Porcine reproductive and respiratory syndrome virus nonstructural protein 2 promotes the autophagic degradation of adaptor protein SH3KBP1 to antagonize host innate immune responses by enhancing K63-linked polyubiquitination of RIG-I.

Non-structural protein 2 (NSP2) of PRRSV is highly variable and plays crucial roles in the virus's life cycle. To elucidate the function of NSP2 during PRRSV infection, we identified SH3KBP1 as an NSP2-interacting host protein using mass spectrometry. Exogenous SH3KBP1 expression significantly inhibited PRRSV replication by enhancing IFN-I and related ISGs production. Conversely, SH3KBP1 knockdown promoted viral replication by downregulating IFN-I and ISGs levels. In vivo experiments revealed that Sh3kbp1^-/- mice were more susceptible to VSV infection, exhibiting reduced serum IFN-β levels. Further investigation showed that SH3KBP1 enhances RIG-I signal transduction by increasing K63-linked polyubiquitination through interaction with the E3 ubiquitin ligase TRIM25. We also found that PRRSV infection and NSP2 overexpression induce the autophagic degradation of SH3KBP1, counteracting the host's innate immune response. A critical interaction site was identified within the third proline-rich motif in NSP2 (⁴⁵³PVPAPR⁴⁵⁸). Recombinant PRRSV lacking this motif displayed reduced virulence and decreased SH3KBP1 degradation. This study advances our understanding of how PRRSV interferes with the host immune response and offers valuable insights for development novel attenuated vaccines against PRRSV. Author summary: PRRS is one of the most devastating diseases affecting the global pig industry. The genome of the PRRS virus (PRRSV) exhibits high variability, characterized by rapid mutation and frequent recombination, particularly in the NSP2 gene. NSP2 plays crucial roles in viral replication, virulence, and immune regulation. This study reveals a novel strategy used by NSP2 to counteract innate immunity. We found that SH3KBP1 enhances the K63-linked polyubiquitination of RIG-I by recruiting TRIM25, thereby stabilizing RIG-I and promoting the production of IFN-I. Mice lacking SH3KBP1 were more susceptible to VSV infection and had lower survival rates. NSP2 directly binds to SH3KBP1 and induces its degradation through the autophagic pathway, aiding viral replication. Additionally, mutations in the amino acid sequences at positions 453–458 within the hypervariable domain of PRRSV NSP2 led to attenuated virulence. This research uncovers a new function for the adaptor protein SH3KBP1 and provides deeper insights into how PRRSV evades host immune responses. [ABSTRACT FROM AUTHOR]