单纯疱疹病毒Ⅰ型VP26蛋白在病毒感染过程中的分子生物学功能分析

发布时间：2018-05-15 11:04

本文选题：单纯疱疹病毒Ⅰ + 质膜蛋白tetraspanin　；参考：《北京协和医学院》2008年博士论文

【摘要】：单纯疱疹病毒工(HSV-1)是一种结构复杂的双链DNA病毒,具有较强传染性,在细胞内的复制过程中与宿主细胞之间表现有多种相互作用。HSV-1与细胞的相互作用是研究HSV-1感染不同细胞后会形成不同的感染过程及结果的基础。以这个角度来看,对于病毒蛋白及其细胞作用蛋白在病毒感染周期中的功能分析,在探索细胞对病毒感染产生相应反应的机理,和由此机理所导致的各种可能的生物学意义,以及由此确定的临床治疗或诊断方法等领域内的应用都具有很大的意义。 HSV-1 VP26是核衣壳组成中最小的蛋白,其功能至今尚未阐明。本实验的研究重点是分析在病毒感染周期中病毒核衣壳蛋白VP26的功能及寻找与其相互作用的细胞蛋白。首先构建了UL35-EGFP重组病毒,利用该重组病毒与野毒株的生长曲线的差异分析,发现重组病毒出现了出胞膜阻滞的现象,提示VP26的功能可能与病毒感染周期中的出质膜环节相关。随后通过酵母双杂交系统筛查到VP26和细胞膜上的蛋白——CMTP-7有相互作用,免疫共沉淀和双荧光细胞共定位分析验证了二者在完整细胞中存在生理性相互作用。随后采用siRNA技术和实时定量PCR方法进行了进一步的研究,在敲除CMTP-7蛋白的细胞内,病毒复制装配未受影响,但出现病毒出胞阻滞的现象,检测病毒滴度发现病毒生长量低于正常细胞内的所得数量,运用透射电镜也观察到了在近细胞膜的胞质外围处出现了堆积成团的病毒。而流式细胞仪分析发现在病毒出细胞时CMTP-7也随之减少。这些结果表明CMTP-7的功能和HSV-1出质膜这一过程相关。HSV-1在CMTP-7蛋白敲除的细胞内和UL35-EGFP重组病毒在正常细胞内所表现出的特性极为相似。以上结果强烈提示二者通过相互作用在完成同一项功能。即VP26具有与病毒包膜化和有效出胞膜的相关功能,并且是通过CMTP-7相互作用来完成这一对病毒感染周期至关重要的过程。而在荧光显微镜下还观察到,CMTP-7可以随病毒再次感染其它细胞。并且在新生病毒粒上检测出了CMTP-7蛋白。这些结果似乎表明CMTP-7和VP26作用后,对于病毒的意义远不止是体现在出胞释放这一个过程中。至此,我们据本实验研究结果对VP26和CMTP-7的相互作用在HSV-1感染周期中所起的作用提出初步假设。核衣壳在细胞核内装配成熟后,经过胞质运输到达质膜,VP26蛋白通过和CMTP-7的相互作用,触发了HSV-1包膜化的进程,并且在完成HSV-1的包膜化的同时进行有效出胞的过程。CMTP-7也结合在病毒粒核衣壳外层被膜中并且跟随病毒参与新一轮细胞感染。
[Abstract]:Herpes simplex virus (HSV-1) is a complex double-stranded DNA virus with strong infectivity. The interaction between HSV-1 and host cells in the process of intracellular replication. The interaction between HSV-1 and cells is the basis for the study of different infection processes and results of HSV-1 infection with different cells. From this point of view, the functional analysis of viral proteins and their cell-acting proteins in the viral infection cycle is used to explore the mechanism of cell response to virus infection and the possible biological implications of the mechanism. And the clinical treatment or diagnostic methods are of great significance. HSV-1 VP26 is the smallest protein in the nucleocapsid composition, and its function has not been elucidated. The focus of this study was to analyze the function of viral nucleocapsid protein (VP26) in the viral infection cycle and to find the cell proteins interacting with it. Firstly, the UL35-EGFP recombinant virus was constructed. By analyzing the difference between the growth curve of the recombinant virus and that of the wild strain, it was found that the recombinant virus appeared the phenomenon of cell membrane block, which suggested that the function of VP26 might be related to the plasma membrane in the virus infection cycle. Then the interaction between VP26 and CMTP-7 was detected by yeast two-hybrid system. The results of immunoprecipitation and bifluorescence cell co-localization showed that there was physiological interaction between VP26 and CMTP-7 in intact cells. SiRNA technique and real-time quantitative PCR were used to further study. In the cells knockout CMTP-7 protein, the replication and assembly of the virus was not affected, but the phenomenon of virus exocytosis was found. Detection of virus titer showed that the virus growth was lower than that of normal cells, and a cluster of viruses was observed near the cytoplasm of the cell membrane by transmission electron microscopy (TEM). Flow cytometry showed that CMTP-7 also decreased when the virus came out of the cell. These results suggest that the function of CMTP-7 is closely related to the process of HSV-1 exudation. HSV-1 exhibits very similar characteristics in CMTP-7 protein knockout cells and UL35-EGFP recombinant virus in normal cells. The above results strongly suggest that the two perform the same function by interacting with each other. That is to say, VP26 has the function of virus encapsulation and effective exfoliation, and it is a very important process of virus infection cycle through CMTP-7 interaction. Under fluorescence microscope, CMTP-7 could reinfect other cells with the virus. CMTP-7 protein was detected on the neovascularization virus. These results suggest that the effect of CMTP-7 and VP26 on the virus is much more than the process of cell release. So far, we hypothesized the role of VP26 and CMTP-7 interaction in HSV-1 infection cycle. After the nucleocapsid was assembled and matured in the nucleus, the VP26 protein was transported to the plasma membrane through the cytoplasmic transport, which triggered the process of HSV-1 encapsulation through the interaction with CMTP-7. CMTP-7 also binds to the outer membrane of the nucleocapsid of the virus and follows the virus to participate in the new round of cell infection.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R373

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