Functional Analyses Of The Stalk/Head Interface Of Human Parainfluenza Virus Type 3 Hemagglutinin-neuraminidase Protein

Background:In order to deliver enveloped viruses' genetic material to host cells during infection,specialized proteins are used on their surfaces that bind cellular receptors and induce fusion of the viral and host membranes.The Paramyxoviridae are enveloped,negative-strand RNA viruses that infect both humans and animals.There are many representatives of Paramyxoviridae,such as human parainfluenza viruses,Newcastle disease virus and parainfluenza virus type 5.Their attachment and fusion machinery is composed of two separate proteins:hemagglutinin-neuraminidase(HN)and fusion(F)protein,which interact each other to affect membrane fusion.Hemagglutinin-neuraminidase(HN)protein plays multiple roles in viral entry and diffusion,including binding to sialic acid on host cell,activating the fusion(F)protein to switch on membrane fusion and viral entry,and hydrolyzes these receptors during viral egress.HN is an oligomeric type ? transmembrane protein with a short cytoplasmic domain and a large ectodomain comprising a long helical stalk and large globular head domain containing the enzymatic functions.The stalk forms a parallel tetrameric coiled-coil bundle(4HB).In addition,stalk region contains specificity determinants for F interactions and activation.Recently,a structure of hPIV3 HN ectodomain revealed the heads in a "4-heads-down" conformation whereby two of the heads form a symmetrical interaction with two sides of the stalk.The interface includes stalk residues implicated in triggering F,and the heads sterically shield these residues from interaction with F(at least on two sides).The structure supports a model in which the heads of HN transition from down to up upon receptor binding thereby releasing steric constraints and facilitating the interaction between critical HN-stalk residues and F protein.Structures of the HN NA domains have been solved previously,however,there is a lack of research on the structure of the stalk region,and function of amino acids at stalk/head interface of hPIV3 HN protein in membrane fusion process has remained elusive.In this study,we selected 7 residues from the global head and 8 residues from 4HB stalk that form the stalk/head interface of hPIV3 HN,and then mutated these sites to alanine.Then we detected the abilities to form syncytia,neuraminidase activity,receptor-binding ability and protein surface expression.Objectives:To explore the roles of amino acids at stalk/head interface of hPIV3 HN protein in membrane fusion process,to illustrate the mechanisms of this domain affecting membrane fusion and try to locate the key amino acid sites.Methods:Primary sequences of attachment proteins of PIV5,NDV and hPIV3 were compared and then the homology modeling of the hPIV3 HN protein was generated based on the NDV HN crystal structure(PDB ID:3TIE)in the"4-heads-down"conformation.The residues that comprise this stalk/head interface in hPIV3 and NDV HN align based on primary sequence alignment.1.Seven residues from the global head and eight residues from 4HB stalk that form the stalk/head interface of hPIV3,were mutated to alanine by site-directed mutagenesis.And then wild-type(wt)HN and all mutated HN proteins were used in the vaccinia virus-T7 RNA polymerase expression system to be expressed.2.Giemsa staining assay,the report gene assay and the dye transfer assay were employed to analyze the fusogenic activity at different stages of membrane fusion.3.The receptor binding activity of each mutant was detected by the ability to adsorb human red blood cells(RBCs).4.The lysates were assayed for neuraminidase activity according to the procedures of Neuraminidase Assay Kit(Beyotime Biotechnology).Fluorescence was measured with excitation and emission wavelengths of 360nm and 460nm,respectively.5.Qualitative and quantitative analysis of expression efficiency of mutations by indirect immunofluorescence assay and fluorescence-activated cell sorter.6.Statistical analysis:All results were expressed as means x±SD of at least 3 independent experiments.Statistical analysis was performed using Student's t test,and P<0.05 was considered as statistically significant.Results:1.This homology model was generated based on the NDV HN crystal structure(PDB ID:3TIE).The selected amino acids at the hPIV3 stalk/head interface with 8 residues from stalk helices(I112,Q116,M118,D120,R122,K123,S126,E127)and 7 residues from the head(R141,1142,D145,G147,1148,L151,N152)were individually mutated to alanine successfully.2.The ability of each mutant to promote cell fusion was evaluated with Giemsa staining,a content-mixing assay,and a lipid-mixing assay.The result of hemifusion and dye transfer assay was shown almost mirrored the Giemsa staining of wt or mutated HN and F protein mutants(like I112A,R122A,I148A and L151A)that almost lost the ability to form syncytia can just be detected only very littlefluorescence dequenching.In contrast,mutants(like R141A,I142A and G147A)that enhance the fusion ability had strong fluorescence.As for other 8 mutants(Q116A,M118A,D120A,K123A,S126A,E127A,D145A,N152A),although the ability to form syncytia was preserved,the number and area of syncytia formation were lower than those of the wt HN,and the ability of the hemifusion and dye transfer also,to some extent,decreased.3.The results of FACS suggested that expression efficiency of most mutants were quite comparable to that of wt HN from 86.31%to 106.54%,except for L151A.The MFI of L151A decreased to 38.09%as much as that of wt HN.4.Five mutants(E127A,R141A,I142A,D145A,G147A)retained a similar receptor binding ability as the wt hPIV3 HN(from 88.33%to 92.78%).I48A and L151A significantly affected the HAD ability(32.49%and 24.61%,respectively).The other 8 mutants directed approximately half the levels of receptor binding ability of that of the wt HN.5.Neuraminidase activity of 14 mutant proteins decreased,to some extent,compared with that of wt HN protein,except for R141A,which enhanced NA activity.Conclusions:Amino acids at stalk/head interface of hPIV3 HN protein have important influence on the membrane fusion process.Mutations at this interface play an important role in membrane fusion,receptor binding ability and neuraminidase activity without affecting F protein triggering.Then,L151A of the HN stalk domain affects protein surface expression.Finally,functional studies suggest that specific residues of the HN stalk domain(I112,D120 and R122)are related to the activation of F protein.