口蹄疫病毒利用未知受体入侵宿主细胞的内吞路径研究
发布时间：2018-08-04 21:12
【摘要】：口蹄疫是由口蹄疫病毒(FMDV)引起的一种主要侵害偶蹄兽的急性热性高度接触性传染病。FMDV是小RNA病毒科,口蹄疫病毒属的典型成员,为无囊膜单股正链RNA病毒,其基因组大约含有8400个核苷酸。至少存在七种血清型,亚洲1型、A型、O型、C型和南非1、2和3型,型间无交叉保护性。即使同一血清型内不同病毒的抗原性也有差异,为防制和消灭口蹄疫带来一系列艰巨而复杂的问题。病毒首先通过受体结合位点(receptor-binding site,RBS)与细胞表面的受体结合起始感染,然后被内化到Qg吞囊泡中,再通过未知机制被转移到细胞核内体中。目前已知的FMDV受体包括与FMDV结构蛋白VP1的G-H环上RGD基序相互作用的四种整联蛋白受体αvβ3、αvβ6、αvβ1和αvβ8;与FMDV结构蛋白VP3上56位精氨酸相互作用的硫酸乙酰肝素(HS)受体;以及仍未被鉴定的第三受体。整联蛋白结合型病毒利用网格蛋白介导的内吞路径感染细胞,并通过早期核内体转运至循环核内体释放病毒。硫酸乙酰肝素结合型病毒通过小窝蛋白介导的内吞途径入侵细胞。进入核内体的FMDV依赖低pH环境诱导病毒衣壳裂解,并通过未知机制穿过病毒诱导的核内体膜上的小孔来释放病毒遗传物质。另外,发动蛋白可促使内吞囊泡与细胞膜的分离,在网格蛋白和小窝蛋白介导的内吞中发挥主要作用。本课题以不含整联蛋白β3、β6、β1和β8,和硫酸乙酰肝素的中国乳仓鼠肾细胞CHO-677为研究对象,通过细胞内过表达显性负突变体、RNA干扰和化学药物阻断等方法,系统分析了FMDV利用未知受体入侵宿主细胞的内吞路径。本研究首先建立了FMDV感染CHO-677细胞的模型,并通过病毒学实验,分析了FMDV入侵宿主细胞的生物学特性。然后,通过药物抑制试验发现,FMDV的入侵可被网格蛋白内吞路径的特异性抑制剂氯丙嗪所抑制;同时,抑制小窝路径的药物菲律宾菌素和染料木黄铜也可显著病毒的复制。并进一步通过外源转染特异性的显性负突变体或RNA干扰产品,破坏内吞途径中的关键分子,证明了FMDV利用未知受体入侵宿主细胞可同时利用网格蛋白介导的内吞和小窝蛋白介导的内吞。在以上研究基础上,通过Dynamin-2的显性负突变体验证了发动蛋白对于病毒入侵宿主细胞的重要作用;通过药物NH4Cl破坏核内体酸性环境证明了病毒入侵细胞对核内体低pH环境的依赖。总之,本研究证明了FMDV利用未知受体入侵宿主细胞时可同时利用网格蛋白依赖型内吞途径和小窝蛋白依赖型内吞路径。病毒的入侵依赖发动蛋白的活动和核内体酸性环境。病毒的多种内吞途径和方式可能与病毒未知受体的复杂性或多样性有关。本研究为深入研究FMDV入侵宿主细胞的方式和致病机制提供新的思路和依据。
[Abstract]:Foot-and-mouth disease (FMD) is an acute thermal highly contact infectious disease caused by foot-and-mouth disease virus (FMDV). FMDV is a typical member of the genus FMDV in small RNA family. It is a single-stranded positive strand RNA virus without envelope. Its genome contains about 8400 nucleotides. There were at least seven serotypes, Asian type 1, type A, type O, and South Africa type 1, type 2 and type 3, and there was no cross-protection between them. Even the antigenicity of different viruses in the same serotype is different, which brings about a series of difficult and complex problems for the prevention and control and elimination of foot-and-mouth disease. The virus was first infected by receptor binding site (receptor-binding site), then internalized into the QG vesicle, and then transferred to the nucleus via unknown mechanism. The known FMDV receptors include four integrin receptors, 伪 v 尾 3, 伪 v 尾 6, 伪 v 尾 1 and 伪 v 尾 8, which interact with RGD motif on G-H ring of FMDV structural protein VP1, and the (HS) receptor of acetyl sulfate interacting with arginine at 56 position on FMDV structural protein VP3, and 伪 v 尾 3, 伪 v 尾 6, 伪 v 尾 1 and 伪 v 尾 8, which interact with RGD motif on G-H ring of FMDV structural protein VP1. And a third receptor that has not yet been identified. The integrin binding virus infects the cells via the endocytosis pathway mediated by griddle protein and releases the virus into the circulating nucleus via the early nuclear endosome. Heparin-binding virus sulfate invades cells through the endocytosis pathway mediated by fossa proteins. The FMDV that enters the nucleus depends on the low pH environment to induce the virus capsid cleavage and release the virus genetic material through the holes in the nucleosome membrane induced by the virus through the unknown mechanism. In addition, activation proteins can promote the separation of endocytic vesicles from cell membranes and play a major role in endocytosis mediated by grid proteins and fossa proteins. In this study, CHO-677 of Chinese neonatal hamster renal cells without integrin 尾 3, 尾 6, 尾 1 and 尾 8 and heparin sulfate were studied by overexpression of dominant negative mutants and blocking of chemical drugs. The endocytosis pathway of FMDV invading host cells using unknown receptors was systematically analyzed. In this study, the model of FMDV infection on CHO-677 cells was established, and the biological characteristics of FMDV invading host cells were analyzed by virological experiments. Then, it was found that the invasion of FMDV was inhibited by chlorpromazine, a specific inhibitor of the endocytosis pathway of griddle protein, and that the drugs of Philippines and genistein, which inhibited the path of the nest, could also significantly replicate the virus. Furthermore, the key molecules in the endocytosis pathway were destroyed by exogenous transfection of specific dominant negative mutants or RNA interference products. It is proved that FMDV invades the host cells by unknown receptors and simultaneously endocytosis mediated by grid protein and fossa protein. On the basis of the above studies, the dominant negative mutants of Dynamin-2 were used to verify the important role of initiating proteins in invading host cells, and the dependence of invading cells on low pH environment was demonstrated by the destruction of acid environment in nucleus by drug NH4Cl. In conclusion, this study demonstrated that FMDV can use both the grid protein-dependent endocytosis pathway and the nest protein-dependent endocytosis pathway when invading host cells with unknown receptors. The invasion of the virus depends on the activity of the initiating protein and the acidic environment in the nucleus. The multiple endocytosis pathways and modes of viruses may be related to the complexity or diversity of virus unknown receptors. This study provides a new idea and basis for the further study of the way and pathogenesis of FMDV invading host cells.
