PRRSV Nsp9 hijacks BNIP3-mediated autophagy to provide membrane platforms for viral replication complexes

Porcine reproductive and respiratory syndrome virus (PRRSV) is a major pathogen threatening the global swine industry. The viral nonstructural protein 9 (Nsp9) harbors RNA-dependent RNA polymerase activity and acts as a core component of the viral replication complex, yet its interplay with host factors remains incompletely understood. In this study, we identified the mitochondrial protein BCL2 interacting protein 3 (BNIP3) as a specific binding partner of Nsp9. This interaction is mediated by the transmembrane (TM) domain of BNIP3 and the N-terminal 1-449 region of Nsp9. Functional assays demonstrated that BNIP3 facilitates PRRSV replication via its TM domain and LC3-interacting region (LIR). Mechanistically, BNIP3 recruits Nsp9 to mitochondria, where Nsp9 blocks autophagic flux and triggers incomplete mitophagy. This process leads to the accumulation of BNIP3-associated autophagic membranes, which serve as platforms for viral replication complex assembly. Moreover, simultaneous knockdown of BNIP3 and ATG7 results in further suppression of viral replication compared to knockdown of either gene alone. Rescue experiments showed that BNIP3 overexpression partially restored viral replication in ATG7-deficient cells, while ATG7 overexpression failed to rescue the replication defect caused by BNIP3 knockdown, indicating that BNIP3 possesses ATG7-independent platform functions. Collectively, our findings reveal that PRRSV hijacks BNIP3 to reprogram autophagy and generate membrane platforms for replication, which may serve as a promising target for antiviral development.IMPORTANCEPorcine reproductive and respiratory syndrome virus (PRRSV) is one of the most economically significant viral pathogens affecting the global swine industry, yet the specific host factors that support viral replication remain incompletely defined. In this study, we identify BNIP3, a known mitochondrial protein, as a previously unrecognized interactor of the PRRSV RNA-dependent RNA polymerase Nsp9. We demonstrate that PRRSV Nsp9 exploits BNIP3 to block autophagic flux and create a specialized membrane microenvironment that serves as a membrane platform for viral replication complex assembly. Our findings reveal a mechanism by which PRRSV, a positive-sense RNA virus, utilizes a mitophagy receptor to generate replication-permissive membrane platforms, which advances our understanding of how viruses manipulate autophagy. The identification of BNIP3 as a critical host factor that promotes viral replication and the elucidation of its membrane platform function open new avenues for understanding PRRSV replication and may inform the development of novel antiviral strategies.