Novel Model for HIV Latency in Primary Memory T Cells

J. David Gladstone Institutes, San Francisco CA

Investigators

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Abstract

DESCRIPTION (provided by applicant): 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 (naïve 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.