TOLLIP suppresses picornavirus internalization by targeting and degrading VP1 protein through selective autophagy

Foot-and-mouth disease virus (FMDV) represents a major threat to global livestock production. The capsid protein VP1 is crucial for infection; however, the host factors and mechanisms responsible for VP1 restriction remain poorly understood. We previously identified the host chaperone DNAJA3 as a host restriction factor that inhibits FMDV infection by promoting VP1 degradation through the autophagy-lysosomal pathway. Here, we elucidate the molecular mechanism by which DNAJA3 mediates degradation of VP1. We demonstrate that DNAJA3 recruits the autophagy cargo receptor TOLLIP to facilitate selective autophagic degradation of VP1. Mechanistically, TOLLIP directly interacts with the 1-37 amino acid (aa) region of VP1 through its N-terminal and C-terminal domains, and full-length TOLLIP is required for efficient VP1 degradation. Furthermore, DNAJA3 recruits the E3 ubiquitin ligase TRIM21 to promote VP1 polyubiquitination through K27-, K48-, and K63-linked ubiquitination. TOLLIP additionally restricts FMDV internalization by modulating early endosomal trafficking in an autophagy-dependent manner. In vivo, tollip-deficient suckling mice exhibit increased susceptibility to FMDV infection. Moreover, TOLLIP exerts antiviral activity against multiple picornaviruses, including Senecavirus A and Enterovirus 71. Notably, FMDV downregulates endogenous TOLLIP expression through the protease activity of the viral 3C protein, enabling the virus to evade host autophagic surveillance. Collectively, our study identifies a novel DNAJA3-TRIM21-TOLLIP axis that restricts FMDV infection through selective autophagy, establishes TOLLIP as a host restriction factor against picornaviruses, and reveals potential molecular targets for antiviral intervention.