Study On The Mechanism Of Apoptosis Induced By Porcine Reproductive And Respiratory Syndrome Virus Infection

Porcine reproductive and respiratory syndrome (PRRS), which is characterized by respiratory disease in piglets and severe reproductive failure in sow, is a highly contagious and immunosuppressive disease in pigs caused by PRRS virus (PRRSV). Although PRRSV infection-induced host cell apoptosis has been established, the related mechanism is still unclear.In this study, we investigated the ability of PRRSV proteins to induce apoptosis. We showed that PRRSV nonstructural protein4(NSP4) was a critical apoptosis inducer. By transfecting viral protein genes into cells, we demonstrated that PRRSV NSP4could induce apoptosis in various cell lines, including Marc-145cells, Hela cells, and COS-1cells. NSP4could activate caspase-3,-8, and-9, which is in accordance with PRRSV infection. Blocking caspase-8activation by its inhibitor, z-IETD-FMK, led to a nearly complete inhibition of PRRSV-induced or NSP4-induced apoptosis, implying that the crosstalk between intrinsic and extrinsic pathway may exist in apoptosis caused by PRRSV infection or NSP4.Subsequently, we focused on the mechanism of apoptosis induction by NSP4. PRRSV NSP4protein is a22.4kDa protein, which consists of three domains. Using truncated constructs without different domains in NSP4, we showed that the apoptosis-inducing abilities of deletion mutants of NSP4were nearly abolished when compared to wild-type NSP4. Taken together, the full-length of NSP4structure was required for its apoptosis-inducing activity. NSP4is a3C-like serine protease that contains the canonical catalytic triad of His39-Asp64-Ser118. His39, Asp64, and Ser118were substituted to Ala, and three single-point mutants were generated separately. We showed that3C-like serine protease activity of NSP4was abolished by single-point mutation. Meanwhile the ability of each mutant to induce apoptosis was significantly impaired. We demonstrated that His39, Asp64, and Serl18were essential for NSP4to trigger apoptosis, and NSP4-induced apoptosis was dependent on its3C-like serine protease activity.In conclusion, our present work showed that PRRSV NSP4could induce apoptosis in host cells and might be partially responsible for the apoptosis induced by PRRSV infection. PRRSV3C-like protease-mediated apoptosis represents the first report in the genus Arterivirus, family Arteriviridae.