Viral Modulation of Epigenetic Mechanisms

 描述(由申请人提供)：本提案的总体目标是确定表观遗传性组蛋白翻译后修饰(PTM)在人类巨细胞病毒(HCMV)感染过程中的意义。巨细胞病毒是已知的最大的人类疱疹病毒，众所周知，很大比例的人类群体(60%)感染了巨细胞病毒。巨细胞病毒感染会导致出生缺陷；对于免疫功能低下的人群来说，这些感染往往会危及生命。病毒直接早期启动子(MIEP)控制病毒IE1和IE2蛋白的表达，这些蛋白与多种核蛋白相互作用。这些相互作用最终调节病毒和细胞的基因表达，部分是通过表观遗传机制。表观遗传学是指在不改变DNA序列的情况下调节基因表达模式的机制，包括DNA甲基化、小ncRNAs和组蛋白PTM。关于巨细胞病毒如何利用表观遗传机制的有限图景为理解巨细胞病毒的发病机制和开发针对这些染色质因子的新型抗病毒治疗提供了重要障碍。在此之前，我们确定了HCMV感染会导致组蛋白上单个PTM位点的几个改变，其中一个修饰位点特别是H3K79me2及其相应的酶DOT1L影响了HCMV的生长。在这里，我们旨在回答以下问题：HCMV如何利用组蛋白上的组合PTM来控制宿主和病毒的基因表达模式和病毒复制？有没有病毒生长所需的非组蛋白染色质蛋白？当某些蛋白赖氨酸甲基转移酶在巨细胞病毒感染过程中上调时，是否有其他非组蛋白蛋白在感染过程中发生甲基化，是病毒复制所必需的？我们将使用生物学和定量相结合的方法来解决这些问题。我们将开发增强质谱学方法来定量检测在HCMV感染时间过程中来自MRC5细胞的组合组蛋白H3和H4 PTM。基因组范围的方法将被用来识别宿主和病毒基因，这些基因含有显著的组合PTM。将评估耗尽负责最丰富的组合PTM变化的酶对宿主和病毒基因表达以及对病毒生长和复制的影响。使用生化和亲和方法，我们将分离全球病毒染色质和MIEP特异的病毒染色质，以确定有助于调节病毒基因转录的染色质因子。最后，我们将使用我们的新的亲和定量蛋白质组学方法来鉴定在病毒感染过程中赖氨酸甲基化的非组蛋白。预计这些实验将在一个未被充分研究的HCMV生物学领域产生新的基础性见解，并可能导致开发用于治疗HCMV的新的表观遗传药物疗法。

 DESCRIPTION (provided by applicant): The overall objective of this proposal is to determine the significance of epigenetic histone post-translational modifications (PTMs) during human cytomegalovirus (HCMV) infection. HCMV is the largest known human herpes virus and it is well recognized that a large percentage of the human population (>60%) is infected with HCMV. HCMV infections cause birth defects; and for immunocompromised populations, these infections are often times life threatening. Upon infection of cells and chromatinization of the virus in the host nucleus, the major immediate-early promoter (MIEP) controls the expression of the viral IE1 and IE2 proteins which interact with many nuclear proteins. These interactions ultimately modulate both viral and cellular gene expression, in part through epigenetic mechanisms. Epigenetics refers to mechanisms that can regulate gene expression patterns without involving changes in DNA sequence, and include DNA methylation, small ncRNAs and histone PTMs. The limited picture of how HCMV harnesses epigenetic mechanisms presents a significant barrier to understanding HCMV pathogenesis and developing novel antiviral therapy to target these chromatin factors. Previously, we determined that HCMV infection results in several changes to single PTM sites on histones, and that one modification site in particular H3K79me2 and its corresponding enzyme DOT1L affected HCMV growth. Here we aim to answer the following questions: How does HCMV utilize combinatorial PTMs on histones for controlling both host and viral gene expression patterns and viral replication? Are there any non- histone chromatin proteins that are needed for viral growth? As certain protein lysine methyltransferases are upregulated during HCMV infection, are there other non-histone proteins methylated during infection that are needed for viral replication? We will address these questions using a combination of biological and quantitative methods. We will develop enhanced mass spectrometry methods to quantify combinatorial histone H3 and H4 PTMs from MRC5 cells during an HCMV infection time-course. Genome-wide approaches will be employed to identify both host and viral genes harboring significant combinatorial PTMs. The effect of depleting the enzymes responsible for the most abundant combinatorial PTM changes on both host and viral gene expression, and on viral growth and replication will be assessed. Using biochemical and affinity approaches, we will isolate both global viral chromatin and MIEP specific viral chromatin to determine the chromatin factors that contribute to regulation of viral gene transcription. Lastl, we will use our novel affinity quantitative proteomics approach to identify non-histone proteins that are lysine methylated during viral infection. It is expected that these experiments will produce new fundamental insights in an understudied area of HCMV biology, and could lead to development of new epigenetic drug therapy for treatment of HCMV.