【作者】 王艳红；

【导师】 萧飒；

【作者基本信息】 西北农林科技大学 ， 预防兽医学， 2018， 博士

【摘要】 新城疫病毒(Newcastle disease virus,NDV)是一种单股、不分节段、含有囊膜的负链RNA病毒,能够引起禽类新城疫(Newcastle disease,ND),给全球养禽业带来巨大的经济损失。NDV的致病性主要是由病毒的融合蛋白裂解位点(Fusion protein cleavage site,Fcs)氨基酸序列决定的。一般情况下,强毒和中等毒力毒株的Fcs包含多个碱性氨基酸,基序(Motif)为“R/K-R-Q/R/K-R/K-R↓F”;弱毒株Fcs则包含1个或2个碱性氨基酸,基序为“G/E-K/R-Q-G/E-R↓L”。Fcs中的氨基酸种类、性质及其所处的位置因毒株的不同而呈现一定的多样性。虽然已有报道Fcs中单个或多个氨基酸的突变会影响诱导细胞融合能力及病毒的致病性。但是,Fcs中所呈现的氨基酸序列多样性对病毒的影响至今不明。另外,有研究发现,虽然一些毒株的Fcs中氨基酸序列一致,但对诱导细胞融合能力及宿主的致病性有较大差异,说明除了Fcs外,F蛋白其它区段或位点的氨基酸也起着重要的作用。因此,本研究针对Fcs的氨基酸序列通过分析其多样性,分别以强毒株和弱毒株的F基因为骨架,构建Fcs突变体,利用体外细胞实验,探索Fcs的氨基酸序列多样性及特殊Fcs情况下F蛋白其它区段对诱导细胞融合能力的影响。具体研究内容及结果包括以下几个方面:1.NDV自然分离株Fcs氨基酸序列多样性的广泛性分析通过GenBank数据库及文献报道收集了1572株NDV的相关信息,利用软件Lasergene7.1对Fcs氨基酸序列多样性进行统计分析,我们将Fcs分为强毒Fcs(VFcs)和弱毒Fcs(AFcs)2大类,VFcs包含1073个毒株,AFcs包含499个毒株。其中,将VFcs又分为8种类型,毒株基本都属于Class II类基因型;将AFcs分为10种类型,毒株大多属于Class I类基因型,部分属于Class II的基因型I、II和X。通过分析毒株的流行病学,我们发现Fcs与禽的种类、时空分布之间存在显著的关联性,其中一些Fcs类型具有禽类种属依赖性及地域特性。2.Fcs氨基酸序列多样性对F蛋白诱导细胞膜融合能力的影响利用NDV强毒株F48E9和弱毒株La Sota的F蛋白为骨架,将其Fcs氨基酸序列分别突变为10种AFcs和8种VFcs,通过将F突变体和HN质粒共转染BHK-21细胞观察对诱导细胞膜融合活性的影响。结果显示,在VFcs中,当P3位是谷氨酰胺(Q)时,该F蛋白能显著提高细胞融合活性。在连续5个碱性氨基酸情况下,赖氨酸(K)对于细胞融合活性的增强起着重要的作用。VFcs-1和VFcs-2是F蛋白诱导细胞膜融合活性最强的两种类型,VFcs的8种类型对促细胞膜融合活性的作用依次为:VFcs-1、-2>VFcs-3、-4>VFcs-5、-6、-7、-8。在AFcs中,无胰蛋白酶存在时,除AFcs-10类型外,其它几种类型均不能引起合胞体产生;有胰蛋白酶存在时,除了含有P4位为中性氨基酸Q的AFcs-9类型的F蛋白不能诱导细胞膜融合现象,其它均产生程度相似的细胞膜融合效率。3.Fcs为“RRQRR↓L”的F蛋白其它区段对诱导细胞膜融合能力的影响在对Fcs氨基酸序列多样性研究时发现,Fcs“RRQRR↓L”为弱毒裂解位点(AFcs),通常认为拥有AFcs的无论强毒株还是弱毒株F蛋白都不能有效诱导细胞膜融合,但是,我们发现拥有此Fcs的强毒株F48E9的F蛋白具有很高的细胞膜融合诱导能力,而拥有此Fcs弱毒株LaSota的F蛋白不能有效诱导细胞膜融合。此结果表明除了Fcs,强毒株F蛋白的其它区域触发了细胞膜融合。为了寻找此F蛋白的具体作用区域,我们将F蛋白的主要功能域划分成9个区段,然后将这些区段在强、弱毒株F蛋白之间进行相互替换,通过细胞转染实验分析F蛋白突变体之间对宿主细胞膜融合效率的影响,并根据鉴定结果进一步进行替换或突变,最终明确了在Fcs为“RRQRR↓L”时,F48E9-F蛋白上影响细胞膜融合能力的2个关键氨基酸,即D479,S486。4.一种检测NDV诱导细胞融合新方法的建立NDV感染可引起宿主细胞合胞体的产生,其诱导细胞融合的程度可作为一种衡量毒力强弱的指标。目前对细胞融合程度进行评估时,普遍采用人工计算合胞体中细胞核的平均数量的方法对其进行判定。此法虽然比更早采用的计算合胞体面积判断法要更精确,但费时费力,主观性强易出现较大的误差。为了探索更方便、有效的定量检测NDV诱导细胞融合效率的方法,本研究运用慢病毒转导技术成功筛选出两种荧光细胞系BHK-21/GFP和BHK-21/tRFP。将此两种细胞等比例混合后感染病毒,通过流式细胞技术检测形成合胞体(双荧光阳性)的数量来判定细胞的膜融合效率。结果表明,在不同剂量的NDV感染混合细胞系后,双荧光阳性细胞数所占比例能很好地反应出病毒感染剂量与形成合胞体数量之间的线性关系。本研究为开发方便、有效的定量检测细胞融合效率提供了新的技术手段。综上所述,本研究较为详细的阐明了NDV Fcs氨基酸序列多样性对细胞融合活性的影响,为研究Fcs氨基酸序列多样性对宿主致病性提供必要的理论依据。其次,除Fcs外,F蛋白其它区域在诱导细胞膜融合能力方面也起着重要作用,为NDV F蛋白触发的细胞膜融合作用提供了新的见解,并为设计抗病毒多肽药物提供了理论基础。更多还原

【Abstract】 Newcastle disease virus(NDV)is an enveloped virus with a single-stranded,non-segmented,negative-sense RNA genome.It can cause Newcastle disease(ND)in avian species,which leads to substantial economic losses in the poultry industry worldwide.The amino acid sequence at the F protein cleavage site is a major determinant of NDV virulence.In general,the F protein cleavage site(Fcs)sequence of velogenic and mesogenic strains contains multi-basic amino acids with the motif “(R/K)-R-(Q/R/K)-(R/K)-R↓F”.Lentogenic Fcs contains a single or two basic amino