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）感染了所有退伍军人的一半，并威胁 免疫系统受损。即使在具有正常免疫系统的退伍军人中，阴险的重新激活 病毒征税的免疫系统，促进低水平的炎症，并可能加速衰老和 缩短寿命。 HCMV是最常见的出生缺陷原因。没有HCMV疫苗 抗病毒药在效力，毒性和抗药性方面存在问题。远程目标 这项研究是为了确定病毒转录-DNA复制周期中的关键枢轴点 脆弱的治疗干预目标。该提议基于我们在 了解病毒早期转录如何使病毒DNA复制和病毒DNA复制成为病毒 晚期转录限制了我们设计病毒疾病治疗治疗的能力。我们已经发展了 修改了Pro-Seq和Pro-CAP方法，以确定Pol II和Pol II和 它附着的新生成绩单位于该核苷酸的何种频率下 感染期间的位置。这使我们能够精确确定启动转录的位置，程度 启动子螺旋pol II的暂停以及生产转录伸长的程度 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 在早期和晚期感染中利用宿主的pol II伸长控制，但HCMV发展了不同的策略 促进病毒转录并将其与病毒DNA复制联系起来。经常使用的病毒启动子更多 通常包含上游的塔塔元素，而不是宿主启动子和病毒引发剂元素 核苷酸偏好的宿主。在晚期感染中，HCMV而不是宿主使用Tatt作为Pol II定位 元素，大概需要病毒后期转录因子（LTF）的作用。我们也发现 在病毒裂解起源的某个位置的非编码长RNA4.9基因中发现了近20％的暂停POL II 对于Orilyt函数必不可少的复制（Orilyt）；强大的增强子转录是世界的基础 著名的病毒主要直接启动子；病毒转录普遍存在，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，建立含义并机械理解这些发现。为了促进这些目标， 我们已经改编了一种新开发的方法，可以在推测上进行专门和快速复制病毒蛋白 参与病毒转录。作为概念证明，我们观察到多个病毒RNA水平的变化 在6小时的时间范围内消除晚期感染中所有病毒IE2蛋白同工型 这些病毒蛋白同工型中的一种或多种的作用可能对病毒转录具有总体影响。 我们将将这一技术与亲seq和Pro-CAP相结合，以确定IE2同工型的作用和 病毒晚期转录中的UL79 LTF（推定的晚期病毒转录伸长因子）。在做这些 研究，我们将检验以下假设：HCMV篡夺Pol II计划和伸长控制以导向病毒 DNA复制，在裂解和潜在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。我们的研究是专门设计的，以确定 转录和结构元素在HCMV复制起源功能中的作用（AIM 1）；决定 病毒转录后期需要的核心DNA元素和病毒因子（AIM 2）；并确定如何 静止感染中的转录差异并响应激活（AIM 3）。该提案整合了 Meier的相关和广泛的专业知识和病毒学和转录的价格实验室。 这项研究不能仅由任何一个实验室完成。 Meier和Price Labs拥有强大的基础 构建和完成该研究计划的生产性合作。