Although highly active retroviral therapy (HAART) dramatically reduces viral load and restores CD4 T-cell counts in HIV-infected individuals, its success is hampered by the fact that HIV can establish a latent proviral reservoir within long-lived resting memory CD4 lymphocytes. The molecular underpinnings of HIV latency involve multiple host factors and pathways actively repressing HIV transcription and maintaining the proviral genome in a transcriptionally inert state. Many host factors have been implicated based on genome-wide screens in cell-line based models of latency. But the exact role of these candidate genes in HIV latency occurring within resting primary CD4 T cells, remain unknown. The evaluation of these genes in resting primary CD4 T cells is hampered by the fact that shRNAs cannot be introduced into these cells using lentiviral vectors. In this collaboration among three CARE investigators (Warner C. Greene, Jonathan Karn, Vincente Planelles), we now propose to utilize a novel technology based on HIV-2 Vpx to render resting CD4 T cells permissive to lentivirus infection. This technology will permit efficient knockdown and evaluation of the candidate regulatory genes in resting primary CD4 T cells. This approach first involves pretreating cells with virion like particles (VLP) encapsulating Vpx to remove anti-viral reverse transcription block mediated by SAMHD1 followed by shRNA lentiviral transduction. A number of potential positive and negative regulators of HIV latency identified in CARE-sponsored studies will be systematically tested including the genome organizer and transcription repressor CTCF, select receptor subunits in the immunosuppressive PDCD1 and pro-inflammatory interleukin IL22RA pathways, and a putative Tat cofactor termed TRIM32. The evaluation of these cellular genes in primary CD4 T cell models of HIV latency formed in memory CD4 T cells, Th17 CD4 T cells and Treg CD4 T cells promises to provide key insights into the role of these genes in HIV latency. These studies could lay the foundation for finding small molecules that when combined will promote effective purging of the latent reservoir in patients while not producing a general state of cellular activation.

Public Health Relevance

Although highly active retroviral therapy (HAART) can dramatically extend the life expectancies of HIVinfected individuals, these drugs cannot cure patients because of the presence of a latent reservoir of druginsensitive virus. We propose testing the involvement of various gene candidates in primary CD4 T cells using a novel Vpx-based technology to introduce lentiviral encoded shRNAs into these cells. These studies promise to provide key insights into the molecular underpinnings of HIV latency that will inform the search for small molecule cocktails capable of effectively purging latent HIV proviruses.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Program--Cooperative Agreements (U19)
Project #
5U19AI096113-03
Application #
8725378
Study Section
Special Emphasis Panel (ZAI1-JBS-A (M1))
Project Start
Project End
Budget Start
2013-07-01
Budget End
2014-06-30
Support Year
3
Fiscal Year
2013
Total Cost
$23
Indirect Cost
$7,971
Name
University of North Carolina Chapel Hill
Department
Type
DUNS #
608195277
City
Chapel Hill
State
NC
Country
United States
Zip Code
27599
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