口蹄疫病毒利用未知受體入侵宿主細(xì)胞的內(nèi)吞路徑研究
發(fā)布時(shí)間：2018-08-04 21:12
【摘要】：口蹄疫是由口蹄疫病毒(FMDV)引起的一種主要侵害偶蹄獸的急性熱性高度接觸性傳染病。FMDV是小RNA病毒科,口蹄疫病毒屬的典型成員,為無囊膜單股正鏈RNA病毒,其基因組大約含有8400個(gè)核苷酸。至少存在七種血清型,亞洲1型、A型、O型、C型和南非1、2和3型,型間無交叉保護(hù)性。即使同一血清型內(nèi)不同病毒的抗原性也有差異,為防制和消滅口蹄疫帶來一系列艱巨而復(fù)雜的問題。病毒首先通過受體結(jié)合位點(diǎn)(receptor-binding site,RBS)與細(xì)胞表面的受體結(jié)合起始感染,然后被內(nèi)化到Qg吞囊泡中,再通過未知機(jī)制被轉(zhuǎn)移到細(xì)胞核內(nèi)體中。目前已知的FMDV受體包括與FMDV結(jié)構(gòu)蛋白VP1的G-H環(huán)上RGD基序相互作用的四種整聯(lián)蛋白受體αvβ3、αvβ6、αvβ1和αvβ8;與FMDV結(jié)構(gòu)蛋白VP3上56位精氨酸相互作用的硫酸乙酰肝素(HS)受體;以及仍未被鑒定的第三受體。整聯(lián)蛋白結(jié)合型病毒利用網(wǎng)格蛋白介導(dǎo)的內(nèi)吞路徑感染細(xì)胞,并通過早期核內(nèi)體轉(zhuǎn)運(yùn)至循環(huán)核內(nèi)體釋放病毒。硫酸乙酰肝素結(jié)合型病毒通過小窩蛋白介導(dǎo)的內(nèi)吞途徑入侵細(xì)胞。進(jìn)入核內(nèi)體的FMDV依賴低pH環(huán)境誘導(dǎo)病毒衣殼裂解,并通過未知機(jī)制穿過病毒誘導(dǎo)的核內(nèi)體膜上的小孔來釋放病毒遺傳物質(zhì)。另外,發(fā)動(dòng)蛋白可促使內(nèi)吞囊泡與細(xì)胞膜的分離,在網(wǎng)格蛋白和小窩蛋白介導(dǎo)的內(nèi)吞中發(fā)揮主要作用。本課題以不含整聯(lián)蛋白β3、β6、β1和β8,和硫酸乙酰肝素的中國乳倉鼠腎細(xì)胞CHO-677為研究對象,通過細(xì)胞內(nèi)過表達(dá)顯性負(fù)突變體、RNA干擾和化學(xué)藥物阻斷等方法,系統(tǒng)分析了FMDV利用未知受體入侵宿主細(xì)胞的內(nèi)吞路徑。本研究首先建立了FMDV感染CHO-677細(xì)胞的模型,并通過病毒學(xué)實(shí)驗(yàn),分析了FMDV入侵宿主細(xì)胞的生物學(xué)特性。然后,通過藥物抑制試驗(yàn)發(fā)現(xiàn),FMDV的入侵可被網(wǎng)格蛋白內(nèi)吞路徑的特異性抑制劑氯丙嗪所抑制;同時(shí),抑制小窩路徑的藥物菲律賓菌素和染料木黃銅也可顯著病毒的復(fù)制。并進(jìn)一步通過外源轉(zhuǎn)染特異性的顯性負(fù)突變體或RNA干擾產(chǎn)品,破壞內(nèi)吞途徑中的關(guān)鍵分子,證明了FMDV利用未知受體入侵宿主細(xì)胞可同時(shí)利用網(wǎng)格蛋白介導(dǎo)的內(nèi)吞和小窩蛋白介導(dǎo)的內(nèi)吞。在以上研究基礎(chǔ)上,通過Dynamin-2的顯性負(fù)突變體驗(yàn)證了發(fā)動(dòng)蛋白對于病毒入侵宿主細(xì)胞的重要作用;通過藥物NH4Cl破壞核內(nèi)體酸性環(huán)境證明了病毒入侵細(xì)胞對核內(nèi)體低pH環(huán)境的依賴�？傊�,本研究證明了FMDV利用未知受體入侵宿主細(xì)胞時(shí)可同時(shí)利用網(wǎng)格蛋白依賴型內(nèi)吞途徑和小窩蛋白依賴型內(nèi)吞路徑。病毒的入侵依賴發(fā)動(dòng)蛋白的活動(dòng)和核內(nèi)體酸性環(huán)境。病毒的多種內(nèi)吞途徑和方式可能與病毒未知受體的復(fù)雜性或多樣性有關(guān)。本研究為深入研究FMDV入侵宿主細(xì)胞的方式和致病機(jī)制提供新的思路和依據(jù)。
[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, 偽 v 尾 3, 偽 v 尾 6, 偽 v 尾 1 and 偽 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 偽 v 尾 3, 偽 v 尾 6, 偽 v 尾 1 and 偽 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.
【學(xué)位授予單位】：甘肅農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：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, 偽 v 尾 3, 偽 v 尾 6, 偽 v 尾 1 and 偽 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 偽 v 尾 3, 偽 v 尾 6, 偽 v 尾 1 and 偽 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.
【學(xué)位授予單位】：甘肅農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：S852.65
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