Identifying proteins involved in virus DNA replication

 描述（由申请人提供）：病毒操纵细胞过程以创造有利于其自身复制的条件。由于病毒拥有小的基因组，不能编码DNA复制所需的所有因子，它们依赖于宿主细胞蛋白来繁殖它们的基因组。细胞蛋白主动募集到病毒复制区室的功能是促进病毒基因表达和复制。相反，病毒早期蛋白靶向抗病毒细胞DNA传感器和转录阻遏物，以防止它们进入病毒基因组。研究病毒复制和病毒-宿主相互作用一直受到缺乏技术来识别参与病毒DNA复制的细胞蛋白质的阻碍。我们在此提议中的贡献将是应用一种新技术来鉴定病毒和细胞蛋白， 与复制病毒基因组有关。我们的中心假设是，细胞DNA复制/修复蛋白被募集到病毒基因组中，与病毒编码的复制因子一起发挥作用，并且早期病毒蛋白阻止抗病毒因子进入病毒基因组，对病毒复制产生有害结果。我们建议采用最近描述的技术，通过将新生DNA上的蛋白质分离（iPOND）与质谱（MS）耦合来识别复制DNA上的蛋白质。我们将使用iPOND-MS来鉴定在感染过程中与病毒DNA相互作用的蛋白质。为了测试这项创新技术的可行性，我们选择了单纯疱疹病毒1型（HSV-1）。疱疹病毒非常适合这种方法，因为它们阻断宿主细胞DNA复制，并在裂解复制过程中产生大量的病毒基因组。HSV-1编码立即早期蛋白ICP 0，其促进病毒基因表达并且还克服固有宿主防御。在强有力的初步数据的指导下，我们将通过追求两个具体目标来检验我们的假设，这两个目标将：（i） 鉴定由HSV-1病毒DNA复制特异性利用的细胞蛋白，和（ii）确定 病毒ICP 0如何改变与HSV基因组相关的蛋白质谱。将复制病毒基因组上鉴定的蛋白质与活跃细胞复制叉上的蛋白质清单进行比较，将揭示病毒和细胞DNA复制之间的差异。病毒特异性募集的蛋白质可能是抗病毒药物的潜在靶点， 病毒繁殖比较与野生型HSV-1和ICP 0突变体基因组相关的蛋白质将揭示ICP 0如何招募因子以促进基因表达，同时还操纵细胞反应以防止识别和抑制病毒基因组。我们的方法是创新的，因为它代表了一种全新的大规模方法来识别被招募来帮助病毒转录和复制的蛋白质。我们预计这项新技术将广泛适用于许多其他DNA病毒。识别通常用于不同病毒家族的病毒DNA复制的细胞蛋白质将表明潜在的 用于开发新的广泛作用的抗病毒治疗剂的靶点。

 DESCRIPTION (provided by applicant): Viruses manipulate cellular processes to create conditions conducive to their own replication. Since viruses possess small genomes that do not encode all the factors required for DNA replication, they rely on host cell proteins to propagate their genomes. Active recruitment of cellular proteins to viral replication compartments functions to promote virus gene expression and replication. In contrast, viral early proteins target antivira cellular DNA sensors and transcriptional repressors to prevent their access to viral genomes. Studying viral replication and virus-host interactions has been hampered by the lack of technologies to identify cellular proteins involved in viral DNA replication. Our contribution in tis proposal will be the application of a novel technology to identify viral and cellular proteins that associate with replicating viral genomes. Our central hypothesis is that cellular DNA replication/repair proteins are recruited to viral genomes to function with viral- encoded replication factors, and that early viral proteins prevent access to viral genomes by antiviral factors with detrimental outcomes for viral replication. We propose to employ a recently described technology that identifies proteins on replicating DNA by coupling Isolation of Proteins on Nascent DNA (iPOND) with Mass Spectrometry (MS). We will use iPOND-MS to identify proteins that interact with viral DNA during infection. To test the feasibility of this innovative technology we have chosen herpes simplex virus type 1 (HSV-1). Herpesviruses are ideally suited to this approach because they block host cell DNA replication, and generate large numbers of viral genomes during lytic replication. HSV-1 encodes the immediate early protein ICP0 that promotes viral gene expression and also overcomes intrinsic host defenses. Guided by strong preliminary data, we will test our hypothesis by pursuing two Specific Aims that will (i) identify cellular proteins specifically exploited by HSV-1 viral DNA replication and (ii) determine how viral ICP0 alters the spectrum of proteins associated with HSV genomes. Comparing proteins identified on replicating viral genomes to the inventory of proteins on active cellular replication forks will reveal differences between viral and cellular DNA replication. Proteins recruited specifically by viruses could represent potential targets for antivirals that would block virus propagation. Comparing proteins associated with the genomes of wild-type HSV-1 and ICP0 mutants will reveal how ICP0 recruits factors to promote gene expression, while also manipulating cellular responses to prevent recognition and inhibition on viral genomes. Our approach is innovative because it represents a completely new large-scale approach to identify proteins that are recruited to aid viral transcription and replication. We anticipate this new technology will be broadly applicable to many other DNA viruses. Identifying cellular proteins commonly exploited for viral DNA replication across different virus families will suggest potential targets for development of novel broadly acting antiviral therapeutics.