Mechanisms regulating cytomegalovirus

巨细胞病毒的调节机制

• 批准号: 10421243
• 负责人: JEFFERY L MEIER
• 金额: --
• 依托单位: IOWA CITY VA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-10-01 至 2022-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10421243
• 关键词: Address; Aging; Algorithm Design; Algorithms; Antiviral Agents; Biochemical Genetics; Bioinformatics; Biological; Cell model; Chromatin; Collaborations; Congenital Abnormality; Cost Savings; Cytomegalovirus; Cytomegalovirus Infections; Cytomegalovirus Vaccines; DNA; DNA Polymerase II; DNA biosynthesis; Data; Disease; Drug resistance; Early Promoters; Elements; Enhancers; Exhibits; Foundations; Frequencies; Gene Expression; Genes; Genetic Transcription; Goals; Health; Health Care Costs; Human; Immune system; Impairment; Infection; Infection prevention; Inflammation; Institute of Medicine (U.S.); Knowledge; Life; Link; Location; Longevity; Lytic; Methods; Modeling; Mutation; Nucleotides; Outcome Study; Pattern; Pharmacology; Positioning Attribute; Price; Protein Isoforms; Proteins; Replication Origin; Research; Research Design; Role; Site; Taxes; Techniques; Testing; Therapeutic; Therapeutic Intervention; Toxic effect; Transcript; Transcription Elongation; Transcriptional Elongation Factors; Untranslated RNA; Veterans; Viral; Viral Drug Resistance; Viral Genome; Viral Proteins; Virus; Virus Diseases; base; cell type; design; genetic approach; human disease; human model; insight; mortality risk; new therapeutic target; preference; prevent; programs; promoter; transcription factor; viral DNA; viral RNA; virology

Human cytomegalovirus (HCMV) infects over half of all Veterans and threatens the lives of those with impaired immune systems. Even among Veterans with normal immune systems, the insidious reactivation of the virus taxes the immune system, incites low-level inflammation, and possibly accelerates aging and shortens lifespan. HCMV is the most common infectious cause of birth defects. There is no HCMV vaccine and the antiviral drugs have problems with potency, toxicity, and drug-resistance. The long-range goal of this research is to identify critical pivot points in the viral transcription-DNA replication cycle that are vulnerable new targets for therapeutic intervention. This proposal is based on the premise that our gap in knowledge of how viral early transcription begets viral DNA replication and viral DNA replication begets viral late transcription limits our ability to design therapeutic treatments for the viral disease. We have developed modified PRO-Seq and PRO-Cap methods to determine exactly where on the viral genome that Pol II and its attached nascent transcript is located and at what frequency the engaged Pol II is at that nucleotide position during the infection. This allows us to precisely determine where transcription is initiating, the extent of promoter-proximal Pol II pausing, and the degree to which productive transcription elongation is taking place. We designed bioinformatics algorithms to analyze this data. Our preliminary studies show that HCMV utilizes host Pol II elongation control in early and late infection, but HCMV evolved different strategies in promoting the viral transcription and linking it to viral DNA replication. Frequently used viral promoters more often contain upstream TATA elements than do host promoters and viral initiator elements differ from that of the host in nucleotide preference. In late infection, HCMV and not the host uses TATT as a Pol II positioning element, which presumably requires the actions of viral late transcription factors (LTFs). We also find that nearly 20% of paused Pol II is found in the non-coding long RNA4.9 gene at a location in the viral lytic origin of replication (oriLyt) that is essential for oriLyt function; robust enhancer transcription underlies the world renown viral major immediate-early promoter; and viral transcription is pervasive and exhibits a pattern predictive of that arising from an unchromatinzed DNA template. Our research plan is designed to further validate, establish meaning of, and mechanistically understand these findings. To advance these objectives, we have adapted a newly developed approach to specifically and rapidly deplete viral proteins putatively involved in viral transcription. As proof of concept, we observe changes in levels of multiple viral RNAs after eliminating all the viral IE2 protein isoforms in late infection over a 6-hr timeframe, suggesting that the actions of one or more of these viral protein isoforms may have an overarching effect on viral transcription. We will combine this technique with PRO-Seq and PRO-Cap to determine the roles of the IE2 isoforms and the UL79 LTF (a putative late viral transcription elongation factor) in viral late transcription. In doing these studies, we will test the hypothesis that HCMV usurps Pol II initiation and elongation control to direct viral DNA replication, coordinate the virus’s gene expression program in lytic and latent-like infections, and contend with the threat of chromatin invasion. Our studies have been specifically designed to determine the role of transcription and structural elements in the function of the HCMV replication origin (Aim 1); determine the core DNA elements and viral factors required in late viral transcription (Aim 2); and determine how transcription differs in a quiescent infection and responds to activation (Aim 3). This proposal integrates the pertinent and extensive expertise of the Meier and the Price labs in virology and transcription, respectively. This research could not be done by either lab alone. The Meier and Price labs have a strong foundation of productive collaboration on which to build and complete this research plan.

