【作者】 张文强；

【导师】 王志玉；

【作者基本信息】 山东大学 ， 流行病与卫生统计学， 2009， 博士

【摘要】 副粘病毒是一类对人和动物致病的重要病毒,在世界范围内流行,分为两个亚科:副粘病毒亚科和肺病毒亚科。副粘病毒亚科包括副粘病毒属、麻疹病毒属和腮腺炎病毒属;副粘病毒属主要包括人副流感病毒(hPIV)、新城疫病毒(NDV)和仙台病毒(SV),麻疹病毒属主要包括麻疹病毒(MV),腮腺炎病毒属主要有流行性腮腺炎病毒(MuV)。肺病毒亚科只包括肺病毒属,主要有人和牛呼吸道合胞病毒(RSV)。副粘病毒能够引起人类呼吸系统和生殖系统疾病,如上呼吸道感染、支气管炎、毛细支气管炎、肺炎、结膜炎、睾丸炎、卵巢炎等等,严重时甚至引起死亡。其中,MV、MuV、RSV等能引起儿童常见病,如麻疹、流行性腮腺炎以及呼吸道感染等。人副流感病毒3型(hPIV3)是婴幼儿毛细支气管炎和肺炎的第二大病因,还可引起婴幼儿的哮吼和喉炎。目前尚无有效的抗hPIV3治疗措施。除感染人类外,副粘病毒还能感染动物如禽类、马类、猪类等。NDV是重要的禽类传染病病原体,往往引起灾难性的、广泛流行的疾病。此外,NDV尚有一定的临床意义,如诱导干扰素(IFN)、免疫调节、肿瘤治疗等。近年发现的一些病毒,如尼帕病毒、亨德拉病毒、Salem病毒、鸟类肺炎病毒(APV)、人类后肺炎病毒(hMPV)等,都属于副粘病毒科。这些病毒能引起严重的人类和动物疾病,可以感染许多器官中的血管和血管外实质,还能引起严重的中枢神经系统疾病。临床上主要表现为发烧、头痛,病死率极高。1998年新加坡和马来西亚暴发类亨德拉病毒感染,导致上百人死亡。这种病毒来源于猪,和来源于马的亨德拉病毒相似。细胞融合是多种病毒增殖、扩散和致病过程中不可缺少的一步。除了副粘病毒以外,许多人类重要的致病病毒都含有细胞融合作用的包膜糖蛋白,如单纯疱疹病毒、人类免疫缺陷病毒等。因此,对副粘病毒细胞融合进行研究具有广泛的意义。副粘病毒属于单股负链RNA病毒,核衣壳外有包膜。基因组为15kb左右的不分节段RNA,主要编码6种病毒蛋白:核衣壳蛋白(NP)、磷蛋白(P)、内膜蛋白(M)、融合蛋白(F)、吸附蛋白、大分子量RNA聚合酶(L)。其中NP、P、L蛋白与基因组有关,共同参与病毒RNA的转录,以形成有活性的mRNA;吸附蛋白、F、M与病毒包膜有关,吸附蛋白和F蛋白形成刺突,是重要的保护性抗原,M蛋白构成病毒包膜内表面的支撑物。吸附蛋白在不同病毒属具有不同的活性:在hPIV、NDV、MuV具有血凝素(HA)和神经氨酸酶(NA)两种活性,称为HN蛋白;在MV只有HA活性,没有NA活性,称为H蛋白:在RSV既无HA活性,也无NA活性,称为G蛋白。HN蛋白为同源四聚体,基因长度大约为2 100个核苷酸(nt),编码的蛋白以N-末端插入膜内,主要分为4部分:亲水的胞内尾巴(CT)、疏水的穿膜区(TM)、颈部和头部;主要负责3种功能:(1)受体结合活性;(2)NA活性;(3)促细胞融合活性。副粘病毒感染宿主细胞过程中,膜融合是整个过程的第一步,也是至关重要的一个环节,由HN和F蛋白共同负责完成。感染过程中病毒通过HN蛋白与宿主细胞表面受体结合,引起HN蛋白构型改变。与HN蛋白相连的F蛋白构象随之发生变化,七重复基元(HR)HR1与HR2相互作用形成稳定复合体,使病毒包膜和宿主细胞膜靠近,同时融合肽(FP)暴露并嵌入到宿主细胞膜中而启动膜融合。在这个过程中,F蛋白是主要的调节蛋白,但单独的F蛋白并不能引起膜融合,必需有同源性HN蛋白辅助。HN蛋白的促细胞融合作用在副粘病毒中具有极强的特异性,即F蛋白只有与同源性HN共表达时才能完成细胞融合这一过程,而与异源性HN共表达时则不能引起细胞融合。例如,NDV F和NDV HN共表达或hPIV3 F和hPIV3 HN共表达时,有细胞融合发生;而NDV F和hPIV3HN共表达或hPIV3 F和NDV HN共表达时,则不能发生细胞融合。这表明在HN蛋白和F蛋白间存在着特异性的相互作用。HN蛋白促细胞融合区域是副粘病毒研究热点之一。为了寻找副粘病毒HN蛋白分子中与同源F蛋白相互作用的特异性区域,弄清HN蛋白促细胞融合作用的分子机制,我们采用基因定点突变和基因重组的方法对NDV HN和hPIV3 HN的cDNA进行定点突变并构建各种嵌合体(chimera),然后与同源或异源性F蛋白于真核细胞BHK21内共表达,测定细胞表面HN蛋白的表达和细胞融合情况。结果表明hPIV3 HN蛋白中促细胞融合区域位于TM区中部到胞外区起始的第82个氨基酸间的区域。再次研究发现嵌合时引入了1个糖基化位点,此糖链对hPIV3HN蛋白促细胞融合活性有抑制作用。除去此糖基化位点后再次嵌合将HN蛋白促细胞融合区域缩短了10个氨基酸,定位于TM区中部到胞外区起始的第72个氨基酸间的区域。NDV HN蛋白促细胞融合区域也定位于相同的区域。研究发现副粘病毒HN蛋白促细胞融合区域两端各含有一个保守的亮氨酸拉链,一个位于TM区,另一个位于颈部近膜区。通过定点突变发现NDV HN蛋白的这两个亮氨酸拉链对于蛋白的促细胞融合作用有重要影响。TM区亮氨酸拉链中的保守氨基酸L30、L37和L44突变后对蛋白转运并没有影响,但蛋白的促细胞融合活性都有所降低,尤其是L44突变和L30、L37联合突变。颈部近膜区亮氨酸拉链中的保守氨基酸L96、L103和L110突变后HN蛋白促细胞融合能力同样明显降低。在hPIV3 HN蛋白的促细胞融合区域两端同样存在两个亮氨酸拉链,但目前尚未见相关的研究报道。本研究在原有工作的基础上,利用定点突变技术构建hPIV3 HN蛋白促细胞融合区域两端亮氨酸拉链中的保守氨基酸突变体,然后检测突变体与同源F蛋白共表达后的促细胞融合活性以及受体结合活性、细胞表面表达效率,从而确定亮氨酸拉链对hPIV3 HN蛋白功能的影响。一、TM区亮氨酸拉链突变体对促细胞融合活性的影响hPIV3 HN蛋白TM区亮氨酸拉链由氨基酸L36～I50组成,其中L36、L43和I50等氨基酸在副粘病毒中高度保守,采用同源重组PCR方法进行定点突变构建突变体。每个突变体需要设计两对含同源序列的引物,进行两次PCR。获得的两个PCR产物含有短同源末端序列,将其同时转染大肠杆菌TG1后,二者会自动重组形成完整的含突变位点的质粒。为避免引入额外突变,引物中未引入酶切位点。转染后提取质粒,电泳结果正确,经测序证实引入突变,成功将各保守氨基酸突变为丙氨酸(A)。由此得到3个单独突变体,分别命名为:L36A、L43A和I50A。在单独突变的基础上,构建1个联合突变体,命名为L36A-L43A。将突变体分别转染痘苗病毒vTF7-3预感染的BHK21细胞,采用定性(Giemsa染色)和定量(指示基因法)两种方法检测细胞融合情况。细胞融合功能经校正后,消除了蛋白表达效率的影响。结果显示,与野生型(wt)hPIV3 HN基因相比,各单独突变体促细胞融合活性有不同程度的降低。