NSP4 promotes replication of porcine reproductive and respiratory syndrome virus-2

Porcine reproductive and respiratory syndrome (PRRS) is one of the most detrimental contagious swine ailments worldwide. Currently, no effective drugs are available for its treatment. Targeting the structural and non-structural proteins (NSP) of the type 2 PRRS virus (PRRSV-2) with small interfering RNA (siRNA) is an effective approach to inhibit PRRSV replication. NSP4, which is highly conserved and possesses 3C-like serine protease activity (3CLSP), can cleave PRRSV self-proteins, thereby contributing to viral replication. To investigate the mechanism by which NSP4 regulates PRRSV-2 replication and screen for effective siRNA inhibitors of PRRSV-2 replication, the recombinant plasmid pEGFP-C1-NSP4 was constructed, and a control siRNA pair and two siRNA pairs targeting the PRRSV-2 NSP4 gene (shRNA-ctr, shRNA150, and shRNA536) were synthesized and cloned into the pSilencer4.1-CMV-neo vector. After 24 h of incubation, Marc-145 cells were transfected with recombinant plasmids, and subsequently infected with PRRSV. Subsequently, the effects of NSP4 overexpression, shRNA on PRRSV-2 replication were evaluated by assessing cytopathic effects (CPE), TCID50, immunofluorescence assays (IFA), and western blotting. The data from these CPE, TCID50, and IFA experiments revealed conclusive evidence of effective inhibition of PRRSV-2 replication in Marc-145 cells by shRNAs. NSP4 overexpression significantly enhanced PRRSV-2 replication. Western blotting results demonstrated that NSP4 overexpression enhanced the expression of PRRSV-2 NSP4 and N proteins and that shRNA150 and shRNA536 effectively suppressed the expression of PRRSV-2 NSP4 and N proteins, indicating that shRNAs could serve as candidate molecules for fundamental research on PRRSV-2.