Vaccinia DNA Replication

痘苗 DNA 复制

• 批准号: 9117163
• 负责人: Paula Traktman
• 金额: $ 26.16万
• 依托单位: MEDICAL UNIVERSITY OF SOUTH CAROLINA
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-07-25 至 2017-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9117163
• 关键词: A20 protein; ATP phosphohydrolase; Address; Antiviral Therapy; Base Excision Repairs; Biochemical; Biological; Biological Models; Biological Process; Cell Nucleus; Cells; Cellular Membrane; Complex; Coupled; Cytoplasm; D4 protein; DNA; DNA Ligases; DNA Primase; DNA Viruses; DNA biosynthesis; DNA-Directed DNA Polymerase; Development; Endoplasmic Reticulum; Ensure; Evaluation; Figs - dietary; Fright; Funding; Genetic; Genetic Materials; Genome; Genome Stability; Genomics; Holoenzymes; Infection; Investigation; Knowledge; Length; Lesion; Libraries; Ligase; Mediating; Membrane; Membrane Protein Traffic; Modeling; Monkeypox virus; Okazaki fragments; Plague; Polymerase; Poxviridae; Primer Extension; Process; Property; Proteins; Role; Scanning; Site; Small Interfering RNA; Smallpox; Structure; Testing; Vaccination; Vaccinia; Vaccinia virus; Vaccinium; Viral; Viral Genome; Virus; Work; helicase; human morbidity; human mortality; insight; mutant; nuclease; recombinational repair; repaired; scaffold; uracil-DNA glycosylase; viral DNA; weapons

DESCRIPTION (provided by applicant): The faithful duplication of the genetic material is arguably the most fundamental of all biological processes. For DNA viruses as for cells, a complex repertoire of proteins mediates DNA synthesis and coordinates replication with recombination and repair. In this proposal, we present our plan for continued investigation of how vaccinia virus, the prototypic poxvirus, accomplishes genome replication. Poxviruses are among the only DNA viruses whose infectious cycle is restricted to the cytoplasm of infected cells. This autonomy from the host nucleus poses unusual challenges, in that the virus must encode a complete replication apparatus and establish dedicated cytoplasmic sites for replication. Poxviruses represent intriguing model systems in addition to being of significant biomedical importance. The impact of our studies is further strengthened by the importance of the viral replication proteins as targets for the development of rational antiviral therapies. During the next funding period, we will utilize genetic, biochemical and cell biological approaches to further elucidate the replication of vaccinia virus by focusing on the following three aims: AIM I: Analysis of the E9/A20/UDG polymerase holoenzyme in replication and repair. We will continue our studies of the DNA polymerase complex, which is comprised of the E9 DNA Pol and a heterodimeric processivity factor consisting of the A20 and D4 proteins. We will focus on understanding the mechanism whereby UDG/A20 confers processivity on Pol. Because D4 is an active uracil DNA glycosylase (UDG), we will investigate how replication-associated base excision repair is accomplished by viral and cellular proteins. AIM II: Analysis of the mechanism of DNA replication, and investigation of the contributions of the D5 primase/helicase, the A50 ligase, and the G5 nuclease to this process. We will revisit the mechanism of poxvirus replication and assess the possible involvement of both leading and lagging strand synthesis. In this context, we will continue our analysis of the essential D5 ATPase, which is likely to have both primase and helicase activity. We will also investigate the roles of the viral DNA ligase and the G5 protein, which has homology to FEN1 nucleases, in the replication and maturation of the viral genome. AIM III: Analysis of the assembly and structure of viral replication factories. Viral replication takes place in dedicated cytoplasmic regions known as replication factories. These factories are delimited by, and associated with, membranes derived from the endoplasmic reticulum (ER). We will test our hypothesis that the abundant H5 protein serves as a scaffold within the factories. Furthermore, we will use a targeted siRNA library to identify cellular membrane-trafficking proteins that contribute to the reorganization of the ER for the purpose of replication factory assembly.

描述（由申请人提供）：遗传物质的忠实复制可以说是所有生物过程中最基本的。对于DNA病毒和细胞来说，一个复杂的蛋白质库介导DNA合成，并协调复制与重组和修复。在这个建议中，我们提出了我们的计划，继续研究痘苗病毒，原型痘病毒，如何完成基因组复制。痘病毒是仅有的感染周期局限于受感染细胞细胞质的DNA病毒之一。这种来自宿主细胞核的自主性带来了不同寻常的挑战，因为病毒必须编码一个完整的复制装置，并建立专门的细胞质位点进行复制。痘病毒除了具有重要的生物医学意义外，还代表了有趣的模型系统。病毒复制蛋白作为开发合理抗病毒疗法的靶标的重要性进一步加强了我们研究的影响。在接下来的资助期内，我们将利用遗传学、生物化学和细胞生物学的方法进一步阐明牛痘病毒的复制，重点关注以下三个目标：AIM I：分析E9/A20/UDG聚合酶的复制和修复。我们将继续研究DNA聚合酶复合物，该复合物由E9 DNA Pol和由A20和D4蛋白组成的异二聚体加工因子组成。我们将重点了解UDG/A20赋予Pol处理能力的机制。由于D4是一种活性的尿嘧啶DNA糖基酶（UDG），我们将研究病毒和细胞蛋白如何完成复制相关的碱基切除修复。AIM II：分析DNA复制的机制，并研究D5引物酶/解旋酶、A50连接酶和G5核酸酶在这一过程中的作用。我们将重新审视痘病毒复制的机制，并评估前导链和后链合成的可能参与。在这种情况下，我们将继续分析必需的D5 atp酶，它可能同时具有引物酶和解旋酶活性。我们还将研究病毒DNA连接酶和与FEN1核酸酶同源的G5蛋白在病毒基因组复制和成熟中的作用。AIM III：病毒复制工厂的组装和结构分析。病毒复制发生在被称为复制工厂的专用细胞质区域。这些工厂由内质网（ER）衍生的膜划分并与之相关。我们将验证我们的假设，即丰富的H5蛋白在工厂内充当支架。此外，我们将使用靶向siRNA文库来鉴定有助于内质网重组的细胞膜运输蛋白，以实现复制工厂组装。