USF2 restricts enterovirus replication and transmission by inhibiting autophagy and vesicle-mediated viral spread

Enteroviruses pose a substantial global health burden, and a complete understanding of host defense mechanisms is still evolving. Through proteomic profiling of coxsackievirus B3 (CVB3)-infected cells, we identified the host factor USF2 (upstream transcription factor 2) as significantly upregulated, a process that may be associated with the viral capsid protein VP4. Here, we characterize USF2 as a novel, infection-induced restriction factor. Functional studies demonstrate that USF2 knockdown enhances viral RNA replication without affecting entry, while its overexpression suppresses replication and cytopathic effects. Mechanistically, USF2 acts as a transcriptional repressor of autophagy-related genes, thereby inhibiting autophagosome formation. Crucially, USF2 depletion promotes the release of MAP1LC3/LC3-positive extracellular vesicles carrying infectious virus, defining its role in blocking a key route of vesicle-mediated viral dissemination. USF2 exhibits broad antiviral activity against multiple enteroviruses and other RNA viruses that depend on cytoplasmic membrane remodeling. Our findings reveal that the host deploys a virus-triggered transcriptional regulator to restrict enterovirus spread by suppressing macroautophagy/autophagy-dependent viral dissemination.Abbreviations: BafA1: bafilomycin A₁; CQ: chloroquine; CPE: cytopathic effect; CVB3: coxsackievirus B3; dpi: days post infection; DEGs: differentially expressed genes; EV-A71: enterovirus A71; EVs: extracellular vesicles; GFP: green fluorescent protein; LAMP1: lysosomal-associated membrane protein 1; TR: LysoTracker Red; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MOI: multiplicity of infection; TFEB: transcription factor EB; USF2: upstream transcription factor 2; UPR: unfolded protein response; VP1: viral capsid protein 1; VP4: viral capsid protein 4.