at different steps in HIV-1 expression and indicate the requirement for another posttranscriptional factor in murine cells for full HIV replication.
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PMID:Additive activity between the trans-activation response RNA-binding protein, TRBP2, and cyclin T1 on HIV type 1 expression and viral production in murine cells. 1458 7


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