PROJECT SUMMARY People living with HIV (PLWH) on anti-retroviral therapy (ART) still suffer from HIV-associated non-AIDS complication (HANA) such as neurocognitive disorders. Although chronic inflammation has been thought to be the driver of HANA, what causes chronic immune activation in PLWH on ART remains unclear. Despite successful ART, cell-associated HIV RNA has been detected in peripheral and tissues in PLWH on ART. We have demonstrated that cytoplasmic expression of HIV intron-containing RNAs, even those from defective HIV proviruses, induce pro-inflammatory responses in primary human macrophages including brain-resident microglia. Since the current ART regimens cannot prevent expression of HIV RNA, it is plausible that persistent HIV RNA expression in infected cells contributes to chronic inflammation in PLWH on ART. In fact, we have found in our cohort that there is a correlation between the expression level of cell-associated HIV RNA and the degree of systemic inflammation, supporting our hypothesis that cell-associated HIV RNA drives innate immune activation. However, the molecular mechanism that makes HIV RNA immunostimulatory remains unclear. Increasing lines of evidence have suggested that post-transcriptional base modification of viral RNA plays a role in HIV-1 replication. Interestingly, our preliminary data demonstrate that (1) there is a link between HIV RNA stability and its immunostimulatory potential, (2) HIV RNA has a different RNA modification landscape compared to a non-immunostimulatory HIV mutant RNA, and (3) RNA modification reading proteins (RNA “readers”) play an important role in innate immune activation in HIV-infected monocyte-derived macrophages. Therefore, we hypothesize that the interaction of unique modified bases on HIV RNA and “proinflammatory” RNA readers makes HIV RNA stable and immunostimulatory, which contributes to chronic inflammation in PLWH on ART. To test this hypothesis, in this proposal, we will determine the role of HIV RNA modifications in chronic inflammation in PLWH on ART. Specifically, we will identify the molecular mechanisms that link “proinflammatory” RNA readers to innate immune activation in HIV-infected macrophages. In addition, we will identify HIV RNA modifications which are associated with innate immune activation using nanopore direct RNA sequencing. Moreover, we will identify the unique RNA modification landscape associated with inflammation in PLWH on ART in our cohort at Boston Medical Center. Then, we will develop strategies to alter proinflammatory RNA modification landscape to reduce HIV RNA-induced inflammation in PLWH on ART. Through this work, we expect to better understand the role of post-transcriptional modifications of HIV RNA in innate immune responses and to develop strategies to prevent proinflammatory HIV RNA modifications. This would ultimately allow us to develop novel therapeutics as adjunct therapy to ART to dampen chronic inflammation in PLWH and reduce the ris...