# The role of RNA m6A modification in the regulation of HIV latency and reactivation

> **NIH NIH R61** · CASE WESTERN RESERVE UNIVERSITY · 2024 · $715,479

## Abstract

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.

## Key facts

- **NIH application ID:** 10814972
- **Project number:** 5R61AI169629-03
- **Recipient organization:** CASE WESTERN RESERVE UNIVERSITY
- **Principal Investigator:** JONATHAN KARN
- **Activity code:** R61 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $715,479
- **Award type:** 5
- **Project period:** 2022-04-01 → 2025-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10814972

## Citation

> US National Institutes of Health, RePORTER application 10814972, The role of RNA m6A modification in the regulation of HIV latency and reactivation (5R61AI169629-03). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10814972. Licensed CC0.

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