# 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

## TL;DR

This study reveals how porcine astrovirus causes cell death and weakens immune responses, offering new targets for antiviral treatments.

## Contribution

The study identifies the nsP1a/3 protease's role in inducing apoptosis and suppressing interferon responses in porcine astrovirus.

## Key findings

- PAstV induces apoptosis via the mitochondrial pathway, enhancing viral replication.
- The nsP1a/3 protease cleaves MAVS and suppresses the type I interferon response.
- Inhibiting the nsP1a/3 protease activity reduces apoptosis and viral replication.

## Abstract

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 (His459, Asp487, and Ser549) 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.

Porcine astrovirus (PAstV) is widespread in pigs, posing a threat to the pig industry’s sustainability, with no effective drugs or vaccines available. This study explored the mechanisms by which PAstV modulates apoptosis and innate immunity. Our findings indicate that PAstV infection triggers apoptosis through the mitochondrial pathway, which subsequently enhances viral replication during the later stages of infection. Additionally, we identified the viral protein nsP1a/3 as a critical factor that interacts with MAVS, localizes to mitochondria, and promotes apoptosis. Notably, our observations revealed that nsP1a/3 expression results in MAVS cleavage and a subsequent suppression of the type I IFN response. Importantly, the processes of nsP1a/3-induced apoptosis and MAVS cleavage were found to be independent. Site-directed mutagenesis of the catalytic triad (His459, Asp487, and Ser549) within the 3C-like serine protease domain of nsP1a/3 to alanine significantly reduced its capacity to induce apoptosis, cleave MAVS, and inhibit type I IFN production. Moreover, the application of a serine protease inhibitor significantly curtailed PAstV replication, highlighting the critical role of nsP1a/3 protease activity in both apoptosis induction and type I IFN suppression. These findings offer valuable insights into the pathogenesis of PAstV and establish a foundation for the development of innovative antiviral strategies.