Localization Of Key Amino Acids In Cytoplasmic Tail Of Newcastle Disease Virus Fusion Protein And Their Effects On Cell Autophagy

Background:Newcastle disease virus,a single-stranded negative-strand RNA virus with envelope,belongs to the Paramyxoviridae,Paramyxivirinae,Avian Paramyxovirus.The hemagglutinin-neuraminidase protein(HN)and fusion protein(F)on the NDV envelope play a crucial role in the membrane fusion process and pathogenicity.Recent studies have shown that NDV has a specific killing effect on tumor cells.F protein and HN protein are also indispensable for the oncolytic process.The NDV F protein consists of 553 amino acids with five glycosylation sites,all of which are located in the in the ectodomain.The F protein,a trimer structure,has five functional domains:a cleavage site and a fusion peptide(FP),two heptad repeat domains(HR),a transmembrane region(TM),and a cytoplasmic tail(CT).The CT consists of 31 amino acids(amino acid position 523-553).It was found that the length and amino acid type of CT among several paramyxoviruses were different.Studies have shown that the deletion of NDV F protein CT reduces the formation of syncytia significantly.It is well known that the ectodomain of the F protein have important implications for cell fusion,studies on other type I glycoproteins,such as retroviruses,herpesviruses and other paramyxovirus fusion proteins,have also shown that CTs affect viral entry,F protein cleavage and fusion activity.In addition,studies have shown that CTs are also involved in the process of transporting newly synthesized glycoproteins from the endoplasmic reticulum to different intracellular compartments and cell surfaces.At present,there are few studies on site-directed mutagenesis of amino acids in the CT of NDV F protein over the world.The deletion study of Sergel T et al showed that the deletion of 540-546 aa in the posterior segment of the CT had a greater effect on fusion function than the deletion of 525-532 aa in the anterior segment of the CT.However,the key amino acids are still unknown.Therefore,in this study,we mutate this part of the amino acid to alanine(A)from the posterior segment of the CT with the site-directed mutagenesis and vivo homologous recombination method to locate the key amino acids that affect the function of the F protein in the CT.In the future research,reverse genetics can be used to rescue mutant viruses with significantly decreased and increased fusion function,which can provide better candidate virus strains for the research of Newcastle disease recombinant vaccine and NDV oncolytic function.There is a complex relationship between autophagy and viruses.Autophagy can not only resist the invasion of certain viruses,but also provide favorable conditions for the replication of some viruses.Studies have shown that the Newcastle disease Beaudette C strain can induce autophagy production and facilitate its replication in U251 glioma cells and chicken fibroblasts(DF-1).The researches on the structural proteins NP and P of NDV suggested that it can induce autophagy through the endoplasmic reticulum stress pathway,but the molecular mechanism of NDV-induced autophagy and the effects of envelope glycoproteins HN and/or F on autophagy are still needed further research and exploration.Objectives:1.To explore the effect of NDV F cytoplasmic tail on the process of promoting cell fusion.2.To locate key amino acids in the cytoplasmic tail of NDV F.3.To explore the effect of NDV envelope glycoprotein on autophagy in different cells.Methods:1.Mutant construction:Seven NDV F CT mutants,G540 A,N541 A,N542A,L544A,G545A,Q546A,and M547A,were obtained using site-directed mutagenesis and homologous recombination technology to start from the posterior CT segment that has an obvious effect on F function.2.Protein expression:T7 transient expression system of recombinant vaccinia virus was used to transfect each mutant into BHK-21 cells for protein expression.3.Detection of protein expression efficiency:The indirect immunofluorescence method and flow cytometry were used to qualitatively and quantitatively detect the protein expression efficiency of each mutant.4.Detection of mutant fusion activity:The R18 dye transfer experiment,indicator gene method,and Giemsa staining were used for qualitative and quantitative analysis of the three phases of each mutant protein promoting cell fusion.5.Detection of mutant cleavage activity:Western blot was used to detect whether or not the mutation function was related to the cleavage activation of F protein.6.Detection of autophagy marker proteins:Western blot was used to detect autophagy marker proteins LC3? and p62 to determine autophagy status.7.Detection of autophagosomes:The formation of autophagosomes in cells under different treatment conditions was detected by immunofluorescence,and the effects of NDV and its envelope glycoprotein on autophagy in different cells were analyzed.Results:1.Sequencing results showed that all 7 NDV F mutants were successfully constructed and named as:G540A,N541 A,N542A,L544A,G545A,Q546A,M547A.2.Except for the reduced expression level of M547A(66.3%of wild type),the expression level of F protein of other mutants on the cell surface is between 91.3%and 111.4%of wild type F(P>0.05).3.Compared with wild type F,the semi-fusion activities of mutants N542A,L544A,and M547A all changed significantly,which were 189.8%,58.5%,and 17.3%of wild type,respectively,while those of mutants G540A,N541A,G545A,Q546A were between 80.1%and 117.5%of the wild type(P>0.05).The contents mixing abilities of mutants N542A,L544A,and M547A were 248.5%,62.2%,and 12.6%of the wild type,respectively,while those of other mutants were between 95.8%and 119.9%of wild type F protein(P>0.05).M547A almost lost its ability to form syncytia,while mutant N542A's ability to form syncytia increased significantly.4.The cleavage activity of each mutant was similar to that of wild-type F,and the expression of N547A decreased(the same as in the above result 2),but the expressed mutant protein could still be activated by cleavage.5.Western blot results showed that there was no significant difference in p62 protein levels between the three treatment groups after 24 hours and the blank control(P>0.05).LC3II in cells treated with rapamycin and infected with NDV BJ strain for 24 hours was significantly higher than that in the blank group,and the difference was statistically significant(P<0.05).The expression of wt HN protein alone in BHK-21 and Vero cells had no effect on the content of LC3II,but when wt F/N542A was expressed alone and co-expressed wt F+wt HN/N542A+wt HN,the LC3II content was significantly higher than that of the blank control The difference was statistically significant(P<0.05).LC3II content was significantly higher in DF-1 cells when N542A was expressed alone and co-expressed wt F+wt HN/N542A+wt HN compared with the blank control(P<0.05).6.Immunofluorescence results show that cells can observe significant green dot-like fluorescence production in cells 24 hours after infection with NDV BJ strain and when they express wt F/N542A alone or co-express wt HN+wt F/wt HN+N542A.Conclusions:1.NDV F CT is very important for the membrane fusion process,and amino acid mutations in this region have varying degrees of effect on cell fusion activity.2.N542,L544 and M547 are the key amino acids of NDV F CT,which play an important role in the fusion activity of F.3.NDV F can induce cell autophagy alone in BHK-21 and Vero cells,and has a stronger ability to induce autophagy when it interacts with HN.4.The ability of NDV envelope glycoprotein to induce autophagy is positively correlated with the fusion activity of F.