acids with the motif “(G/E)-(K/R)-Q-(G/E)-R↓L”.The type,property and position of amino acids at the Fcs present diversity due to the strains.Although a large number of mutations in single or multiple amino acids at Fcs have been reported to affect the ability to induce cell fusion and viral virulence,the role of the diversity of amino acid sequences at the Fcs remains unknown.In addition,some studies have found that although the strains have same amino acid sequence in the Fcs,their ability to induce cell fusion and cause pathogenicity are quite different.This indicated that in addition to Fcs,the amino acids in other regions of F protein also plays an important roles in the fusogenic activity and viral virulence.In this study,we analyzed the diversity of amino acid sequence of NDV Fcs.The Fcs mutants were constructed on the base of the F genes of virulent and avirulent strains,respectively.Using in vitro assay,we explore the effect of the amino acid sequence diversity of the Fcs and other regions of F protein under specific Fcs motif on fusogenic activity.The contents and results of our studies are following:1.Comprehensive analysis of the diversity of Fcs in natural isolatesThe 1572 F gene sequences of NDV natural isolates were collected from the GenBank database and previous reports,and the diversity of Fcs was analyzed by software Lasergene7.1.Based on the pathogenicity of these natural isolates,the Fcs was classified into eight types of virulent Fcs(VFcs)with 1073 isolates,which belonged to Class II.Ten types of avirulent Fcs(AFcs)with 499 isolates,which mostly belonged to Class I,partly belonged to the genotype I,II and X of Class II.By analyzing the epidemiology of the strains,we foundthat there was a significant correlation between Fcs and bird species,temporal and spatial distribution.Some of them were species-dependent and region-specific characteristics.2.Effect of the Fcs amino acid sequence diversity on cell membrane fusionThe F gene of the virulent F48E9 strain and avirulent LaSota strain were used as a backbone to contruct 10 types of AFcs mutants and 8 types of VFcs mutants,respectively.The effect of inducing cell fusion efficiency was observed by co-transfected BHK-21 cells with F mutants and HN plasmids.The result showed that the neutral residue Q at the P3 position of the VFcs played an enhancing role compared to the basic residues R and K.The single residue K is more efficient of fusogenic activity at VFcs with five basic residues.The types VFcs-1and VFcs-2 are the most efficient for cell-cell membrane fusion activity.The contribution of these VFcs to fusion efficacy occurs in the order VFcs-1 > VFcs-3 and VFcs-4;VFcs-2 >VFcs-4 > VFcs-5,VFcs-6,VFcs-7 and VFcs-8.The AFcs mutants couldn’t induce syncytium formation in the absence of trypsin,except for the type AFcs-10,and could induce syncytium