【作者】 余娴；

【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， 2009， 博士

【摘要】 1型单纯疱疹病毒(Herpes simplex virus type 1, HSV-1)是一类对人类具有很强感染性的病毒,人类是其唯一的宿主,世界各地约45%以上的人曾感染过HSV-1。HSV-1原发感染后,机体虽然能够产生特异性免疫反应,但并不能彻底消除病毒,病毒以潜伏感染的形式终生存在于宿主神经细胞内,在一定状态下可以被重新激活而启动新一轮的裂解性感染。HSV-1基因以级联反应的方式表达,根据基因表达时序的不同,病毒基因被分为立即早期基因(α基因)、早期基因(β基因)和晚期基因(γ基因)。由于HSV-1基因表达方式的特殊性,它已被作为研究真核基因表达调控的模型和工具。除了DNA核心、核衣壳、包膜三种结构外,HSV-1还具有一种特殊的结构,即无定形的蛋白层——间层。虽然这20余种间层蛋白约占病毒体积的50%,并且许多间层蛋白以高拷贝数存在(1000～2500个／病毒颗粒),但是人们对这类蛋白的系统认识仅处于起步阶段。其中,VP22便作为一种在HSV-1中以高拷贝数存在的间层蛋白(2400个／病毒颗粒)而受到研究者的关注。迄今为止,对VP22的研究结果表明VP22对HSV-1的装配以及病毒颗粒的形成具有重要的支持意义。本研究以间层蛋白VP22的体外转录调控功能分析为切入点,首次揭示了VP22的普遍转录抑制效应,并初步探讨了VP22普遍转录抑制效应的可能机制；此外,还首次证明了VP22能显著抑制α蛋白ICP0的转录促进活性,并深入探讨了VP22、ICP0、组蛋白乙酰化酶(histone acetyltransferases, HAT) PCAF三者间的相互作用与VP22抑制ICP0的转录促进作用之间的密切联系。实验结果显示,VP22高表达在HSV-1感染Vero细胞后的一定时间内抑制HSV-1α、β和γ基因的转录；同时,VP22高表达在HSV-1感染Vero细胞后的一定时间内也抑制HSV-1的生长增殖能力。体外转录调控功能分析显示,VP22在一定范围内呈剂量效应关系普遍抑制HSV-1α、β和γ基因启动子的转录活性；此外,VP22也可以在一定范围内呈剂量效应关系,消除ICP0利用PCAF的有关功能而针对β、γ基因的转录促进作用。而VP22羧基端(151～301aa)是VP22行使其转录抑制功能的决定性功能域。初步探讨发现,募集组蛋白去乙酰化酶(histone deacetyltransferases, HDAC)并非为VP22普遍抑制病毒启动子转录的机制。进一步研究VP22、ICP0、PCAF三者间的相互作用表明,VP22能分布于细胞核与细胞质中,VP22通过其羧基端(151～301aa)与ICP0的环指区发生相互作用,VP22与PCAF间不存在相互作用。此外,VP22能竞争性抑制PCAF与ICP0环指区的结合。利用染色质免疫沉淀技术,我们进一步发现,在HSV-1感染过程中,VP22本身不能改变病毒基因启动子区组蛋白H3第14位赖氨酸(H3K14)的乙酰化状态,但却能显著抑制ICP0通过其环指区促进病毒基因启动子区组蛋白H3K14乙酰化修饰的功能。然而ICP0与VP22对Vero细胞总组蛋白H3K14的乙酰化修饰均无影响。基于上述的实验结果,我们建立了一个关于调控病毒基因转录的VP22、ICP0、PCAF三种蛋白间相互作用的模型：ICP0通过其环指区与PCAF结合而增强病毒基因启动子区组蛋白的乙酰化修饰,继而引发ICP0依赖PCAF的转录促进作用；而VP22羧基端与ICP0环指区的相互作用能竞争性抑制PCAF与ICP0环指区的结合,结果导致VP22显著抑制ICP0通过其环指区与PCAF结合而增强的病毒基因启动子区组蛋白的乙酰化修饰,最终造成VP22显著抑制ICP0依赖PCAF促进病毒基因转录的生物学效应。更多还原

【Abstract】 Herpes simplex virus type 1 is a kind of highly infectious pathogen for human which is the only host. And at least 45 percentage of the world’s population are infected with HSV-1. In its primary infection, HSV-1, which is able to induce the specific immune response of individual, can keep its long time existence in neurons of host through latent state. It will be reactivated to a lytic infection again under some certain conditions. During its replication, this viral gene expression is coordinately regulated and sequentially ordered in a cascade manner. According the sequence of gene expression, HSV-1 genes are designated asagenes,βgenes andygenes. Based on this understanding, HSV-1 is an important model for expressional regulation of eukaryotic genes.Besides core, capsid and envelope, HSV-1 has an unstructured proteinacous layer called the tegument. Although these more than 20 different kinds of tegument proteins are about 50 percentage of the viral volume and many tegument proteins have a large number of copies (1000-2500 molecules/virion), there is little systematic knowledge regarding these tegument proteins. VP22 has received notable attention for its large number of molecules in each polypeptide per virion (2400 molecules/virion). At present, VP22 is confirmed to be important for virus assembly and virus structure. Based on findings of its transcriptional regulatory functions in chloramphenicol acetyltransferase (CAT) assay, this study revealed the global transcriptional inhibitory effect of VP22 for the first time, and then analyzed the mechanisms. In addition, this study also illuminated VP22 repressing a protein ICPO transcriptional activation that attributed to the interactions of VP22, ICPO and histone acetyltransferases (HAT) PCAF. The results are shown as follow. During HSV-1 infection, overexpressed VP22 inhibited the transcriptions of HSV-1α,βand y genes during a period of time, and inhibited HSV-1 replication in Vero cells during a period of time. The CAT assay indicated that VP22 exerts a dose-dependent transcriptional inhibitory effect on HSV-1α,βand y gene promoters, and VP22 is capable of eliminating the ICPO transcriptional activation mediated by PCAF onβandγgene promoters in a dose-dependent manner. Carboxyl terminus of VP22 was absolutely required for its transcriptional inhibitory functions. And VP22 transcriptional repressive functions were not involved in recruiting histone deacetyltransferases. In analysis of interactions of VP22, ICPO, and PCAF, the results indicated that VP22 localizes in cytoplasm and nuclei, and carboxyl terminus of VP22 interacts with the RING finger domain of ICPO, but not PCAF. It was important that VP22 is able to competitively inhibit the interaction of PCAF and the ICPO RING finger domain. Chromatin Immunoprecipitation analyses revealed that VP22 do not impact the levels of acetylated histone H3K14 at virus promoters during HSV-1 infection, but VP22 can repress the enrichment of acetylated histones H3K14 at virus promoters dependent on the the ICPO RING finger domain. However, both ICPO and VP22 didn’t alter global pattern of acetylated histones H3K14 in Vero cells.Based on our studies, we summarize a protein-protein interaction model of VP22, ICPO and PCAF. On one hand, enrichment of acetylated histones at virus promoters mediated by interaction of the ICPO RING finger domain and PCAF leads to transcriptional activation. On the other hand, VP22 competitively inhibits the interaction of PCAF and the ICPO RING finger domain, which contributes to VP22 represses ICPO and PCAF-mediated enrichment of acetylated histones at virus promoters. And as a result, VP22 eliminates the ICPO transcriptional activation dependent on PCAF.更多还原