Localization Of The Key Amino Acids In The Transmembrane Domain Of NDV Fusion Protein And Its Effect On The Polarization Of Human Macrophages

Background:Newcastle disease virus(NDV)belongs to the Paramyxoviridae,Paramyxovirinae,Paramyxovirus.It can cause highly infectious diseases in poultry and cause serious economic losses,classified as a Class B infectious disease by the World Organization for Animal Health.The disease develops rapidly in poultry infected with NDV,with a high mortality rate,mainly manifested by symptoms such as reduced egg numbers,respiratory distress,neurological disorders,and diarrhea.Human beings may be infected and develop conjunctivitis or lymphadenitis when exposed to sick poultry and live vaccines,mainly manifested as self-limiting flu-like symptoms.Variant strains are constantly appearing in various places,and the types of Paramyxoviruses are also increasing.Further exploration of NDV as a typical representative of paramyxovirus can not only provide the theoretical basis for NDV vaccine research and antiviral drugs,but also provide some references for researches on other paramyxoviruses.Cell fusion is the key step for NDV's invading its host cells.The entry of NDV into host cells is mediated by two kinds of surface glycoproteins,hemagglutinin-neuraminidase(HN)protein responsible for the binding of the virus to the cell's receptor and fusion(F)protein that mediates the fusion of the virus with the host's membrane.The NDV F protein is synthesized into precursor F0,which is activated by the host protease into a disulfide-linked F1 and F2 polypeptide.F protein is a trimeric structure,divided into head,stalk,transmembrane domain,and cytoplasmic tail.Many studies have found that the transmembrane domain not only acts a part in membrane anchoring in F protein,but also affects F protein's structure and function.Studies showed that replacing the TM region with a glycosylphosphatidylinositol(GPI)anchor will cause the protein to fail to complete membrane fusion.The L486 and 1488 of the parainfluenza virus(PIV5)F protein TM domain play a key role in the fusion function.Replacing the transmembrane domain of NDV with the measles virus and Sendai virus can cause defects in fusion activity,but the key amino acids in this domain are not yet clear.Therefore,in this study gene-directed mutagenesis technology and homologous recombination technology were used to mutate L501,1502,1505,1510,V513,V516,L519,and V520 to alanine respectively,except A500 that is mutated to T,so as to find the pivotal amino acids that have an effect on the structure and function of F protein in the transmembrane domain.Newcastle disease virus,a promising oncolytic virus,can also play an oncolytic role by direct killing or causing cancer cells to immunogenic death.Macrophages act as an important part of the immune system and have polarizing properties.NDV may also exert an oncolytic effect on the polarization of human macrophages.Therefore,NDV BJ strain,La Sota strain,NDV HN and F were used to study the effect of the NDV on human macrophage polarization.Objectives:1.To determine the key amino acids in the transmembrane domain of NDV F protein.2.To analyze the effect of NDV F protein transmembrane domain on membrane fusion function.3.To study the effects of velogenic strain,lentogenic strain,HN,and F protein on the polarization of human macrophages.Methods:1.Mutant construction:site-directed mutagenesis and homologous recombination techniques were used to construct mutants.2.Protein expression:each mutant protein was expressed in BHK-21 cells with the help of T7 polymerase provided by recombinant vaccinia virus.3.Detection of protein expression efficiency:qualitative indirect immunofluorescence and quantitative flow cytometry were utilized to verify the expression of the mutant protein.4.Detection of mutant fusion activity:indicator gene assay was used to detect the mixing ability of the mutant proteins,R18 dye transfer experiment was used to detect the semi-fusion activity of the mutant proteins,and Giemsa stain was used to detect the abilities of the mutant proteins to form syncytia.5.Detection of mutant cleavage activity:Western blot was used to analyze whether or not the functional changes of the mutants were related to the cleavage activity of F protein.6.Detection of polarization markers of human macrophages:real-time fluorescent quantitative PCR and Western Blot were applied separately to detect the expression of macrophage markers.Results:1.Sequencing results showed that nine mutants were successfully constructed and named A500T,L501A,1502A,I505A,1510A,V513A,V516A,L519A,and V520A.2.The expression of L519A and V520A mutant proteins is seriously defective,while the cell surface expression level of the other mutant proteins is between 70%?90%of the wild-type F protein(P>0.05).And the Western blot experiment revealed that the mutant protein was successfully cleaved into F1 and F2 with fusion activity except for L519A and V520A.3.The experiment results showed that the mutants L519A and V520A have no fusion function due to protein expression defects.In the semi-fusion stage,the remaining mutants showed a degree of semi-fusion similar to that of the wild type.However,in the mixing stage of the contents,the mixing capacity of the A500T,1505A,V513A,and V516A mutants decreased,which were 85.38%,67.05%,55.38%,and 51.13%of the wild type respectively(P<0.05),while the mutants L501A,I502A,and I510A were 86.69%,83.92%,and 71.24%of the wild-type F protein(P>0.05).Finally,at the syncytial stage,the abilities of L501A and I502A mutants to form syncytia are 100%and 104%of the wild-type(P>0.05),while I510A mutant is 76%of the wild-type(P>0.05).The number and area of syncytia formed by A500T,1505A,V513A and V516A are reduced,only 50%?74%of the wild type(P<0.05).4.After the cells were infected by BJ strain and La Sota strain,the expression of iNOS mRNA showed an overall upward trend and then a downward trend,and the difference was statistically significant at 24 hours(P<0.01).The expression of M1 marker gene TNF-? of the BJ strain was the highest at 4h,which was significantly different from that of the La Sota strain(P<0.01),and then the expression gradually declined.The La Sota strain increased first and then decreased,reaching the highest point at 12h.The expression of CD206 of human macrophages infected by B J strain and La Sota strain was almost the same after infection for 4h?12h.After 24h,the CD206 expression of BJ strain significantly exceeded that of La Sota strain(P<0.01).After BJ strain and La Sota strain infected human macrophage cells,the expression of IL-10 showed a gradual upward trend,and showed a statistical difference at 48h(P<0.01).After HN protein was transfected into THP-1 cells,the expression of iNOS was always higher than that of F protein,statistically significant at 24h and 72h(P<0.05;P<0.01),while the expression of TNF-? of F protein was higher than HN protein.For the expression of M2 marker factor CD206,HN protein was higher than F protein at 12h,48h,and 72h(P<0.01,P<0.05,P<0.01),and the expression of IL-10 of F protein was higher than HN protein at 48h(P<0.05).Conclusions:1.The amino acids in the transmembrane region of ND V F protein are very fundamental to the fusion function.2.A500T,I505A,V513A,and V516A are the key amino acids in the transmembrane region of NDV F protein,which affect the fusion activity of F protein,while L519A and V520A have an impact on the transport of F protein.3.BJ strain induces a stronger M1/M2 mixed phenotype of macrophages than La Sota strain.Compared with the strains,HN and F proteins induce a more moderate M1/M2 mixed phenotype,and there is no obvious difference between HN and F.