PROJECT SUMMARY Human immunodeficiency virus (HIV) is a highly manageable infection when treated with combination anti-retroviral therapy (cART). However, it is not yet curable and people living with HIV (PLWH) taking cART suffer from suboptimal immune health and reduced quality of life long-term. A leading cause of ongoing health concerns is persistent, residual infection. Persistently infected T-cells of PLWH that take cART still produce viral RNA and proteins, as well as defective proviruses, that can chronically activate the immune system despite undetectable viral loads. A better understanding of the biology and metabolism of HIV RNA and gene expression in PLWH taking cART could unlock additional therapeutic strategies to reduce viral gene products and improve quality of life. Cellular RNA and viral RNA can be subjected to a variety of post-transcriptional chemical modifications. These modifications modulate or fine-tune molecular interactions and thereby control gene expression and function through RNA processing, turnover, localization, or translation. Covalent base modification and its effects on RNA regulation at a broad level beyond the four canonical bases is often termed epitranscriptomics. The intimate relationship between RNA modification and RNA metabolism is becoming better appreciated. However, manipulating this process for therapeutic treatment of HIV and persistent infection in PLWH taking cART requires a deeper understanding of HIV epitranscriptomics. Here we propose systematic characterization and manipulation of HIV-1 RNA transcripts in model and patient T-cells. In the R61 phase, we will utilize three powerful sequencing methodologies (total RNA-seq, ribosome profiling, and nanopore direct RNA sequencing) to globally characterize the abundance, processing, translation, and modification status of both viral and host RNA in T-cells during cART. These experiments will include selection of persistently infected T-cells from PLWH taking cART. We will target several known modifications via enzyme knockouts and nanopore sequencing. These experiments will characterize HIV-1 RNA chemical modifications of potential significance and identify those with high a likelihood of impacting HIV RNA and protein loads in T-cells. In the R33 phase, we will use chemically modified oligonucleotides to alter modifications of high interest by blocking or directing site-specific modification. This will include oligonucleotides that sterically block modification or guide new modification, both therapeutic modalities that are maturing rapidly. Chemically modified oligonucleotides will provide a proof-of-concept for epiptranscriptome manipulation as a potential therapeutic approach while offering nucleic acid therapeutic candidates.