以wt HN基因促细胞融合活性为100%,突变体L36A融合活性为wt HN基因的66.66%,突变体L43A融合活性为wt HN基因的60.60%,突变体I50A融合活性为wt HN基因的57.60%。联合突变体L36A-L43A转染后未见细胞融合形成。hPIV3 HN蛋白颈部近膜区的亮氨酸拉链由氨基酸L114～I128组成,其中L114、L121和I128在副粘病毒中高度保守。采用同源重组PCR方法进行定点突变。突变后提取质粒后电泳结果正确,经测序证实成功引入突变。由此得到3个单独突变体,分别命名为:L114A、L121A和I128A。在单独突变的基础上,构建1个联合突变体,命名为L121A-I128A。将突变体分别转染痘苗病毒vTF7-3预感染的BHK21细胞,采用定性(Giemsa染色)和定量(指示基因法)两种方法检测细胞融合情况。受体结合活性经校正后,消除了蛋白表达效率的影响。以wt hPIV3 HN基因促细胞融合活性为100%,L114A促细胞融合活性为wt HN基因的53.00%,L121A促细胞融合活性为wt HN基因的59.10%,I128A促细胞融合活性为wt HN基因的90.90%。联合突变体L121A-I128A转染后未观察到细胞融合。颈部亮氨酸拉链附近及内部的氨基酸P111、I112和I125在副粘病毒中亦是保守位点。同样以同源重组PCR方法获得3个单独突变体,分别命名为:P111A、I112A和I125A。将突变体分别转染痘苗病毒vTF7-3预感染的BHK21细胞,采用定性(Giemsa染色)和定量(指示基因法)两种方法检测细胞融合情况。促细胞融合活性经校正后,消除了蛋白表达效率的影响。以wt hPIV3 HN基因促细胞融合活性为100%,P111A为wt HN的43.90%,I112A为wt HN的39.40%,I125A为wt HN的34.80%。各突变体分别进行3次平行实验,所得实验数据采用秩和检验进行分析;秩和检验统计值H=28.78,P＜0.05,表明各突变体促细胞融合活性差别有统计学意义,其中突变体I125A活性最低,突变体I128A活性最高。FACS分析表明,各突变体都成功在细胞表面表达,但与wt HN基因相比,表达效率有所降低。二、亮氨酸拉链突变体对受体结合活性的影响将上述获得的11个突变体L36A、L43A、150A、L114A、L121A、I128A、P111A、I112A、I125A、L36A-L43A和L121A-I128A分别转染痘苗病毒vTF7-3预感染的BHK21细胞,采用血吸附实验检测突变体的受体结合活性。受体结合活性经校正后,消除了蛋白表达效率的影响。结果表明各突变体对受体结合活性影响较小。与wt hPIV3 HN基因相比,TM区亮氨酸拉链的3个突变体中,突变体L36A受体结合活性和wt HN基因一样,为100%,而突变体L43A和I50A受体结合活性稍低,分别为wt HN基因的98.99%和91.92%。颈部近膜区亮氨酸拉链的3个突变体L114A、L121A和I128A受体结合活性分别为wt hPIV3 HN的95.96%、92.93%和100%。此外,突变体P111A为wt HN的97.98%,I112A为wt HN的95.96%,I125A为wt HN的85.86%。两个联合突变体受体结合活性也与wt HN基因接近,L36A-L43A受体结合活性为wt HN基因的95.96%,L1121A-I128A为wt HN基因的93.94%。各突变体分别进行3次平行实验,所得实验数据采用秩和检验进行分析。秩和检验统计值H=17.99,P＞0.05,表明各突变体受体结合活性差别不存在统计学意义。三、F蛋白对HN蛋白受体结合活性的影响将上述11个突变体分别与F蛋白共转染痘苗病毒vTF7-3预感染的BHK21细胞,采用血吸附实验检测突变体的受体结合活性。以wt HN基因受体结合活性为100%,突变体L36A受体结合活性最高,为wt HN基因的99.14%,突变体I125A受体结合活性最低,为wt HN的84.48%。采用秩和检验分析HN单独表达(血吸附HN组)与HN和F蛋白共表达(血吸附HN+F组)两组受体结合活性的差别,结果H=108.00,P＜0.05,表明两组存在差别,血吸附HN组受体结合活性比血吸附HN+F组高。从本实验结果可得出结论:本实验成功构建了hPIV3 HN蛋白TM区和颈部近膜区两个亮氨酸拉链中保守氨基酸的突变体,共获得突变体11个,其中9个单独突变体,分别为L36A、L43A、I50A、L114A、L121A、I128A、P111A、I112A和I125A:在单独突变的基础上构建了两个联合突变体,L36A-L43A和L121A-I128A。TM区和颈部近膜区两个亮氨酸拉链对于hPW3 HN蛋白促细胞融合活性具有重要的作用。其中的保守氨基酸突变后,促细胞融合活性均有不同程度的降低,最低的为突变体I125A,仅为wt HN的34.80%,而突变体I128A融合活性最高,为wt HN的90.90%。其它突变体融合活性在39.40%～66.66%之间。而两个联合突变体转染后未见细胞融合形成。这进一步证明hPIV 3 HN蛋白促细胞融合区域不是位于头部,而是位于TM区和颈部近膜区,并在氨基酸I125结尾,I125是对促细胞融合活性有着关键作用的氨基酸残基;亮氨酸拉链结构的完整性是副粘病毒细胞融合所必需的,对HN蛋白促细胞融合作用有着重要的影响。HN蛋白促细胞融合区域两端亮氨酸拉链对受体结合活性并无影响。各突变体受体结合活性与wt HN非常接近;突变体I125A受体结合活性为wt HN的85.86%,其它突变体在91.92%～100%之间。与HN蛋白单独表达相比,F蛋白和HN蛋白共表达能够一定程度的降低HN蛋白的受体结合活性。更多还原

【Abstract】 Paramyxoviruses are a group of important viruses that are pathogenic to human beings and animals.They can be epidemic all over the globe.The family of paramyxoviridae is divided into two subfamilies:paramyxovirinae and pneumovirinae. Paramyxovirinae is divided into three genuses:paramyxovirus,morbillivirus and rubulavirus.Paramyxovirus includes human parainfluenza virus(hPIV),Newcastle disease virus(NDV) and Sendai virus(SV);morbillivirus consists of measles virus (MV);and rubulavirus contains