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

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

A small animal model would be very valuable for HIV/AIDS vaccine testing, investigating HIV pathophysiology, and exploring anti-HIV therapeutics. Unfortunately, HIV does not replicate in mouse cells. Provision of mouse cells with human CD4, CCR5 and cyclin T1 (cycT1) has uncovered a block to HIV assembly or release. Since mouse-human cell fusions allow viral replication, mouse cells lack at least one critical factor that permits completion of the viral life cycle. To identify this factor(s) we are employing 2 similar genetic approaches. Each cell line of a panel of monochromosomal mouse-human somatic cell hybrids was individually transduced with an HIV vector encoding both cycT1 and blasticidin resistance (HIV-CIB). Each was then transfected with vesicular stomatitis virus (VSV) G protein and measurable virus was recovered from only the hybrid-containing chromosome 2. This was verified with an M-tropic envelope and was shown to be specific to HIV. In addition, the amount of p24 release from that hybrid was substantially greater than that from the parent. A second cell line expressing chromosome 2 had a similar phenotype. CycT1 has been introduced into one chromosome 2 line to monitor the spread of HIV. In a related but separate approach, an entire collection of approximately 500 mouse-human microcell hybrids was transduced with HIV-CIB and broken down into manageable pools. Virus was similarly recovered as above from a few of the pools. Those pools were then broken down to clones and several cell clones have been identified that allow virus release. Revertants that no longer have the human chromosome are now being tested for loss of phenotype. Clones will then be tested for ability to support both HIV replication and Gag processing. Human chromosomal content of the clones of greatest interest will be determined by STS content analysis. Results from the 2 approaches are expected to be in agreement and may provide direction for an expression cloning approach.
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PMID:Development of a mouse model for HIV/AIDS. 1284 74

The oligomerization chain reaction (OCR) strategy is a recently described technique for inactivation of target proteins that function as homoassociate complexes. This novel strategy is based on the fusion of self-associating coiled-coil (CC) domain of the nuclear factor promyelocytic leukemia (PML) to target proteins. Here, we present the successful application of the OCR strategy for inactivation of the heterodimeric Cdk9/cyclin T1 complex. Cyclin T1/Cdk9 (P-TEFb) complex is a positive regulator of gene transcription, whose function is underlined by the ability to phosphorylate the carboxyl-terminal domain (CTD) of the RNA polymerase II conferring productive transcript elongation. Fusion of the CC domain to Cdk9 leads to the formation of high molecular complexes to which the endogenous cyclin T1 is recruited. The CC-Cdk9 chimera effectively inhibits HIV-1 Tat activation, whose transcription activity is exquisitely dependent upon cyclin T1/Cdk9 function. Furthermore, expression of CC-Cdk9 protein inhibits cell proliferation, as shown by colony-formation assay. Collectively, our findings add further support to the OCR strategy for functional inactivation of hetero-associated factors such as the Cdk9/cyclin T1 complex, and highlight a putative function of Cdk9 in cell growth control.
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PMID:Functional inactivation of Cdk9 through oligomerization chain reaction. 1289 30

Positive transcription elongation factor b (P-TEFb) hyperphosphorylates the carboxy-terminal domain of RNA polymerase II, permitting productive transcriptional elongation. The cyclin T1 subunit of P-TEFb engages cellular transcription factors as well as the human immunodeficiency virus type 1 (HIV-1) transactivator Tat. To identify potential P-TEFb regulators, we conducted a yeast two-hybrid screen with cyclin T1 as bait. Among the proteins isolated was the human I-mfa domain-containing protein (HIC). HIC has been reported to modulate expression from both cellular and viral promoters via its C-terminal cysteine-rich domain, which is similar to the inhibitor of MyoD family a (I-mfa) protein. We show that HIC binds cyclin T1 in yeast and mammalian cells and that it interacts with intact P-TEFb in mammalian cell extracts. The interaction involves the I-mfa domain of HIC and the regulatory histidine-rich region of cyclin T1. HIC also binds Tat via its I-mfa domain, although the sequence requirements are different. HIC colocalizes with cyclin T1 in nuclear speckle regions and with Tat in the nucleolus. Expression of the HIC cDNA modulates Tat transactivation of the HIV-1 long terminal repeat (LTR) in a cell type-specific fashion. It is mildly inhibitory in CEM cells but stimulates gene expression in HeLa, COS, and NIH 3T3 cells. The isolated I-mfa domain acts as a dominant negative inhibitor. Activation of the HIV-1 LTR by HIC in NIH 3T3 cells occurs at the RNA level and is mediated by direct interactions with P-TEFb.
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PMID:The human I-mfa domain-containing protein, HIC, interacts with cyclin T1 and modulates P-TEFb-dependent transcription. 1294 66

