PROJECT SUMMARY Although HAART treatment is successful to block active replication of HIV in AIDS patients, it does not completely eradicate the infection. HIV latent reservoirs remain as a major obstacle for complete elimination of HIV viruses and cure of the infection. Investigation of host machineries that regulate HIV proviral expression will help to improve the understanding of the stage of HIV latent infection. It will also provide new strategies to perturb host regulatory factors for eliminating latent HIV. We characterized that one of NSUN RNA m5C methyltransferases (m5C-MTases), NSUN1/NOP2, restricts HIV replication, suppresses HIV proviral expression, and promotes viral latency. The impact of m5C methylation catalyzed by NSUN m5C-MTases (NSUN1-7) on HIV replication still remains largely unknown but starts to unfold. In this proposal, we will initiate the in-depth examination of NSUN m5C-MTases as regulators of HIV proviral expression. For aim 1, we will employ the ultra-sensitive reverse transcription droplet digital PCR (RT-ddPCR) assays to measure various HIV transcripts in cells depleted of NSUN m5C-MTases, which will provide a high-resolution profiling of their effect on HIV proviral expression. Furthermore, we will confirm whether the enzymatic activity of NSUN m5C- MTases is required. We will also determine whether NSUN m5C-MTases interferes with HIV post- transcriptional events, including HIV mRNA stability and translation. For aim 2, we will investigate the impact of NSUN m5C-MTases on the activation of P-TEFb and RNA Pol-II that play a critical role in promoting HIV transcription. Our results showed that the loss of NSUN1 reduces m5C methylation of HIV TAR RNA and that its MTase catalytic domain (MTD) prevents HIV Tat-TAR interaction, indicating that m5C methylation of TAR may regulate its interaction with Tat directly. We will identify the m5C methylation site(s) of TAR catalyzed by NSUN m5C-MTases and further determine its role in modulating Tat-TAR interaction. For aim 3, we will investigate the role of NSUN m5C-MTases in regulation of HIV 5’ and 3’ UTRs’ viral functions. We will determine whether NSUN m5C-MTases bind with HIV 5’ and 3’ UTRs as well as contribute to their m5C methylation. Since 5’ and 3’ UTRs of HIV mRNA play a critical role in regulation of HIV proviral expression epi- transcriptionally, we will determine whether m5C methylation of HIV 5’ and 3’ UTRs affects mRNA stability and protein translation. Furthermore, it has been recently shown that NSUN m5C-MTases also play an important role in regulating host cellular epi-transcriptomics. Thus, we will determine their functional impact on host gene expression in HIV latently infected CD4+ T cells. Overall, this proposal will comprehensively investigate NSUN m5C-MTases as novel regulators of HIV proviral expression. We believe that these studies will significantly improve our understanding of host-HIV interactions. From these studies, we will confirm whether memb...