Studies On The Molecular Mechanism That Equine Arthritis Virus Non-structural Protein 4 Inhibits IFN-β Production

EAV is an enveloped virus with a positive-sense,single-stranded RNA genome. Equine viral arthritis(EVA), an infectious disease, is caused by equine arthritis virus(EAV) in horses. The genome encodes 8 structural proteins and at least 13 non-structural proteins, among which nsp4 has chymotrypsin-like serine protease(SP) activity. The three dimensional structure of EAV nsp4 indicated that nsp4 adopts the smallest known chymotrypsin-like fold and a canonical catalytic triad of Ser-120, His-39, and Asp-65.Host cells could produce a variety of cytokines and chemokines during virus infection, among which type I IFN is one of the most potent antiviral cytokines. Studies have demonstrated that EAV has an ability of inhibiting the production of IFN-β, an important member of type I interferon in infected cells. However, the specific mechanism is not clear. According to the reports, the 3C protease of FMDV and HAV could inhibit IFN-β production. Based on its 3C-like serine protease activity, we wondered if EAV nsp4 could also antagonize IFN-β induction.To investigate the role of EAV nsp4 in IFN-β production and explain the molecular mechanism, we carried out the studies as follows: 1. Overexpression EAV nsp4 blocks the expression of IFN-β and ISGs induced by SeV.HEK293-T cells were transfected with the eukaryotic expression plasmid of EAV nsp4. Through dual luciferase reporter assay and Real-time PCR, we validated that EAV nsp4 blocked the production of SeV-induced IFN-β and attenuated the transcription of SeV-induced ISGs. 2. EAV nsp4 inhibits the IFN-β production by disrupting IRF3 and NF-κB signal cascades.The transcription factors IRF3 and NF-κB were known to play a vital role in the expression of IFN-β. Thus, HEK293-T cells were co-transfected with EAV nsp4, IFN-β-luc, NF-κB-luc or IRF3-luc.Then we found that the activation of these three promoters was inhibited in a dose-dependent manner. Next, we detected the phosphorylation levels of IRF3 and NF-κB-p65 induced by Se V after transfected with EAV nsp4. Western Blot assay showed that both of the phosphorylation levels of IRF3 and NF-κB-p65 were inhibited by EAV nsp4. Therefore, we concluded that EAV nsp4 antagonized the IFN-β production by inhibiting the promoters of IRF3 and NF-κB. 3. The inhibition of IFN-β induction by EAV nsp4 depends on its 3C-like serine protease activity.EAV nsp4 is a 3C-like serine proteases, containing a canonical catalytic triad of His39–Asp65–Ser120. To explore whether the serine protease activity was involved in blocking IFN-β production, three single-point mutants which lost its 3C-like serine protease activity, namely nsp4-H39 A, nsp4-D65 A and nsp4- S120, were constructed and transfected into HEK293-T. Our results showed that wild-type nsp4 overexpression led to the inhibition of IFN-β, IRF3 and NF-κB transcription activities, whereas nsp4-H39 A, nsp4-D65 A and nsp4-S120 A partially lost the IFN inhibition ability. 4. EAV nsp4 inhibits the IFN-β induction by cleaving NEMO.Given that RIG-I/MDA5 signal pathway plays a key role in IFN-β production, HEK293-T were co-transfected with nsp4 and RIG-I, MDA5, IPS1, TBK1 or NEMO. The results of dual luciferase reporter assay and western blot showed that EAV nsp4 inhibited the induction of IFN-β by cleaving NEMO. 5. The serine protease activity is responsible for EAV nsp4 cleaving NMEO.It has been confirmed that the serine protease activity was responsible for EAV nsp4 blocking IFN-β production, thus, we speculated it was also necessery for the cleavage of NEMO. The results showed that nsp4-H39 A, nsp4-D65 A and nsp4-H39 A could not cleave NEMO.