Mechanisms regulating cytomegalovirus

巨细胞病毒的调节机制

• 批准号: 10047701
• 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/10047701
• 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; Structure; 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.

人类巨细胞病毒(HCMV)感染了超过一半的退伍军人，并威胁到患有 免疫系统受损。即使在免疫系统正常的退伍军人中，潜伏的重新激活 这种病毒会加重免疫系统的负担，引发低水平的炎症，并可能加速衰老和 缩短寿命。人巨细胞病毒是导致出生缺陷的最常见的感染原因。目前还没有人巨细胞病毒疫苗 而抗病毒药物在效力、毒性和耐药性方面都存在问题。的长远目标 这项研究旨在确定病毒转录-DNA复制周期中的关键支点 治疗干预的新靶点易受攻击。这项提议是基于这样一个前提，即我们在 关于病毒早期转录如何产生病毒DNA复制和病毒DNA复制如何产生病毒的知识 转录延迟限制了我们为这种病毒疾病设计治疗方法的能力。我们已经开发出 改进的PRO-Seq和PRO-Cap方法以确定Pol II和PRO-Cap在病毒基因组上的确切位置 其附加的新生转录本被定位，以及参与的POL II在该核苷酸的频率是多少 在感染期间的位置。这使我们能够准确地确定转录在哪里开始，范围 启动子-Pol II近端暂停，以及生产性转录延伸的程度 地点。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，人巨细胞病毒 在早期和晚期感染中利用宿主POL II延长控制，但HCMV在 促进病毒转录，并将其与病毒DNA复制联系起来。常用病毒启动子更多 通常含有上游TATA元件比宿主启动子和病毒启动子元件不同 宿主在核苷酸偏好上。在晚期感染中，人巨细胞病毒而不是宿主使用START作为POLII定位 元素，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POL II是在病毒裂解起源的非编码长RNA4.9基因中发现的 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；病毒转录是普遍存在的，并呈现出一种模式 由未显色的DNA模板所产生的预测。我们的研究计划旨在进一步 验证、确定其含义，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来特异性地、快速地消耗病毒蛋白。 参与病毒转录。作为概念证明，我们观察了多种病毒RNA水平的变化 在6小时内消除晚期感染的所有病毒IE2蛋白亚型，这表明 这些病毒蛋白亚型中的一个或多个的作用可能对病毒转录产生重要影响。 我们将把这项技术与PRO-Seq和PRO-Cap结合起来，以确定IE2亚型和 病毒晚期转录中的UL79 LTF(可能是病毒晚期转录延伸因子)。在做这些事情的时候 研究，我们将检验假设，巨细胞病毒篡夺Pol II的启动和延伸控制，以指导病毒 DNA复制，在裂解性和潜伏性感染中协调病毒的基因表达程序，以及 应对染色质入侵的威胁。我们的研究是专门为确定 转录和结构元件在巨细胞病毒复制起点功能中的作用(目标1)；确定 病毒后期转录所需的核心DNA元件和病毒因子(目标2)；并确定如何 转录在静止感染时不同，对激活有反应(目标3)。这项提议整合了 梅尔实验室和普赖斯实验室分别在病毒学和转录方面拥有相关和广泛的专业知识。 这项研究不能仅靠任何一个实验室来完成。梅尔和普莱斯实验室拥有坚实的基础 富有成效的协作，在此基础上构建和完成此研究计划。