Screens for host DNA replication and repair factors involved in viral infection

描述（由申请方提供）：宿主细胞途径在生产性感染期间被病毒利用和灭活。体外复制系统对于研究病毒DNA复制机制是有用的，但是它们未能捕获细胞环境的全部复杂性以及空间调节和细胞内宿主防御的重要性。从最近的工作中可以清楚地看出，细胞DNA损伤机制影响病毒感染。虽然已经鉴定了一些参与调节病毒-宿主相互作用的细胞蛋白，但尚未对宿主复制和修复因子进行全面分析。RNA干扰（RNAi）的出现和短干扰RNA（siRNA）文库的开发使得能够系统地分析参与病毒过程的宿主基因。在本申请中，我们提出使用针对参与DNA复制、修复和重组的细胞蛋白的靶向siRNA文库。我们将比较和对比四种在细胞核中复制的特征良好的DNA病毒。这些不同病毒家族和基因组的代表是：腺病毒（Ad）和单纯疱疹病毒1型（HSV-1）的线性双链DNA（dsDNA）基因组，细小病毒腺相关病毒（AAV）的单链DNA（ssDNA）基因组，以及猿猴病毒40型（SV 40）的环状dsDNA基因组。我们将使用野生型病毒或表达报告蛋白的重组载体开发和验证四种病毒系统的转导、复制和病毒生产的高通量功能测定。从我们的筛选中出现的命中将通过额外的siRNA来确认以排除脱靶效应，以及在RNA和蛋白质水平上的敲除验证。这些筛选将产生关于调节病毒感染的宿主因子的重要信息数据集，并将为进一步研究控制病毒感染的机制提供平台。平行比较多个病毒系统将揭示具有不同基因组的病毒所采用的统一方法和替代策略，并将确定积极和消极调节病毒感染的宿主因素。了解这些主题将提供对细胞DNA修复途径的深入了解，以确保宿主基因组的完整性，并可能确定抗病毒方法的新靶点。公共卫生相关性：病毒依赖于宿主细胞机制来复制其遗传物质和产生后代，因此提供了研究细胞过程的有力工具。我们建议筛选四种不同的DNA病毒（腺病毒，疱疹病毒，腺相关病毒和猿猴病毒40）对小干扰RNA的图书馆，针对参与DNA复制，重组和修复的细胞蛋白质。这些筛选将确定调节病毒感染的细胞因子，将提供对病毒-宿主相互作用和基本细胞过程的见解，并可能为抗病毒治疗提供靶点。

DESCRIPTION (provided by applicant): Host cell pathways are harnessed and inactivated by viruses during productive infection. In vitro replication systems have been useful for studying viral DNA replication mechanisms, but they fail to capture the full complexity of the cellular environment and the importance of spatial regulation and intracellular host defenses. It is clear from recent work that the cellular DNA damage machinery impacts virus infection. Although some cellular proteins involved in regulating virus-host interactions have been identified, a comprehensive analysis of host replication and repair factors has not yet been undertaken. The advent of RNA interference (RNAi) and the development of short interfering RNA (siRNA) libraries, enables a systematic analysis of host genes involved in viral processes. In this application we propose to use a targeted siRNA library against cellular proteins involved in DNA replication, repair and recombination. We will compare and contrast four well- characterized DNA viruses that replicate in the nucleus. These representatives of different viral families and genomes are: the linear double-stranded DNA (dsDNA) genomes of Adenovirus (Ad) and Herpes Simplex Virus type 1 (HSV-1), the single-stranded DNA (ssDNA) genome of the parvovirus Adeno-Associated Virus (AAV), and the circular dsDNA genome of Simian Virus type 40 (SV40). We will develop and validate high- throughput functional assays for transduction, replication, and virus production for the four viral systems, using either wild-type viruses or recombinant vectors that express reporter proteins. Hits that emerge from our screens will be confirmed by additional siRNAs to rule out off-target effects, together with verification of knock- down at the RNA and protein level. These screens will yield an important dataset of information about host factors that regulate virus infection and will provide a platform for further studies into the mechanisms that control virus infection. Comparing multiple viral systems in parallel will reveal both unifying approaches and alternative strategies employed by viruses with different genomes, and will identify host factors that both positively and negatively regulate virus infection. Understanding these themes will provide insights into cellular DNA repair pathways that ensure the integrity of the host genome, and may identify new targets for antiviral approaches. PUBLIC HEALTH RELEVANCE: Viruses are dependent upon the host cell machinery for replication of their genetic material and progeny production, and therefore present powerful tools to study cellular processes. We propose to screen four different DNA viruses (adenovirus, herpesvirus, adeno-associated virus and simian virus 40) against a library of small interfering RNAs targeted to cellular proteins involved in DNA replication, recombination and repair. These screens will identify cellular factors that regulate viral infection, will provide insights into virus-host interactions and fundamental cellular processes, and may suggest targets for anti-viral therapies.