Studies On Molecular Mechanism That Nonstructural Protein 4 Of Prrsv Regulated IFN-β Signaling

Porcine reproductive and respiratory syndrome(PRRS), a highly infectious disease caused by PRRS virus(PRRSV), leads to enormous economic losses in swine production worldwide. The prevalent clinical manifestations of PRRS are severe reproductive failure in sows and gilts and respiratory problems in pigs of all ages. PRRSV has developed various strategies to evade the host innate immune response, including the suppression of type I interferon(IFN) activation and non-structural proteins play significant roles in IFN suppression pathways. Nsp4 is a 3C-like serine protease(3CLSP) that processes pp1a/pp1 ab of the virus into 10 mature nonstructural proteins(Nsp3–Nsp12). Molecular mechanism that Nsp4 inhibits type I IFN expression is not clear.In this study we revealed that PRRSV Nsp4 acts as a multifunctional protein that antagonizes type I IFN activation by cleave MAVS and NEMO. MAVS cleavage is specifically attributable to the protease activity of 3CLSP and independent of the apoptosis and proteasome pathways MAVS and NEMO cleavage by PRRSV 3CLSP presents an alternative mechanism to mediate innate immune. The following is the main content of our research. 1. PRRSV Nsp4 disrupts RIG-I/MDA5 signaling by inactivation of both IRF3 and NF-κB.We reveal that PRRSV Nsp4 inhibits SEV-induced type IFN-β expression by luciferase reporter system. Then, we show that PRRSV Nsp4 disrupts RIG-I/MDA5 signaling by inactivation of both IRF3 and NF-κB in a dose-dependent manner and catalytic activity-dependent manner. 2. PRRSV 3CLSP blocks IFN-β induction by cleaving MAVS and NEMONsp4 is the major protease of PRRSV, which plays an important role during virus replication. The catalytic activity is essential for Nsp4 inhibition of IFN-β production. To investigate whether PRRSV Nsp4 blocks IFN-β induction by interaction with adaptor protein of RIG-I signal pathway, HEK-293 T cells were cotransfected with nsp4 and MAVS or NEMO expression plasmids. Western blotting was performed and we found that PRRSV Nsp4 was able to cleave MAVS and NEMO in catalytic activity-dependent manner.. Then we made a further study of MAVS cleavage and found that MAVS cleavage is specifically attributable to the protease activity of 3CLSP and independent of the apoptosis and proteasome pathways. 3. PRRSV Nsp4 cleaves MAVS at Glu-268Previous studies have indicated that 3CLSP preferentially cleaves glutamic acid-glycine(E-G), glutamic acid-alanine(E-A), and/or glutamic acid-serine(E-S) bonds of viral polyproteins and cellular targets. To examine the residues of MAVS for potential PRRSV Nsp4 cleavage sites, we construct MAVS mutants(MAVS-E268 A, MAVS-E274 A, MAVS-E288A) and cotransfected MAVS mutants along with Nsp4 into HEK-293 T cells. Western blotting was performed and we found that E268 A was resistant to Nsp4-mediated cleavage in HEK293 cells. Compared with the full-length MAVS, neither fragment(1-268 and 269-540) of MAVS was capable of efficiently activating any of the three promoters.