S146 and M148 within the mature chain domain of PSMB4 are crucial for degrading PRRSV nsp1α

Porcine reproductive and respiratory syndrome virus (PRRSV) is a single-stranded positive-sense RNA virus with an envelope. It-encoded non-structural protein 1α (nsp1α) plays a key role in evading host immune responses. Exploring the interaction between host factors and PRRSV nsp1α is crucial for understanding the mechanism of virus immune escape and virus control. Here, we constructed a cDNA library using porcine lung tissues and identified 33 potential host proteins interacting with viral nsp1α using yeast two-hybrid (Y2H) screening. These interactions were further analyzed using Gene Ontology and KEGG pathway analysis. Confocal microscopy revealed that proteasome subunit beta type-4 (PSMB4), carnosine dipeptidase 2 (CNDP2) and poly(rC) binding protein 1 (PCBP1) colocalized with viral nsp1α. The interaction between PSMB4 and nsp1α was further confirmed by Y2H and co-immunoprecipitation. PRRSV infection did not affect PSMB4 expression in both Marc-145 cells and porcine alveolar macrophages (PAMs). Overexpression of PSMB4 reduced nsp1α protein levels in a dose-dependent manner and decreased the accumulation of both viral N and nsp1α proteins in the context of PRRSV infection, while its knockdown promoted PRRSV replication. These data suggest that PSMB4 is a host restriction factor for PRRSV. Structure prediction and truncated mutant assays found that S146 and M148 within the mature chain domain of PSMB4 were crucial for binding and degrading nsp1α. These findings suggest that PRRSV nsp1α interacts with host proteins, with PSMB4 specifically binding to degrade nsp1α, thereby inhibiting PRRSV replication.