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

The human positive transcription elongation factor P-TEFb is composed of two subunits, cyclin T1 (hCycT1) and CDK9, and is involved in transcriptional regulation of cellular genes as well as human immunodeficiency virus type 1 (HIV-1) mRNA. Replication of HIV-1 requires the Tat protein, which activates elongation of RNA polymerase II at the HIV-1 promoter by interacting with hCycT1. To understand the cellular functions of P-TEFb and to test whether suppression of host proteins such as P-TEFb can modulate HIV infectivity without causing cellular toxicity or lethality, we used RNA interference (RNAi) to specifically knock down P-TEFb expression by degrading hCycT1 or CDK9 mRNA. RNAi-mediated gene silencing of P-TEFb in HeLa cells was not lethal and inhibited Tat transactivation and HIV-1 replication in host cells. We also found that CDK9 protein stability depended on hCycT1 protein levels, suggesting that the formation of P-TEFb CDK-cyclin complexes is required for CDK9 stability. Strikingly, P-TEFb knockdown cells showed normal P-TEFb kinase activity. Our studies suggest the existence of a dynamic equilibrium between active and inactive pools of P-TEFb in the cell and indicate that this equilibrium shifts towards the active kinase form to sustain cell viability when P-TEFb protein levels are reduced. The finding that a P-TEFb knockdown was not lethal and still showed normal P-TEFb kinase activity suggested that there is a critical threshold concentration of activated P-TEFb required for cell viability and HIV replication. These results provide new insights into the regulation of P-TEFb function and suggest the possibility that similar mechanisms for monitoring protein levels to modulate the activity of proteins may exist for the regulation of a variety of other enzymatic pathways.
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PMID:Inhibition of human immunodeficiency virus type 1 replication by RNA interference directed against human transcription elongation factor P-TEFb (CDK9/CyclinT1). 1496 54

HIV replication occurs principally in activated CD4+ T cells and macrophages. The HIV-1 Tat protein is essential for HIV replication and requires a cellular protein kinase activity termed TAK/P-TEFb, composed of CDK9 and cyclin T1, for its transactivation function. This article reviews recent work indicating that under some circumstances TAK/P-TEFb is likely to be limiting for HIV replication in CD4+ T cells and macrophages, and discusses mechanisms of regulation of the TAK/P-TEFb subunits in these cell types. In resting CD4+ T lymphocytes, TAK/P-TEFb function is low. Following lymphocyte activation, even under conditions of minimal activation in which activation markers and cellular proliferation are not induced, both CDK9 and cyclin T1 mRNA and protein levels are increased, leading to an induction of TAK/P-TEFb kinase activity that correlates with increased viral replication. In macrophages, regulation of TAK/P-TEFb involves mechanisms distinct from those in lymphocytes. In freshly isolated monocytes, CDK9 protein levels are high, while cyclin T1 protein levels are low to undetectable. Cyclin T1 protein expression is up-regulated during early macrophage differentiation by a mechanism that involves post-transcriptional regulation. Later during differentiation, cyclin T1 expression becomes shut off by a post-transcriptional mechanism, and this correlates with a decrease in Tat transactivation. Interestingly, cyclin T1 can be re-induced with lipopolysaccharide (LPS). These findings suggest that changes in cyclin T1 expression can influence HIV-1 replication levels in monocytes and macrophages. Important areas for future research on Tat and TAK/P-TEFb function are discussed.
Curr HIV Res 2003 Oct
PMID:Regulation of TAK/P-TEFb in CD4+ T lymphocytes and macrophages. 1504 26

Human immunodeficiency virus type 1 (HIV-1) gene expression and transcription is an essential step in the viral life cycle, which is considered to be a possible target for inhibition of HIV-1 replication. Among the factors involved in this step, the cellular transcription factor nuclear factor (NF)-kappaB is the most potent inducer of HIV-1 gene expression, while the viral transactivator protein Tat seems to play a central role in sustaining a high level of HIV-1 replication. Another important mechanism of HIV-1 gene expression is the nuclear export control of viral mRNA conducted by the viral regulatory protein Rev. Various attempts have been undertaken to discover selective inhibitors of HIV-1 gene expression and transcription. Several small-molecule compounds were reported to inhibit Tat functions though blocking either the Tat/TAR RNA interaction or the kinase activity of cellular cofactors, such as cyclin T1/CDK9. In the case of Rev inhibitors, it appears to be more difficult to find them than Tat inhibitors, and only a few compounds have been identified as Rev inhibitors. However, the selectivity of these Tat and Rev inhibitors was not high enough to eliminate the cytotoxicity to the host cells. Since the signal transduction pathways leading to NF-kappaB activation are redox regulated, several antioxidants have been shown to block HIV-1 transcription. Although some of them have progressed into clinical trials in HIV-1-infected patients, the results were not conclusive. In addition, various compounds have been identified as inhibitors of HIV-1 gene expression and transcription, yet their precise mechanisms are still unknown.
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PMID:Inhibitors of HIV-1 gene expression and transcription. 1513 46

The macrophage is an important cell type in the pathophysiology of human immunodeficiency virus type 1 (HIV-1) infection. Macrophages both support viral replication and are capable of attracting and activating lymphocytes, thus rendering CD4+ T lymphocytes highly permissive for infection. The viral Tat protein, whose function is mediated by the cellular cyclin T1 protein complexed with CDK9, is required for efficient transcription of the integrated HIV-1 provirus by RNA polymerase II. Cyclin T1 expression is highly regulated during macrophage differentiation, and this has important implications for HIV-1 replication. In monocytes isolated from healthy blood donors, cyclin T1 protein expression is low and is induced to high levels within the first few days of differentiation by a post-transcriptional mechanism. After 1-2 weeks of macrophage differentiation, however, cyclin T1 expression is shut off. Treatment of macrophages with lipopolysaccharide (LPS) can re-induce cyclin T1, indicating that the activation status of macrophages can regulate cyclin T1 expression. Recent results indicate that HIV-1 infection is able to induce cyclin T1 expression in macrophages. Future studies of cyclin T1 regulation in macrophages may suggest means of manipulating expression of this crucial cellular co-factor for therapeutic benefit in HIV-1 infected individuals.
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PMID:HIV-1 infection and regulation of Tat function in macrophages. 1518 43


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