Mishra, T.; Edwards, A.; Wu, L.

N6-methyladenosine (m6A) is a major epitranscriptomic modification that regulates RNA metabolism and affects the replication and latency reversal of human immunodeficiency virus type 1 (HIV-1) in cells. Methyltransferase-like 3 (METTL3) is the principal catalytic enzyme responsible for m6A deposition, and its pharmacological inhibition has emerged as a potential therapeutic strategy for cancer and viral infections. However, the relative potency of METTL3 inhibitors in reducing m6A levels and their effects on HIV-1 latency reversal remain undefined. Here, we compared three commercially available METTL3 inhibitors (STM2457, STM3006, and STC-15) to evaluate their ability to reduce RNA m6A levels, suppress HIV-1 latency reversal, and affect cell viability in latently infected J-Lat cells and primary CD4+ T cells. In J-Lat cells, STM3006 and STC-15 were more potent than STM2457 in reducing RNA m6A levels at 24 and 48 hours post-treatment, as reflected by lower half-maximal inhibitory concentrations (IC50). However, STM3006 and STC-15 exhibited significant cytotoxicity at concentrations above 2 M at 48 hours post-treatment, whereas STM2457 displayed minimal toxicity across all tested doses. In primary CD4+ T cells from three healthy donors, all three inhibitors reduced RNA m6A levels but induced greater cytotoxicity compared with J-Lat cells, with comparable effects at optimized concentrations. Notably, reduced RNA m6A levels correlated with diminished HIV-1 latency reversal in both J-Lat cells and a primary central memory CD4+ T cell model. Together, these findings demonstrate differential potency and cytotoxicity among METTL3 inhibitors and support a critical role for m6A RNA modification in regulating HIV-1 latency reversal.