Transcription is a key regulatory step in the HIV-1 life cycle that remains largely unexplored as therapeutic target. We have identified factor acetylation as a critical step in the regulation of HIV-1 transcription. Acetylation is the reversible transfer of an acetyl group (14 Da) to the -amino group of lysines. It is mediated by acetyl transferases (HAT) and reversed by the activity of deacetylases (HDACs). Inhibitors of HDACs are currently being explored as potential therapeutics to flush the latent reservoir in HIV-infected patients. Three factors stand out in the regulation of HIV transcription: Tat, NF-B and cyclin T1/P-TEFb. Notably, the functions of Tat and NF-B are importantly regulated by reversible acetylation of these proteins. We now report that cyclin T1 in P-TEFb is also acetylated at four defined acetyl acceptor sites. Cyclin T1 acetylation activates P-TEFb by promoting its dissociation from ribonucleoprotein complexes containing 7SK RNA and Hexim1 and importantly regulates the activity of the HIV LTR. In addition, we have identified a novel posttranslational modification in Tat, monomethylation of lysine 51 by Set9 that regulates Tat acetylation and activates Tat transcriptional activity. We propose to examine the impact of these two new findings on the HIV transcriptional process. We hypothesize that reversible cyclin T1 acetylation is an important regulatory mechanism that controls the activity of the HIV promoter during active and latent HIV infection. In addition, we speculate that Tat monomethylation is a critical step to promote Tat acetylation thereby activating Tat transcriptional activity.
Our specific aims are designed to test these hypotheses and to define the mechanisms associated with cyclin T1 acetylation and Tat methylation/acetylation in actively or latently HIV-infected T cells. Our focus lies on 1. Assessing the role of cyclinT1 acetylation in HIV replication. We propose to examine whether acetylation- deficient mutants of cyclinT1 can support HIV replication in CD4+ T cells and whether cyclin T1 acetylation regulates the establishment and maintenance of post-integration latency. Our preliminary results show that cyclin T1 acetylation is an important regulatory mechanism for the activity of the HIV LTR while it may not affect Tat transactivation. Since the basal activity of the HIV LTR is critical at the beginning of the infectious process or during reactivation from latency when the Tat protein is not yet produced, we speculate that cyclin T1 acetylation might be a critical determinant of HIV transcription at these times. 2. Characterizing how cyclin T1 acetylation regulates P-TEFb function at the HIV promoter. We propose to test the hypothesis that acetylated lysines in cyclin T1 interact with bromodomains in Brd4. Brd4 is a factor that can recruit P-TEFb to the HIV promoter in the absence of Tat and contains two bromodomains, which are bona fide binding domains for acetyl lysines. We speculate that cyclin T1 acetylation activates HIV LTR activity by recruiting Brd4 into the active P-TEF complex. Similarly, we will examine how acetylated cyclin T1 interacts with Tat and TAR RNA. 3. Studying how lysine methylation cooperates with Tat acetylation to regulate HIV transcription. We propose to study the potential crosstalk between Tat monomethylation by Set9 and Tat acetylation by p300. Our preliminary data show that Tat monomethylation is an early step in the Tat transactivation cycle and precedes Tat acetylation. We will test whether monomethylated Tat or Set9 recruits p300 to Tat. We will also examine how differently modified Tat species accumulate in HIV-1-infected T cells using modification-specific Tat antibodies generated in my laboratory. We anticipate that these studies will increase our molecular understanding of the biology of HIV transcription and provide novel insight into the therapeutic potential of HDAC inhibitors in HIV infection.

Public Health Relevance

We seek to identify and characterize novel regulatory mechanisms controlling HIV-1 transcription that might be exploited as new therapeutic targets. Our proposed studies characterize the role of reversible factor acetylation (cyclin T1 and Tat) in the control of active and latent HIV infection. These studies are directly relevant to HIV/AIDS and may contribute to the development of novel antiviral drugs that will address public need.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI083139-05
Application #
8490286
Study Section
AIDS Molecular and Cellular Biology Study Section (AMCB)
Program Officer
Lawrence, Diane M
Project Start
2009-07-15
Project End
2014-06-30
Budget Start
2013-07-01
Budget End
2014-06-30
Support Year
5
Fiscal Year
2013
Total Cost
$351,935
Indirect Cost
$167,676
Name
J. David Gladstone Institutes
Department
Type
DUNS #
099992430
City
San Francisco
State
CA
Country
United States
Zip Code
94158
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Boehm, Daniela; Calvanese, Vincenzo; Dar, Roy D et al. (2013) BET bromodomain-targeting compounds reactivate HIV from latency via a Tat-independent mechanism. Cell Cycle 12:452-62
Schröder, Sebastian; Herker, Eva; Itzen, Friederike et al. (2013) Acetylation of RNA polymerase II regulates growth-factor-induced gene transcription in mammalian cells. Mol Cell 52:314-24
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Ott, Melanie; Geyer, Matthias; Zhou, Qiang (2011) The control of HIV transcription: keeping RNA polymerase II on track. Cell Host Microbe 10:426-35
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Pagans, Sara; Kauder, Steven E; Kaehlcke, Katrin et al. (2010) The Cellular lysine methyltransferase Set7/9-KMT7 binds HIV-1 TAR RNA, monomethylates the viral transactivator Tat, and enhances HIV transcription. Cell Host Microbe 7:234-44