人巨细胞病毒（HCMV）感染了超过一半的退伍军人，并威胁着那些患有巨细胞病毒的人的生命。 免疫系统受损即使在免疫系统正常的退伍军人中， 这种病毒会加重免疫系统的负担，引发低水平的炎症，并可能加速衰老， 缩短寿命HCMV是导致出生缺陷的最常见的感染性原因。没有HCMV疫苗 并且抗病毒药物存在效力、毒性和耐药性的问题。长期目标是 这项研究旨在确定病毒转录-DNA复制周期中的关键支点， 治疗干预的脆弱新目标。这一建议是基于这样一个前提，即我们在 病毒早期转录如何引起病毒DNA复制，病毒DNA复制如何引起病毒 晚期转录限制了我们设计病毒性疾病治疗方法的能力。我们已经开发 改进的PRO-Seq和PRO-Cap方法，以确定Pol II和 定位其附着的新生转录物，以及接合的Pol II在该核苷酸处的频率 感染期间的位置。这使我们能够精确地确定转录起始的位置， 启动子近端Pol II暂停的程度，以及生产性转录延伸的程度 地方我们设计了生物信息学算法来分析这些数据。我们的初步研究表明，HCMV 在感染早期和晚期，HCMV利用宿主Pol II延伸控制，但在感染早期和晚期， 促进病毒转录并将其连接到病毒DNA复制。常用的病毒启动子更多 与宿主启动子和病毒启动子元件不同， 宿主对核苷酸的偏好。在晚期感染中，HCMV而不是宿主使用TATT作为Pol II定位 元件，这可能需要病毒晚期转录因子（LTF）的作用。我们还发现 近20%的暂停的Pol II存在于病毒裂解起点的非编码长RNA 4.9基因中 复制（oriLyt）是oriLyt功能所必需的;强大的增强子转录是世界的基础 著名的病毒主要立即早期启动子;病毒转录是普遍的，并表现出一种模式 预测由未染色的DNA模板产生的。我们的研究计划旨在进一步 验证，建立意义，并机械地理解这些发现。为了推进这些目标， 我们采用了一种新开发的方法， 参与病毒转录。作为概念的证明，我们观察到多种病毒RNA水平的变化， 在6小时的时间范围内消除了晚期感染中的所有病毒IE 2蛋白同种型，这表明 一种或多种这些病毒蛋白质同种型的作用可能对病毒转录具有总体影响。 我们将联合收割机将这种技术与PRO-Seq和PRO-Cap相结合，以确定IE 2亚型的作用， UL 79 LTF（一种推定的晚期病毒转录延伸因子）在病毒晚期转录中的作用。做这些 研究中，我们将测试HCMV篡夺Pol II起始和延伸控制以指导病毒 DNA复制，在裂解性和潜伏样感染中协调病毒的基因表达程序， 与染色质入侵的威胁作斗争。我们的研究是专门设计来确定 转录和结构元件在HCMV复制起点（Aim 1）功能中的作用;确定 晚期病毒转录所需的核心DNA元件和病毒因子（目标2）;并确定如何 转录在静止感染中不同，并响应于激活（Aim 3）。该提案将 Meier和Price实验室分别在病毒学和转录方面的相关和广泛的专业知识。 这项研究不能由任何一个实验室单独完成。Meier和Price实验室拥有强大的基础， 建立和完成这项研究计划的富有成效的合作。