Study On The Role And Mechanism Of IFITMs Against Newcastle Disease Virus Infection In Chickens

Host Restriction factor(HRFs)Interferon induced transmembrane proteins(IFITMs)are small transmembrane proteins produced by interferon induction,which have become a hot topic in recent years due to their broad-spectrum antiviral activity and unique ability to inhibit viral IFITMs are small transmembrane proteins produced by interferon induced transmembrane proteins.Since the first report of their antiviral activity in 1996,IFITMs have been shown to limit infection by a variety of viruses,including influenza A virus,Ebola virus,severe acute respiratory syndrome virus,HIV and Zika virus,some of the major viruses that pose a serious threat to human health and social stability,but there is no known association between IFITMs and Newcastle disease virus(NDV).However,there are no reports of IFITMs being associated with Newcastle disease virus(NDV).Studies have shown that IFITMs exert antiviral activity mainly during virus entry,in particular by interfering with the fusion of the envelope and endosomal membrane,or by forming fusion micropores to prevent virus entry into the cytoplasm.However,the exact mechanism by which they do so is unclear.In the present study,we investigated the inhibition of NDV infection by chIFITMs in detail and systematically using chicken interferon-induced transmembrane proteins(chicken IFITMs,chIFITMs)and NDV,and resolved the mechanism.Firstly,three chicken-derived IFITMs(chIFITM1,chIFITM2 and chIFITM3)were successfully amplified using DF-1 cells,and the cloned fragments were analysed.The sequencing results showed that the target genes were successfully obtained with the same sequence as the reference genes;homology analysis showed that the amino acid homology of IFITMs of all species was above 20%,and the amino acid sequence homology between mammalian IFITMs was above 50%,while the homology between chIFITMs and mammals was generally low(around 30%),suggesting that although they belong to the same class of proteins,the protein sequence analysis showed that chIFITMs have similar structures to other species,including NTD,IMD,CIL,TMD,CTD and other structural domains,and have many similar conserved sites and sequences,such as Y20 and Gxxx G,etc.The similar functional sites and functional structures suggest that chIFITMs may play similar roles The genetic evolutionary results reflect the differences in IFITMs proteins among species,with IFITMs in mammals such as human,mouse and pig clustered in the same larger branch,while chIFITMs are located in another branch that distinguishes them from other species,a result that may be related to the fact that mammalian genomes have undergone rapid evolution over the same period of time,while avian genomes have experienced significant gene loss during the evolutionary process The topological,secondary and tertiary structure predictions found that the topological,secondary and tertiary structure predictions of chIFITM1 were generally consistent,but the secondary structure prediction of chIFITM2 showed three helix structures while the tertiary structure prediction showed two helix structures;the secondary structure prediction of chIFITM3showed three helix structures and the tertiary structure prediction showed three The above predictions are revealing for our understanding of chIFITMs,but their true structures need to be further determined by our experiments.The current studies on chIFITMs are mostly focused on genetic evolution,sequence analysis and antiviral phenotypes,and there is a lack of analysis and comparison of the antiviral systems of chIFITM1,chIFITM2 and chIFITM3,the main members of this family,as well as the exploration of antiviral mechanisms.Therefore,we constructed recombinant eukaryotic plasmids expressing chIFITMs based on the amplified target fragments,and analyzed their antiviral effects by transient overexpression.The results showed that the transient overexpression of chIFITMs significantly inhibited the proliferation of various viruses,including NDV,in DF-1 cells,and the antiviral effects of chIFITM1,chIFITM2 and chIFITM3 were not significantly different from each other;meanwhile,the antiviral effects of chIFITMs were dose-dependent;in addition,the overexpression of chIFITMs in heterologous cells In addition,chIFITMs overexpressed in heterologous cells also showed antiviral effects but with cellular variability,with the viral inhibition rate in the order of porcine>murine>monkey>human.In order to further confirm the antiviral effect of chIFITMs and elucidate their mechanism,chIFITM1,chIFITM2 and chIFITM3 inducible cell lines were constructed using the Tet-On system,and the positive cells were selected for mass culture after double antibiotic stress screening,and the optimal expression conditions were explored by setting different inducer concentrations and induction times.The optimal Dox induction conditions for chIFITM1,chIFITM2 and chIFITM3 were 2.5μg/m L for 24h,2.5μg/m L for 24 h and 