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.

项目摘要 细胞已经进化了复杂的机械，以复制DNA和修复误差 脱氧核糖核酸。单纯疱疹病毒1（HSV）是一种大的双链DNA病毒，在该病毒中复制 宿主单元的核和指挥官的一些主机复制机制。尽管 数十年的研究，HSV DNA复制的机制仍然鲜为人知。这 传入的基因组包含划痕和间隙，尚不清楚何时或是否修复这些缝隙 与DNA合成的时机有关。 HSV编码七个必需的DNA复制蛋白 包括起源结合蛋白，单链DNA（ssDNA）结合蛋白（ICP8），三个 亚基解旋酶/原始酶（UL5/UL8/UL52）和两亚基聚合酶（UL30/UL42）。在 此外，HSV还编码由5'-3'外切酶（UL12）组成的两单位重组酶 具有ICP8的功能。新生DNA（ipond）上蛋白质的分离是研究DNA的强大工具 复制是因为它允许将复制叉的特定纯化从散装中移开 染色质。当与SILAC（细胞培养中氨基酸的稳定同位素标记）结合使用时 定量蛋白质组学，IPOND-SILAC-MS方法提供了强大的，无偏见的发现 通过确定脉冲样品中蛋白的强度来识别叉子相关蛋白的工具 与追逐样本相比。我们已经利用IPOND-SILAC-MS生成强大的数据集 HSV复制叉的蛋白质组成和缺乏UL12的复制叉。在 不存在UL12的细胞去泛素化酶USP15，并未募集到复制前叉。 USP15与UL12直接相互作用，是有效的HSV复制所必需的。除了病毒 蛋白质，许多细胞蛋白都富含病毒复制叉。在十二个ipond-silac- MS我们已经鉴定出富含HSV DNA复制叉上的200-300蛋白（病毒和宿主）。 该研究建议的总体目标是确定与 病毒DNA以更完整地了解HSV DNA的机制 复制。为此，我们将解决我实验室中的三个重叠研究领域：1） 表征UL12与USP15的物理相互作用，2）识别人类的重生 HSV DNA复制所需的蛋白质，3）确定划痕和间隙的命运 传入的病毒基因组。