Analysis Of Gene Mutation In The Interaction Domain Of Head And Stalk Of Newcastle Disease Virus Hemagglutinin-Neuraminidase Glycoprotein

Background:Newcastle disease virus(NDV)belongs to the genes avian rubulavirus of the family Paramyxoviridae,a kind of non-segmented,single-stranded,negative-sense RNA virus.NDV is a pathogenic factor for Newcastle disease(ND),a fulminating infectious viral disease endangering the development of poultry that progressing rapidly with a high mortality rate.The major symptoms of ND are mucosal hemorrhage,diarrhea,dyspnea and nervous disorder,etc.People might suffer mild symptoms such as conjunctivitis,transient influenza-like illness after the infection of NDV.Although the NDV vaccines alleviate the large-scale outbreak to some extent,the research and development of antiviral drugs for NDV also encountered great challenges as the mutants of NDV constantly appearing in avian.The main difficulty is that the specific fusion mechanism of viral envelope and membrane of NDV infected host cell is not clear.NDV is a typical representative of paramyxovirus,understanding the cell fusion-promotion mechanism of NDV will provide reference for the study of other paramyxoviruses.Cell fusion is critical for the entry and proliferation of NDV,and also the typical pathological feature for NDV infecting host cells.The membrane fusion between the viral envelope and cell membrane caused by NDV require coordinate expression of hemagglutinin-neuraminidase(HN)protein with its cognate fusion(F)protein,indicating that there is must be a certain signal communication or interaction between HN and F protein,A majority of researches hold that a certain signal generated and is transduced to the interaction area of HN with F protein after the binding of globular head of HN with sialic acid receptors.As a result,the interaction between HN and F protein can activate F protein by promoting proteolysis reaction and conformational change.Structure forms of four-heads-up and four-heads-down position are proposed by Welch BD based on the X-ray crystal structure,which indicates that the HN heads are in a pendulous position before binding to the sialic acid receptors.The drooping homologous tetramer heads conceal the amino acid residues in the upper segment of the HN 4HB(49-78)that hindered the interaction between HN and F protein.Next model is that globular heads rise after binding to the sialic acid receptors,which exposed the interaction domain of HN to activate F protein.Here the conformational change of NDV HN involves two interaction domains that are contained in 4HB stalk.One is the interaction domain of HN stalk with F protein(49-78),which take an active participate in the activation of F protein.The other one is the interaction domain of HN stalk with its head(91-112),which plays an important role in maintaining the normal transition of head.Up to now,studies about HN stalk are mainly focused on the interaction domain between H-N and the F protein.However,study on the interaction domain between the HN head and stalk has not been reported yet.In this study,NDV HN was used as the research object.By comparing the NDV HN(91-112)amino acid sequence with that similar amino acid sequence of hPIV3 HN,MeV H,RSV G,the deletion mutants and chimeras of NDV HN(91-112)aa were constructed.By detecting the changes in the activity and function of the mutant HN protein,we further understand the biological function of NDV HN(91-112)aa and the possible mechanism of promoting cell fusion.Objectives:To study the influence of the mutants of NDV head and stalk interaction domain on the biological activity and cell fusion promotion,analyze the possible mechanism for the effect of head and stalk interaction domain on cell fusion,in order to provide theoretical basis for the research and development of safe and effective antiviral drugs and construct recombinant tumor vaccines for anti-tumor treatment.Methods:1.Construction of NDV HN mutants:the amino acid sequence of NDV HN(91-112)was compared with amino acid sequences of hPIV3 HN,MeV H and RSV G,respectively,to determine the similar gene sequences of hPIV3 HN,MeV H and RSV G.PCR fragment mutation and homologous recombination technique were combined to construct the deletion mutants De(HN)and chimera Ch(hPIV3),Ch(MeV),Ch(RSV).2.Expression of NDV HN mutant proteins:cationic liposome transfection agent ExFect2000 was used to transfect HN mutant plasmid and wide-type F plasmid into baby hamster kidney(BHK-21)cells.The recombinant vaccinia virus vTF7-3 transient expression system was created to express mutant proteins.3.To detect the function of NDV HN mutant proteins:the qualitative and quantitative results of the cell fusion promotion activities of NDV HN mutant proteins were measured by Giemsa stain and reporter gene method.Dye transfer method was used to analyze the semi-fusion activity of mutant proteins.The receptor recognition activity was analyzed by hemadsorption assay.The fluorescence method was used to determine neuraminidase(NA)activity.4.To detect the expression of NDV HN mutant proteins on cell surface of BHK-21:the qualitative expression of mutant protein was detected by indirect immunofluorescence(IIFA).The quantitative analysis of the expression efficiency on cell surface of BHK-21 was conducted by flow cytometry(FCM).5.Statistical analysis:Statistical analysis was conducted by SPSS20.0 software.The multiple activities of NDV HN proteins were expressed as the form of mean±standard deviation(x±s)and were analyzed by T-test.P<0.05 means the difference was statistically significant.Results:1.The results of DNA sequencing showed that NDV HN(91-112)amino acids were deleted and substituted respectively and delete mutation De(HN)and chimera Ch(hPIV3),Ch(MeV),Ch(RSV)were all constructed successfully.2.Cell surface expression level of chimeras Ch(hPIV3),Ch(MeV),Ch(RSV)were 75.65%,82.20%,70.16%of that of wild-type(wt)HN,respectively(P>0.05).But cell surface expression level of De(HN)protein decreased dramatically,only 9.04%of that of wt HN.3.Fusion promotion activity of De(HN),Ch(hPIV3),Ch(MeV),Ch(RSV)were 3.83%?57.84%?24.76%,29.42%of that of wt HN,respectively(P<0.05).The fusion promotion activity of De(HN)was almost disappeared and syncytium couldn't be found under the microscope.4.Hemadsorption activity(HAD)of De(HN),Ch(hPIV3),Ch(MeV),Ch(RSV)proteins were 13.48%,65.22%,36.25%,34.93%of that of wt HN,respectively(P<0.05),which were consistent with the fusion promotion activity of mutant proteins.5.Neuraminidase activity of De(HN),Ch(hPIV3),Ch(MeV),Ch(RSV)proteins were 10.81%,60.35%,54.42%,50.13%of that of wt HN,respectively(P<0.05).6.Semi-fusion activity of De(HN),Ch(hPIV3),Ch(MeV),Ch(RSV)proteins decreased to different levels.This activity of Ch(hPIV3)was a little higher than Ch(MeV),Ch(RSV),as for De(HN),this activity was almost lost.Conclusion:1.NDV HN(91-112)domain plays an important role in receptor recognition activity and fusion promotion activity of HN protein.2.The loss of fusion promotion activity of De(HN)protein was related to the failure of cell surface expression of the mutant,while the decreases of this activity of chimeras were less likely influenced by the cell surface expression level of all the mutants.3.The decreases of fusion promotion activity of mutant proteins were consistent with the decreases of their HAD activity,and there was positivecorrelation with the change of NA activity.4.The attenuation of transmit signal of chimeras in the process of binding and removing sialic acid receptor might relate to the decline of their HAD or NA activity,for which the F protein and cell fusion couldn't be fully activated.