Porcine reproductive and respiratory syndrome virus N protein-mediated viral replication enhancement via interaction with host caspase-6.

Porcine reproductive and respiratory syndrome virus (PRRSV) poses a persistent threat to the global swine industry, owing to its high genetic variability and immune evasion capabilities. However, the mechanisms by which PRRSV manipulates host cell apoptosis to promote its own replication and evade the host immune response remain inadequately understood. This study reveals, for the first time, a critical role for caspase-6-a cysteine-aspartic protease of the apoptotic cascade-in PRRSV infection. We demonstrated that caspase-6 specifically cleaves the viral nucleocapsid (N) protein at aspartate residue 94 (D94), generating N-terminal and C-terminal fragments that subsequently inhibit the activation and nuclear translocation of the key host transcription factor, namely, interferon (IFN) regulatory factor 3 (IRF3). This leads to a significant reduction in the expression of host IFN-β, thereby promoting viral replication. Further investigation confirmed that this cleavage site is highly conserved across different PRRSV strains, suggesting it as a potential target for the development of broad-spectrum antiviral therapeutics. Moreover, a D94A mutant virus (PRRSV-D94A), constructed using reverse genetics, exhibited significantly attenuated replication and pathogenicity. This mutant induced a more robust host antiviral immune response, characterized by markedly elevated levels of IFNs and inflammatory cytokines, indicating its potential as an ideal live attenuated vaccine candidate. This study elucidated, from the perspective of a host protease, a novel molecular mechanism by which PRRSV evades the host immune response and promotes viral replication, providing a vital theoretical basis and a new strategy for PRRSV vaccine design and antiviral drug development.IMPORTANCEPorcine reproductive and respiratory syndrome virus (PRRSV) remains one of the most economically devastating pathogens in the swine industry, largely due to its ability to evade innate immunity and persist within its host. This study identifies, for the first time, a host-virus mechanism in which PRRSV recruits caspase-6 to cleave its N protein at a conserved D94 site, thereby suppressing type I IFN signaling and enhancing viral replication. We further demonstrate that disrupting this cleavage through the D94A mutation attenuates viral replication, enhances innate immune activation, and reduces pathogenicity in pigs. These findings not only reveal a previously unrecognized immune-evasion strategy of PRRSV but also highlight caspase-6 and the N-protein cleavage site as promising targets for host-directed antiviral interventions and rational vaccine design.