mumps virus(MuV).Pneumovirinae only contains pneumovirus which includes respiratory syncytial virus(RSV).Paramyxoviruses mainly induce diseases of respiratory and reproduction systems in human beings,including upper respiratory tract infection,bronchitis,bronchiolitis, pneumonia,conjunctivitis,orehitis and ovaritis,etc.Sometimes,it can cause death. Some virus,such as MV,MuV and RSV,may cause familiar disease in children including morbilli,parotitis and respiratory infection,hPIV3 is the second important pathogen of bronchiolitis and pneumonia in infants.Besides these,it may cause croup and laryngitis.Up to date,no effective anti-hPIV3 measure was found.Besides human beings,paramyxovirus can also infect animals such as poultry,horses and swine.For NDV,an avian virus that is a serious agricultural problem in many regions of the world at present,it is an important pathogen of animal disease.NDV can cause avian and cattle diseases,usually disastrous and wide epidemic.NDV is of other clinical significances.For example,it can induce interferon and can be used to treat tumors.Some viruses are emerging ones,including Nipah virus,Hendra virus,Salem virus,avian pneumovirus and human metapneumovirus belonging to paramyxoviridae. They can cause severe human and animal diseases,infecting many organs or tissues, especially central nervous system.Clinically,fever and headache are common,and the fatality rate is very high.Infection of virus that is similar to Hendra virus bursted out in Singapore and Malaysia.This kind of virus originated from swine,which is familiar to horse Hendra virus.Cell fusion is a critical step in virus multiplication,spread and pathogenesis. Besides paramyxoviridae,many other human pathogenic viruses have envelope glycoprotein that can cause cell fusion,such as herpes simplex virus,human immunodeficiency virus(HIV),etc.Thus,studies on cell fusion of paramyxoviruses possess wide significances in viral theory and practice.The paramyxoviruses are a group of enveloped,single negative-stranded, non-segmented RNA viruses.There occurs envelope outside the viral capsid.The non-segmented genome,with the length of about 15 000 base pairs(bp),encodes 6 kinds of protein,including nucleocapsid protein(NP),phosphoprotein(P),Matrix Protein(M),fusion pritein(F),attachment protein and large molecule protein(L). Among these protein,NP,P and L protein are related with viral genome,which transcribe viral RNA and form active mRNA together.F,M and attachment protein are involved in viral envelope.F and attachment protein,the important protective antigen,compose the viral spike;M protein forms underprop inside the viral envelope. The activity of attachment protein varies in different paramyxovirus.For hPIV,NDV and MuV,their attachment protein possesses both neuraminidase and hemagglutinin acitivity,which were named hemagglutinin-neuraminidase protein(HN).The attachment protein of MV only has the