Mechanisms Regulating Cytomegalovirus

巨细胞病毒的调节机制

• 批准号: 10481050
• 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 2026-09-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10481050
• 关键词: Acute; Address; Affect; Antiviral Agents; Binding; Biological Assay; Cell Line; Cell model; ChIP-seq; Chromatin; Chromatin Structure; Collaborations; Complex; Congenital Abnormality; Cost Savings; Cytomegalovirus; Cytomegalovirus Infections; Cytomegalovirus Vaccines; DNA Polymerase II; DNA Sequence; DNA biosynthesis; Data; Disease; Drug resistance; Environment; Fibroblasts; Gene Expression; Gene Expression Regulation; Genes; Genetic Engineering; Genetic Transcription; Genome; Genomic approach; Goals; Health; Health Care Costs; Herpesviridae; Hour; Human; Human Herpesvirus 4; Human Herpesvirus 6; Immune system; Impairment; Individual; Infection; Infection prevention; Inflammation; Institute of Medicine (U.S.); Knowledge; Life; Maps; Mass Spectrum Analysis; Modeling; Nucleosomes; Oncogenic; Output; Pathway interactions; Pattern; Population; Predisposition; Price; Productivity; Promoter Regions; Protein Isoforms; Regulation; Research; Resolution; Role; Signal Transduction; Site; System; Taxes; Techniques; Technology; Testing; Therapeutic; Therapeutic Intervention; Toxic effect; Transcript; Transcription Initiation; Veterans; Viral; Viral Drug Resistance; Virus; Virus Activation; Virus Diseases; bioinformatics pipeline; cell type; design; functional genomics; gammaherpesvirus; genetically modified cells; genome-wide; genomic locus; human disease; human model; member; mortality risk; new therapeutic target; novel therapeutics; pharmacologic; prevent; programs; promoter; sarcoma; tool; transcription factor; viral DNA; virology

Human cytomegalovirus (HCMV) infects over half of all Veterans and threatens the lives of those with impaired immune systems. HCMV is the leading 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 points in the viral transcription-DNA replication cycle that would serve as new targets for therapeutic intervention. This proposal is based on the premise that our gap in knowledge of how viral early transcription produces viral DNA replication and how viral DNA replication results in viral late transcription limits our ability to design new therapeutic treatments for the viral disease. By customizing advanced technologies and developing new tools, we have used integrated functional genomics (dTag system, PRO-Seq, ChIP-Seq, genetically engineered test viruses, and promoter function assays) to determine where and when Pol II initiates transcription, identify sites of viral transcription factor binding genome-wide, and quantify change in Pol II nascent transcripts from individual promoters in relation to core promoter sequences, transcription factor loss, stage of infection, and viral DNA replication. We find that there are three distinct pathways to viral late transcription. Two of these pathways involve the HCMV IE2 and late transcription factor (LTF) group members. The individual role of each of the 3 different IE2 isoforms (IE2-86, IE2-60, and IE2-40) in viral late transcription is unknown. The six-member set of LTFs bind to Pol II and a DNA sequence signature in gene promoters, forming a preinitiation complex (PIC) that drives transcription. Diversity in sequence signature pattern likely determines the amount of individual promoter output. It is unknown precisely when and how the LTF complex assembles on viral promoters and how the LTF assembly engages Pol II in transcription. Our use of a new high-resolution ChIP-Seq technique and bioinformatics pipeline to map genomic locations of nucleosomes, as well as IE2 and LTF PICs, suggests that LTF PICs occupy genome regions not occupied by nucleosomes. This new ChIP-Seq technique will strengthen our integrated functional genomics approach to further determining the mechanisms controlling viral promoter transcription in relation to chromatin structure. Our preliminary data indicate that: 1) the early-late transcription switch lags many hours behind the onset of viral DNA replication; 2) the HCMV promoter population members that are active differs by cell type and condition, and this difference may involve IE2 and LTF functions; and 3) the HCMV promoter population that is active during viral reactivation in the NT2 model differs from that in acute productive infection. We will test the hypothesis that HCMV transcription factors usurp host Pol II that navigates a modified chromatin environment suited to bring about viral late transcription (Aims 1 and 2) and to coordinate the viral transcription program in diverse cell types (Aim 2) and under cellular conditions supporting quiescent and reactivation infections in the NT2 model (Aim 3). We will apply a multifaceted approach to each of the specific aims to: 1) elucidate the regulators of the early-late transcription switch, 2) determine the mechanistic basis for cell type differences in viral transcription, and 3) determine how activation of quiescent infection changes viral transcription. Our proposal integrates the expertise of the Meier and the Price labs in virology and transcription, respectively. We will build on this productive collaboration to complete the proposed research plan. The discoveries coming from these studies will identify generalizable features of gene regulation that pertain to other members of beta- and gammaherpesvirus subfamilies, which include human herpesvirus 6 and the oncogenic herpesviruses, Epstein-Barr Virus and Kaposis sarcoma-associated herpesvirus.

