PRMT3 restricts porcine epidemic diarrhea virus replication by disrupting the interaction between VAPA and the viral nucleocapsid protein

Porcine epidemic diarrhea virus (PEDV) represents a severe threat to the global swine industry. Its infection process involves intricate virus–host interactions and immune evasion mechanisms, but effective therapeutic targets remain elusive. In this study, we identified protein arginine methyltransferase 3 (PRMT3) as a novel regulatory factor that significantly modulates PEDV infection via genome-wide CRISPR/Cas9 knockout library screening. Knockout or inhibition of PRMT3 markedly enhanced PEDV infection in multiple cell lines, including LLC-PK1, IPEC-J2, and primary porcine intestinal epithelial cells. Mechanistic investigations revealed that PRMT3 can restrict PEDV infection by interacting with vesicle-associated membrane protein-associated protein A (VAPA). Further analysis revealed that VAPA facilitates cholesterol transport through binding to oxysterol-binding protein (OSBP) and inhibits the autophagic degradation of the viral nucleocapsid (N) protein, with both processes being critical for promoting PEDV infection in host cells. A detailed analysis revealed that K52 within its major sperm protein (MSP) domain interacts with D404 and D405 in the two phenylalanines in an acidic tract (FFAT)-like motifs of the N protein, and these interactions proved essential for PEDV infection. In summary, this is the first study to identify and validate the PRMT3–VAPA–N protein autophagic degradation axis as a key pathway through which PRMT3 suppresses PEDV infection, with VAPA acting as an essential host factor for PEDV pathogenesis. These findings uncover novel signaling pathways and molecular targets for the development of anti-PEDV therapeutics.Author summary: Porcine epidemic diarrhea virus (PEDV) severely hampers global pig production, and few effective therapeutic targets have been identified. Using genome-wide CRISPR/Cas9 screening, we identified protein arginine methyltransferase 3 (PRMT3) as a novel anti-PEDV–host restriction factor. We found that PRMT3 knockout or inhibition markedly boosted PEDV replication in diverse porcine cell lines. Mechanically, we revealed that PRMT3 can restrict PEDV by interacting with the proviral factor vesicle-associated membrane protein-associated protein A (VAPA). We further demonstrated that VAPA promotes viral entry via oxysterol-binding protein-dependent cholesterol transport and suppresses autophagic degradation of the viral nucleocapsid (N) protein to facilitate replication, relying on the VAPA major sperm protein (MSP) domain and D404 in N protein for mutual binding. Our study establishes the critical PRMT3–VAPA–N protein–autophagy axis against PEDV and provides candidate targets for antiviral development.