Porcine IFITM3 Inhibited The Early Infection Of PK15 Cells By TGEV And Its Mechanism

Interferon-inducible transmembrane proteins（IFITMs）,as a host restriction factor,played an important antiviral effect in innate immunity.IFITMs restricted a variety of enveloped RNA viruses from invading host cells,and inhibited virus replication by preventing the release of viral nucleic acid into the cytoplasm.Coronavirus is a positive-strand RNA virus,which seriously threatened public health safety and the development of the breeding industry.Transmissible gastroenteritis virus（TGEV）is a member of the coronavirus.Its infection caused piglet diarrhea and huge economic losses to the pig industry.There is no research to explore whether IFITM3 has an antiviral effect on TGEV infection and related mechanisms.Therefore,this study aims to explore the antiviral mechanism of pig-derived IFITM3 against TGEV.The main research contents are as follows:1.TGEV infection stimulated the natural immune response of PK15 cells.PK15 cells were infected with TGEV by the same MOI.RNA was harvested at different times,and Real-time PCR was used to detect the transcription level of cytokine mRNA related to natural immunity in the cells after reverse transcription.The results showed that TGEV infection caused significant increases in IFNs,TNF-α,IL-1β,IL-6,IL-18,IFITM1、IFITM2、IFITM3 and Mx1 mRNA levels in host cells.Later,Western Blot was used to detect the expression level of endogenous IFITM3 protein caused by TGEV infection.The results showed that the protein expression level of IFITM3 increased significantly with the increase of infection time,which was basically consistent with the results of Real-time PCR.2.Successfully constructed cell lines that stably overexpress porcine IFITM3 or stably interfere with endogenous IFITM3.The PK15 cell line（PK15-IFITM3）that stably overexpressed porcine IFITM3 and the PK15 cell line（PK15-IFITM3KD）that stably interfered with the expression of endogenous IFITM3 were constructed by lentiviral expression system and RNAi technology.Indirect immunofluorescence assay（IFA）and Western Blot were used to detect the localization and expression of IFITM3 in PK15-IFITM3 cells.The results showed that the overexpression of IFITM3 protein was distributed in the cytoplasm,and compared with the control group,its expression level was Significantly improved;Real-time PCR results also showed that the mRNA expression level of IFITM3 in PK15-IFITM3KD cells was down-regulated by nearly 77%.Further MTT assay results showed that the cell activity of PK15-IFITM3 and PK15-IFITM3KD was not significantly different from that of normal PK15 cells.3.IFITM3 has important amino acid residues on the antiviral mechanism.PK15-IFITM3 and PK15-IFITM3KD cells were infected with TGEV by the same MOI.the copies of TGEV and the expression of inflammatory cytokines in the cells were detected by Real-time PCR.The results showed that compared with the normal PK15 group,the copies of TGEV in IFITM3 cells decreased significantly,and the transcription level of inflammatory cytokine mRNA also decreased significantly.In contrast,the copies of TGEV in PK15-IFITM3KD cells was significantly increased.Further studies found that IFITM3mainly inhibited the adsorption of TGEV to cells.In addition,¹⁸TYEMLK²⁵ in the N-terminal domain of IFITM3 was respectively mutated to alanine.It was found that the IFITM3 Y20A and M22A mutants promoted TGEV replication,whereas the K24A mutants improved the inhibitory effect of IFITM3 on TGEV.4.IFITM3 cooperated with endosomal inhibitors to inhibit TGEV infection.Our study infected PK15-IFITM3 or PK15-IFITM3KD cell lines with TGEV by the same MOI,and treated the cells with endosome acidification inhibitor NH₄Cl or Chloroquine Phosphate（CQ）.Real-time PCR was used to detect the copies of TGEV and the mRNA expression of inflammatory cytokines.It was found that endosomal inhibitors can promote the antiviral effect of IFITM3 on TGEV infection.In addition,the inflammatory cytokine mRNA level in the cells treated by CQ or NH₄Cl was also significantly lower than that in the control group.Conversely,endosomal inhibitors can compensate to a certain extent for the loss of antiviral effect caused by the loss of IFITM3.