The general transcription factor P-TEFb, consisting of Cdk9 and cyclin T, strongly stimulates RNA polymerase II elongation. It is also a host cell cofactor for Tat activation of HIV-1 transcription. Accumulating evidence suggests that Tat and the TAR RNA, located at the 5'end of all viral transcripts, not only recruit P-TEFb to the HIV-1 LTR but also cause the activation of Cdk9 kinase. For general transcription of cellular genes, data obtained during the current funding period indicate that P-TEFb is recruited to chromatin templates by the bromodomain protein Brd4. In addition, a major reservoir of nuclear P-TEFb is sequestered in the inactive 7SK snRNP. Further analyses indicate that in response to Ca2+-signaling, P-TEFb is released from 7SK snRNP upon the dephosphorylation of the conserved Cdk9 T-loop by PP1? and PP2B. The dephosphorylated P- TEFb is preferentially bound by Brd4, which recruits it to the transcription pre-initiation complex. As the phosphorylation of Cdk9 T-loop is essential for P-TEFb activity, the T-loop is expected to undergo rephosphorylation by an as yet unidentified Cdk activating kinase (CAK) at a later stage in order to restore full activity to P-TEFb. Given that P-TEFb is essential for productive HIV-1 infection, the objective of this proposal is to examine how the various modes of P-TEFb regulation exerted by its associated factors, a putative Cdk9-specific CAK and the HIV-1 Tat/TAR will impact HIV-1 transcription and replication. Proposed are experiments to investigate whether the expression and activity of various P-TEFb-associated factors can be manipulated to control HIV-1 replication and latency. A combination of targeted investigations and comprehensive, unbiased screens will be employed to identify the Cdk9-specific CAK and elucidate the mechanism and functional significance of its phosphorylation of P-TEFb. To determine the mechanism of Tat/TAR activation of P-TEFb, the impact of Tat/TAR on phosphorylation status of the Cdk9 T-loop at different stages of HIV-1 transcription, the possible existence of novel components within the Tat-TAR-P-TEFb complex, and the ability of TFIIH and TAF7 to inhibit P-TEFb activation will be examined. These experiments will offer an exciting opportunity to identify novel factors that contribute to the activation of P-TEFb and HIV-1 transcription and provide fresh insights into how P-TEFb stimulates transcription of both HIV-1 and cellular genes. A better understanding of the mechanism by which P-TEFb controls HIV-1 replication and latency and the versatility of Tat/TAR in modulating this process will be informative toward the identification of new targets for anti-HIV therapy.

Public Health Relevance

The Cdk9-cyclin T1 heterodimer of P-TEFb is a host cell cofactor for Tat activation of HIV-1 transcription. The proposed experiments will allow us to investigate how the P-TEFb-dependent HIV-1 transcription and replication can be controlled by: (1) the various P-TEFb-associated cellular factors;(2) a putative Cdk9 activating kinase;and (3) the HIV-1 Tat protein and TAR RNA that can do more than simply recruiting P-TEFb to the viral LTR. These experiments will lead to the identification of novel factors that contribute to the activation of P-TEFb and HIV-1 transcription and provide fresh insights into how P-TEFb controls transcriptional elongation of both HIV-1 and cellular genes. A better understanding of the mechanism by which P-TEFb regulates HIV-1 replication and latency and the versatility of Tat/TAR in modulating this process may lead to the identification of new targets for anti-HIV therapy.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI041757-15
Application #
8423360
Study Section
Special Emphasis Panel (ZRG1-AARR-H (02))
Program Officer
Lawrence, Diane M
Project Start
1997-08-01
Project End
2015-02-28
Budget Start
2013-03-01
Budget End
2015-02-28
Support Year
15
Fiscal Year
2013
Total Cost
$382,694
Indirect Cost
$133,382
Name
University of California Berkeley
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
124726725
City
Berkeley
State
CA
Country
United States
Zip Code
94704
Zhao, Yang; Karijolich, John; Glaunsinger, Britt et al. (2016) Pseudouridylation of 7SK snRNA promotes 7SK snRNP formation to suppress HIV-1 transcription and escape from latency. EMBO Rep 17:1441-1451
Li, Zichong; Lu, Huasong; Zhou, Qiang (2016) A Minor Subset of Super Elongation Complexes Plays a Predominant Role in Reversing HIV-1 Latency. Mol Cell Biol 36:1194-205
Schulze-Gahmen, Ursula; Echeverria, Ignacia; Stjepanovic, Goran et al. (2016) Insights into HIV-1 proviral transcription from integrative structure and dynamics of the Tat:AFF4:P-TEFb:TAR complex. Elife 5:
Lu, Huasong; Xue, Yuhua; Xue, Yuahua et al. (2015) Compensatory induction of MYC expression by sustained CDK9 inhibition via a BRD4-dependent mechanism. Elife 4:e06535
Wang, Cong; Yang, Shuiyuan; Lu, Huasong et al. (2015) A Natural Product from Polygonum cuspidatum Sieb. Et Zucc. Promotes Tat-Dependent HIV Latency Reversal through Triggering P-TEFb's Release from 7SK snRNP. PLoS One 10:e0142739
Lu, Huasong; Li, Zichong; Zhang, Wei et al. (2015) Gene target specificity of the Super Elongation Complex (SEC) family: how HIV-1 Tat employs selected SEC members to activate viral transcription. Nucleic Acids Res 43:5868-79
Ji, Xiaodan; Lu, Huasong; Zhou, Qiang et al. (2014) LARP7 suppresses P-TEFb activity to inhibit breast cancer progression and metastasis. Elife 3:e02907
Schulze-Gahmen, Ursula; Lu, Huasong; Zhou, Qiang et al. (2014) AFF4 binding to Tat-P-TEFb indirectly stimulates TAR recognition of super elongation complexes at the HIV promoter. Elife 3:e02375
Karijolich, John; Zhao, Yang; Peterson, Bret et al. (2014) Kaposi's sarcoma-associated herpesvirus ORF45 mediates transcriptional activation of the HIV-1 long terminal repeat via RSK2. J Virol 88:7024-35
Lu, Huasong; Li, Zichong; Xue, Yuhua et al. (2014) AFF1 is a ubiquitous P-TEFb partner to enable Tat extraction of P-TEFb from 7SK snRNP and formation of SECs for HIV transactivation. Proc Natl Acad Sci U S A 111:E15-24

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