HSV-1感染相关细胞分子HTRP的转录调控功能分析

发布时间：2018-04-25 23:12

本文选题：Ⅰ型单纯疱疹病毒(HSV-1) + HTRP　；参考：《中国协和医科大学》2010年硕士论文

【摘要】： Ⅰ型单纯疱疹病毒(HSV-1)是具有较强传染性,严重危害人类健康的病原体,人类是其唯一的宿主,至今尚无相应疫苗问世。作为一种结构复杂,而且在基因转录复制过程中表现明确级联效应的双链DNA病毒,HSV-1在与细胞的相互作用过程中呈现出了不同层次的特点。对于HSV-1病毒结合受体所导致的细胞早期基因产物功能的分析,不仅能加深对HSV-1和细胞之间相互作用的理解,也有助于阐明细胞中未知功能蛋白的作用方式及其分子机理。在此领域中,我们的前期研究发现HSV-1与人成纤维细胞KMB-17株表面受体结合后,能够刺激细胞基因表达水平发生差异性的改变。同时,我们利用mRNA差异显示和蛋白2D差异技术所特异诱导的细胞相关分子做了较为深入的研究,并从中筛选克隆到了一些新基因。本研究的htrp基因,即为上述研究工作中所克隆的功能未知的基因之一(基因登录号：AF442486)。htrp所编码蛋白HTRP(human transcription regulator protein)作为一个与细胞转录调控相关的蛋白分子,表现出能够与转录辅助抑制复合物mSin3A的成分之一SAP30 (mSin3 A Association Protein, SAP30)之间相互作用,并由此促进HSV-1感染细胞死亡的生物学效应。本课题围绕着HTRP分子与SAP30分子在细胞与宿主之间的相互作用中的生物学功能及分子机制展开研究,揭示了HTRP对HSV-1病毒的普遍转录抑制效应,进一步证实了HTRP与SAP30的转录抑制协同效应,并深入探讨了两者相互作用与转录抑制的密切联系及其分子机制。首先我们利用上调表达HTRP的细胞环境来研究其对病毒复制增殖的影响,结果显示HTRP可以抑制感染不同时期的病毒TK、gC基因的转录活性,且随着时间的延长而抑制越明显。接着,在HTRP和SAP30同时表达上调的细胞环境下,进一步证实了两者对TK、gC基因的转录抑制表现出协同效应。有关细胞分子生物学的研究资料已经证明,SAP30作为细胞内转录调控的辅抑制复合物中的重要结构成分之一,具有介导组蛋白去乙酰化酶(hi stone deacetyltransferases, HDAC)结合至该复合物的功能,我们的实验表明HTRP和SAP30则可以通过使染色质组蛋白的去乙酰化途径实现对酶基因的转录抑制。为了进一步验证HTRP与SAP30对病毒基因的协同抑制作用的分子机制,在双荧光素酶检测系统中加入HDACs抑制剂TSA的实验证明,这种协同作用是通过HDAC酶活性的改变而实现的。同时,使用染色质免疫沉淀(Chromatin Immunoprecipitation, ChIP)实验表明,由于HTRP与SAP30的结合而导致HDACs活性的改变,使得HDACs酶增强了组蛋白H3分子第14和9位赖氨酸的去乙酰化水平。基于上述实验结果,我们可以针对HSV-1感染人成纤维细胞所特异上调的细胞蛋白分子HTRP的具体分子生物学作用机理提出如下模型：HTRP作为一种细胞在HSV-1感染时反应性上调的转录调控分子,可以通过和SAP30的结合,增加经辅抑制复合物Sin3A而发挥功能的HDACs酶的活性,从而使得结合HSV-1基因启动子区域的组蛋白成分H3的第9、14位赖氨酸去乙酰化水平上调,其结果在一定程度上抑制了HSV-1相关基因的转录过程,并影响病毒的复制增殖。这一模型的建立,为我们更深入地理解HSV-1与宿主细胞相互作用通过细胞蛋白修饰酶系统来影响病毒基因转录调控,从而导致HSV-1感染细胞所致的多种病理学结果提供了实验依据。
[Abstract]:Herpes simplex virus type I (HSV-1) is a pathogen with strong contagion and serious harm to human health. Human is the only host and there is no corresponding vaccine yet. As a complex structure, and in the process of gene transcription, the double chain DNA virus that shows clear cascade effect, HSV-1 in the process of interaction with cells. The analysis of the function of the early gene products caused by the HSV-1 virus binding receptor not only deepens the understanding of the interaction between HSV-1 and cells, but also helps to elucidate the mode of action of the unknown functional proteins in the cells and their molecular mechanisms. In this field, our previous study found that HS After the combination of V-1 with the surface receptor of human fibroblast KMB-17 strain, it can stimulate the difference in the level of cell gene expression. At the same time, we have made a more in-depth study by using mRNA differential display and protein 2D differentially induced cell related molecules, and screened some new genes from it. The HTRP of this study. Gene, HTRP (human transcription regulator protein), one of the genes (human transcription regulator protein) encoded by one of the unknown functions cloned in the above research work (AF442486), is a protein molecule associated with cell transcription regulation, showing SAP30 (mSin3 A) that is one of the components of the transcriptional assisted inhibition complex mSin3A. The interaction between Association Protein and SAP30 promotes the biological effects of HSV-1 infection on cell death.
