Proteomics of HSV1 Replication

HSV1 复制的蛋白质组学

• 批准号: 10684243
• 负责人: Kareem N Mohni
• 金额: $ 39.75万
• 依托单位: MAYO CLINIC ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-15 至 2027-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10684243
• 关键词: Amino Acids; Antiviral Therapy; Area; Binding Proteins; Blindness; Cell Culture Techniques; Cell Nucleus; Cells; Chromatin; Complex; Coupled; DNA; DNA Maintenance; DNA Primase; DNA biosynthesis; DNA replication fork; Data Set; Deubiquitinating Enzyme; Disease; Double Stranded DNA Virus; Eye Infections; Genital; Genitalia; Genome; Goals; Herpesvirus 1; Human; Laboratories; Methods; Physiologic pulse; Polymerase; Population; Proteins; Proteomics; Research; Research Proposals; SS DNA BP; Sampling; Set protein; Stable Isotope Labeling; Ulcer; Viral; Viral Genome; Viral Proteins; Virus Replication; design; helicase; oral lesion; pathogen; recombinase; repaired; spleen exonuclease; tool; ubiquitin isopeptidase; viral DNA

PROJECT SUMMARY Cells have evolved complex machinery for both the replication of DNA and for repairing errors in DNA. Herpes Simplex Virus 1 (HSV) is a large double strand DNA virus that replicates in the nucleus of the host cell and commandeers some of this host replication machinery. Despite decades of study, the mechanisms of HSV DNA replication are still poorly understood. The incoming genome contains nicks and gaps, and it is not known when or if these are repaired in relation to the timing of DNA synthesis. HSV encodes seven essential DNA replication proteins including an origin binding protein, a single strand DNA (ssDNA) binding protein (ICP8), a three- subunit helicase/primase (UL5/UL8/UL52), and a two-subunit polymerase (UL30/UL42). In addition, HSV also encodes a two-unit recombinase consisting of a 5'-3' exonuclease (UL12) that functions with ICP8. Isolation of proteins on nascent DNA (iPOND) is a powerful tool to study DNA replication because it allows for the specific purification of replication forks away from bulk chromatin. When coupled with SILAC (stable isotope labeling of amino acids in cell culture)-based quantitative proteomics, the iPOND-SILAC-MS method provides a robust, unbiased discovery tool to identify fork associated proteins by determining the intensity of proteins in a pulse sample compared to a chase sample. We have utilized iPOND-SILAC-MS to generate robust data sets of the protein composition of HSV replication forks and replication forks lacking UL12. In the absence of UL12 a cellular deubiquitinating enzyme, USP15, is not recruited to replication forks. USP15 interacts directly with UL12 and is required for efficient HSV replication. In addition to viral proteins, many cellular proteins are enriched on viral replication forks. In twelve iPOND-SILAC- MS we have identified 200-300 proteins (viral and host) enriched on HSV DNA replication forks. The overall goal of this research proposal is to identify the host proteins associated with viral DNA to generate a more complete understanding of the mechanisms of HSV DNA replication. To this end we will address three overlapping research areas in my laboratory: 1) Characterize the physical interaction of UL12 with USP15, 2) Identify the human replisome proteins required for HSV DNA replication, 3) Determine the fate of the nicks and gaps in the incoming viral genome.

项目概要 细胞已经进化出复杂的机制来复制DNA和修复DNA错误。 脱氧核糖核酸。单纯疱疹病毒 1 (HSV) 是一种大型双链 DNA 病毒，可在体内复制 宿主细胞的细胞核并霸占了一些宿主复制机制。尽管 经过几十年的研究，人们对HSV DNA复制的机制仍然知之甚少。这 传入的基因组包含切口和间隙，并且不知道这些何时或是否会在 与DNA合成的时间有关。 HSV 编码七种必需的 DNA 复制蛋白 包括起始结合蛋白、单链 DNA (ssDNA) 结合蛋白 (ICP8)、三链 亚基解旋酶/引物酶 (UL5/UL8/UL52) 和双亚基聚合酶 (UL30/UL42)。在 此外，HSV 还编码由 5'-3' 核酸外切酶 (UL12) 组成的双单位重组酶， 具有 ICP8 功能。新生 DNA 上的蛋白质分离 (iPOND) 是研究 DNA 的强大工具 复制，因为它允许对复制叉进行特定纯化以使其远离批量 染色质。当与基于 SILAC（细胞培养中氨基酸的稳定同位素标记）结合时 定量蛋白质组学，iPOND-SILAC-MS 方法提供了稳健、公正的发现 通过确定脉冲样本中蛋白质的强度来识别叉相关蛋白质的工具 与追逐样本相比。我们利用 iPOND-SILAC-MS 生成可靠的数据集 HSV 复制叉和缺乏 UL12 的复制叉的蛋白质组成。在 缺乏 UL12 时，细胞去泛素化酶 USP15 不会被招募到复制叉中。 USP15 直接与 UL12 相互作用，是有效 HSV 复制所必需的。除了病毒传播 蛋白质，许多细胞蛋白质在病毒复制叉上富集。十二个iPOND-SILAC- MS 我们已经鉴定出 200-300 种在 HSV DNA 复制叉上富集的蛋白质（病毒和宿主）。 该研究计划的总体目标是鉴定与相关的宿主蛋白 病毒 DNA 可以更全面地了解 HSV DNA 的机制 复制。为此，我们将在我的实验室中解决三个重叠的研究领域：1） 表征 UL12 与 USP15 的物理相互作用，2) 识别人类复制体 HSV DNA 复制所需的蛋白质，3) 确定切口和间隙的命运 传入的病毒基因组。