Abstract
The persistence of latent HIV-1 reservoirs remains a critical barrier to functional curing AIDS, as current latency-reversing agents (LRAs) exhibit limited clinical efficacy. While RNA modifications like N⁶-methyladenosine (m⁶A) regulate viral replication, their role in maintaining HIV-1 latency is poorly defined. Here, we identify the RNA-binding protein RBM39 as a scaffold organizing an m⁶A-dependent silencing complex that enforces viral latency. Through proteomic and functional analyses, we demonstrate that RBM39 recruits the m⁶A reader YTHDC1 and the RNA helicase DDX5, forming a tripartite complex that accelerates Tat RNA decay and enforces viral quiescence. Genetic or pharmacological degradation of RBM39 (using the clinically explored molecular glue indisulam) potently reactivates latent HIV-1 in J-Lat cell models, primary CD4⁺ T cells from people living with HIV-1 (PLWH), and synergizes with established LRAs (Bryostatin-1, JQ-1, SAHA) to broadly activate proviral reservoirs. Our work reveals a previously unrecognized host pathway in which RBM39-organized RNA decay complexes silence HIV-1 through epitranscriptomic regulation of Tat. In addition to establishing RBM39 as a promising therapeutic target for addressing the limitations of current "shock and kill" strategies, our findings establish a novel mechanistic framework for m⁶A-dependent regulation of viral gene expression. This framework may serve as a valuable reference for investigating similar regulatory mechanisms in other latent viral infections or oncogenic processes where RNA methylation plays a pivotal role.