Antiviral Functional Study Of N-terminal Domain Of Swine Interferon-induced Transmembranes

Interferon-induced transmembranes(Interferon-induced transmembranes,IFITMs)belong to a class of small transmembrane proteins,which have been widely distributed in vertebrates.In the course of evolution,IFITMs are highly conservative.Among them,IFITM1,IFITM2,and IFITM3 are related to the body’s natural anti-viral immune response.They limit virus infection through multiple pathways and mechanisms.Existing research believes that IFITMs are the only type of effector that blocks the process of viral membrane fusion.A large number of studies have reported the antiviral activity of IFITMs.A variety of enveloped viruses and some non-enveloped viruses are located in the antiviral spectrum of IFITMs,but the exact antiviral mechanism of action still needs further study to determine.This article mainly analyzes and explores the genetic evolution relationship,structure,function and other characteristics of pig-derived IFITMs.1.In order to verify the genetic evolution relationship and structural characteristics of pig-derived IFITMs,this study completed the cloning of swine IFITM1,2,and 3 genes,and analyzed and predicted IFITMs using bioinformatics methods.The results showed that the IFITMs gene coding region consists of two Exon composition,the encoded proteins are all stable proteins,showing overall hydrophobicity,the theoretical isoelectric point is located in a slightly alkaline environment;IFITMs are highly conserved in different species,and the conserved regions are mainly located in the middle of the protein molecule CD225 Domains;porcine IFITM1,2,and 3 genes have the highest homology with chimpanzees,with homology of 70.2%,67.7%,and 70.5%,respectively;predictions show that IFITM1,IFITM2,and IFITM3 are also type 2 transmembrane proteins and are located at The plasma membrane,endosomal membrane and other structures are partially exposed to the cytoplasm without signal peptides and enzyme binding sites;homology modeling shows that the IFITMs molecules are rich in α-helix structures,and the N-terminus of IFITM2 and IFITM3 curls irregularly.There may be an endocytotic sorting signal,an amphiphilic helix structure and a hydrophobic region in the CD225 domain;combined with comparative genomics studies,screening and prediction Y19,K23,C70 and some other functional residues and structural motifs.2.Using E.coli and affinity chromatography to express and purify the proteins of pig IFITM1,2,3,and mix the protein with adjuvant to immunize the animal,and successfully prepare the cloned antibody of rabbit-derived anti-IFITMs.The results show that the antibody It has a good specific reaction with IFITMs.3.To further clarify the antiviral effects of IFITMs in various swine viral infections,this study explored the interaction between IFITMs and TGEV,PRV,and PCV2 at the cellular level.The results showed that after 24 hours of infection of PK15 cells with TGEV and PCV2,IFITM1 and The m RNA level of IFITM3 was up-regulated and further increased after 36 h.In PRV-infected PK15 cells,the transcription level of IFITM1 and 3 showed a short-term increase and then significantly decreased;overexpression of IFITM1 on TGEV,PRV and PRV infected PK15 cells All have different degrees of inhibition.Overexpression of IFITM3 also reduced the replication level of TGEV and PCV2,but PRV infection lower than that of the empty vector control group could not be detected in cells overexpressing IFITM3.In addition,the results of truncated mutagenesis analysis of IFITMs NTD domain show that the amino acid residues 19 to 56 of IFITM3 NTD domain(19YEMLKEEHEVAVLGAPQTSAPVATTVINIRSETSVPDH56)have an important contribution to the anti-TGEV activity of the entire protein molecule,and The N-terminal structure of IFITM1 is not indispensable for its antiviral function.In summary,this study provides clues for a deeper understanding of the structure and genetic evolution of IIFTMs in pigs,confirms the inhibitory effect of IFITMs on TGEV replication in vitro,and also analyzes functional residues on the NTD domain of IFITMs.These results will be The research on the mechanism of action of swine IFITMs laid a preliminary foundation.