Viral Modulation of Genetic Stability

 描述（由申请人提供）：病毒通过利用或灭活细胞机制来创造有利于其自身复制的细胞条件。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。鉴定促进或限制病毒DNA复制的宿主因素一直受到缺乏技术的阻碍，只有有限数量的细胞蛋白质被证明会影响病毒复制。我们的长期目标是了解细胞如何对病毒基因组做出反应，并确定早期病毒蛋白质克服细胞限制以促进病毒复制的方式。在本申请中，我们使用腺病毒感染作为模型系统，并采用一种新的技术来鉴定与复制病毒DNA基因组相关的细胞蛋白。我们的中心假设是，病毒蛋白选择性地招募细胞DNA复制/修复因子到病毒基因组上，与病毒编码的复制因子一起发挥作用，同时也靶向特定的宿主抗病毒因子，以防止它们进入病毒基因组。我们建议，病毒早期E1 b和E4基因产物克服宿主反应，并通过影响招募到病毒基因组的细胞蛋白质来实现其多效性功能。这些病毒早期蛋白促进病毒DNA复制、RNA加工、蛋白表达和后代产生，但目前对其底物的了解甚少。我们采用新的蛋白质组学方法来识别与这些病毒早期蛋白质相互作用的宿主因子，并确定它们如何影响识别病毒DNA的宿主因子的景观。在强有力的初步数据的指导下，我们提出了三个特定的目标，以确定病毒早期蛋白招募或灭活的宿主因子，并确定它们对病毒DNA复制的影响。目的1：确定与复制病毒基因组相关的宿主因素如何影响感染。目的2：定义Ad家族的细胞反应。目的3：确定E1 b55 K相互作用蛋白如何影响病毒感染。这些研究采用创新的方法，提供了一个全面的看法，细胞反应的外源DNA基因组复制在宿主细胞核。我们预期我们的研究结果将确定蛋白质招募，以帮助病毒复制，以及wys病毒蛋白质操纵主机的反应。鉴定通常用于不同病毒的病毒DNA复制的细胞蛋白质将为开发新的广泛作用的抗病毒治疗剂提供潜在的靶点。我们的研究提供了对外源DNA的细胞传感器的见解，这些将指导改进的基因递送载体、病毒溶瘤剂和抗病毒疗法的开发。

 DESCRIPTION (provided by applicant): Viruses create cellular conditions conducive to their own replication by harnessing or inactivating cellular machinery. 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 replication. In contrast, viral early proteins target antiviral cellular DNA sensors and transcriptional repressors to prevent their access to viral genomes. Identifying host factors that facilitate or limit virus DNA replication has been hampered by the lack of technologies, and only a limited number of cellular proteins have been shown to affect virus replication. Our long-term goal is to understand how cells respond to virus genomes, and to define ways that early viral proteins overcome cellular restrictions to promote virus replication. In this application we use Adenovirus infection as a model system, and employ a novel technology to identify cellular proteins that associate with replicating viral DNA genomes. Our central hypothesis is that viral proteins selectively recruit cellular DNA replication/repair factors onto viral genomes to function with viral-encoded replication factors, while also targeting specific host antiviral factors to prevent their access to viral genomes. We propose that viral early E1b and E4 gene products overcome host responses and achieve their pleiotropic functions by influencing the cellular proteins recruited to virus genomes. These viral early proteins facilitate viral DNA replication, RNA processing, protein expression, and progeny production, but their substrates are currently poorly understood. We employ novel proteomic approaches to identify host factors that interact with these viral early proteins, and determine how they impact the landscape of host factors that recognize viral DNA. Guided by strong preliminary data, we propose three Specific Aims to identify host factors recruited or inactivated by viral early proteins and determine their impact on virus DNA replication. Aim 1: To determine how host factors associated with replicating viral genomes impact infection. Aim 2: To define cellular responses across the Ad family. Aim 3: To determine how E1b55K interacting proteins impact virus infection. These studies employ innovative approaches to provide a comprehensive view of cellular responses to foreign DNA genomes replicating in the host cell nucleus. We anticipate that our results will identify proteins recruited to aid viral replication, as well as wys viral proteins manipulate host responses. Identifying cellular proteins commonly exploited for viral DNA replication across different viruses will suggest potential targets for development of novel broadly acting antiviral therapeutics. Our studies provide insights into cellular sensors of foreign DNA, and these will guide development of improved gene delivery vectors, viral oncolytics, and antiviral therapies.