人巨细胞病毒蛋白UL80.5和UL84与宿主细胞因子相互作用及EGS抑制HSV-1病毒基因表达和增殖的研究

发布时间：2018-03-03 16:42

本文选题：HCMV　切入点：UL80.5装配蛋白前体　出处：《武汉大学》2016年博士论文　论文类型：学位论文

【摘要】：人巨细胞病毒(Human cytomegalovirus, HCMV)与单纯疱疹病毒1型(Herpes simplex virus, HSV-1)都是属于疱疹病毒家族的DNA病毒,在人群中的感染率很高。HCMV是导致新生儿先天性畸形和智力障碍的最主要病毒因素,目前抗病毒药物主要针对的是DNA聚合酶UL54,容易产生抗药性。为了扩展新抗病毒药物的研究思路,本文从病毒颗粒组装和免疫逃逸这两个病毒增殖过程中不可或缺的环节研究了病毒-宿主之间的相互作用。HSV-1病毒可在体内引起长期潜伏感染,立即早期ICP4基因是调控其基因表达引发增殖性感染的关键激活因子。我们通过对靶向HSV-1病毒ICP4基因mRNA的EGSs研究探索了这种新型抗病毒技术抑制病毒基因表达及增殖的效果。人巨细胞病毒组装蛋白前体UL80.5在病毒颗粒组装中起着不可或缺的作用,与多种病毒衣壳蛋白相互作用,起始并调控整个组装过程。本研究通过酵母双杂筛选发现SUMO化(Small Ubiquitin-like Modify, SUMOylation,小泛素样修饰)E2蛋白连接酶UBC9可以与UL80.5蛋白相互作用；UL80.5蛋白之后被证明可以被SUMO化,SUMO化位点为K371赖氨酸位点；通过竞争性免疫共沉淀实验发现增强UL80.5蛋白的SUMO化修饰可以抑制其与衣壳蛋白UL86的结合；UL80.5蛋白的SUMO化修饰缺失对病毒增殖略有抑制,但并不是重要影响因素。这些结果暗示HCMV病毒可能通过利用宿主细胞SUMO化系统帮助自身进行衣壳组装,但这种需要并不是必不可少的。人巨细胞病毒UL84蛋白被认为是与裂解性复制的起始有关的重要蛋白,它通过靶向结合oriLyt RNA/DNA杂交区域的茎-环RNA序列发生作用。UL84蛋白除调控DNA复制外,根据其DExD/H-box家族同源的结构分析还被认为有转录调控、RNA转运等功能的可能性。本研究通过酵母双杂筛选发现转录辅助因子FHL2可以与UL84蛋白相互作用,且共定位于细胞核；UL84蛋白与FHL2蛋白的结合区域为400-586位氨基酸,该区域包含有DExD/H-box家族同源的walker B结构域；双荧光实验显示UL84蛋白对IFNP(干扰素β)的转录有抑制作用,而FHL2蛋白对IFNβ转录有增强作用,且这两种结果相互拮抗。我们构建了靶向HSV-1 (herpes simplex virus 1)主要转录调节子ICP4蛋白mRNA的EGS,ICP4蛋白对于病毒早期和晚期基因表达及病毒增殖是必不可少的。EGS(External guide sequence)是一类特异识别与目标mRNA形成与tRNA前体相似二级结构的RNA序列,从而诱导内源的核酶P切割靶向mRNA.本研究发现根据EGS筛选系统构建的C468-A诱导核酶P切割ICP4基因mRNA的效率比根据天然前体tRNA结构构建的SER-A高60倍。在表达C468-A和SER-A的HSV-1病毒感染细胞中,ICP4基因表达分别下降大约97%和75%,病毒增殖抑制分别约7000和500倍。
[Abstract]:Human cytomegalovirus (HCMV) and herpes simplex virus type 1 (HSV-1) are DNA viruses belonging to the herpesvirus family. At present, antiviral drugs are mainly aimed at DNA polymerase UL54, which is easy to produce drug resistance. In order to expand the research ideas of new antiviral drugs, In this paper, we studied the interaction between virus and host. HSV-1 virus can cause long-term latent infection in vivo from the aspects of virus particle assembly and immune escape. Immediate and early ICP4 gene is the key activator to regulate its gene expression and induce proliferative infection. We explored this new antiviral technique to inhibit and increase the expression of ICP4 gene of HSV-1 virus by means of EGSs research on the target ICP4 gene of HSV-1 virus. Human cytomegalovirus assembly protein precursor UL80.5 plays an indispensable role in viral particle assembly. Interacting with a variety of viral capsid proteins, In this study, we found that SUMO small Ubiquitin-like modify, SUMOylation, small ubiquitin modified E 2 protein ligase UBC9 could interact with UL80.5 protein, and it was proved that the ligase UBC9 could be SUMO modified with UL80.5 protein. K371 lysine site; By competitive immunoprecipitation assay, it was found that enhancing the SUMO modification of UL80.5 protein could inhibit the SUMO modification deletion of capsid protein UL86 and inhibit the virus proliferation slightly. These results suggest that the HCMV virus may be helping to assemble its capsid by using the host cell SUMO system. But this need is not essential. The human cytomegalovirus UL84 protein is thought to be an important protein involved in the initiation of lytic replication. In addition to regulating the replication of DNA, it acts on the RNA sequence of stem loop by targeting the region of oriLyt RNA/DNA hybridization. According to the homologous structural analysis of its DExD/H-box family, it is also believed that it is possible to regulate the transcriptional translocation of UL84. In this study, we found that the transcriptional cofactor FHL2 can interact with UL84 protein by yeast double heterogeneity screening. Moreover, the binding region of FHL2 protein to UUL84 protein was 400-586 amino acids, which contained the walker B domain homologous to the DExD/H-box family, and the double fluorescence assay showed that UL84 protein could inhibit the transcription of IFNP- 尾. However, FHL2 protein enhanced IFN 尾 transcription. And these two kinds of results are antagonistic. We constructed the ICP4 protein mRNA, the main transcriptional regulator of HSV-1 herpes simplex virus 1, which is essential to the early and late gene expression and virus proliferation of the virus. Recognition of RNA sequences with target mRNA forming secondary structures similar to those of tRNA precursors. In this study, we found that the efficiency of C468-A induced ribozyme P cleavage of ICP4 gene mRNA was 60 times higher than that of SER-A constructed based on the structure of natural precursor tRNA. The expression of ICP4 gene in HSV-1 infected cells decreased by about 97% and 75, respectively, and the inhibition of virus proliferation was about 7000and 500-fold, respectively.
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R373

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