I型单纯疱疹病毒衣壳蛋白UL25与细胞骨架微管蛋白的相互作用及其生物化学分析

发布时间：2018-10-31 18:18

【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。
[Abstract]:Herpes simplex virus type I (Herpes Simplex Virus 1) is a common pathogen, and human is its only natural host. The virus infects the host through damaged skin and mucous membranes, proliferates in the epithelium, further invades neurons from the nerve terminals of the infected epithelium, and after initial infection in the trigeminal ganglion, Latent infection with lifetime in the dorsal root ganglion and other sites is established. HSV-1 UL25 gene encodes an important viral capsid protein located near the apical angle of the outer surface of the capsid. At present, it has been found that this protein is involved in many important biological processes of virus infection, including virus transport, proliferation, assembly and so on. Using yeast two-hybrid screening, we found that UL25 interacts with different structural and functional proteins of host cells, one of which is the interaction with cytoskeletal tubulin. The analysis of galactosidase activity showed that the interaction between galactosidase and galactosidase was specific. UL25 was recombined into green fluorescent report vector pEGFP-N2 and the constructed UL25-EGFP fusion protein was analyzed by intracellular fluorescence analysis. The results showed that UL25 was distributed in the cytoplasm and co-located with the cytoskeletal tubulin stained by immunofluorescence. Further using colchicine to destroy the microtubule structure of cytoskeleton, it was found that the distribution of UL25 and the disordered tubulin also basically coincided with each other, indicating that there was a clear interaction between the two proteins in space. Because HSV virus moves along the cytoskeleton microtubules in infected cells, based on the above two-hybrid experiments and fluorescence analysis, it is speculated that UL25 may play an important role in the transport of virus capsid cells. The results showed that UL25 135-353aa and its carboxyl terminal 360-580aa were similar to UL25 protein, while UL25 amino-terminal 140aa was located in cytoplasm and tubulin. They are also located in the nucleus. In combination with yeast two-hybrid experiments, the fact that UL25 interacted with intracellular transcription regulatory proteins such as transcriptional extenders suggests that UL25 proteins are involved in some unknown functions in the nucleus. The amino terminal may be an important region of its interaction with different proteins.
【学位授予单位】：中国协和医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R373

【共引文献】