BRD4溴结构域抑制剂促进人单纯疱疹病毒复制及其机理研究

发布时间：2017-12-28 16:31

本文关键词：BRD4溴结构域抑制剂促进人单纯疱疹病毒复制及其机理研究　出处：《南京大学》2017年博士论文　论文类型：学位论文

【摘要】：人单纯疱疹病毒(human herpes simplex virus,HSV)是一类具有囊膜结构的线性双链DNA病毒,是引起人类皮肤性疾病最常见的病原体。根据血清型不同,该病毒可分为Ⅰ型和Ⅱ型(HSV-1和HSV-2)。HSV-1主要感染面部的皮肤和黏膜,引起口龈炎、疱疹性角膜炎、皮肤疱疹性湿疹。HSV-2主要感染生殖器部位的皮肤和黏膜,导致生殖器疱疹,是性传播疾病(STD)的主要病原体,还能显著增加HIV(human immunodeficiency virus)感染的风险。据世界卫生组织(World Health Organization,WHO)统计发现,截至2012年,全球50岁以下人口中,约有2/3人数感染过HSV-1,而在15-49岁人群中,有超过5.5亿人有生殖器疱疹病毒感染。HSV感染具有溶细胞感染(lyticinfection)和潜伏感染(latentinfection)两种形式。溶细胞感染,也称裂解性感染,是指HSV感染上皮细胞后,进行复制,引起细胞发生病变(cytopathiceffect,CPE)直至裂解死亡。与此同时,部分病毒沿轴突上行至神经节潜伏,在神经元细胞中建立潜伏感染。潜伏期的HSV在宿主细胞中不进行复制,但过度劳累或免疫力下降等因素可以将潜伏的HSV病毒激活,导致病毒迁移至上皮细胞造成再次感染,这是造成HSV反复感染的主要原因。HSV如何建立和维持潜伏感染,以及再激活的机制都尚不清楚。HSV感染必须借助宿主细胞的转录系统以实现自身基因的表达,而这一过程自然会受到宿主转录系统的调控。文献中对于宿主转录系统调控病毒基因表达的作用机制虽然有比较细致的描述,但是对于HSV感染以及潜伏—再激活转换的分子机理缺乏了解。在溶细胞感染过程中,病毒非核小体DNA被注入细胞核,并快速环化成有组蛋白修饰的染色质,主要表现为基因组的甲基化和乙酰化,并且与病毒溶细胞感染过程中的复制以及病毒潜伏状态的维持密切相关。因此组蛋白乙酰化或者甲基化因素可以显著影响HSV复制和感染。为探究表观遗传调控对HSV感染的影响,本课题选择筛选表观遗传学小分子化合物库这一策略来寻找能够对溶细胞感染过程中病毒感染复制产生影响的表观遗传调控因素。我们发现两类结构互异的小分子化合物可以显著促进HSV复制。除已经报道的组蛋白去乙酰化酶抑制剂HDACi(Histone deacetylases inhibitor,HDACi)外,我们还发现 BET 家族蛋白溴结构域(bromodomain,BD)抑制剂,包括 PFI-1,(+)-JQ1,I-BET-762 等能够显著促进 HSV-1 和 HSV-2 溶细胞感染。JQ-1 是BRD4(Bromdomain-containing protein4,BRD4)选择性抑制剂,其通过与BRD4的BD区(bromodomain,BD)结合,阻断BRD4与乙酰化赖氨酸结合,从而抑制宿主细胞基因转录。作为一个多结构域蛋白,BRD4的C末端结构域能够与正性转录延伸因子(Positive transcription elongation factor,P-TEFb)结合形成具有转录活性的复合物。为了进一步研究BRD4溴结构域抑制剂促HSV复制的机制,我们首先研究了 BRD4在HSV溶细胞感染中的作用。用siRNA抑制细胞BRD4蛋白表达,我们发现抑制BRD4表达能够明显抑制病毒复制,提示BRD4是HSV溶细胞感染所需要的宿主因子;通过免疫共沉淀以及免疫荧光染色方法,我们发现HSV感染诱导BRD4与P-TEFb亚单元CDK9结合,并且与DNA依赖性RNA聚合酶(RNAPII)形成复合物,JQ-1处理可以增加这些复合物的形成,表明JQ-1可能通过BRD4及蛋白复合物形成而加速病毒mRNA延伸过程;最后,通过染色质免疫沉淀(ChIP)实验我们发现该转录复合物能够定位于病毒基因启动子区,与之相对应的是JQ1处理能够增加该复合物在HSV基因启动子区富集。由于JQ-1是作用机制相对清楚的小分子探针,其主要通过阻断BD域与乙酰化赖氨酸结合,影响组蛋白乙酰化修饰及宿主细胞基因转录过程,因此我们研究了过表达BRD4结构域在HSV感染中的作用,发现过表达BD1(aa1-196),BD1/2(aa 1-640)以及C端结构域(aa 197-1362)能不同程度地促进HSV感染。这些结果表明BRD4在基因转录方面调控HSV溶细胞感染过程。化学生物学是90年代新兴的一门学科,其特点之一是将小分子作为工具发现生命过程中的复杂生物学问题。小分子化合物探针具有作用靶点专一、作用机制相对清楚的特点,因此可以用于发现调控生命过程的重要分子靶点、构建蛋白作用网络。本研究通过筛选表观遗传学小分子化合物库发现BRD4蛋白溴结构域抑制剂能够促进人单纯疱疹病毒的溶细胞感染,并以小分子抑制剂JQ1为工具,同时结合病毒学、细胞分子生物学以及化学生物学的研究方法,首次发现并证明了在HSV溶细胞感染过程中,BRD4蛋白参与了病毒基因的转录调控,并初步探明了 JQ1能够促进HSV溶细胞感染复制的作用机制。这些工作为今后深入地理解和研究细胞对HSV基因表达的调控机制提供了新的思路和实验基础。
[Abstract]:Human herpes simplex virus (HSV) is a class of linear double stranded DNA viruses with envelope structure. It is the most common pathogen causing skin diseases in humans. According to the different serotypes, the virus can be divided into type I and type II (HSV-1 and HSV-2). HSV-1 mainly infects the facial skin and mucous membrane, cause gingivitis, herpes simplex keratitis, skin eczema. HSV-2 mainly infects skin and mucosa of genital organs, leading to genital herpes. It is the main pathogen of sexually transmitted disease (STD), and it can also significantly increase the risk of HIV (human immunodeficiency virus) infection. According to statistics from WHO (World Health Organization, WHO), by the year 2012, about 50 of the world's population under the age of 15-49 had been infected with HSV-1, while in the age of 15-49, more than 550 million people had genital herpes virus infection. HSV infection has two forms of cytolytic infection (lyticinfection) and latent infection (LATENTINFECTION). Cytolytic infection, also known as cleavage infection, refers to the replication of HSV infected epithelial cells, causing cell lesions (cytopathiceffect, CPE) until lysis and death. At the same time, some viruses move along the axon to the ganglion, and the latent infection is established in the neuron cells. The latent period of HSV does not replicate in host cells, but excessive fatigue or decreased immunity can activate the latent HSV virus, leading to the migration of virus to epithelial cells, resulting in re infection, which is the main reason for HSV repeated infection. It is not clear how HSV establishes and maintains latent infection, and the mechanism of reactivation. HSV infection must use the transcriptional system of the host cell to realize the expression of its own genes, and this process is naturally regulated by the host transcriptional system. Although there is a detailed description of the mechanism of host