The role of RNA m6A modification in the regulation of HIV latency and reactivation - Background. This proposal, which is submitted in response to RFA-AI-21-021 “Understanding Post-Transcriptional Regulation of Intact and Defective HIV RNA”. N6-methyladenosine (m6A), is the most common RNA modification and is known to regulate RNA stability, splicing and nuclear export. m6A modification of HIV transcripts is crucial for the early stages of HIV infection during acute infection of primary T cells, but it is an open question whether m6A modification controls HIV latency and reactivation in ART-suppressed patients. Our goal. Our multidisciplinary team has extensive experience in studies of HIV latency and reactivation in patients and in reliable primary cell models, studies of RNA m6A modification, and cutting-edge technologies such as NGS sequencing and scRNA-seq analysis. To overcome the challenge of measuring m6A in RNA recovered from the extremely low numbers of HIV+ cells present in patient samples, we will develop a sensitive next-generation sequencing assay for the profiling and quantification of m6A modification in different HIV transcripts from patient samples. This assay, which we call MeRIP-EDITS combines methylated RNA immunoprecipation with the EDITS assay, which has been used in multiple clinical studies to measure the inducible HIV reservoir. We will use the MeRIP-EDITS assay to characterize m6A modification of different HIV transcripts at different reactivation kinetic points of latent HIV and examine changes of the m6A pathway during HIV latency and reactivation. In parallel we will perform mechanistic studies on the m6A pathway using the QUECEL primary cell model of HIV latency. We will use the model to develop a sensitive nanopore RNA-sequencing assay which can subsequently be applied to patient samples. We will also inhibit the activity of the m6A writer METTL3 and the erasers FTO and ALKBH5 by knocking out the expression of these genes by using the CRISPR gene editing technology. High resolution mRNA FISH experiments, which distinguish between spliced and partially spliced HIV mRNA transcripts will be used to study the colocalization of m6A readers and HIV mRNAs. How will we advance the field? Demonstration of a central role of m6A in the control of HIV latency would immediately suggest pharmacological strategies to incorporate into HIV cure regimens. To date, it has been impossible to efficiently reverse HIV latency using agents that are designed for “kick and kill” strategies for an HIV cure. Using the sensitive assays described above, we will evaluate the impact of inhibitors of m6A erasers as part of a “kick and kill” strategy for HIV latency reversal. As a complementary approach we will also evaluate whether inhibitors of m6A writers can inhibit HIV reactivation and lead to long term silencing, as part of a “block and lock” strategy.
