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（由适用提供）：该提案的总体目的是确定人类巨细胞病毒（HCMV）感染期间翻译后修饰（PTMS）的重要性。 HCMV是最大的已知人类疱疹病毒，人们众所周知，大部分人口（> 60％）感染了HCMV。 HCMV感染会导致先天缺陷；对于免疫功能低下的人群，这些感染通常会危及生命。细胞感染并在宿主核中染色体后，主要的即时启动子（MIEP）控制了与许多核蛋白相互作用的病毒IE1和IE2蛋白的表达。这些相互作用最终通过表观遗传机制部分调节病毒和细胞基因表达。表观遗传学是指可以调节基因表达模式的机制，而无需涉及DNA序列的变化，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情况为理解HCMV发病机理和开发新型抗病毒疗法以靶向这些染色质因子的重要障碍。以前，我们确定HCMV感染会导致组蛋白上的单个PTM位点的几次变化，并且一个尤其是H3K79me2的修饰位点及其相应的酶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来控制宿主和病毒基因表达模式和病毒复制？是否有任何非历史染色质蛋白需要用于病毒生长？由于在HCMV感染期间某些蛋白质赖氨酸甲基转移酶被上调，因此在感染过程中是否还有其他非固定蛋白甲基化的病毒复制？我们将结合生物学和定量方法解决这些问题。我们将开发增强的质谱法，以在HCMV感染时间过程中量化MRC5细胞的组蛋白H3和H4 PTM的组合组蛋白H3和H4 PTM。将聘请全基因组方法来识别具有重要组合PTM的宿主和病毒基因。耗尽负责对宿主和病毒基因表达以及病毒生长和复制的最丰富组合PTM变化的酶的影响将得到评估。使用生化和亲和力方法，我们将分离全局病毒染色质和MIEP特异性病毒染色质，以确定有助于调节病毒基因转录的染色质工厂。最后，我们将使用新型的亲和力定量蛋白质组学方法来鉴定在病毒感染期间赖氨酸甲基化的非固定蛋白。预计这些实验将在HCMV生物学领域中产生新的基本见解，并可能导致新的表观遗传药物治疗用于治疗HCMV。