Investigating RNA polymerase III driven mechanisms in regulating HIV latency - ABSTRACT Resting CD4+ T cells harbor majority of latent Human Immunodeficiency Virus (HIV) during infection. Elimination of this reservoir is an extremely challenging task because of involvement of multiple mechanisms in regulating HIV latency. Therefore, perhaps, none of the currently promising latency reversing agents (LRAs) were able to reduce the size of the latent proviral reservoir in patients. Hence, there is a dire need to find novel mechanisms and therapeutic targets that can be targeted to purge the heterogenous latent reservoir. RNA Polymerase III (RNA Pol III) appears to be a master regulator with unexplored potential of regulating latency via mediating distinct mechanisms, such as i) regulating the expression from neighboring RNA Pol II gene promoters and ii) transcription of novel noncoding RNAs with potential to regulate expression of cellular/viral genes. Our preliminary studies suggest the enrichment of Pol III transcribed noncoding RNAs in latent cells, namely 7SK, 21A and BC200 that are interspersed among Alu repeats. This is highly relevant to HIV latency because the HIV genome is found to preferentially integrate near Alu repeats. Consequently, use of an RNA Pol III inhibitor, ML60218, resulted in an unprecedented reactivation (up to 90%) of latent cell lines J89GFP and THP89GFP, in a dose- dependent manner (25 μM-50 μM). Further, we observed a high degree of cell death specifically in HIV infected cells due to viral cytopathic effects, whereas uninfected cells maintained survival even at a very high concentration of RNA Pol III inhibitor (100 μM). These exciting findings and corroborating reports will be leveraged to test the hypothesis that RNA Pol III plays a crucial role in the establishment of HIV latency and targeting novel intermediate effectors of RNA Pol III driven mechanisms may enhance the efficacy of cure strategies. This study is divided into two specific aims that will be focused on investigating RNA Pol III driven direct (by genomic occupancy) and indirect (by ncRNAs) mechanisms that may regulate HIV latency. In Aim 1, effect of RNA Pol III inhibition/knockdown will be tested in ex vivo cultured primary CD4+ T cell model. Further in Aim 1.2 we will investigate if physical presence of RNA Pol III in proximity can modify chromatin landscape at HIV 5´ LTR. In Aim 2, we will identify RNA Pol III transcribed noncoding RNAs involved in latency by employing RNA-seq in combination with RNA Pol III ChIP-Seq. Finally, gene knockdown studies will be performed for select noncoding RNAs alone or in combination to investigate their role in promoting repressed chromatin state at HIV 5´ LTR. Our study is highly innovative as we aim to identify novel epigenetic modulators that can be synchronously targeted to overcome challenges associated with shock and kill strategy of HIV cure. Successful completion of this study will provide critical mechanistic information which may address the heterogeneity among latent reservoirs.
