The crosstalk between ubiquitination and GlcNAcylation of CHAF1A regulates HIV-1 latency and reactivation.

The persistence of latent HIV-1 reservoirs remains a critical barrier to cure. Current "shock and kill" strategies are limited by ineffective latency-reversing agents (LRAs) and poor understanding of epigenetic regulation. Here, we identify chromatin assembly factor 1 subunit A (CHAF1A), a histone chaperone enforcing HIV-1 latency, as a therapeutic target regulated by antagonistic post-translational modifications: ubiquitination promotes its degradation, while O-GlcNAcylation stabilizes it. We demonstrate that trifluridine, a Food and Drug Administration-approved antiviral drug, reactivates latent HIV-1 by disrupting O-GlcNAcylation, triggering CHAF1A ubiquitination and proteasomal degradation. Notably, CHAF1A expression increases with age in CD4+ T cells (>60 years), correlating with deeper proviral reservoirs. This age-dependent accumulation inversely associates with reduced O-GlcNAcase levels, suggesting O-GlcNAcylation-mediated stabilization in aging. Our findings establish CHAF1A as both a therapeutic target and an age-stratifying biomarker, advancing trifluridine as a translatable LRA to enhance reservoir clearance in aging populations-a demographic increasingly impacted by HIV-1 persistence.IMPORTANCEHIV-1 latency continues to represent a significant barrier to achieving a cure, particularly in aging populations characterized by expanded viral reservoirs and compromised immune recovery-a challenge further intensified by the absence of therapies specifically designed to target age-related mechanisms. Current latency-reversing agents (LRAs) are insufficient in addressing the metabolic and epigenetic dysregulation that sustains viral persistence in older individuals. In this study, we reveal a dynamic interplay between ubiquitination and O-GlcNAcylation that regulates the stability of CHAF1A, a histone chaperone essential for maintaining HIV-1 latency. We identify trifluridine as a novel LRA capable of disrupting O-GlcNAcylation to degrade CHAF1A, thereby effectively reversing latency in primary cells. This research bridges a critical gap between fundamental virology and clinical gerontology. These findings establish a robust foundation for refining strategies aimed at HIV-1 eradication, with a focus on targeting host metabolic-epigenetic networks to address latency in underserved aging populations.