Viral Transactivators Specifically Target Distinct Cellular Protein Kinases That Phosphorylate the RNA Polymerase II C-Terminal Domain

Herrmann, Christine; Gold, Moses O.; Rice, Andrew P.

doi:10.1093/nar/24.3.501

Cited by 64 publications

(48 citation statements)

References 79 publications

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“…2) suggests that the post-TBP recruitment function of Tat is primarily at the level of RNAP II elongation-clearance from the promoter. This is consistent with recent biochemical evidence for an association between Tat and a kinase that phosphorylates the carboxylterminal domain of the large subunit of RNAP II (12,21,38) and with the large body of literature ascribing to Tat a role in efficient transcriptional elongation (e.g., references 15, 40, and 62 and references therein). The recent finding that Tat might exist in a preinitiation complex (13,16) raises the interesting possibility that commitment to promoter clearance is determined at both pre-and post-TBP recruitment steps.…”

Section: Discussionsupporting

confidence: 90%

Promoter Activity of Tat at Steps Subsequent to TATA-Binding Protein Recruitment

Xiao

Lis

Jeang

1997

Molecular and Cellular Biology

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Artificial recruitment of TATA-binding protein (TBP) to many eukaryotic promoters bypasses DNA-bound activator function. The human immunodeficiency virus type 1 (HIV-1) Tat is an unconventional activator that up-regulates transcription from the HIV-1 long terminal repeat (LTR) through binding to a nascent RNA sequence, TAR. Because this LTR and its cognate activator have atypical features compared to a standard RNA polymerase II (RNAP II) transcriptional unit, the precise limiting steps for HIV-1 transcription and how Tat resolves these limitations remain incompletely understood. We thus constructed human TBP fused to the DNA-binding domain of GAL4 to determine whether recruitment of TBP is one rate-limiting step in HIV-1 LTR transcription and whether Tat functions to recruit TBP. As a control, we compared the activity of the adenovirus E1b promoter. Our findings indicate that TBP tethering to the E1b promoter fully effected transcription to the same degree achievable with the potent GAL4-VP16 activator. By contrast, TBP recruitment to the HIV-1 LTR, although necessary for conferring Tat responsiveness, did not bypass a physical need for Tat in achieving activated transcription. These results document that the HIV-1 and the E1b promoters are transcriptionally limited at different steps; the major rate-limiting step for E1b is recruitment of TBP, while activation of the HIV-1 LTR requires steps in addition to TBP recruitment. We suggest that Tat acts to accelerate rate-limiting steps after TBP recruitment.DNA-bound activator proteins dictate many aspects of transcription by RNA polymerase II (RNAP II) in vivo (37). While many details remain to be fully characterized, much is understood about how activators stimulate transcription complex assembly at the promoter (9, 42, 54) and egress of RNAP II from the promoter (7, 59). For productive transcription to occur, it is believed that activators bound to upstream enhancers influence recruitment to the TATA promoter of TATAbinding protein (TBP) and TBP-associated factors which then modulate docking of RNAP II holoenzyme (8,29) to the initiator site (reviewed in reference 52). Consistent with this picture, DNA-bound activators have been found to associate with components of the general transcription machinery that include TBP (50), TBP-associated factors (54), transcription factor IIA (TFIIA) (28), TFIIB (33), TFIIF (26), and TFIIH (57) (see reference 54 for a review). In many systems, a direct correlation between transcription and the interactive affinity of activators for TBP has been established, suggesting that activator-TBP recruitment and/or activator-TBP-TATA stabilization are critical steps in promoter activation (for example, see reference 23).Several recent studies have shown that artificial recruitment of TBP through a heterologous DNA-binding domain to many yeast promoters confers high transcriptional activity (11,27,56). This finding suggests that in yeast, recruitment of TBP is a single in vivo rate-limiting step accelerated by DNA-bound activators. Howeve...

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Section: Discussionsupporting

confidence: 90%

Promoter Activity of Tat at Steps Subsequent to TATA-Binding Protein Recruitment

Xiao

Lis

Jeang

1997

Molecular and Cellular Biology

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“…Protein concentrations were determined by a Bio-Rad protein assay, and 20 g of total protein was loaded onto 10% sodium dodecyl sulfate (SDS)-polyacrylamide gels. The procedure for immunoblotting using enhanced chemiluminescence for detection was described previously (16). The Ab against ␤-actin was purchased from Sigma and used at a dilution of 1:3,000.…”

Section: Cellsmentioning

confidence: 99%

Transient Induction of Cyclin T1 during Human Macrophage Differentiation Regulates Human Immunodeficiency Virus Type 1 Tat Transactivation Function

