The 3C-like serine protease activity of porcine astrovirus nsP1a/3 mediates mitochondrial apoptosis and MAVS cleavage to facilitate viral replication and antagonize type I interferon response

Porcine astrovirus (PAstV) is globally prevalent in swine and is associated with diarrhea and encephalitis in piglets, posing a threat to porcine health. However, its pathogenic mechanisms remain poorly understood. This study used the PAstV1-GX1 strain to infect PK-15 cells, revealing that the virus induces significant apoptosis, with late apoptotic cells reaching 41.2% at 24 hours post-infection. The infection activates caspase-9 and caspase-3, but not caspase-8, and causes mitochondrial damage, indicating apoptosis via the mitochondrial pathway. The apoptosis inhibitor Z-VAD-FMK reduced viral replication, while apoptosis inducer ABT-263 enhanced it at later stages. The nsP1a/3 protein, which interacts with MAVS and localizes to mitochondria, was identified as key in inducing apoptosis. Its 3C-like serine protease domain likely mediates this interaction. Knocking down MAVS reduced apoptosis and increased early-stage replication but decreased it later. Overexpressing MAVS increased apoptosis and decreased replication. Furthermore, we observed that the expression of nsP1a/3 resulted in the cleavage of MAVS and the suppression of the type I interferon (IFN) response. Notably, treatment with Z-VAD-FMK did not influence nsP1a/3-mediated MAVS cleavage or type I IFN inhibition, suggesting that the induction of apoptosis and MAVS cleavage are distinct processes. By employing site-directed mutagenesis to substitute alanine for the catalytic triad residues (His 459 , Asp 487 , and Ser 549 ) of the 3C-like serine protease, we significantly reduced the ability of nsP1a/3 to induce apoptosis, cleave MAVS, and suppress the type I IFN response, underscoring the essential role of protease activity in these functions. Furthermore, the use of a serine protease inhibitor markedly decreased PAstV replication. These findings provide significant insights into the pathogenesis of PAstV and establish a foundation for the development of novel antiviral therapies.