hemagglutinin activity,named hemagglutinin protein(H).RSV attachment protein,named G protein,possesses neither neuraminidase nor hemagglutinin activity.HN protein,penetrating into viral envelope with its N-terminal domain,is homologous tetramer encoded by the gene containing 2 100 nucleotides.HN glycoprotein is divided into head,stalk,transmembrane domain (TM) and cytoplamic tail(CT),responsible for three kinds of function,receptor binding activity,NA activity and cell membrane fusion promotion activity.A flexible site had been observed in head domain that comprised 6β-sheets.It is a bifunctional site,responsible for NA and receptor binding activity,and can switch between them under different conditions.Cell fusion,a key step in paramyxovirus infection,is mediated by HN and F protein together.During viral infection,HN configuration is altered aider the attachment of HN protein to viral receptor on host cell surface,which induces the configuration change of F protein.Heptad repeat motif 1(HR1) and HR2 form steady complex which results in the adjacency of viral envelope and host cell membrane.At the same time,hydrophobic fusion peptides(FP) are released into cell membranes, and the cell fusion process is triggered.During this process,F protein is directly responsible for fusion of the viral and cellular membranes.But F protein can not induce cell fusion alone,which needs the cooperation between F protein and homologous HN protein.Furthermore,the requirement for the HN protein in fusion is virus specific,meaning that F protein can only induce the cell fusion when coexpressed with the homologous HN protein,but not with the heterologous HN preotein.For example,cell fusion will take place when NDV F and NDV HN or hPIV3 F and hPIV3 HN are coexpressed.Otherwise,there will be no cell fusion observed.The specific membrane fusion has been interpreted as evidence for a necessary interaction between the two kinds of protein,which,in some way,activates the fusion activity of the F protein.The interaction domain between F and HN protein is one of the focuses on envelope glycoproteins of paramyxoviridae in the world.In order to localize an active domain that interacts with homogenous F on HN protein of paramyxoviruses and to understand the molecular mechanism of cell fusion promotion,site-directed mutagenesis and gene recombination were used to get chimeric HN protein.Thus, F-specificity domain of hPIV3 HN protein was localized at a domain that extended from the middle of the membrane anchor to the 82th residue in the ectodomain. Further study showed that hPIV3 chimeric HN protein restored a glycosylation site present in NDV HN,but not in hPIV3 HN.After deletion of this glycosylation site, further mutagenesis and chimeric HN genes analysis showed glycosylation site near the top of hPIV3 HN stalk modulated fusion and shortened the F-specificity domain by 10 amino acids,from the middle of the membrane anchor