Study Of Two Newcastle Disease Viruses HN Protein Mediating The Difference Of Membrane Fusion Activity

Newcastle disease（ND）is an infectious disease caused by Newcastle disease virus（NDV）.It can be divided into three strains,velogenic,mesogenic and lentogenic,and the velogenic can cause 100%lethal infection of poultry.The hemagglutinin-neuraminidase（HN protein）of NDV is a glycoprotein,which located on the surface of virus.HN protein mainly plays receptor binding,membrane fusion promotion and neuraminidase activities in the life cycle of NDV.However,some articles report that the pathogenicity and membrane fusion activity of NDV were also related to HN protein.Two NDV strains（NDV-Blackbird and NDV-Dove）were isolated from wild birds in the early stage,and their genomes were very similar.Their F proteins were completely identical and the cleavage motifs were¹¹²R-R-Q-R-R↓F¹¹⁷（the motif of velogenic and mesogenic）,and there were only five mutant amino acids on the HN protein（AAs:54 110 116 469 and 522）.However,after determined the virulence,it found that NDV-Blackbird was velogenic and NDV-Dove was lentogenic,and there was significant difference in the syncytia produced by them.In order to explore the effects of five mutant amino acids on the membrane fusion activity of two NDV strains and the influencing pathways,so this thesis contained the following aspects:1. Biological activities analysis of two NDV strainsAfter the two NDV strains were infected with BHK21 cells for 24 h,the diameters of40 syncytia produced by two strains were measured by Image J software,and those results were used to compare the difference in membrane fusion activity.It was found that the syncytia produced by NDV-Blackbird were significantly larger than that of NDV-Dove.The plaque experiment was used to measure the virus titers at different time points after inoculation,and those results were used to analysis their proliferation in DF-1 cells.It was found that the proliferation rate of NDV-Blackbird was significantly faster than that of NDV-Dove.At the same time,the hemadsorption（HAd）assay and neuraminidase（NA）detection kit were used to detect the receptor binding activity and neuraminidase activity,respectively.The study had shown that the receptor binding and neuraminidase activities of NDV-Blackbird were significantly higher than NDV-Dove.In summary,these results indicated that the difference of membrane fusion activity between the two NDV strains may be related to the difference of proliferation ability and the HN protein functional activity.2. Differential site analysis of HN proteins in two NDV strainsThe different amino acids in the encoded structural proteins of two NDV strains were counted,and the three-dimensional structural models of HN protein were constructed using Py MOL software,and then the models were uesed to analyze the positions of different amino acids.At the same time,the sequence information of 763 NDV strains from Gen Bank were collected,and their cleavage motifs were ¹¹²R/K-R-Q-R/K-R↓F¹¹⁷.The lasergene7.1 software was used to analyze their complete HN protein sequences,and the main amino acid species at five positions were determined.The study had shown that five mutant amino acids were located in different positions of HN protein,and these amino acid mutations occur between the main amino acids.3. Analysis of membrane surface expression efficiency of HN mutant proteinsAfter HN mutant and wild-type plasmids were transfected into BHK21 cells for 24 h,the indirect immunofluorescence（IIFA）assay and flow cytometry（FCM）assay were uesd to analyze the expression levels and membrane surface expression efficiencies of HN proteins.It found that all of the HN mutant and wild-type proteins were successfully expressed,and there were no significant differences in express efficiency on the cell membrane surface.4. Biological activities analysis of HN mutant proteinsTo rule out the effect of virus proliferation on membrane fusion activity,the overlapping PCR was used to construct five HN single mutant plasmids,and then co-transfect them with F plasmid for 36 h,respectively.The areas of 40 syncytia produced by co-transfection were measured by Image J software,and those results were used to compare the difference in membrane fusion promotion activity.It was found that NDV-Blackbird HN protein had stronger membrane promotion activity than NDV-Dove,and then T522I had no influence,while G54S,F110L,G116R and A469V mutants had different degrees of influence,among them that F110L and G116R had greater influence.The western blotting was used to detect the cleavage level of F protein,and it was found that there were no differences in the cleavage degrees of F protein,indicating that there were no differences in the stimulation degrees of HN mutant proteins to the F protein cleavage.After 24 h of the HN mutant plasmids were transfected,the HAd and NA activities were detected.It was found that the HAd and NA activities of two NDV HN proteins were significant different.Five mutations leaded to different effects on HAd activity,and then F110L,G116R,A469V and T522I significantly affected the NA activity,while G54S had no effect.The above results indicated that five mutations had specific effects on the HN protein function activities,and the difference in the membrane fusion activity of the two NDV strains may be related to these functional activity changes,but not to the stimulation of HN protein to F protein cleavage.In summary,it found that the HN protein had important influence on membrane fusion activity of NDV.Five mutant amino acids of HN protein may make receptor binding,neuraminidase and membrane fusion promotion activities reach a balance state at the protein and virus levels,and finally caused the difference in membrane fusion activity between two NDV strains.And the L110,R116,V469 and I522 were key amino acids to affect the corresponding functional activity of HN protein.These results will provide new idea for the subsequent study of NDV pathogenicity and the development of attenuated vaccine.