The long-term persistence of HIV in a latent state in memory T cells in patients treated with HAART prevents the eradication of HIV and forces patients to remain on HAART for their whole life. While the transcriptional regulation of HIV has been extensively studied in transformed cell lines, our understanding of how latent HIV infection occurs in primary memory CD4 T lymphocytes is rudimentary. The purpose of this application is to develop new single cell technology to examine the transcriptional status of HIV in single primary lymphoid cells over time after an infection in vitro. These studies will bridge the two research fields of human immunology and HIV molecular virology. Understanding HIV latency in primary lymphocytes may lead to the identification of cellular proteins that control the entry of HIV in latency, the maintenance of latency or its reactivation. Such cellular targets could represent new avenues for the treatment of HIV/AIDS among drug abusers and possibly lead to the eradication of infection. I propose to use a novel live cell, time-lapse fluorescence microscopy combined with cell trapping via microfluidic chips and the use of HIV expressing recombinant fluorescent protein (destabilized GFP) to study the kinetics of HIV transcription at the single cell level. This novel technique will allow the fate of HIV expression to be followed in live individual cells over time. Human lymphoid cells will be activated in vitro, infected with an HIV expressing a fluorescent protein, activation signals will be removed and HIV transcription will be followed over time. We anticipate that HIV transcription will be restricted in a subset of cells returning to quiescence. The time separating removal of activation signal to infection is likely to be critical in allowing infection to proceed until HIV integration while restricting HIV transcriptional activation. The nature of the activation signal could also prove critical. Experiment will eventually focus on highly enriched human lymphoid cells (nave vs. memory) and on the use of R5 HIV envelope to closely mimic the situation observed in HIVinfected patients and to develop an in vitro model that closely mimics the situation in patients. When an in vitro model for HIV latency has been established, I will study the role of the FOXO3A transcription factor. Our hypothesis is that FOXO3A represents a master regulator of HIV latency in memory T cells. FOXO3A is critical for memory T cell maintenance and survival, strongly represses NF-?B and is therefore likely to repress HIV expression and to contribute to latency establishment or maintenance. We will study the PI3K/AKT cellular activation pathway for FOXO3A and the effect of SIRT1 and SIRT3 on FOXO3A function and HIV latency. Finally, we will study the effect of recently identified polymorphisms in the FOXO3A gene that affect its function on the size of the latent pool in patients infected with HIV and on the establishment of latency in vitro.

Public Health Relevance

HIV persists in patients treated with current antiretroviral drugs, including in drug abusers infected with HIV. The virus is able to persist in a state of dormancy, called latency, in specific memory lymphocytes. Because of the long-term persistence of these latently infected lymphocytes, HIV can reactivate, come out of latency, and reinitiate an infection when anti-HIV drugs are interrupted. Latently infected cells therefore are forcing HIV-infected patients, including drug abusers, to be maintained on anti-HIV drugs for their whole life. The goal of this proposal is to study the mechanism of HIV latency in memory lymphocyte cells isolated from humans. We propose to develop a novel approach that will allow us to study rare memory lymphocyte populations directly isolated from normal individuals and to study the fate of HIV infection in single cells. By studying the mechanism used by HIV to establish latency, we hope to be able to devise novel strategies aimed at eliminating latently infected cells. If successful, such strategies might lead to the elimination of latent HIV infection or to a restriction of the latent pool to a size that can be controlled by the immune system in the absence of drug treatment.

Agency
National Institute of Health (NIH)
Type
NIH Director’s Pioneer Award (NDPA) (DP1)
Project #
5DP1DA031126-05
Application #
8706838
Study Section
Special Emphasis Panel (ZDA1)
Program Officer
Satterlee, John S
Project Start
Project End
Budget Start
Budget End
Support Year
5
Fiscal Year
2014
Total Cost
Indirect Cost
Name
J. David Gladstone Institutes
Department
Type
DUNS #
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
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
Shirakawa, Kotaro; Chavez, Leonard; Hakre, Shweta et al. (2013) Reactivation of latent HIV by histone deacetylase inhibitors. Trends Microbiol 21:277-85
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