The Mechanism Of Antiviral Protein Viperin In Porcine Epidemic Diarrhea Virus Infection

The body’s innate immunity is the first line of defense against pathogens.In the early stage of foreign pathogen infection,host cells activate the PRRs signaling pathway to induce the activation of interferon-stimulated genes(ISGs),and exert antiviral effects by generating a series of mechanisms including expression of antiviral proteins.The innate antiviral protein Viperin is one of the broad-spectrum antiviral active endoplasmic reticulum-associated proteins that affects virus proliferation through different mechanisms.Porcine epidemic diarrhea(PED)is a viral infection caused by porcine epidemic diarrhea virus(PEDV),which has caused huge losses to the domestic pig industry in recent years.This article aims to explore the effects of Viperin on PEDV and its mechanism of action,thus opening up new ideas for effective prevention and control of the disease.The main progress is as follows:(1)Genetic evolution analysis of the antiviral protein Viperin: Bioperinology was used to compare the Viperin gene in 31 species including 21 mammals,2 species of birds,6 species of fish and 2 species of mollusks.Through the gene phylogenetic tree analysis,we found that different species of veprin gene homology is above 81%,protein homology is above 83%.(2)Cloning,expression and localization analysis of porcine Viperin gene: Viperin gene was extracted and amplified from porcine alveolar macrophages,prokaryotic expression vector was constructed,and purified Viperin protein was obtained.The polyclonal antibody was obtained by immunizing mice.Eukaryotic expression vector was constructed.Cell transfection and confocal analysis by different deletion mutants showed that the Viperin protein was mainly localized to the endoplasmic reticulum organelle,and the 1-50 amino acids of Viperin were closely related to its location in the endoplasmic reticulum.(3)The role and mechanism of porcine Viperin in PEDV infection: The expression of type I interferon and Viperin were increased in PEDV-infected cells;construction of eukaryotic expression vector of veprin,we used porcine intestinal epithelial cells(IPEC-J2)model to study the effect about oversxpression of Viperin on PEDV,the overexpression or knockdown experiments of Viperin indicated that Viperin can affect PEDV viral load;Co-IP experiments showed that Viperin protein can interact with PEDV N protein,and deletion mutant transfection experiments showed that its S-adenosylmethionin domain is a key region for interaction with PEDV N protein.This study laid the foundation for further research on the antiviral innate immune mechanism of Viperin.In summary,this study successfully cloned the porcine Viperin gene,and obtained porcine Viperin recombinant protein.The 1-50 amino acid of porcine Viperin is an endoplasmic reticulum targeting sequence that allows Viperin to anchor to the endoplasmic reticulum.The porcine Viperin has a colocalization and interaction with the structural protein N of PEDV,and its S-adenosylmethioninase domain is a key region for interaction with PEDV N protein.After PEDV infection,the expression level of interferon was down-regulated in IPEC-J2 cells,and the expression level of antiviral protein Viperin was up-regulated.Indicated that Viperin may exert antiviral effects by up-regulating expression and its interaction with N proteins affecting the assembly and proliferation of viruses.