Identifying proteins involved in virus DNA replication

鉴定参与病毒 DNA 复制的蛋白质

• 批准号: 9034220
• 负责人: Matthew D. Weitzman
• 金额: $ 21万
• 依托单位: CHILDREN'S HOSP OF PHILADELPHIA
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-01-01 至 2017-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9034220
• 关键词: Address; Affect; Antiviral Agents; Antiviral Therapy; Binding Proteins; Biochemical; Bioinformatics; Biological Assay; Cell Nucleus; Cell physiology; Cells; Cellular Structures; Chromatin; Complement; Complex; Coupling; DNA; DNA Viruses; DNA biosynthesis; DNA replication fork; Data; Development; Environment; Equipment and supply inventories; Family; Fluorescence; Gene Expression; Genetic Transcription; Genome; Global Change; Goals; Herpesviridae; Herpesvirus 1; Host Defense; Human; Immediate-Early Proteins; Infection; Integration Host Factors; Lead; Lytic Phase; Mass Spectrum Analysis; Measures; Methodology; Modeling; Outcome; Precipitation; Process; Production; Proteins; Proteome; Proteomics; RNA Interference; RNA interference screen; Recruitment Activity; Research; Role; Simplexvirus; Techniques; Technology; Testing; Therapeutic; Transcription Repressor/Corepressor; Viral; Viral Genes; Viral Genome; Viral Proteins; Virus; Virus Diseases; Virus Replication; innovation; innovative technologies; insight; lytic replication; mutant; new technology; novel; pathogen; prevent; public health relevance; reactivation from latency; repaired; response; sensor; ubiquitin-protein ligase; viral DNA; virology; virus host interaction

 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.

 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。由于病毒拥有较小的基因组，无法编码 DNA 复制所需的所有因子，因此它们依赖宿主细胞蛋白来传播其基因组。细胞蛋白主动募集到病毒复制区室的作用是促进病毒基因表达和复制。相比之下，病毒早期蛋白以抗病毒细胞 DNA 传感器和转录抑制子为目标，以阻止它们接触病毒基因组。由于缺乏鉴定参与病毒 DNA 复制的细胞蛋白的技术，研究病毒复制和病毒与宿主相互作用受到了阻碍。我们在该提案中的贡献将是应用一种新技术来识别病毒和细胞蛋白， 与复制病毒基因组有关。我们的中心假设是，细胞 DNA 复制/修复蛋白被招募到病毒基因组中，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，从而对病毒复制产生不利结果。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) 相结合来识别复制 DNA 上的蛋白质。我们将使用 iPOND-MS 来识别感染期间与病毒 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 1 型单纯疱疹病毒 (HSV-1)。疱疹病毒非常适合这种方法，因为它们阻止宿主细胞 DNA 复制，并在裂解复制过程中产生大量病毒基因组。 HSV-1 编码立即早期蛋白 ICP0，该蛋白可促进病毒基因表达并克服宿主的内在防御。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： 识别 HSV-1 病毒 DNA 复制专门利用的细胞蛋白，并 (ii) 确定 病毒 ICP0 如何改变与 HSV 基因组相关的蛋白质谱。将复制病毒基因组时鉴定的蛋白质与活跃细胞复制叉上的蛋白质库存进行比较，将揭示病毒和细胞 DNA 复制之间的差异。病毒专门招募的蛋白质可能代表抗病毒药物的潜在靶点，从而阻断 病毒传播。比较与野生型 HSV-1 和 ICP0 突变体的基因组相关的蛋白质将揭示 ICP0 如何招募因子来促进基因表达，同时操纵细胞反应以防止对病毒基因组的识别和抑制。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。识别不同病毒家族中常用于病毒 DNA 复制的细胞蛋白将表明其潜力 开发新型广泛作用的抗病毒疗法的目标。