A New In Vitro Model for HIV Latency

J. David Gladstone Institutes, San Francisco CA

Investigators

Abstract

[unreadable] DESCRIPTION (provided by applicant): The presence of latent reservoirs has prevented the eradication of Human Immunodeficiency Virus (HIV) from infected patients successfully treated with antiretroviral therapy. HIV latency is poorly understood, partly because of the lack of an in vitro model. We reported that the chromatin environment at the site of integration of HIV into the genome plays an important role in its transcriptional activity. We predicted that integration into unique regions of chromatin could suppress HIV provirus expression. We have used an HIV retroviral vector expressing green fluorescent protein (GFP) under the control of the HIV promoter to highly enrich for latently infected cells after infection. We observed that HIV latency occurred reproducibly with low frequency (1.5%), during infection. Clonal cell lines derived from this latent population showed no basal expression, but could be induced after treatment with biological and pharmacological agents. Direct sequencing of integration sites demonstrated that HIV frequently integrated in alphoid repeat elements in latent clones in contrast to acute infection where alphoid repeats are disfavored as a site for integration. Here, we propose to establish and characterize cell lines latently infected with a full-length HIV genome containing the reporter GFP (either in lieu of Nef or as a fusion with the env protein) in Jurkat cells using FACS. We will explore the role of different pathways for HIV latency activation. Our analysis indicates that the use of phorbol esters as an activation signal in our preliminary experiments led to the selection of a subset of latently infected cells. To examine the full spectrum of latently infected cells, we propose to use other activating agents, including histone deacetylase inhibitors, DNA methylation inhibitors and a variety of cytokines and T cell activating agents. Latent cell lines obtained after activation with each of these agents will be analyzed and compared. Finally, to increase the relevance of our observations to HIV latency in vivo, we propose to apply the same experimental strategy for the isolation of latently infected cells after infection of peripheral blood mononuclear cells and primary human CD4 T cell clones. These studies should further establish the validity of our model for the study of HIV latency in patients. [unreadable] [unreadable]