人巨细胞病毒相互作用蛋白的系统筛选和功能研究

发布时间：2018-10-25 12:49

【摘要】：人巨细胞病毒(human cytomegalovirus,HCMV)是导致先天性流产和新生儿智力低下的主要病毒病因,并且在人免疫缺陷病毒(human immunodeficiency virus,HIV)感染者中引发严重和致死性的并发症。为探讨HCMV病毒感染和复制的分子机制,本文从3个方面对HCMV病毒的相互作用蛋白进行了系统筛选和功能研究,分别是:(1)运用酵母双杂交技术系统筛选8个HCMV病毒DNA复制蛋白(UL44,UL54,UL57,UL70,UL84,UL102,UL105,UL114)的相互作用细胞蛋白,部分结果进一步进行免疫共沉淀验证。由此发现了包括UBC9、SNAPAP、PIAS3、FHL2、DNAJB6、CTBP2、POLD2、ATRX、PFAAP5 和 ZMYND19 等在内的 130 个能同上述病毒蛋白相互作用的细胞蛋白和154个不同的相互作用关系。结合来源于文献的数据,该结果为全面了解涉及到HCMV病毒DNA复制的宿主-病毒蛋白间相互作用网络通路的复杂性提供了基础,同时也描绘出一个病毒蛋白"由基因至功能"的全过程草图。(2)运用酵母交配技术系统筛选174个HCMV病毒蛋白同16个HIV病毒蛋白间可能的相互作用关系。由此发现了 7个HIV病毒蛋白同7个HCMV病毒蛋白间存在的 12 对相互作用,它们分别是 PR-RL1、Nef-RL1、p17-RL1、p24-RL1、Tat-RL1、p6-RL1、p6-UL119、p6-UL125、IN-RL7、IN-UL24、IN-UL45 和 IN-UL77。这些相互作用的发现,为发展出控制HCMV-HIV病毒共感染、致病的策略和手段提供了一个全新的视角。(3)运用激光共聚集、His pull down和免疫共沉淀等实验手段,从体外和体内证实了 HCMV的ppUL44蛋白同细胞小泛素样修饰E2结合酶UBC9的相互作用,确认了 ppUL44的11-16位和260-269位的这两段氨基酸短区域对结合至关重要。并进一步证实了 ppUL44是一个新发现的小泛素样修饰蛋白(sumo)底物,sumo-1、-2、-3蛋白均可以共价结合到病毒蛋白上,ppUL44位于ψKxE保守序列中的410位赖氨酸是ppUL44主要的小泛素样修饰位点。这些结果证明了 UBC9蛋白同ppUL44的结合、及小泛素样修饰在调节ppUL44的功能和HCMV病毒的复制方面的重要作用。上述3个方面的研究,将有利于阐明病毒基因在致病和复制中的功能,进而深入理解巨细胞病毒的感染机制。
[Abstract]:Human cytomegalovirus (human cytomegalovirus,HCMV) is the main cause of congenital abortion and neonatal mental retardation, and it can cause severe and fatal complications in (human immunodeficiency virus,HIV infection. In order to investigate the molecular mechanism of HCMV infection and replication, the interaction proteins of HCMV virus were systematically screened and their functions were studied from three aspects. The results were as follows: (1) eight interacting cellular proteins of DNA replication protein (UL44,UL54,UL57,UL70,UL84,UL102,UL105,UL114) of HCMV virus were screened by yeast two-hybrid technique, and some of the results were further verified by immunoprecipitation. A total of 130 cellular proteins, including UBC9,SNAPAP,PIAS3,FHL2,DNAJB6,CTBP2,POLD2,ATRX,PFAAP5 and ZMYND19, were found to interact with the virus proteins and 154 different interactions were found. Combined with data from the literature, the results provide the basis for a comprehensive understanding of the complexity of the host-virus protein interaction network pathway involved in the replication of HCMV virus DNA. At the same time, a whole process sketch of viral protein "from gene to function" was described. (2) the possible interaction between 174 HCMV proteins and 16 HIV virus proteins was screened by yeast mating technique. 12 pairs of interactions between 7 HIV virus proteins and 7 HCMV virus proteins were found. They are PR-RL1,Nef-RL1,p17-RL1,p24-RL1,Tat-RL1,p6-UL119,p6-UL125,IN-RL7,IN-UL24,IN-UL45 and IN-UL77.. The discovery of these interactions provides a new perspective for the development of strategies and means to control co-infection and pathogenesis of HCMV-HIV virus. (3) Experimental methods such as laser co-aggregation of, His pull down and immunoprecipitation are used. In vitro and in vivo, the interaction of HCMV ppUL44 protein with small ubiquitin modified E2 binding enzyme (UBC9) was confirmed. The short amino acid regions at the 11-16 and 260-269 positions of ppUL44 were confirmed to be crucial for the binding. It is further confirmed that ppUL44 is a newly discovered (sumo) substrate of a small ubiquitin like modified protein, and sumo-1,-2,-3 protein can be covalently bound to the viral protein. Lysine at the 410th position in the conserved sequence of ppUL44 in 蠄 KxE is the main small ubiquitin like modified site of ppUL44. These results indicate that the binding of UBC9 protein to ppUL44, and the role of ubiquitin like modification in regulating the function of ppUL44 and the replication of HCMV virus. The above three studies will be helpful to clarify the function of viral gene in pathogenesis and replication, and to further understand the mechanism of cytomegalovirus infection.
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R373

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