Project Summary/Abstract Despite highly effective anti-retroviral therapy (ART), the latent HIV-1 reservoir in resting CD4+ T cells is the major barrier to a functional cure of HIV-1 infection. Our overall objectives of this R61/R33 bi-phasic project are: to understand the mechanisms of post-transcriptional regulation of HIV-1 RNA in HIV-1-infected individuals who are on ART (Exploratory R61 phase), and to develop a novel therapeutic strategy to alter RNA post- transcriptional modifications as a potential therapeutic platform for inhibiting HIV-1 replication (Developmental R33 phase). Our multidisciplinary group is uniquely poised to address several key questions highlighted in this funding opportunity. My lab was among three groups that independently discovered that N6-methyladenosine (m6A) modifications of HIV-1 RNA modulate viral replication in CD4+ T cells in vitro. Using CD4+ T cell lines and primary CD4+ T cells from healthy donors, we investigated the mechanisms by which m6A modifications modulate HIV-1 infection. We also found that m6A modifications of HIV-1 RNA inhibit innate antiviral immune responses in primary macrophages from healthy donors. Our in vitro studies suggested that m6A modifications of HIV-1 RNA play a critical role in viral replication and innate immune responses to viral infection. However, the role of m6A modifications of HIV-1 RNA in regulating viral replication in HIV-1-infected individuals on ART remains unknown. We aim to fill this important knowledge gap and to translate the findings into potential anti- HIV-1 therapeutics. We hypothesize that m6A modifications of HIV-1 RNA help establish and maintain viral latency in CD4+ T cells and avoid innate antiviral immune responses in HIV-1 infected individuals on ART. To test this hypothesis and to facilitate the development of a novel strategy for HIV-1 cure, we designed three specific aims in two phases: (1) R61 phase (years 1-3): Aim 1. To determine m6A profile of HIV-1 RNA in subsets of CD4+ T cells from ART- treated patients; Aim 2. To identify cellular targets in the m6A pathway important for HIV-1 reactivation in primary CD4+ T cells; and (2) R33 phase (years 4-5): Aim 3. To examine anti-HIV-1 effects of small molecules inhibiting m6A modifications in primary CD4+ T cells. Overall Impact: These studies will reveal how m6A modifications of HIV-1 RNA regulate viral latency in ART- treated patients. The studies in the R61 phase will define new mechanisms of HIV-1 persistence and identify potential therapeutic targets. The R33 phase study will develop an m6A-specific strategy to inhibit HIV-1 replication in primary CD4+ T cells.