Study Of IFITM1 Restriction Mechanism On PEDV Invasion Into IPEC-J2 Cell

Interferon induced transmembrane proteins(IFITMs)play an important role in the innate immune response caused by virus infection.Previous studies have shown that IFITM1,2,and 3 are involved in innate immunity of the body and can inhibit a variety of encapsuled RNA virus infections.Porcine epidemic diarrhea virus(PEDV)is a pathogen that causes intestinal infectious diseases in pigs,the high fatality rate of infected piglets causes serious harm to pig industry in China.PEDV is a single stranded RNA virus with a capsule structure.This study found that endogenous IFITM1 expression was induced after PEDV infection of IPEC-J2 cells,while overexpression of IFITM1 could inhibit the infection of PEDV on host cells.Further investigation demonstrated that it plays a role in the invasion stage of viral infection.This study aims to explore the effects of IFITMs on PEDV infection in susceptible cells.The main research contents are as follows:1.PEDV infection induced changes in immune-related cytokine expression in IPEC-J2 cells.The proliferation of PEDV in IPEC-J2 cells infected with PEDV(MOI=0.1)at different time points and the m RNA levels of IFNs,interleukin and IFITMS were examined by RT-PCR.2.To study the effect of IFITM1 on PEDV infection.IPEC-J2 cell lines with overexpessed or downregulated expression of IFITM1 were established through lentivirus transduction system and RNAi interference technology.Intriguingly,the results from RT-PCR revealed that the overexpression of IFITM1 could inhibit the proliferation of PEDV in IPEC-J2,while the interference of IFITM1 had opposite effect,suggesting that IFITM1 affects the proliferation of PEDV in host cells.3.To explore specific stages of anti-PEDV action of IFTM1 and the important amino acid residues involved,the eukaryotic expression plasmids including p CINEO-IFITM1 and those of IFITM1 mutants were constructed and introduced to achieve transient expression in IPEC-J2 cells,followed by the infection of MOI=0.1 PEDV.The localization of IFITM1 and PEDV in cells after PEDV infection was revealed by Immunofluorescence assay(IFA),the results showed that IFITM1 was observed in the cytoplasm mainly surrounding the nuclei of the transfected cells,and the co-localization of it with PEDV could be identified.Real-time PCR results showed that: Transient expression of IFITM1 did not affect PEDV adsorption,while the C-terminal domain(CTD)mutations of IFITM1 inhibited PEDV adsorption.Specifically,the CTD mutations of IFITM1 reduced its inhibitory function on PEDV proliferation,with the amino acid residues 113 to 117(113q MLER117)in the CTD of IFITM1 having a pronounced effect.In summary,this study confirmes an inhibitory role for IFITM1 in vitro replication of PEDV,and determines the functional residues on the CTD of IFITM1 being associated with this action.Therefore,these findings provide a theoretical foundation for elucidating the mechanism of inhibition of PEDV infection by porcine IFITM1.