人巨细胞病毒蛋白pUL23影响宿主蛋白RACK1与STAT1间的相互作用

发布时间：2018-01-05 11:00

本文关键词：人巨细胞病毒蛋白pUL23影响宿主蛋白RACK1与STAT1间的相互作用　出处：《暨南大学》2012年硕士论文　论文类型：学位论文

【摘要】：人巨细胞病毒（Human cytomegalovirus HCMV）属疱疹病毒科β亚属（β疱疹病毒）,在人群中的感染率十分普遍。HCMV像其它β疱疹病毒一样，不能被宿主体内的免疫系统完全清除，它会以一种较低的病毒水平保持对宿主的持续感染或者是以一种非活性状态持续潜伏于宿主体内，因而对于免疫功能正常的人，HCMV可长期潜伏而不致病。但是当宿主免疫功能低下时，HCMV则可大量繁殖，进而引起严重的致死并发症。随着目前多个HCMV病毒株完整基因组核酸序列的分析完成，HCMV基因组结构已基本被阐明。通过对病毒临床株基因的分析和缺失突变等发现，在200多个开放阅读框中（Open Reading Frame，ORF），仅有45到57个ORFs为病毒在人成纤维细胞中繁殖所必要，余下的都为非必需基因。研究表明，部分病毒蛋白参与了协调与宿主间细胞关系，，以达到自身与宿主细胞长期共生的目的。 UL23基因是HCMV US22基因家族的成员，它能编码一个大小为33kD的病毒皮层蛋白pUL23,聚集在细胞质中的核周边区域。然而对于pUL23蛋白的功能目前了解甚少。为了进一步阐明pUL23的潜在功能，本实验室前期工作利用酵母双杂交方法，从人胚肾cDNA文库中筛选到多个与pUL23相互作用的宿主蛋白。RACK1[Receptor for activated C kinase1]就是其中之一。RACK1是蛋白激酶C的受体蛋白，因其可以和多种类型的蛋白分子相结合，从而被普遍地认为是一种多功能脚手架蛋白。本课题利用回复性酵母双杂交、免疫共沉淀等实验方法进一步确认了pUL23与RACK1间的相互作用,且pUL23和RACK1共定位于细胞质中。研究发现RACK1能够与非磷酸化的STAT1结合，并作为一个脚手架蛋白发挥了招募STAT1到干扰素受体上的功能。而STAT1与干扰素受体的预先结合是STAT1活化和干扰素信号途径传导必不可少的一步，这说明RACK1与STAT1的结合对STAT1的活化和干扰素信号途径的传导是至关重要的。因此本文利用GSTpull-down实验检测了pUL23与RACK1的结合对RACK1-STAT1相互结合的影响，实验结果表明pUL23减弱了RACK1与STAT1间的亲和力，这说明病毒蛋白pUL23能够更牢固的“抓住”RACK1蛋白，进而导致RACK1、STAT1或许还有干扰素受体在内的复合物的解离。由文献可知，RACK1与STAT1结合的减弱会直接导致STAT1磷酸化水平的下降和干扰素抗病毒信号途径传导的受阻。因此本文的研究结果提高了pUL23在干扰素信号途径中发挥调节作用的可能性，并为今后pUL23蛋白功能的研究提供了诸多有意义的线索。
[Abstract]:Human cytomegalovirus (Human cytomegalovirus HCMV) is a beta herpesvirus subgenus (beta herpesvirus). The infection rate in population is very common.HCMV like other beta herpes virus, can not be completely removed the host immune system in the body, it will be in a low level of virus infection on the host to keep or in a non active state persistent in the host body, and for people with normal immune function, HCMV can lurk without disease. But when the host immune function is low, HCMV can multiply, causing serious complications. With the death of nucleic acid sequence analysis of multiple strains of HCMV complete genome completion HCMV, the genome structure has been elucidated. Through the analysis of clinical strains of virus and lack of gene mutations found in the more than 200 open reading frame (Open Reading Frame, ORF), only 45 to 57 ORFs is necessary for virus propagation in human fibroblasts, and the rest are non essential genes. Studies show that some viral proteins are involved in coordinating cell relationship with host cells, so as to achieve long-term purpose of their own host cells.
UL23 gene is a member of the HCMV US22 gene family encoding, it can a 33kD virus tegument protein pUL23, nuclear surrounding areas gathered in the cytoplasm. However, the function of pUL23 protein is currently poorly understood. In order to further elucidate the potential function of pUL23, the previous work in our laboratory using yeast two hybrid screening methods, from the people embryo kidney cDNA library to multiple interactions with the pUL23.RACK1[Receptor for activated C kinase1] host protein is one of the.RACK1 receptor protein protein kinase C, due to its many types of protein molecules and the combination, which was widely believed to be a multifunctional protein scaffold. The recovery of yeast two hybrid and Co immunoprecipitation experiments further confirmed the interaction between RACK1 and pUL23, and pUL23 and RACK1 were localized in the cytoplasm.
The research found that RACK1 and non phosphorylated STAT1 binding, and as a scaffolding protein play on the recruitment of STAT1 to interferon receptor function. STAT1 and interferon receptor binding is essential for pre STAT1 activation and interferon signal transduction step, which indicates that the combination of RACK1 and STAT1 on activation of STAT1 and interferon signaling the way of transmission is very important. So we use GSTpull-down test to detect effects of combination of pUL23 and RACK1 on RACK1-STAT1 with each other, the experimental results show that pUL23 can decrease the affinity between STAT1 and RACK1, suggesting that the virus protein pUL23 can be more firmly hold RACK1 protein, leading to RACK1, STAT1 may have complex dissociation compound interferon receptor including. By literature, weaken the RACK1 combined with STAT1 will directly lead to the phosphorylation level of STAT1 decreased and interference Therefore, the results of this study improve the possibility of pUL23 regulating the function of IFN signaling pathway, and provide many meaningful clues for the research of pUL23 protein function in the future.

【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R363

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