α属冠状病毒S蛋白的变异对其细胞感染特异性的影响

发布时间：2018-04-03 00:02

本文选题：α属冠状病毒　切入点：S蛋白　出处：《复旦大学》2011年硕士论文

【摘要】：冠状病毒(Coronaviruses, CoV)为有包膜的正链RNA病毒,分为α、β和γ三个属。CoV的RNA基因组发生重组后,相关基因的突变突破种属障碍,实现跨宿主感染。CoV的S蛋白识别细胞受体并介导膜融合,是病毒感染宿主细胞的关键因子。S蛋白S1结构域的受体识别区(Receptor binding domain, RBD)直接参与了受体的识别,该区域的氨基酸变异会导致病毒的种属嗜性和感染特性的变化。由于α属CoV的受体为氨肽酶N(Aminopeptitase N, APN),其氨基酸序列高度保守。因此,S蛋白的变异在很大程度上决定了CoV的种属特异性。本论文选择犬冠状病毒(Canine coroanvirus, CCoV)、猪传染性胃肠炎病毒(Transmissible gastroenteritis virus, TGEV)和猫冠状病毒(Feline coronavirus, FCoV)三种α属冠状病毒为模型,研究三种病毒在不同宿主来源细胞上的感染特性。并构建嵌合异源S基因的重组病毒,探讨CoV种属间传播的分子机制。首先,用CCoV、FCoV和TGEV分别感染犬A72、猫FCWF和猪ST细胞,通过噬斑实验等方法分析病毒复制与细胞病理变化(Cytopathic effect, CPE)差异。研究发现：三种病毒在其天然宿主来源的细胞上均良好增殖,CPE出现的时间较早,细胞病变明显并且典型,噬斑的大小和形状较一致,病毒滴度峰值出现在感染后12小时左右。在犬A72和猫FCWF细胞上三种病毒的噬斑形态存在差异：TGEV在A72细胞上的适应性不如CCoV和FCoV;CCoV和TGEV对FCWF细胞的病变小于FCoV。在猪ST细胞上,CCoV几乎不引起明显的CPE,而FCoV出现CPE较晚,二者均不能形成明显噬斑和有效增殖。另外,CCoV、FCoV和TGEV均不能在小鼠L2细胞上增殖,同样β属MHV也不能感染A72、FCWF和ST细胞。上述结果说明同属冠状病毒容易突破宿主障碍,但不同属间不容易突破。其次,将CCoV、FCoV和TGEV的S基因分别克隆到载体pMH54中替换MHV的S基因,未能获得重组病毒。但将三个病毒的S基因膜外区替换MHV的S基因膜外区,结果显示相应的重组MHV复制特性与野生型相似。此结果证明α属CoV的细胞感染性差异与S基因膜外区有关,而膜内区可能通过与病毒其它蛋白的作用限制S蛋白装配到病毒粒子上。进一步制备α属CoV的S蛋白N端高变区替代的重组病毒,从而更精确地对决定属内跨宿主感染的S蛋白的氨基酸定位是必要的。
[Abstract]:Coronaviruses (CoV) is a positive strand RNA virus with envelope. After recombination of RNA genome of 伪, 尾 and 纬, the mutation of related genes breaks through the obstacle of species, and the S protein of transhomologous infection. CoV recognizes cell receptor and mediates membrane fusion.Receptor binding domain (RBDs), the receptor recognition region of S1 domain of S protein, is a key factor of virus infection in host cells. The amino acid variation in this region will lead to the change of species eosinophilia and infection characteristics.The amino acid sequence of 伪 -genus CoV is highly conserved because its receptor is aminopeptidase N(Aminopeptitase N, APNN.Therefore, the variation of S protein determines the species-specificity of CoV to a great extent.The recombinant virus containing chimeric heterologous S gene was constructed to study the molecular mechanism of interspecies transmission of CoV.Firstly, CCoV FCoV and TGEV were used to infect canine A72, cat FCWF and porcine St cells respectively. The difference of viral replication and cytopathic effectt (CPE) was analyzed by plaque assay.CCoV did not cause significant CPE in St cells, but CPE appeared late in FCoV, neither of them could form plaque and proliferate effectively.In addition, CCoV FCoV and TGEV could not proliferate on L2 cells, and neither 尾 MHV could infect A72 FCWF and St cells.These results suggest that the coronavirus of the same genus can easily break through the host barrier, but it is not easy to break through among different genera.Secondly, the S gene of CCoV FCoV and TGEV were cloned into the vector pMH54 to replace the S gene of MHV, and the recombinant virus was not obtained.However, the S gene extracellular region of MHV was replaced by the S gene extracellular region of the three viruses. The results showed that the replication characteristics of the corresponding recombinant MHV were similar to those of the wild type.The results showed that the difference in cell infectivity of 伪 -genus CoV was related to the extracellular region of S gene, and the intramembrane region might restrict the assembly of S protein to virus particles by interaction with other proteins of the virus.Furthermore, it is necessary to prepare the recombinant virus substituted for the N-terminal hypervariable region of S protein of 伪 -genus CoV, so as to more accurately locate the amino acid of S protein which determines the intrageneric cross host infection.
【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R373.9

【参考文献】