CRISPR and yeast two-hybrid screens identify CD81 as a proviral host factor for PRRSV.

Porcine reproductive and respiratory syndrome virus (PRRSV) remains a major economic burden on the global swine industry. Identifying host factors that mediate viral invasion is critical for developing antiviral strategies. Here, using a CRISPR screen targeting genes encoding cell surface CD proteins combined with yeast two-hybrid screening, we identified the tetraspanin CD81 as a novel host factor that promoted PRRSV infection by directly interacting with viral glycoprotein 5 (GP5). CD81 knockdown reduced PRRSV infection, whereas CD81 overexpression enhanced it. Consistently, antibody-mediated blockade of CD81 restricted infection in both Marc-145 cells and primary porcine alveolar macrophages. Mechanistic analysis showed that CD81 mainly facilitated viral internalization, and its C-terminal transmembrane region was required for GP5 binding and efficient infection. During infection, CD81 underwent autophagic degradation mediated by the selective autophagy receptor NDP52, suggesting a host restriction response that reduced CD81 availability. PRRSV countered this process by promoting NDP52 degradation, thereby stabilizing CD81 and favoring infection. These findings identify CD81 as both a key factor in PRRSV pathogenesis and a potential target for antiviral intervention.IMPORTANCEPorcine reproductive and respiratory syndrome virus (PRRSV) remains one of the most economically important swine pathogens worldwide, yet effective antiviral strategies are lacking because of its extensive genetic diversity and immune evasion. Here, we identified the tetraspanin CD81 as a previously unrecognized host factor that promoted efficient PRRSV infection. CD81 directly interacted with the viral glycoprotein GP5 to facilitate viral internalization, whereas disruption of CD81 by genetic depletion or antibody blockade markedly restricted infection. We further demonstrated that CD81 abundance was controlled by NDP52-mediated selective autophagy as an intrinsic antiviral mechanism, which PRRSV overcame by degrading NDP52 to stabilize CD81 and enhance viral replication. These findings reveal a previously unrecognized virus-host interaction that governs PRRSV entry and identify CD81 as a promising host-directed therapeutic target.