Pharmacological METTL3 inhibition attenuates HIV-1 latency reversal in CD4 ⁺ T cells

ABSTRACT N 6 -methyladenosine (m 6 A) 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 m 6 A 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 m 6 A 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 m 6 A 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 m 6 A levels at 24 and 48 h post-treatment, as reflected by lower half-maximal inhibitory concentrations (IC 50 ). However, STM3006 and STC-15 exhibited significant cytotoxicity at concentrations above 2 µM at 48 h 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 m 6 A levels except STC-15 treatment of one donor’s cells, while STM3006 induced significant cytotoxicity at 24 h post-treatment. Notably, reduced RNA m 6 A 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 m 6 A RNA modification in regulating HIV-1 latency reversal.