to the 72th residue in the ectodomain.F-specificity domain in NDV HN protein was localized at the similar domain.In F-specificity domain of paramyxovirus HN protein,there are two conserved leucine zipper motifs at N-terminal and C-terminal,respectively.Many studies showed conserved leucine zipper motif in this domain had great effects on HN protein. The residues from L30 to L44 compose the C-terminal leucine zipper motif of NDV HN.Site-directed mutation in this domain will markedly reduce the cell fusion promotion activity while there is no effect on protein transport.N-terminal leucine zipper motif consists of residues from L96 to L110,which can markedly reduce the cell fusion promotion activity after mutation.Two leucine zipper motifs also occur in the same domain of hPIV3 HN protein.However,up to date,no functional analysis of conserved leucine zipper motif in hPIV3 HN protein F-specificity domain has been reported.In order to identify the effects of leucine zipper motif on hPIV3 HN protein, site-directed mutagenesis was used to mutate the conserved amino acids in the leucine zipper motifs on the basis of previous experiments results.Mutant HN genes were expressed with homogenous F in eukaryocytes.Their cell fusion promoton and receptor binding ability were analyzed with qualitative and quantitative methods, respectively.1.Effects of leueine zipper motif on cell fusion promotion abilityThe motif in TM domain lies from L36 to I50,in which residues L36,L43 and I50 are highly conserved,aligned with other paramyxovirus.Recombinant PCR was used to generate mutant HN genes with corresponding primers for each mutant.Two pairs of primers were designed for one mutation.After PCR,two products with a short homologous sequence that came from the primers will form a complete plasmid automatically in TG1.In order not to mutate other amino acid except the aimed amino acid,no enzyme restricted site was introduced into the primers.PCR product was identified by sequencing after agarose electrophoresis.Thus,3 mutants were obtained, named L36A,L43A and I50A,respectively.To further determine the roles of leucine zipper motif,combined mutant,L36A-L43A,was constructed on the basis of individual mutations.To determine their cell fusion promotion activity,both qualitative and quantitative method,Giemsa staining and reporter gene method,were performed after the mutants transfected BHK21 cells which were infected with vaccine virus vTF7-3 in advance.After corrected by cell surface expression efficiency, mutant HN possessed 66.66%,60.60%and 57.60%of wt HN activity,respectively.In combined mutant L36A-L43A,cell fusion was rarely found.Residues L114 to I128 consist of the leucine zipper motif in the stalk domain close to viral envelope.Among those,L114,L121 and I128 are highly conserved in paramyxovirus.Mutant HN genes were obtained by recombinant PCR with corresponding primers for each mutant.No enzyme