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 HIV-11 Tat is a viral protein that interacts with transactivation response (TAR) RNA, a hairpin-loop structure at the 5Ј-end of all nascent viral transcripts (reviewed in Refs. 1 and 2). Tat stimulates transcriptional elongation (3), which is regulated by phosphorylation of the largest subunit of RNA polymerase II (RNAPII). The C-terminal domain (CTD) of RNA-PII consists of heptapeptide YSPTSPS repeats, which are phosphorylated on Ser-2 and Ser-5 residues during transcription (reviewed in Ref. 4). CTD is phosphorylated by host cell cycle-dependent protein kinases CDK7 and CDK9 (reviewed in Ref. 5). General transcription factor TFIIH-associated CDK7 phosphorylates Ser-5 during initiation of transcription, whereas positive transcription elongation factor b-associated CDK9 phosphorylates Ser-2 during elongation of transcription (5). Tat associates with the bulge of TAR RNA and also binds to cyclin T1, a cyclin partner of CDK9, which in turn interacts with the loop of TAR RNA (6). This allows CDK9/cyclin T1 to be recruited by Tat to the HIV-1 promoter (7). Tat also modifies the substrate specificity of CDK9 to achieve additional Ser-5 phosphorylation (8).Although the evidence for the role of CDK9/cyclin T1 in Tat-mediated HIV-1 transcription is overwhelming, our recent data suggest that there may be an additional CTD kinase involved in the Tat response. We have purified a Tat-associated T-cell-derived kinase (TTK) that phosphorylates CTD (9 -11). TTK stimulates Tat transactivation in vitro (11) and in vivo (10, 11). TTK containes CDK2, which phosphorylates CDK7 (11).In the work presented here, we analyze the effect of Tat on CTD phosphorylaton by CDK2/cyclin E and the function of CDK2 in transcription assays of HIV-1 promoter in vitro. Our finding demonstrated that interaction of Tat with CTD and a dynamic interaction with CDK2/cyclin E stimulated CTD phosphorylation by CDK2. Also we showed that CDK2 was part of transcription complex and was required for Tat-dependent transcription in vitro.

Infection with human T-cell leukemia virus type 1 (HTLV-1) results in adult T-cell leukemia and HTLV-1-associated myelopathy/tropical spastic paraparesis. Tax, a 40-kDa protein, regulates viral and cellular transcription, host signal transduction, the cell cycle, and apoptosis. Tax has been shown to modulate cellular CREB and NFB pathways; however, to date, its role in binding to various host cellular proteins involved in tumorigenesis has not been fully described. In this study, we describe the Tax-associated proteins and their functions in cells using several approaches. Tax eluted from a sizing column mostly at an apparent molecular mass of 1800 kDa. Following Tax immunoprecipitation, washes with high salt buffer, two-dimensional gel separation, and mass spectrometric analysis, a total of 32 proteins was identified. Many of these proteins belong to the signal transduction and cytoskeleton pathways and transcription/chromatin remodeling. A few of these proteins, including TXBP151, have been shown previously to bind to Tax. The interaction of Tax with small GTPase-cytoskeleton proteins, such as ras GAP 1m , Rac1, Cdc42, RhoA, and gelsolin, indicates how Tax may regulate migration, invasion, and adhesion in T-cell cancers. Finally, the physical and functional association of Tax with the chromatin remodeling SWI/SNF complex was assessed using in vitro chromatin remodeling assays, chromatin remodeling factor BRG1 mutant cells, and RNA interference experiments. Collectively, Tax is able to bind and regulate many cellular proteins that regulate transcription and cytoskeletal related pathways, which might explain the pleiotropic effects of Tax leading to T-cell transformation and leukemia in HTLV-1-infected patients.Infection with human T-cell leukemia virus type 1 (HTLV-1) 1 results in adult T-cell leukemia and HTLV-1-associated myelopathy/tropical spastic paraparesis. The Tax protein encoded by HTLV-1 plays a central role in the development of both adult T-cell leukemia and HTLV-1-associated myelopathy/tropical spastic paraparesis. Although Tax itself does not bind to DNA directly or function as an enzyme, its ability to regulate multiple cellular responses is conferred by its protein-protein interactions with various host cellular factors. Importantly, HTLV-1-mediated activation of the host T-cell is induced primarily by the viral protein Tax, which influences transcriptional activation, signal transduction, cell cycle control, and apoptosis. Therefore, understanding how Tax controls these pathways is of significant importance. Tax targets several transcriptional pathways including CREB/activating transcription factor, NFB, and multiple other factors including cell cycle regulators, such as cyclins D2 and D3, the mitotic checkpoint regulator MAD1, the cyclin-dependent kinases (Cdk) Cdk4 and Cdk6, Cdk inhibitors (p16/INK4A and p21/Waf1), and the tumor suppressor p53 (1). Moreover, Tax regulation is observed at both nuclear (e.g. CREB-dependent) and cytoplasmic (e.g. NFB-dependent) levels, functioning through shuttl...

The human immunodeficiency virus type 1 (HIV-1) potent transactivator Tat protein mediates pleiotropic effects on various cell functions. Posttranslational modification of Tat affects its activity during viral transcription. Tat binds to TAR and subsequently becomes acetylated on lysine residues by histone acetyltransferases. Novel protein-protein interaction domains on acetylated Tat are then established, which are necessary for both sustained transcriptional activation of the HIV-1 promoter and viral transcription elongation. In this study, we investigated the identity of proteins that preferentially bound acetylated Tat. Using a proteomic approach, we identified a number of proteins that preferentially bound AcTat, among which p32, a cofactor of splicing factor ASF/SF-2, was identified. We found that p32 was recruited to the HIV-1 genome, suggesting a mechanism by which acetylation of Tat may inhibit HIV-1 splicing needed for the production of full-length transcripts. Using Tat from different clades, harboring a different number of acetylation sites, as well as Tat mutated at lysine residues, we demonstrated that Tat acetylation affected splicing in vivo. Finally, using confocal microscopy, we found that p32 and Tat colocalize in vivo in HIV-1-infected cells.

No abstract