Control of HIV-1 latency and reservoir persistence in primary cells

About This Grant

ABSTRACT The latent HIV-1 reservoir that persists despite the effect antiretroviral therapy (ART) comprises infected cells with intact integrated proviruses that are transcriptionally near silent. The existence and maintenance of this reservoir in essentially all ART-treated individuals is a major barrier to curative interventions in HIV-1 infection, and necessitates the lifelong administration of ART. In the preliminary studies that underpin this proposal we developed a xenograft based model system in NSG mice that reliably generates populations of human primary memory CD4+ T-cells that contain latent proviruses carrying reporter genes. Our methods overcome some of the key limitations of existing approaches to study HIV-1 latency. We determined hundreds of HIV-1 integration sites in active and latent cell populations and uncover relationships between provirus genomic location, infected cell clonal expansion, and the establishment of HIV-1 latency after engraftment. Uniquely, we have recovered and cultivated in large numbers primary memory CD4+ T cell single-cell clones each harbouring a unique HIV-1 integration site in which latency was (or was not) established in vivo. Comparison of these clones with the small number of CD4+ T cell clones that harbor intact HIV-1 proviruses that have been cultivated from ART-treated individuals reveals features in common that suggest that the model system we have developed more accurately recapitulates features of HIV- 1 latency than those deployed heretofore. In Aim 1, we will build on our preliminary studies by deriving a diverse collection of primary CD4+ T-cell single cell clones that harbor latent or active HIV-1 proviruses. Therein, we will investigate the stability, dynamics an mechanisms of transitions between active and latent states at defined integration sites and, whether the new DNA synthesis that accompanies cell proliferation enables these transitions. We will determine relationships between the epigenetic profile of loci containing proviruses and whether proviruses are transcriptionally active or latent. We will further assess how the physiologic status of T-cell clones conspires with epigenetic profile of loci containing proviruses to impact the establishment of latency and transitions between active and latent states. In Aim 2 we will manipulate epigenetic modifications at or surrounding the site of provirus integration in the genome of primary memory CD4+ T-cell clones and determine their effects on HIV-1 latency. We will build a custom, targeted sgRNA library, focused on epigenetic modifiers and gene expression regulators and identify genes that affect HIV-1 latency in various genomic contexts. We will further conduct genome wide CRISPR screens in primary memory CD4+ T-cell clones to identify genes that regulate latency therein. Ultimately, we will aim to provide mechanistic insights into HIV-1 latency and evidence about how manipulation of latency might be accomplished, such that HIV-1 infection might be cured.