Proteomics of HSV1 Replication

项目总结 细胞已经进化出复杂的机制，既可以复制DNA，也可以修复错误 DNA单纯疱疹病毒1型(HSV)是一种大的双链DNA病毒，可在体内复制 宿主细胞的细胞核，并侵占这种宿主复制机制的一部分。尽管 经过几十年的研究，单纯疱疹病毒DNA复制的机制仍然知之甚少。这个 传入的基因组中含有缺口和缺口，目前尚不清楚这些缺口何时或是否被修复。 与DNA合成的时间有关。单纯疱疹病毒编码七种必需的DNA复制蛋白 包括一种起源结合蛋白、一种单链DNA结合蛋白(ICP8)、一种三链结合蛋白 亚基解旋酶/启动酶(UL5/UL8/UL52)和两个亚基聚合酶(UL30/UL42)。在……里面 此外，HSV还编码由5‘-3’外切酶(UL12)组成的两个单位的重组酶 使用ICP8的功能。新生DNA上蛋白质的分离(IPOND)是研究DNA的有力工具 复制，因为它允许对复制分叉进行特定的净化，而不是批量 染色质。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方法提供了一个强有力的、公正的发现 通过测定脉冲样本中蛋白质的强度来识别分叉相关蛋白质的工具 与Chase样本进行比较。我们已经使用iPOND-SILAC-MS来生成健壮的数据集 HSV复制叉和缺少UL12的复制叉的蛋白质组成。在 缺少UL12，一种细胞去泛素酶，USP15，不会被招募到复制叉子中。 USP15直接与UL12相互作用，是有效复制HSV所必需的。除了病毒之外 蛋白质，许多细胞蛋白质富含在病毒复制叉子上。在十二个iPOND-SILAC中- MS我们已经鉴定了200-300个蛋白质(病毒和宿主)，这些蛋白质富含在HSV DNA复制叉上。 这项研究计划的总体目标是确定与 病毒DNA有助于更全面地了解HSV DNA的作用机制 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) 描述UL12与USP15的物理相互作用，2)鉴定人类复制体 HSV DNA复制所需的蛋白质，3)决定了HSV DNA中的缺口和缺口的命运 传入的病毒基因组。

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.