Liou

Herrmann

Rice

2002

J Virol

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The human immunodeficiency virus type 1 (HIV-1) Tat protein is essential for viral replication and stimulates transcription of the integrated provirus by recruiting the kinase complex TAK/P-TEFb, composed of cyclin T1 (CycT1) and Cdk9, to the viral TAR RNA element. TAK/P-TEFb phosphorylates the RNA polymerase II complex and stimulates transcriptional elongation. In this report, we investigated the regulation of TAK/P-TEFb in primary human macrophages, a major target cell of HIV infection. While Cdk9 levels remained constant, CycT1 protein expression in freshly isolated monocytes was very low, increased early during macrophage differentiation, and, unexpectedly, decreased to very low levels after about 1 week in culture. The kinase activity of TAK/P-TEFb paralleled the changes in CycT1 protein expression. RNA analysis indicated that the transient induction of CycT1 protein expression involves a posttranscriptional mechanism. In transient transfection assays, the ability of Tat to transactivate the HIV long terminal repeat (LTR) in the late differentiated macrophages was greatly diminished relative to its ability to transactivate the HIV LTR in early differentiated cells, strongly suggesting that CycT1 is limiting for Tat function in late differentiated macrophages. Interestingly, lipopolysaccharide, a component of the cell wall of gram-negative bacteria, reinduced CycT1 expression late in macrophage differentiation. These results raise the possibility that regulation of CycT1 expression may be involved in establishing latent infection in macrophages and that opportunistic infection may reactivate the virus by inducing CycT1 expression.The two major cell types targeted by human immunodeficiency virus type 1 (HIV-1) are helper T cells and macrophages, as these cell types express CD4 and a chemokine receptor, either CCR5 or CXCR4, on their surfaces. CD4 is the primary receptor for the virus, while CCR5 or CXCR4 serves as a coreceptor that mediates fusion of the enveloped virion with the cell membrane. In most individuals, HIV-1 infection leads to an inevitable erosion of the immune system and development of AIDS. Analyses of patients treated with drugs that effectively block HIV-1 replication in vivo have revealed that infected CD4 ϩ T cells produce the majority (Ͼ90%) of virus during the prolonged infection period before the onset of AIDS (reviewed in reference 4). These infected CD4 ϩ T cells are short-lived, with a half-life estimated to be Ͻ1 day due to direct cytopathic effects of infection and to clearance of infected cells by immune-mediated mechanisms, especially that of cytotoxic CD8 ϩ T lymphocytes. A second population of productively infected cells has a half-life estimated to be 1 to 4 weeks; this population produces a few percent of virus in infected individuals prior to AIDS. Although the nature of this longer-lived population of infected cells is uncertain, macrophages are a likely candidate cell type, as they are resistant to the cytopathic effects of infection in vitro and the half-life of tissue ma...

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“…Protein concentrations were determined by the Bio-Rad protein assay, and 25 g of total protein was analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Immunoblotting was performed by standard procedures using enhanced chemiluminescence for detection as described previously (24). Antibodies for detection of Cdk9, Cdk7, cyclin T2b, and cyclin T1 were obtained from Santa Cruz Biotechnology.…”

Section: Activation Of Cellsmentioning

confidence: 99%

Induction of TAK (Cyclin T1/P-TEFb) in Purified Resting CD4 ⁺ T Lymphocytes by Combination of Cytokines

Ghose

Liou

Herrmann

et al. 2001

J Virol

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Combinations of cytokines are known to reactivate transcription and replication of latent human immunodeficiency virus type 1 (HIV-1) proviruses in resting CD4؉ T lymphocytes isolated from infected individuals. Transcription of the HIV-1 provirus by RNA polymerase II is strongly stimulated by the viral Tat protein. Tat function is mediated by a cellular protein kinase known as TAK (cyclin T1/P-TEFb) that is composed of Cdk9 and cyclin T1. We have found that treatment of peripheral blood lymphocytes and purified resting CD4 ؉ T lymphocytes with the combination of interleukin-2 (IL-2), IL-6, and tumor necrosis factor alpha resulted in an increase in Cdk9 and cyclin T1 protein levels and an increase in TAK enzymatic activity. The cytokine induction of TAK in resting CD4؉ T lymphocytes did not appear to require proliferation of lymphocytes. These results suggest that induction of TAK by cytokines secreted in the microenvironment of lymphoid tissue may be involved in the reactivation of HIV-1 in CD4؉ T lymphocytes harboring a latent provirus.

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Viral Transactivators Specifically Target Distinct Cellular Protein Kinases That Phosphorylate the RNA Polymerase II C-Terminal Domain

Cited by 64 publications

References 79 publications

Promoter Activity of Tat at Steps Subsequent to TATA-Binding Protein Recruitment

Promoter Activity of Tat at Steps Subsequent to TATA-Binding Protein Recruitment

Transient Induction of Cyclin T1 during Human Macrophage Differentiation Regulates Human Immunodeficiency Virus Type 1 Tat Transactivation Function

Induction of TAK (Cyclin T1/P-TEFb) in Purified Resting CD4 ⁺ T Lymphocytes by Combination of Cytokines

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Viral Transactivators Specifically Target Distinct Cellular Protein Kinases That Phosphorylate the RNA Polymerase II C-Terminal Domain

Cited by 64 publications

References 79 publications

Promoter Activity of Tat at Steps Subsequent to TATA-Binding Protein Recruitment

Promoter Activity of Tat at Steps Subsequent to TATA-Binding Protein Recruitment

Transient Induction of Cyclin T1 during Human Macrophage Differentiation Regulates Human Immunodeficiency Virus Type 1 Tat Transactivation Function

Induction of TAK (Cyclin T1/P-TEFb) in Purified Resting CD4 + T Lymphocytes by Combination of Cytokines

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Induction of TAK (Cyclin T1/P-TEFb) in Purified Resting CD4 ⁺ T Lymphocytes by Combination of Cytokines