Genetics of HSV DNA Replication

HSV DNA 复制的遗传学

• 批准号: 8631838
• 负责人: SANDRA K WELLER
• 金额: $ 63.94万
• 依托单位: UNIVERSITY OF CONNECTICUT SCH OF MED/DNT
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-12-01 至 2018-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8631838
• 关键词: Antiviral Agents; Architecture; Binding Proteins; Biochemical; Biological; Biological Assay; Cell Nucleus; Cells; Complex; DNA; DNA Primase; DNA biosynthesis; DNA-Directed DNA Polymerase; DNA-Protein Interaction; Development; Filament; Gene Expression; Genetic; Genetic Materials; Genome; Goals; Herpesvirus 1; Human; In Vitro; Individual; Infection; Lead; Macromolecular Complexes; Methods; Modeling; Molecular; Morbidity - disease rate; Mutate; Mutation; Nuclear; Nucleic Acids; Organism; Oxidation-Reduction; Polymerase; Process; Property; Proteins; Reaction; Regulation; Replication Origin; Role; Simplexvirus; Site; Staging; System; Testing; Viral; Virus; Virus Replication; Work; helicase; insight; mortality; mutant; novel; pathogen; prevent; protein protein interaction; public health relevance; reconstitution; research study; stoichiometry; viral DNA

DESCRIPTION (provided by applicant): Herpes Simplex Virus (HSV) is a ubiquitous human pathogen that can cause significant morbidity and mortality in humans. The survival of all organisms including viruses depends on their ability to produce an exact copy of their genetic material. The DNA replication machinery (replisome) functions as a complex association of proteins that must be tightly regulated in order to accomplish faithful and complete DNA replication. Our previous work has led to the identification and characterization of seven HSV replication proteins as well as the compartments in the cell in which DNA replication occurs; however, the mechanisms by which they function during DNA replication are poorly understood. Recent experiments by us and our collaborators suggest that the assembly and function of the HSV-1 replisome is tightly regulated by protein-protein and protein-nucleic acid interactions that we are only now beginning to appreciate. Our central hypothesis is that protein-protein interactions among viral replication proteins are essential for all stages of genome replication. We will examine the roles of specific interactions in the formation of prereplicative sites and replication compartments, initiation of DNA synthesis at viral origins of replication, processive unwinding of the duplex DNA in front of the replication fork and the coordinated regulation of leading and lagging strand DNA synthesis. HSV dramatically reorganizes the infected cell nucleus leading to the formation of large globular replication compartments in which gene expression, DNA replication and encapsidation occur. Although it has long been recognized that ICP8 is required, little is known about how ICP8 exerts its effects on the nuclear architecture. In aim 1 we will use a combination of genetic, biochemical, biophysical and cell biological methods to test the hypothesis that dynamic properties of ICP8 and its ability to assemble into small subassemblies and filaments drive the formation of prereplicative sites and replication compartments. ICP8 and the origin binding protein UL9 are known to work together to unwind the origins during the initiation of DNA synthesis; however, difficulties in reconstituting origin dependent DNA synthesis in vitro have prevented a thorough understanding of the process. In aim 2 we will test the hypothesis that protein-protein and protein-DNA Interactions are essential for origin dependent initiation of viral DNA synthesis. We will use genetic and biochemical approaches to identify the regions of UL9 and ICP8 that are important for various steps in origin unwinding. In aim 3 we use a newly developed assay for coordinated leading and lagging strand synthesis to test the hypothesis that interactions between the HSV helicase/primase and polymerase are required for coordinated leading and lagging strand DNA synthesis.

描述（由申请方提供）：单纯疱疹病毒（HSV）是一种普遍存在的人类病原体，可导致人类显著的发病率和死亡率。包括病毒在内的所有生物体的生存都取决于它们复制其遗传物质的能力。DNA复制机制（复制体）作为一种复杂的蛋白质组合发挥作用，必须严格调控才能实现忠实和完整的DNA复制。我们以前的工作已经导致7 HSV复制蛋白的鉴定和表征，以及在DNA复制发生的细胞中的隔室;然而，它们在DNA复制过程中发挥作用的机制知之甚少。我们和我们的合作者最近的实验表明，HSV-1复制体的组装和功能受到蛋白质-蛋白质和蛋白质-核酸相互作用的严格调控，我们现在才开始认识到这一点。我们的中心假设是，病毒复制蛋白之间的蛋白质-蛋白质相互作用是基因组复制的所有阶段所必需的。我们将研究特定的相互作用在复制前位点和复制区室的形成中的作用，在病毒复制起点处的DNA合成的起始，在复制叉前面的双链体DNA的进行性解旋以及前导和滞后链DNA合成的协调调节。HSV显著重组感染的细胞核，导致形成大的球状复制区室，其中发生基因表达、DNA复制和胞苷化。虽然人们早就认识到ICP 8是必需的，但对ICP 8如何对核结构产生影响知之甚少。在 目的1我们将使用遗传学、生物化学、生物物理学和细胞生物学方法的组合来测试ICP 8的动态特性及其组装成小的微纤维和细丝的能力驱动复制前位点和复制区室的形成的假设。已知ICP 8和起源结合蛋白UL 9在DNA合成的起始期间一起工作以解开起源;然而，在体外重建起源依赖性DNA合成的困难阻碍了对该过程的透彻理解。在目标2中，我们将检验蛋白质-蛋白质和蛋白质-DNA相互作用对于病毒DNA合成的起源依赖性起始是必不可少的这一假设。我们将使用遗传和生物化学方法来确定UL 9和ICP 8的区域，这些区域对起源解旋的各个步骤都很重要。在目标3中，我们使用一种新开发的用于协调的前导链和滞后链合成的测定来测试HSV解旋酶/引发酶和聚合酶之间的相互作用是协调的前导链和滞后链DNA合成所需的假设。