Proteomics of HSV1 Replication

项目摘要 细胞已经进化出复杂的机制，既可以复制DNA，也可以修复细胞中的错误。 DNA.单纯疱疹病毒1型（HSV）是一种大的双链DNA病毒， 细胞核，并征用一些宿主复制机器。尽管 经过几十年的研究，HSV DNA复制的机制仍然知之甚少。的 传入的基因组包含切口和缺口，并且不知道何时或是否在 与DNA合成的时间有关。HSV编码7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（ICP 8）、三重结合蛋白（T3）、单链DNA（ssDNA）结合蛋白（T4）、单链DNA（ssDNA）结合蛋白（T5）、单链DNA（ssDNA）结合蛋白（T6）、单链DNA（ssDNA）结合蛋白（T7）、单链DNA（ssDNA）结合蛋白（T8）、单链DNA（ssDNA）结合蛋白（T8）。 亚基解旋酶/引发酶（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）组成的两单元重组酶， 与ICP8一起运行。新生DNA上蛋白质的分离（iPOND）是研究DNA的有力工具 复制，因为它允许特定的纯化复制叉远离批量 染色质当与基于SILAC（细胞培养物中氨基酸的稳定同位素标记）的 定量蛋白质组学，iPOND-SILAC-MS方法提供了一个强大的，公正的发现 通过测定脉冲样品中蛋白质的强度来鉴定叉相关蛋白质的工具 与Chase样本相比。我们利用iPOND-SILAC-MS生成了可靠的数据集 HSV复制叉和缺乏UL12的复制叉的蛋白质组成。在 不存在UL12，细胞去泛素化酶USP 15不被募集到复制叉。 USP15直接与UL12相互作用，是HSV有效复制所必需的。除了病毒 蛋白质，许多细胞蛋白质在病毒复制叉上富集。在12个iPOND-SILAC- MS我们已经鉴定了200 - 300种在HSV DNA复制叉上富集的蛋白质（病毒和宿主）。 这项研究计划的总体目标是确定与 病毒DNA产生更完整的了解HSV DNA的机制 复制的为此，我们将在我的实验室解决三个重叠的研究领域：1） 表征UL12与USP 15的物理相互作用，2）鉴定人复制体 HSV DNA复制所需的蛋白质，3）确定HSV DNA中的缺口和缺口的命运， 输入病毒基因组。

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.