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（由申请方提供）：本提案的总体目标是确定人巨细胞病毒（HCMV）感染期间表观遗传组蛋白翻译后修饰（PTM）的意义。HCMV是已知的最大的人类疱疹病毒，并且众所周知，大比例的人类群体（>60%）感染有HCMV。HCMV感染会导致出生缺陷;对于免疫功能低下的人群，这些感染往往会危及生命。在感染细胞和病毒在宿主细胞核中染色质化后，主要立即早期启动子（MIEP）控制与许多核蛋白相互作用的病毒IE 1和IE 2蛋白的表达。这些相互作用最终调节病毒和细胞基因表达，部分通过表观遗传机制。表观遗传学是指可以调节基因表达模式而不涉及DNA序列变化的机制，包括DNA甲基化，小ncRNA和组蛋白PTM。HCMV如何利用表观遗传机制的有限图片提出了一个重要的障碍，了解HCMV的发病机制和开发新的抗病毒治疗，以靶向这些染色质因子。以前，我们确定HCMV感染导致组蛋白上单个PTM位点的几个变化，并且一个修饰位点特别是H3 K79 me 2及其相应的酶DOT 1 L影响HCMV生长。在这里，我们的目标是回答以下问题：HCMV如何利用组合PTM组蛋白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？由于某些蛋白质赖氨酸甲基转移酶在HCMV感染过程中上调，是否有其他非组蛋白在感染过程中甲基化，这是病毒复制所需的？我们将使用生物学和定量方法相结合来解决这些问题。我们将开发增强的质谱方法来定量在HCMV感染时程期间来自MRC 5细胞的组合组蛋白H3和H4 PTM。将采用全基因组方法来鉴定具有显著组合PTM的宿主和病毒基因。将评估消耗负责最丰富的组合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。使用生物化学和亲和方法，我们将分离全球病毒染色质和MIEP特异性病毒染色质，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HCMV生物学的未充分研究领域产生新的基本见解，并可能导致开发用于治疗HCMV的新的表观遗传药物疗法。