5μg/m L for 24 h,respectively;the expression of target genes in chIFITM1,chIFITM2 and chIFITM3 cell lines after Dox induction increased by1.6×10~5,8.7×10~3 and 1.7×10~4 fold respectively after Dox induction.The cell lines were also analysed for antiviral ability using strong and weak strains of NDV,as well as vesicular stomatitis virus(VSV-EGFP)and influenza virus subtypes H3N2 and H9N2,and the results showed that the overexpressed cell lines had good antiviral effects and showed broad-spectrum antiviral effects.To further elucidate the mechanism of action of chIFITMs against NDV infection,the effect of chIFITMs on NDV adsorption and entry was firstly analysed using the classical virus binding-entry assay.The results showed that the virus adsorption process was not affected by Dox induction in the overexpression cell lines,but significantly affected the virus entry process.chIFITMs overexpression group had more than 50%lower virus entry compared to the control group.chIFITM1 and chIFITM2 restricted virus entry to cells at comparable levels,slightly higher than chIFITM3,but no the difference between chIFITMs was not significant.In addition,the relationship between cell membrane fluidity and the antiviral effect of chIFITMs was investigated,and the results showed that the overexpression of chIFITMs reduced cell membrane fluidity,suggesting that they may inhibit NDV entry by affecting cell membrane fluidity.The membrane fluidity of the chIFITM1 and chIFITM3 groups was reduced by about 50%and that of the chIFITM2 group by about 25%.The ultimate goal of chIFITMs to function in cells is to protect them from viral infection and attack and to improve cell survival.To this end,the relationship between chIFITMs and virus-induced cell death was analysed.CCK8 assay results showed that chIFITMs significantly inhibited virus-induced cell death(p<0.001),and the protection rate was not significantly different between chIFITMs,which were all around 30%.crystalline violet staining results were consistent with CCK8 assay results.The chIFITMs group stained darker compared to the empty vector group,indicating a higher cell survival rate.Finally,to further analyse the role of chIFITMs in the interferon-mediated antiviral effect.The expression and preparation of chicken-derived type III interferon(chIFNL3)was first performed using a baculovirus expression system.During the preparation,a previously unidentified novel chIFNL was identified in this study.Genetic evolutionary analysis showed that it belongs to type III interferon with 57.1%homology to chIFNL3,which is located at 1-354 bp of the chIFNL3 locus and shares the same N-terminal sequence with chIFNL3(163 bp,54 aa),unlike chIFNL3,it is not cleaved at 163 bp and has a continuous 354 bp open reading frame(ORF),which was found to induce the expression of ISGs,to be dependent on the IFN III receptor,and to have broad-spectrum antiviral activity.chIFNL3a.On this basis,the role of chIFITMs in the chIFNL-mediated antiviral effect was analysed.The results showed that the ability of chIFNL(chIFNL3 and chIFNL3a)to inhibit NDV replication was significantly reduced by transiently knocking down endogenous chIFITMs using RNAi interference technology,but there were differences in the antiviral activity mediated by different chIFITMs across chIFNL,with the chIFITM3 on the antiviral activity of chIFNL3 The difference in the effect of chIFITM3on the antiviral activity of chIFNL3 was the most significant(P<0.0001),the difference in the effect of chIFITM1 on the antiviral activity of chIFNL3a was the most significant(P<0.001),and the interference of chIFITM2 did not have a significant effect on the antiviral effect of chIFNL3 and chIFNL3a,probably due to its lower background expression in DF-1 cells.The above results show that the different chIFITMs differ in their antiviral effects mediated by different interferons,which may be caused by the induction preferences of different interferons on chIFITMs at the time of action,with chIFITM3 being more important for the chIFNL3-mediated antiviral pathway and chIFITM1 being more important for the chIFNL3a-mediated antiviral pathway.In summary,this study confirmed that exogenous chIFITMs significantly inhibited the proliferation of multiple viruses in DF-1 cells,and there was no significant difference in the antiviral effects of the three chIFITMs.The mechanisms by which exogenous chIFITMs exert their antiviral effects may be as follows:(1)they limit virus replication in cells by affecting the process of virus entry and reduce the level of virus infection;(2)they affect NDV Na virus replication by regulating cell membrane fluidity;(3)chIFITMs can prevent virus-induced cell death and improve cell survival.In addition,endogenous IFITMs could modulate chIFNL-mediated anti-NDV Na virus effects.The reduction of endogenous IFITMs affected the antiviral effects of chIFNL3and chIFNL3a in DF-1 cells,but there were differences in the regulation of antiviral effects of different interferons by different IFITMs.