Functional Analysis Of Interferon-Inducible Transmembrane Protein Family And Viperin In Antiviral Defense Against Muscovy Duck Reovirus Infection

Muscovy duck reovirus(MDRV)causes significant economic losses to waterfowl industry in China.Although it is known that interferon-induced transmembrane proteins(IFITMs)protect host cells from diverse virus infections,their role in defense against MDRV infection remains to be determined.Here,we observed that MDRV infection induced robust expression of IFITM1,IFITM3 and Viperin,and slightly increased the expression of IFITM2 in vitro.To further investigate the functional relevance of IFITMs in MDRV infection,we employed Muscovy duck model.The animal experiments showed that Muscovy ducks displayed obvious clinical symptoms after challenge with MDRV,such as liver discoloration,spleen enlargement and virus-induced ataxia.Similarly,expression of IFITM1,IFITM3 and Viperin was greatly up-regulated by MDRV infection in vivo.Furthermore,we generated stable cell lines specifically silencing IFITM1,IFITM2,IFITM3 or Viperin,and found that disrupting the endogenous expression of IFITM1,IFITM3 or Viperin but not IFITM2 could strongly enhance the MDRV replication.Moreover,overexpression of human or Muscovy duck IFITM1,IFITM3 and Viperin in 293T cells significantly impaired the replication of MDRV,while IFITM2 overexpression had no significant effect on the MDRV infection.Taken together,these data reveal that IFITM1,IFITM3,and Viperin but not IFITM2 play a critical role in host innate immunity against MDRV infection,and suggest that IFITM1,IFITM3 and Viperin proteins may be potential agents for the treatment of MDRV infection.To further determine the function of duck IFITM1 in defense against MDRV infection,we constructed an expression vector pEGX-4T-dIFITM1.The correct recombinant pEGX-4T-dIFITM1 was transformed into Transetta(DE3).The pEGX-4T-dIFITM1 fusion protein was induced by Isopropyl-beta-D-thiogalactoside(IPTG).SDS-PAGE analysis showed that the recombinant protein with a molecular mass about 47 kDa was highly expressed in Transetta.Further studies are required to address the role of recombinant IFITM1 protein.Taken together,these results indicate that MDRV infection can stimulate the innate immune response,including induction of high expression of IFITM1,IFITM3 and Viperin in the host cells,and these host factors play an important role in the anti-MDRV infection process.