This research focuses on the biological functions and molecular mechanisms of HTRP and SAP30 molecules in the interaction between cells and hosts, reveals the universal transcriptional inhibition effect of HTRP on the HSV-1 virus, and further confirms the synergistic effect of transcription inhibition between HTRP and SAP30, and probes into the interaction and transcriptional inhibition of both HTRP and SAP30. Close connection and its molecular mechanism. First, we use the cell environment that up-regulated the expression of HTRP to study its effect on the replication and proliferation of the virus. The results show that HTRP can inhibit the transcriptional activity of TK, gC gene in different stages of infection, and the inhibition is more obvious with the prolongation of time. Then, the up-regulated cells are expressed at the same time in HTRP and SAP30. In the environment, the synergistic effect of both TK and gC gene transcription inhibition is further confirmed. Research data on cell molecular biology have shown that SAP30 is one of the important structural components in the cooppressor complex of intracellular transcriptional regulation, which mediates the histone deacetylase (HI stone deacetyltransferases, HDAC). In combination with the function of the complex, our experiments showed that HTRP and SAP30 could inhibit the transcriptional inhibition of the enzyme gene by making the chromatin histone deacetylation pathway. In order to further verify the molecular mechanism of the synergistic inhibitory effect of HTRP and SAP30 on the virus gene, the HDACs inhibitor TSA was added to the double luciferase detection system. The experiment proved that the synergistic effect was realized through the changes in the activity of HDAC enzyme. At the same time, the use of chromatin immunoprecipitation (Chromatin Immunoprecipitation, ChIP) experiments showed that the activity of HDACs was changed by the combination of HTRP and SAP30, which made the HDACs enzyme enhanced the deacetylation level of the fourteenth and ninth bit lysine of the histone H3 molecule.
Based on the above results, the following model is proposed for the specific molecular biological mechanism of the cell protein molecule HTRP, which is specifically up-regulated by HSV-1 human fibroblasts. HTRP is a transcriptional regulator with a reactive up regulation of a cell in HSV-1 infection. It can be combined with SAP30 to increase the co inhibition compound. The activity of the function of Sin3A and the function of the HDACs enzyme makes the level of lysine deacetylation up-regulated in the 9,14 site of the histone component H3 of the promoter region of the HSV-1 gene, which inhibits the transcriptional process of the HSV-1 related genes and affects the replication and proliferation of the virus. Understanding the interaction of HSV-1 and host cells through the cell protein modified enzyme system affects the regulation of viral gene transcription, which leads to a variety of pathological results caused by HSV-1 infected cells.

【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】：2010
【分类号】：R392

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