【作者】 郭磊；

【导师】 李琦涵； 刘龙丁； 董承红；

【作者基本信息】 中国协和医科大学 ， 免疫学， 2008， 硕士

【摘要】 Ⅰ型单纯疱疹病毒(Herpes Simplex Virus 1,HSV-1)是一种常见的病原体,人是其唯一自然宿主。病毒通过受损皮肤及粘膜感染宿主,在上皮组织增殖,且能进一步从感染的上皮组织的神经末稍入侵神经元,并于初次感染后在三叉神经节、背根神经节等处建立伴随终生的潜伏性感染。HSV-1 UL25基因编码一个重要的病毒衣壳蛋白,位于衣壳外表面五邻体顶角附近。目前研究发现该蛋白参与了病毒感染过程中的许多重要生物学过程,包括病毒在细胞内的转运、增殖、装配等。利用酵母双杂交筛选技术,我们发现UL25与宿主细胞不同的结构性和功能性蛋白存在相互作用,其中之一为与细胞骨架微管蛋白的相互作用。半乳糖苷酶活性分析实验显示两者的相互作用具有特异性。将UL25重组到绿色荧光报告载体pEGFP-N2中,对构建的UL25-EGFP融合蛋白进行细胞内荧光分析,结果表明UL25分布于细胞质中,与经免疫荧光染色的细胞骨架微管蛋白共定位。进一步利用秋水仙碱破坏细胞骨架微管结构后,发现UL25的分布与紊乱的微管蛋白亦基本重合,表明两者在空间上具有明确的相互作用关系。由于HSV病毒在被感染细胞内沿细胞骨架微管移动,基于上述双杂交实验和荧光分析,推测UL25可能在病毒衣壳细胞内转运中具有重要的意义。实验进一步对UL25基因不同片段的缺失突变体进行荧光定位分析,结果发现UL25 135-353aa和其羧基端360-580aa与UL25蛋白定位相似,而UL25氨基端140aa除在胞质内与微管蛋白共定位外,还同时定位于细胞核中。结合酵母双杂交实验筛选到UL25与细胞内转录调控蛋白如转录延伸因子存在相互作用的事实,提示UL25蛋白在核内参与了某些未知的功能作用,氨基端可能是其与不同蛋白相互作用的重要区域。更多还原

【Abstract】 Herpes Simplex Virus 1 (HSV-1) is a common pathogen, homo sapiens are its natural host. HSV-1 infects host through damaged skin or mucous membrane and proliferates in epithelial tissue. It even infects neurons from nerve terminal situated in epithelial tissue. After primary infection, the virus could establish latent infection at trige-minal ganglion and dorsal root ganglion etc.HSV-1 UL25 gene product is an essential capsid protein which locates on the external surface of virus capsid near the vertices. It is required for many crucial biological events during viral infection, including intracellular transport of virions、proliferation and assembling of virions in cell etc. Our research indicated UL25 gene product can interact with distinct structural and functional cell proteins by using yeast two-hybrid screening system. Cytoskeleton microtubule protein - tubulin was one of them.β-D-galactosidase activity assays demonstrated a specific interaction between UL25 and tubulin.UL25 was recombined into pEGFP-N2 to express UL25-EGFP fusion protein. Fluorescence experiment using the EGFP-UL25 fusion protein and an anti-cellular tubulin immunofluorescence antibody demonstrated that UL25 locates in cytoplasm and co-localizes with tubulin. Locations of UL25 were yet consistent with indiscriminate tubulins after treating cell with colchicine which can dissociate microtubule. These results indicate there are interactions between tubulin and UL25 in cell. Base on yeast two-hybrid screening and fluorescence analysis, it is possible that UL25 protein plays an essential role in transport of viral capsids owing to herpesviruses transport along microtubules in cell.Further fluorescence investigations with different fragment deletion mutants of the UL25 gene suggested that the locations of 135-353aa of UL25 and its carboxyl terminus 360-580aa were similar to UL25 protein, and the amino terminus 140aa of UL25 localized to cytoplasm(co-localized with tubulin), as well as to nucleus. Combining with results from yeast two-hybrid screening system that UL25 interacts with transcriptional and regulatory proteins such as eukaryotic translation elongation factor, we infer that UL25 involves in certain unknown functions in nucleus. It is possible the amino terminus is the significant domain of UL25 to interact with different cellular proteins.更多还原