restricted site was introduced in the primers in order to mutate exactly the aimed amino acid.The mutant was identified by sequencing after agarose electrophoresis.Thus,3 mutants were obtained, named L114A,L121A and I128A,respectively.To better determine the roles of leucine zipper motif,combined mutant,L121A-I128A,was constructed on the basis of individual mutations.Their cell fusion promotion activity was determined by Giemsa staining and reporter gene method in the vaccinia-T7 RNA polymerase expression system,too.After corrected by cell surface expression efficiency,mutant HN possessed 53.00%,59.10%and 90.90%of wt HN activity,respectively.Cell fusion was rare in combined mutant L121A-I128A.P111,I112,and I125 are also conserved in leucine zipper motif in F-specificity domain of HN protein.3 mutants were obtained with the same methods,named P111A,I112A,and I125A,respectively.Cell fusion promotion ability was assayed with Giemsa staining and reporter gene method.After corrected by cell surface expression efficiency,the mutants had 43.90%,39.40%,and 34.80%of wt HN cell fusion promotion ability,respectively.Every mutant was examined for 3 times under the same condition.The data were analyzed with rank sum test.The H value of rank sum test was 28.78,P＜0.05, suggesting that there was statistical significant difference between mutants.I125A possessed the lowest cell fusion promotion activity,with 34.80%of fusion promotion activity as compared with wt HN.However,I128A had the highest fusion promotion activity,90.90%of wt HNThe FACS was performed to determine expression efficiency of mutant hPIV3 HN protein.The results of FACS indicated that all mutants were expressed on the cell surface successfully although the expression efficiency of all the mutants was lower than that of their relevant wt HN.2.Effects of leucine zipper motif on receptor binding activityThe receptor binding activity of mutant genes was determined by the ability to absorb guinea pig erythrocytes in the vaccinia-T7 RNA polymerase expression system. The BHK21 cells were transfected with the mutants L36A,L43A,I50A,L36A-L43A, L114A,L121A,I128A,L121A-I128A,P111A,I112A and I125A alone.The results showed mutation in leucine zipper motif possessed slight effect on receptor binding ability.The data was corrected by cell surface expression efficiency.In leucine zipper motif in TM domain,mutant L36A possessed 100%receptor binding activity as compared to wt HN;L43A and I50A had 98.99%and 91.92%,respectively. Combined mutant L36A-L43A is 95.96%of wt HN receptor binding activity.In leucine zipper motif in stalk domain,mutant L114A,L121A and I128A possessed 95.96%,92.93%and 100%of wt HN activity,respectively.Combined mutant L121A-I128A had 93.94%activity,compared to wt HN.In addition,mutant P111A, I112A and I125A had 97.98%,95.96%and 85.86%of the activity of wt HN, respectively. Every mutant was examined for 3 times under the same condition.The