transcriptional system regulating viral gene expression, there is a lack of understanding about the molecular mechanism of HSV infection and latency reactivation transformation. In the process of cytolytic infection, virus non nucleosome DNA was injected into the nucleus, and quickly cyclizes with chromatin histone modification, mainly for genome methylation and acetylation, and viral cytolytic infection during replication and virus latency is closely related to the maintenance of. Therefore, the factors of histone acetylation or methylation can significantly affect HSV replication and infection. To explore the effect of epigenetic regulation on HSV infection, we screened the epigenetic small molecule library strategy to find epigenetic regulation factors that influence the replication of viral infection during lytic infection. We found that two types of small molecular compounds with different structures can significantly promote HSV replication. In addition to the already reported histone deacetylase inhibitor HDACi (Histone deacetylases inhibitor, HDACi), we also found that BET protein family bromodomain (bromodomain, BD) inhibitors, including PFI-1, -JQ1, I-BET-762 (+) HSV-1 and HSV-2 could significantly promote the cytolytic infection. JQ-1 is a selective inhibitor of BRD4 (Bromdomain-containing protein4, BRD4), which blocks the binding of BRD4 to acetylated lysine through binding with BD region (bromodomain, BD) of BRD4, thereby inhibiting the gene transcription of host cells. As a multi domain protein, the C terminal domain of BRD4 can bind to Positive transcription elongation factor (P-TEFb) to form a transcriptional complex. In order to further study the mechanism of BRD4 bromine domain inhibitor to promote HSV replication, we first studied the role of BRD4 in HSV cell infection. The inhibition of BRD4 protein expression by siRNA, we found that inhibition of BRD4 expression can inhibit virus replication, suggesting that BRD4 is a host factor HSV cytolytic infection in need; by CO immunoprecipitation and immunofluorescence staining methods, we found that HSV infection induced by combination of BRD4 and P-TEFb subunits and CDK9 dependent RNA polymerase (RNAPII and DNA) complex formation, formation of JQ-1 treatment can increase these complexes, indicating that JQ-1 may through BRD4 and protein complex formation and accelerated viral mRNA extension process; finally, by chromatin immunoprecipitation (ChIP) experiments we found that the transcription complex to the promoter region of the virus gene localization, corresponding to the JQ1 treatment can increase the complexes in the promoter region of HSV gene enrichment. Because JQ-1 is a small molecule probe mechanism is relatively clear, mainly by blocking the BD domain and the acetyl lysine binding effect, histone modification and host cell gene transcription process, we studied the expression of BRD4 domain in HSV infection, the expression of BD1 (aa1-196). BD1/2 (AA 1-640) and C terminal domain (AA 197-1362) can be promoted HSV infection. These results suggest that BRD4 regulates the process of HSV cell infection in gene transcription. Chemical biology is a newly emerging subject in the 90s. One of its features is the discovery of small molecules as a tool for the discovery of complex biological problems in the life process. Small molecule probes have specific targets and relatively clear mechanisms, so they can be used to identify important molecular targets for regulating life processes and construct protein interaction networks. This study through screening epigenetic small molecular library found BRD4 protein bromodomain inhibitors can promote human herpes virus cytolytic infection, and small molecule inhibitors of JQ1 as a tool, combined with the virology, molecular cell biology and chemical biology research methods, first discovered and proved in HSV cytolytic infection process BRD4, the transcriptional regulation of proteins involved in viral genes, the mechanism and preliminary verified JQ1 can promote HSV cytolytic replication. These work for the future to understand and study the cell to H
【学位授予单位】：南京大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R373

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