Understanding HIV reservoir formation by profiling transcriptomic and epigenetic changes in CD4 T cells following ART initiation

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During HIV infection, a reservoir of latently infected cells forms that persists during therapy, and this reservoir represents the major barrier to an HIV cure. We and others have shown that HIV latency is an epigenetic phenomenon, characterized by distinct changes to chromatin structure and histone modifications that repress HIV gene expression, that can be transmitted through cell division. This heritable property of latency allows the latent phenotype to survive ongoing clonal expansion of infected cells in hosts and sustain the overall reservoir. Furthermore, viral silencing and epigenetic programming is linked to global changes that occur within CD4 T cells as they transition from an activated to a resting state. Fully defining the process by which the reservoir is formed and by which latency is programmed will be essential to guiding novel approaches to prevent reservoir seeding or maintenance. To date, studying the process of reservoir formation during clinical HIV infection has been considered difficult, if not impossible, due to prior observations suggesting that the reservoir forms very early during acute infection. Newer information from several groups, however, has now revealed that the majority of the reservoir is seeded by viruses that are actively replicating at the time of ART initiation, suggesting that ART initiation triggers seeding of the reservoir. This observation raises the exciting possibility that we could potentially study the process of reservoir seeding by longitudinal observation of CD4 T cells from PWH in the immediately post ART period and, by doing so, reveal potential ways to block the reservoir from forming at the time of therapy initiation. In this proposal we aim to comprehensively define epigenetic and chromatin-based changes that occur in CD4 T cells in the post ART period using samples derived from the A5248 cohort. CD4 T cells from these samples will be profiled using a series of cutting-edge assays that have been established and validated in our laboratory. These assays include combined single-cell multi-omic (RNAseq/ATACseq) analysis, and high-resolution genome-wide mapping of multiple key histone modifications through Cleavage Under Targets and Release using Nuclease (CUT&RUN). From this approach we will achieve an integrated understanding of how heritable patterns of chromatin-based changes in CD4 T cells are triggered by ART initiation and generate novel and critical insights into the intracellular conditions that coincide with reservoir seeding in vivo. By achieving this goal, we will reveal mechanisms that promote reservoir seeding and suggest novel approaches to block reservoir formation at the time of ART initiation.