| PRRS(Porcine Reproductive and Respiratory Syndrome), also known as blue ear disease, is an viral epidemic disease among pigs caused by PRRSV(Porcine Reproductive and Respiratory Syndrome Virus) which causes tremendous economic losses to swine industry. The clinical symptoms of PRRS mainly include severe reproductive barriers of sows, lower quality of boars semen, deaths of newborn piglets and respiratory syndrome of pigs in different ages. Innate immune system is the first defense line to resist the viral infection for the host, in reverse, viruses have developed varieties of mechanisms to invade host successfully in the evolutionary process with host immune system. Studies have shown that PRRSV infection can antagonize the induction of type I IFNs(Interferons). And the components of PRRSV also function as the antagonists in IFNs induction, including nonstructural protein nsp1, nsp2, nsp4, nsp11 and structural protein N(Nucleocapsid), but the molecular mechanisms under the fact still needs to be investigated. Nucleocapsid protein is the main structural protein of PRRSV which plays an important role in infection, pathogenesis and immune regulation of PRRSV. Our study concentrates on the molecular mechanism how PRRSV N protein functions as the antagonist of IFN-β and the study is carried out as follows:1. Overdose expression of PRRSV N protein inhibits the activation of IFN-β and IRF3By means of double luciferase reporter assay, we have elucidated that PRRSV N protein can significantly inhibit the promoter activity of IFN-β stimulated by Se V(Sendai Virus). Analysis on the effection to IRF3 by PRRSV N has been conducted in the same strategy. And we concluded that PRRSV N protein can also significantly inhibit the activation of IRF-3 even in a dose-dependent manner.2. Studies on the target protein PRRSV N interacts with in RIG-I/MDA5 signaling pathwayRIG-I/MDA5 signal pathway plays an important role in IFNs expression activated by RNA viruses. To further investigate the target protein how and which PRRSV N interacts with, studies have been carried out by cotransfecting the primary eukaryocyte expression plasmids in RIG-I/MDA5 signaling pathway including receptors(RIG-I, MDA5), adaptors(IPS-1), protein kinase(TBK-1, IKKε) and transcriptional factors(IRF3). Subsequently, the promoter activity of IFN-β was detected by double luciferase reporter assay. We elucidated that PRRSV N can inhibit the activity of IFN-β induced byRIG-I, MDA5, IPS-1, TBK-1, IKKε and IRF3. Additionally, the activity of IFN-β induced by IRF3-5D(positive mutant of IRF3) was also inhibited by PRRSV N, which implied that PRRSV N interacts with the IRF3 or the downstream of IRF3 in RIG-I/MDA5 signaling pathway. IFA(Indirect Immunofluorescence Assay) was conducted to analysis the subcellular location of PRRSV N, the results show that PRRSV N protein spreads in a nuclear-cytoplasmic shuttling manner, which implies PRRSV N protein may interacts with IRF3 in nucleus.3. Study on the domains of PRRSV N protein in inhibiting IFN-βTo further reveal the key domains of PRRSV N protein in inhibiting IFN-β, the ability of different truncated mutants of N(6 N-terminal truncated mutants and 5 C-terminal truncated mutants) in inhibiting the IFN-β has been detected by double luciferase reporter assay. The result shows that aa37-57 and aa69-123 are the key domains of PRRSV N protein in inhibiting the IFN-β activity induced by Se V, and NLS-2 of PRRSV N was included in aa37-57. |
