In the era of HAART, HIV-1 persists as integrated, latent proviruses in reservoir cells of infected individuals, as a major obstacle for cure. Tonsillr CD4+ T cells are a major HIV-1 oral reservoir. Understanding host factors regulating HIV-1 latency in these cells and identify cell-specific latency promoting genes (LPGs) will be useful to develop new latency reversing agents (LRAs) for elimination of HIV-1 oral reservoirs. Our earlier RNAi screens successfully identified LPGs that restrict HIV-1 transcription and facilitate its latency, including BRD4 and SUPT16H. In this proposal, we propose to further characterize these two LPGs in HIV-1 latently infected tonsillar CD4+ T cells (AIM 1 and 2). Their contribution to HIV-1 latency, particularly in oral reservoir cells, is largely unknown and worthy f further characterization. Using our established pipeline, we will also be interested to identify ne LPGs (AIM 3). We believe our proposed study is comprehensive and allows a systematic view how LPGs contribute to HIV-1 latency.
AIM 1 : We are one of the leading groups to identify BRD4 as a LPG, and we will further characterize BRD4 in HIV-1 latently infected tonsillar CD4+ T cells. We will first create a HIV-1 latency cell model using primary tonsillar CD4+ T cells. BRD4 will be depleted to determine how it affects HIV-1 latency/reactivation. Our preliminary results indicated that P-TEFb mediates BRD4's effect, so we will further determine the impact of BRD4 on P-TEFb activity in tonsillar CD4+ T cells. To test the feasibility to target BRD4 pharmacologically for purging latent HIV-1, we will evaluate a set of new promising BET inhibitors to revert HIV-1 latency.
AIM 2 : SUPT16H is our newly identified LPG, so we will study it in HIV-1 oral reservoir cells as well. We will determine whether depletion of SUPT16H in tonsillar CD4+ T cells may affect HIV-1 latency/reactivation. We will also use ChIP-seq approach to determine the genome-wide association of SUPT16H, which may provide clue how SUPT16H facilitates HIV-1 latency. Since SUPT16H is a nucleosome chaperone protein, we will determine whether such activity of SUPT16H may alter for HIV-1 transcription. Our preliminary results suggest that depletion of SUPT16H further enhances a HDAC inhibitor to revert HIV-1 latency, so we will further investigate this result by broadly testing a set of HDACIs.
AIM 3 : Our earlier RNAi screens indicate that unbiased functional genomic tools are capable of systemically identifying previously unappreciated host proteins modulating HIV-1 replication. These screens also suggest that there are other potential LPGs pending for discovery. In this aim, we will use the newly emerged CRISPR/Cas9 functional genomic approach to systematically identify novel LPGs. We will perform comprehensive bioinformatic and biostatistic analyses to filter primary screening results and prioritize a set of the most promising LPGs (10~15 genes) for further validations in tonsillar CD4+ T cells, including confirmation of LPG depletion and re-evaluation of HIV latency reverting phenotype. Our ultimate goal is to translate this understanding to pharmacologically target a handful of LPGs for eradicating HIV-1 oral reservoirs.

Public Health Relevance

Although the antiretroviral therapy is efficient to treat HIV/AIDS and slow the course of the disease, the presence of HIV latent reservoirs, including the one at oral site, remains a major obstacle for HIV cure. We propose to investigate two newly identified latency promoting genes (LPGs), BRD4 and SUPT16H, as well as identify new ones, in HIV latently infected tonsillar CD4 T cells. We aim to acquire a systematic view how LPGs contribute to HIV latency at oral site, and test the feasibility to target LPGs pharmacologically fr an efficient HIV reactivation as the initial step for purging HIV latent reservoirs.

Agency
National Institute of Health (NIH)
Institute
National Institute of Dental & Craniofacial Research (NIDCR)
Type
Research Project (R01)
Project #
5R01DE025447-02
Application #
9110960
Study Section
Special Emphasis Panel (ZDE1)
Program Officer
Gannot, Gallya
Project Start
2015-08-01
Project End
2020-05-31
Budget Start
2016-06-01
Budget End
2017-05-31
Support Year
2
Fiscal Year
2016
Total Cost
Indirect Cost
Name
University of Rochester
Department
Microbiology/Immun/Virology
Type
School of Medicine & Dentistry
DUNS #
041294109
City
Rochester
State
NY
Country
United States
Zip Code
14627
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