formation in the presence of trypsin,except for the type AFcs-9,which has the amino acid Q at the P4 position.3.Effect of other region of F protein with a cleavage motif “RRQRR↓L” on ability of inducing fusogenic activityThe Fcs motif “RRQRR↓L” was found to be an AFcs in above study.In generally,both virulent and avirulent F proteins with AFcs are unable to induce cell fusion.However,we found that the F protein of virulent strain F48E9 with this motif had a high ability of inducing fusogenic activity.Inversely,the F of avirulent strain La Sota with this motif was unable to induce cell fusion.The results suggested that other region of the F protein are involved in fusogenic activity in addition to the Fcs.To identify which region of the F proteins is involved in cell fusion,we divided the F protein into 9 segments based on the functional domains and exchanged them between that of the strains F48E9 and La Sota.Using cell transfection experiments,we analyzed the effect of these F protein mutants on the efficiency of cell fusion.Furthermore,the identifiacton of the F regions were narrowed by replacement or mutation.Finally,we identified that two amino acids(D479,S486)of the virulent strain F protein with this unique motif were critical for fusogenic activity under the condition of Fcs was“RRQRR↓L”.4.Establishment of a new approach for NDV induced cell fusionNDV infection causes syncytia formation in host cells.The extent of cell fusion can be used as a measure of viral pathogenicity.At present,the average number of nuclei in the syncytium is widely used to evaluate the cell fusion.Although this method is more accurate than the earlier determination of syncytia area,time-consuming and strong subjective are easyto be error.In order to explore a more convenient and effectively quantitative detection of the cell fusion,we successfully selected two fluorescent cell lines BHK-21/GFP and BHK-21/tRFP in this study.The two kinds of cells were mixed and then infected with virus,the cell fusion was determined by the number of syncytium(double fluorescence positive cells)through flow cytometry.The result showed that the proportion of double fluorescent positive cells reflected the linear relationship between the dose of virus infection and the number of syncytium after infected with NDV.This study provides a new technical method for the development of convenient and effectively quantitative detection of the cell fusion.In conclusion,our studies comprehensively elucidated the effect of amino acid sequence diversity of NDV Fcs on cell membrane fusion.It would provide the theoretical basis for the Fcs motifs in viral pathogenesis.Next,other regions of F protein also play an important role for inducing fusogenic activity in addition to Fcs.It provides new insights into the cell fusion triggered by F protein and design of antiviral peptides.更多还原