The presence of latent reservoirs has prevented the eradication of Human Immunodeficiency Virus (HIV) from infected patients successfully treated with antiretroviral therapy. We have recently reported that DNA methylation of the HIV promoter plays an important role in the establishment and maintenance of HIV latency. We also found that the inhibitor of DNA methylation, 5-aza-2'deoxycytidine (aza-CdR), synergizes with NF-B activators to reactivate latent HIV. We propose to further test the hypothesis that methylation of the HIV promoter plays a critical role in HIV latency and that inhibition of DNA methylation represents a novel therapeutic approach to induce the reactivation of latent HIV. Since cocaine has emerged as a molecule that can modify epigenetic regulatory processes we also plan to examine its effect in HIV latency. Our specific plans are to:
Aim 1. To understand the mechanism of HIV promoter silencing via DNA methylation. We will use chromatin immunoprecipitation and shRNA-mediated interference to explore the roles in HIV latency of the multiprotein complexes that are recruited to methylated DNA: NuRD/MBD2, SWI/SNF/Sin3/MeCP2 and the SUV39H1/HP1/MBD1 complexes.
Aim 2. To identify small molecules and cellular factors that reactivate latent HIV in synergy with methylation inhibitors. We will screen for small molecules and cDNAs that synergize with aza-CdR to reactivate latent HIV. Agents that synergize with Aza-CdR to reactivate HIV in the J-Lat model of HIV latency will be tested in a primary cell models of latency and in latently infected cells from HIV-positive donors.
Aim 3. To study HIV DNA methylation in primary lymphocytes. To test the hypothesis that HIV integration in or near hypermethylated CpG islands leads to latency, we propose to compare the state of DNA methylation in resting and activated lymphocytes at the sites of latent HIV integration in comparison to productive sites both in the J-Lat model and in latent HIV in primary human lymphoid cells.
Aim 4. To study the size of the latent pool in HIV-infected patients, its reactivation by methylation inhibitors and the effect of cocaine use on these variables. We propose to test for synergistic reactivation of latent HIV in latently infected cells using a primary lymphoid system for HIV latency in vitro (in collaboration with Dr, Vicente Planelles) and in latently-infected cells isolated from HIV-infected patients both cocaine users and non users.

Public Health Relevance

NARRATIVE The identification of DNA methylation and other epigenetic modifications of the latent HIV genome highlights a novel mechanism for the suppression of HIV transcription. The demonstration that the DNA methylation inhibitor, aza-DcR, synergizes with prostratin or TNF- to reactivate latent HIV suggest that epigenetic modification could be used as a drug target in our effort to reactivate latent HIV. Recent experiments also indicate that cocaine use can induce prolonged epigenetic modifications in the brain and possibly in the immune system. Here we propose to study the role of epigenetic modifications in HIV latency and the possible role of drugs that modify epigenetic silencing as a novel therapeutic approach to induce the reactivation of latent HIV.

Agency
National Institute of Health (NIH)
Institute
National Institute on Drug Abuse (NIDA)
Type
Research Project (R01)
Project #
5R01DA030216-03
Application #
8214671
Study Section
Special Emphasis Panel (ZDA1-SXC-E (14))
Program Officer
Satterlee, John S
Project Start
2010-07-01
Project End
2015-02-28
Budget Start
2012-03-01
Budget End
2013-02-28
Support Year
3
Fiscal Year
2012
Total Cost
$441,247
Indirect Cost
$206,179
Name
J. David Gladstone Institutes
Department
Type
DUNS #
099992430
City
San Francisco
State
CA
Country
United States
Zip Code
94158
Manson McManamy, Mary E; Hakre, Shweta; Verdin, Eric M et al. (2014) Therapy for latent HIV-1 infection: the role of histone deacetylase inhibitors. Antivir Chem Chemother 23:145-9
Colin, Laurence; Verdin, Eric; Van Lint, Carine (2014) HIV-1 chromatin, transcription, and the regulatory protein Tat. Methods Mol Biol 1087:85-101
Calvanese, Vincenzo; Chavez, Leonard; Laurent, Timothy et al. (2013) Dual-color HIV reporters trace a population of latently infected cells and enable their purification. Virology 446:283-92
Hakre, Shweta; Chavez, Leonard; Shirakawa, Kotaro et al. (2012) HIV latency: experimental systems and molecular models. FEMS Microbiol Rev 36:706-16
Rafati, Haleh; Parra, Maribel; Hakre, Shweta et al. (2011) Repressive LTR nucleosome positioning by the BAF complex is required for HIV latency. PLoS Biol 9:e1001206