亚洲1型FMDV RGD基序和VP1第154位突变对宿主致病性的影响

发布时间：2018-08-14 11:39

【摘要】：病毒与受体吸附是病毒起始感染的第一步,在致病过程中起着决定性的作用。病毒受体在宿主范围和组织嗜性中也起着重要的作用。野生型口蹄疫病毒(foot-and-mouth disease virus,FMDV)主要通过衣壳蛋白VP1上G-H环中RGD基序吸附整联蛋白受体起始感染宿主和宿主细胞。因此对FMDV受体和受体识别位点的研究将为FMDV致病机制和宿主嗜性以及宿主范围的研究具有重要的意义。先前已有报道,整联蛋白受体识别基序以及RGD+1和+4位氨基酸突变影响FMDV的致病表型。本研究基于Asai1型FMDV出现了规律性的G-H环中RGD基序和RGD+9氨基酸的变化,且该变化与病毒致病表型具有一定的关联性。为了,(1)查明RGD突变对FMDV识别整联蛋白受体的影响;(2)阐释RGD+9位氨基酸突变对FMDV致病性和识别整联蛋白受体的影响;(3)探索FMDV利用非整联蛋白非硫酸乙酰肝素受体入侵宿主细胞的内吞路经;我们进行如下研究:1.RGD突变对FMDV识别猪和牛源整联蛋白受体的影响前期实验室已经拯救了毒株rRGD、rRDD和rRSD,然后比较了这些毒株在宿主细胞上的生物学特性和对宿主动物猪和牛的致病性,也比较了它们对鼠源整联蛋白受体的识别能力。在此,在前期研究的基础上进一步比较它们对猪和牛源整联蛋白受体的识别能力,(1)克隆猪和牛源整联蛋白受体各亚基基因,构建各亚基的真核表达质粒并验证了表达效果。(2)比较了三个毒株对单个整联蛋白受体的识别能力,结果表明,RGD突变改变了Asia1型FMDV对整联蛋白受体的识别能力,而且同一毒株对不同宿主的同一种整联蛋白受体识别能力有差异。2.RGD+9(VP1-154)位氨基酸突变对FMDV致病性和识别整联蛋白受体的影响(1)通过FMDV单质粒反向遗传系统拯救了VP1 S154D突变的毒株。(2)在宿主细胞上生物学特性测定及对乳鼠致病实验结果表明,VP1 S154D突变增强了FMDV在宿主细胞上的生长能力和致病表型以及对乳鼠的致病能力。(3)对猪的致病性实验结果表明,VP1 S154D突变增强了FMDV对猪的致病性。(4)最后比较了两个毒株对猪源整联蛋白受体的利用情况,结果表明,VP1 S154D突变增强了Asia1型FMDV对猪源整联蛋白受体的识别能力,尤其是αvβ6和αvβ8。3.FMDV第三受体介导的内吞路径鉴定近些年,许多报道表明FMDV存在第三受体,但是至今尚未被鉴定。实验室利用Asia1型FMDV感染不含整联蛋白和硫酸乙酰肝素受体的CHO-677细胞模型,在进行第三受体鉴定的同时,也开展了第三受体介导的内吞路径的研究。在这个研究中,通过药物抑制实验、siRNA干扰实验和显性负突变抑制实验,结果表明,FMDV Asia1/HN/CHA/06内吞进入CHO-677细胞时,即利用网格蛋白介导的内吞路径,也利用小窝蛋白介导的内吞路径,同时需要动力蛋白的参与和核内体的酸性环境。综上所述,在本研究中,建立了FMDV猪和牛源整联蛋白受体的瞬时表达系统,并比较了RGD突变对FMDV识别整联蛋白受体的影响,揭示了RGD突变导致致病性变异的可能原因;证明了VP1 S154D突变增强了Asia1/HN/CHA/06毒株的致病性,鉴定了G-H环中又一个重要的位点。探索了FMDV Asia1/HN/CHA/06利用非整联蛋白、非硫酸乙酰肝素受体入侵CHO-677细胞的内吞路径,为将来揭示第三受体的内吞路径奠定了基础。
[Abstract]:Adsorption of viruses to their receptors is the first step in the initiation of viral infection and plays a decisive role in the pathogenesis. Viral receptors also play an important role in host range and tissue tropism. Therefore, the study of FMDV receptor and receptor recognition sites will play an important role in the pathogenesis, host tropism and host range of FMDV. Previous reports have shown that integrin receptor recognition motifs and RGD+1 and+4 amino acid mutations affect the pathogenic phenotype of FMDV. The RGD motif and RGD+9 amino acids in the G-H loop of type 2 FMDV were changed regularly, and the changes were correlated with the pathogenic phenotype of FMDV. We used the non integrin non heparan sulfate receptor to invade the endocytosis pathway of host cells; we conducted the following research: * the 1.RGD mutation has previously rescued the strains rRGD, rRDD and rRSD from the FMDV identification of pig and bovine integrin receptors, and then compared the biological characteristics of these strains on host cells and their active effects on host cells. * the pathogenicity of pig and cattle also compared their ability to identify the integrin receptor of the rat. Based on the previous studies, we further compared their ability to identify the integrin receptor of pig and cattle * * (1) cloning the subunit genes of the integrin receptor from pig and cattle, constructing the eukaryotic expression plasmid of each subunit and verifying the expression. The results showed that RGD mutation changed the recognition ability of Asia1 FMDV to integrin receptor, and the same strain had different recognition ability to the same integrin receptor in different hosts. 2. RGD + 9 (VP1-154) amino acid mutation had different pathogenicity and recognition to FMDV. The effect of integrin receptor (1) rescued the VP1 S154D mutant strain through the FMDV single particle reverse genetic system. (2) the biological characteristics of host cells and the pathogenic experiment on suckling mice showed that VP1 S154D mutation enhanced the growth capacity and pathogenicity phenotype of FMDV on the host cells and the pathogenicity of suckling mice. (3) pigs * The pathogenicity test results showed that VP1 * S154D mutation enhanced the pathogenicity of FMDV to pigs. (4) finally, the utilization of two integrins * receptors in pig origin was compared. The results showed that VP1 * S154D mutation enhanced the ability of Asia1 FMDV to identify the integrin receptor of pig, especially the endocytosis mediated by alpha V beta 6 and alpha v beta 8.3.FMDV third receptor. In recent years, many reports have shown that FMDV has a third receptor, but so far it has not been identified. Laboratory studies on the endocytosis pathway mediated by the third receptor have been carried out in the CHO-677 cell model without integrin and heparan sulfate receptors infected by Asia1 FMDV. The results of drug inhibition, siRNA interference and dominant negative mutagenesis inhibition showed that when FMDV Asia1/HN/CHA/06 enters CHO-677 cells, both reticulin-mediated endocytosis pathway and pit-mediated endocytosis pathway are used, together with the participation of motilin and acidic environment of nucleosome. In this study, we established a transient expression system of FMDV and RGD *, and compared the effect of FMDV mutation on FMDV recognition of integrin receptor, and revealed the possible cause of RGD mutation leading to pathogenicity variation. It was proved that VP1 S154D mutation enhanced the pathogenicity of Asia1/HN/CHA/06 strain and identified another important part of G-H loop. The endocytosis pathway of FMDV Asia1/HN/CHA/06 in CHO-677 cells by non-integrin and non-heparan sulfate receptors was explored, which laid a foundation for revealing the endocytosis pathway of the third receptor in the future.
【学位授予单位】：中国农业科学院
【学位级别】：博士
【学位授予年份】：2015
【分类号】：S852.65

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