人类巨细胞病毒（HCMV）感染了所有退伍军人的一半以上，并威胁 免疫系统受损。 HCMV是出生缺陷的主要传染病。没有HCMV 疫苗和抗病毒药物在效力，毒性和抗药性方面存在问题。远程 这项研究的目标是确定病毒转录DNA复制周期中的关键点 作为治疗干预的新目标。该提议基于我们在 了解病毒早期转录如何产生病毒DNA复制以及病毒DNA复制的知识 导致病毒晚期转录限制了我们为病毒疾病设计新的治疗治疗的能力。 通过自定义高级技术并开发新工具，我们使用了集成的功能 基因组学（DTAG系统，Pro-Seq，Chip-Seq，基因工程测试病毒和启动子功能 测定）要确定Pol II启动转录的何时何地，请识别病毒转录因子的位点 结合基因组的结合，并量化单个启动子的Pol II新生转录本的变化 核心启动子序列，转录因子丢失，感染阶段和病毒DNA复制。我们发现 有三种不同的病毒晚期转录途径。这些途径中的两个涉及HCMV IE2和晚期转录因子（LTF）组成员。三种不同的IE2中的每个人的个体角色 病毒后期转录中的同工型（IE2-86，IE2-60和IE2-40）尚不清楚。由六人组成的LTF 在基因启动子中与POL II和DNA序列签名结合，形成了一种预上络合物（PIC），该复合物 驱动转录。序列签名模式的多样性可能决定了个体的数量 启动子输出。 LTF复合物在病毒启动子和 LTF组件如何使Pol II参与转录。我们使用新的高分辨率芯片序列技术 以及生物信息学管道以绘制核小体的基因组位置以及IE2和LTF图片 表明LTF图片占据了不占核小体占据的基因组区域。这个新的chip-seq 技术将加强我们综合的功能基因组学方法，以进一步确定 控制病毒启动子转录的机制与染色质结构有关。我们的初步数据 表明：1）早期转录开关落后于病毒DNA复制发作的许多小时； 2）主动构件因细胞类型和状况而不同，这 差异可能涉及IE2和LTF功能； 3）在 NT2模型中的病毒重新激活与急性生产感染中的病毒重新激活不同。我们将检验假设 HCMV转录因子篡夺了宿主POL II，该因子导航适合于 带来病毒较晚的转录（目标1和2），并以多种方式协调病毒转录程序 细胞类型（AIM 2）和在细胞条件下支持NT2中的静止和重新激活感染 模型（AIM 3）。我们将对每个特定目的采用多方面的方法来：1）阐明 早期转录开关的调节器，2）确定细胞类型差异的机械基础 在病毒转录中，3）确定静脉感染的激活如何改变病毒转录。我们的 提案分别集成了Meier的专业知识和病毒学和转录中的价格实验室。 我们将基于这一富有成效的合作来完成拟议的研究计划。发现 来自这些研究将确定基因调节的可概括特征，与其他有关 β-和伽马梅氏菌病毒亚家族的成员，包括人类疱疹病毒6和 致癌性疱疹病毒，爱泼斯坦 - 巴尔病毒和卡普病肉瘤相关的疱疹病毒。