Viral Modulation of Epigenetic Mechanisms

表观遗传机制的病毒调节

• 批准号: 8941146
• 负责人: Benjamin A Garcia
• 金额: $ 49.8万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-04-15 至 2020-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8941146
• 关键词: Address; Affect; Affinity; Antiviral Agents; Antiviral Therapy; Area; Biochemical; Biological; Biology; Cell Nucleus; Cells; Cellular Assay; Chromatin; Complex; Congenital Abnormality; Cytomegalovirus; Cytomegalovirus Infections; DNA Methylation; DNA Sequence; Data; Development; Drug Targeting; Early Promoters; Enzymes; Epigenetic Process; Foundations; Gene Expression; Gene Expression Profile; Genetic Transcription; Genome; Genomics; Goals; Growth; HIV; Herpesviridae; Histone H3; Histone H4; Histone-Lysine N-Methyltransferase; Histones; Human; Immune; Immunocompromised Host; Individual; Infection; Lead; Life; Longevity; Lysine; Malignant Neoplasms; Mass Spectrum Analysis; Mediating; Mental Retardation; Methods; Methyltransferase; Modification; Non-Histone Chromosomal Proteins; Nuclear Proteins; Oligonucleotide Probes; Pathogenesis; Patients; Pharmacotherapy; Population; Post-Translational Modification Site; Post-Translational Protein Processing; Proteins; Proteomics; Regulation; Research; Role; Signal Transduction; Site; Therapeutic; Time; Transplantation; Viral; Viral Genes; Viral Genome; Virus; Virus Diseases; base; cellular targeting; chromatin immunoprecipitation; chromatin protein; combinatorial; deafness; epigenome; genome-wide; histone modification; improved; insight; latent infection; methylome; novel; public health relevance; research study; response; small hairpin RNA

 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.

 描述（由申请人提供）：本提案的总体目标是确定人类巨细胞病毒（HCMV）感染期间表观遗传组蛋白翻译后修饰（PTM）的重要性，HCMV 是已知最大的人类疱疹病毒，并且众所周知。很大一部分人群（>60%）感染了 HCMV，HCMV 感染会导致出生缺陷；对于免疫功能低下的人群，这些感染通常会危及生命。病毒在宿主细胞核中的染色质化过程中，主要立即早期启动子 (MIEP) 控制病毒 IE1 和 IE2 蛋白的表达，这些蛋白与许多核蛋白相互作用，这些相互作用最终调节病毒和细胞基因表达。表观遗传学是指在不涉及 DNA 序列变化的情况下调节基因表达模式的机制，包括 DNA 甲基化、小 ncRNA 和组蛋白 PTM。 HCMV 如何利用表观遗传学机制的有限图景。理解 HCMV 发病机制和开发针对这些染色质因子的新型抗病毒疗法的重大障碍之前，我们确定 HCMV 感染会导致组蛋白上单个 PTM 位点发生多种变化，并且一个修饰位点（特别是 H3K79me2 及其相应的酶 DOT1L）会影响 HCMV。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 来控制宿主和病毒基因表达模式以及病毒复制？病毒生长所需的任何非组蛋白染色质蛋白？由于某些蛋白质赖氨酸甲基转移酶在 HCMV 感染期间上调，因此病毒复制过程中是否还有其他甲基化的非组蛋白蛋白？我们将结合使用以下方法来解决这些问题：我们将开发增强的质谱方法来量化 HCMV 感染时间过程中 MRC5 细胞的组合组蛋白 H3 和 H4 PTM。将用于鉴定具有显着组合 PTM 的宿主和病毒基因。将使用生物化学和方法来评估消耗负责最丰富组合 PTM 变化的酶对宿主和病毒基因表达以及病毒生长和复制的影响。亲和力方法，我们将分离全局病毒染色质和 MIEP 特异性病毒染色质，以确定有助于病毒转录基因调节的染色质因子 Lastl，我们将使用我们新颖的亲和力定量蛋白质组学方法。鉴定病毒感染过程中赖氨酸甲基化的非组蛋白蛋白预计这些实验将在 HCMV 生物学的研究领域产生新的基本见解，并可能导致用于治疗 HCMV 的新表观遗传药物疗法的开发。