Structural And Functional Analyses Of F-interaction Region In The Stalk Domain Of Paramyxovirus Hemagglutinin-Neuraminidase

Background:human parainfluenza virus type 3 and Newcastle disease virus are the typical representative of paramyxoviruses.hPIV3 mainly causes human respiratory system diseases and reproductive system diseases,NDV is transmitted by oral mucus and feces of infected poultry,it can also cause diseases of respiratory and nervous system,highly lethal,human exposed to infected poultry may cause lymphadenitis and conjunctivitis.There are no effective measures for the prevention and treatment of these two viral infections since the fusion mechanism of the viral and target cell in pathogenic mechanism is not clear.In paramyxoviruses,the interactions between the fusion protein(F)and hemagglutinin-neuraminidase(HN)are critical in determining host range,virulence and spread of these viruses.Fusion interaction region locates in HN stalk,so far,there is no study on the fragment replacement and conserved amino acids mutants in the F-interaction region of HN.Newcastle disease virus(NDV)and human parainfluenza virus type 3(hPIV3)were selected as the research object in this study.The F-interaction region in HN stalk was replaced by a fragment replacement mutagenesis method to obtain chimeras,then identified two conserved amino acids,Serine(S)and Aspartic acid(D),in the F-interaction regions through the comparison with highly similar sequences in Newcastle disease virus(NDV)and human parainfluenza virus type 3(hPIV3)HN,and mutated them into alanine respectively.We determined the critical sites in F-interaction regions of HN stalk through detection of different activities of each mutant,speculated the main reason of changes and further deepened the understanding of HN stalk region in the fusion mechanism of paramyxoviruses.Objectives:1.To analyze the effects of F-interaction region in HN stalk in mechanism of cell fusion.2.To locate the key amino acids in the F-interaction region of HN stalk.3.To further investigate the role of the HN stalk domain plays in fusion mechanism.Methods:1.Fragment replacement combined with homology recombination method was used to obtain chimeras.2.Site-directed mutagenesis method combined with homology recombination method was used to obtain mutants,which are NDV S51A,NDV D55A,hPIV3 S51A and hPIV3 D55A.3.A transient expression system of the vaccinia virus-T7 RNA polymerase was applied to express the chimeras and mutants.4.Analysis of cell surface expression:flow cytometry analysis was executed to make the quantitative analysis of the cell surface expression level;Indirect immunofluorescene assay(IFA)was used to make the qualitative analysis of the cell surface expression of the chimeras and mutants.5.Analysis of cell fusion promotion activity:Giemsa staining was used to make the qualitative analysis of the formed syncytia to record the fusion of each mutant protein,while reporter gene method was used in quantitative analysis.6.Analysis of receptor recognition activity:The qualitative changes were determined by hemadsorption(HAD)to analyze the receptor binding ability of each chimeras and mutants,and the receptor recognition activity was detected by colorimetric quantification after the adsorbed RBC were lysed.7.Analysis of neuraminidase activity:Neuraminidase assay kit was used to detect the content of neuraminidase through enzymatic reaction.8.Analysis of semi-fusion activity:R18 labeled fresh RBCs were used to determinate the fusion promotion rate of the chimeras and mutants in initial stage of membrane fusion.Results:1.The chimeras C1 and C2 were constructed successfully.The results of DNA sequencing showed that the amino acids designated were mutated into alanine respectively and mutants NDV S51A,NDV D55A,hPIV3 S51A,hPIV3 D55A were constructed successfully.2.Cell surface expression of the chimeras and mutants(C1,C2,NDV S51A,NDV D55A,hPIV3 S51A and hPIV3 D55A)were expressed successfully on the cell surface and the expression efficiency was 82.1%,81.8%,90.2%,88.3%,84.9%and 92.8%of the wt level,respectively.There were no statistic differences compared with wt HN(P>0.05).3.Cell fusion promotion activity of the chimeras and mutants was reduced to significant extent(P<0.05),C1,C2,NDV S51A,NDV D55A,hPIV3 S51A,hPIV3 D55A was 7%,9%,27%,19%,17%and 21%of the wt level,respectively.4.Receptor recognition activity of the chimeras and mutants C1,C2,NDV S51 A,NDV D55A,hPIV3 S51A,hPIV3 D55A decreased to 14.7%,22.3%,35.5%,28.8%,33.9%and 40.2%of the wt level,respectively.There were statistic differences compared with wt HN(P<0.05).5.There were no statistic differences of neuraminidase activity among wild type and each mutant(C1,C2,NDV S51A,NDV D55A,hPIV3 S51A and hPIV3 D55A)(P>0.05),which was 75.9%,80.5%,84.7%,72.7%,75.3%and 73.4%respectively.6.Semi-fusion activity of all the mutants also,to some extent,decreased.Conclusions:1.The F-interaction region in the stalk domain plays a very important role in fusion promotion activity and receptor recognition activity of NDV HN and hPIV3 HN.2.S51 and D55 were identified as the key amino acids in the fusion interaction region in the stalk domain.3.The decreased receptor recognition activity of chimeras and mutants in F-interaction region of HN stalk may decrease the activity of the cell fusion promotion activity by blocking the signal transmission between the head and the stalk of HN,and make the F-interaction region in the stalk can not fully exposed,thus the interaction between HN and F is affected.