data were analyzed with rank sum test,and the H value of rank sum test was 17.99,P＞0.05, suggesting that there was no statistical significant difference between groups.All these results showed that mutation of conserved amino acids in F-specificity domain of hPIV3 had some negligible effects on receptor binding ability of hPIV3 HN protein.3.Effects of F protein on receptor binding activityBHK21 cells,infected with vaccine virus vTF7-3 in advance,were transfected with the 11 mutants described previously,coexpreesed with wt hPIV3 F gene. Absorbing guinea pig erythrocytes in the vaccinia-T7 RNA polymerase expression system was applied to determine the receptor binding activity of mutant genes,too. Mutant I125A possessed the lowest receptor binding activity,84.48%as compared to wt HN gene.And mutant L36A had the highest activity,99.14%ofwt HN.Every mutant was examined for 3 times under the same condition.Rank sum test was used to determine the difference between the group transfected with mutant HN alone and the group transfected with mutant HN and wt F gene together.And the H value of rank sum test was 108.00,P＜0.05,which showed there was difference between the two groups,and the group transfected with mutant HN gene alone possessed higher receptor binding activity.The results suggested as followings:Individual mutation L36A,L43A,I50A,L114A,L121A,I128A,P111A,I112A and I125A were obtained successfully.On the basis of individual mutation,combined mutants L36A-L43A and L121A-I128A were constructed.The leucine zipper motif in TM domain and stalk domain close to viral envelope possessed great effects on cell fusion promotion activity of hPIV3 HN protein.The activity will reduce to different level after the conserved residues were mutated.In those individual mutants,mutant I125A had the lowest cell fusion promotion activity, 34.80%as compared to wt HN gene.However,mutant I128A possessed 90.90%of wt HN gene activity,the highest in individual mutation.The activity of other individual mutation lied from 39.40%to 66.66%.Furthermore,cell fusion was rarely found in the two combined mutants.All these suggested further that the domain specific to HN and F protein interaction was localized in the TM domain and stalk domain close to viral envelope,but not in head domain.And this specific domain ended at the residue I125.Complete leucine zipper motif was prerequisite to fusion promotion of HN protein.I125 is the key amino acid for cell fusion promotion activity of hPIV3 HN protein,which played impotant roles in cell fusion promotion ability.All the mutants possessed negligible effects on receptor binding activity of hPIV3 HN protein.Among those,mutant I125A had 85.86%of wt HN gene activity. And the receptor binding activity of other mutation lied from 91.92%to 100%, similar with wt HN.Compared to HN gene expressed alone,the receptor binding activity reduced when F and HN gene coexpressed together.更多还原