【学位授予单位】：甘肃农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S852.65
[Abstract]:Foot-and-mouth disease (FMD) is an acute thermal highly contact infectious disease caused by foot-and-mouth disease virus (FMDV). FMDV is a typical member of the genus FMDV in small RNA family. It is a single-stranded positive strand RNA virus without envelope. Its genome contains about 8400 nucleotides. There were at least seven serotypes, Asian type 1, type A, type O, and South Africa type 1, type 2 and type 3, and there was no cross-protection between them. Even the antigenicity of different viruses in the same serotype is different, which brings about a series of difficult and complex problems for the prevention and control and elimination of foot-and-mouth disease. The virus was first infected by receptor binding site (receptor-binding site), then internalized into the QG vesicle, and then transferred to the nucleus via unknown mechanism. The known FMDV receptors include four integrin receptors, 伪 v 尾 3, 伪 v 尾 6, 伪 v 尾 1 and 伪 v 尾 8, which interact with RGD motif on G-H ring of FMDV structural protein VP1, and the (HS) receptor of acetyl sulfate interacting with arginine at 56 position on FMDV structural protein VP3, and 伪 v 尾 3, 伪 v 尾 6, 伪 v 尾 1 and 伪 v 尾 8, which interact with RGD motif on G-H ring of FMDV structural protein VP1. And a third receptor that has not yet been identified. The integrin binding virus infects the cells via the endocytosis pathway mediated by griddle protein and releases the virus into the circulating nucleus via the early nuclear endosome. Heparin-binding virus sulfate invades cells through the endocytosis pathway mediated by fossa proteins. The FMDV that enters the nucleus depends on the low pH environment to induce the virus capsid cleavage and release the virus genetic material through the holes in the nucleosome membrane induced by the virus through the unknown mechanism. In addition, activation proteins can promote the separation of endocytic vesicles from cell membranes and play a major role in endocytosis mediated by grid proteins and fossa proteins. In this study, CHO-677 of Chinese neonatal hamster renal cells without integrin 尾 3, 尾 6, 尾 1 and 尾 8 and heparin sulfate were studied by overexpression of dominant negative mutants and blocking of chemical drugs. The endocytosis pathway of FMDV invading host cells using unknown receptors was systematically analyzed. In this study, the model of FMDV infection on CHO-677 cells was established, and the biological characteristics of FMDV invading host cells were analyzed by virological experiments. Then, it was found that the invasion of FMDV was inhibited by chlorpromazine, a specific inhibitor of the endocytosis pathway of griddle protein, and that the drugs of Philippines and genistein, which inhibited the path of the nest, could also significantly replicate the virus. Furthermore, the key molecules in the endocytosis pathway were destroyed by exogenous transfection of specific dominant negative mutants or RNA interference products. It is proved that FMDV invades the host cells by unknown receptors and simultaneously endocytosis mediated by grid protein and fossa protein. On the basis of the above studies, the dominant negative mutants of Dynamin-2 were used to verify the important role of initiating proteins in invading host cells, and the dependence of invading cells on low pH environment was demonstrated by the destruction of acid environment in nucleus by drug NH4Cl. In conclusion, this study demonstrated that FMDV can use both the grid protein-dependent endocytosis pathway and the nest protein-dependent endocytosis pathway when invading host cells with unknown receptors. The invasion of the virus depends on the activity of the initiating protein and the acidic environment in the nucleus. The multiple endocytosis pathways and modes of viruses may be related to the complexity or diversity of virus unknown receptors. This study provides a new idea and basis for the further study of the way and pathogenesis of FMDV invading host cells.
【学位授予单位】：甘肃农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S852.65
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