Bacterial effectors as probes to study (+) RNA virus-host cell biology

 描述(申请人提供)：所有病毒都依靠它们的宿主细胞进行复制。然而，病毒-宿主细胞相互作用的许多方面--例如膜运输在()RNA病毒复制中的作用--仍然知之甚少，部分原因是缺乏足够精确和强大的工具。这项探索性和发展研究提案探索了一种新的策略，通过使用大量细菌效应蛋白作为细胞生物学探针，基于这些蛋白质经常靶向RNA病毒使用的相同细胞途径，来确定病毒与宿主细胞的相互作用。由于细菌效应蛋白是一种遗传编码的酶，已经进化成操纵宿主细胞生物学的高特异性和活性，它们在探测病毒与宿主的相互作用方面比其他试剂有明显的优势，如RNAi或药理试剂。通过使用高通量筛选方法，我们将表达一组435个细菌效应蛋白来操纵宿主细胞生物学，并研究这些扰动如何影响丙型肝炎病毒、登革热病毒和黄热病病毒的复制，这三种病毒是黄病毒科的代表成员。然后，将优先考虑HITS，以进行进一步的机械性表征和后续研究。我们的初步数据证明了这种方法的可行性，并为未来的实验提供了有趣的线索。这些研究的结果将为我们的战略提供概念证明，将产生一套独特的工具来研究一组重要的()RNA病毒，并将与正在进行的细菌效应机制研究合作。我们展望了未来的实验利基市场，在那里，病毒学家和细胞生物学家将能够利用细菌效应蛋白工具箱来探索许多不同的生物学问题。

 DESCRIPTION (provided by applicant): All viruses depend on their host cells for replication. However, many aspects of virus-host cell interaction - such as the role of membrane trafficking in (+) RNA virus replication - are still poorly understood, in part due to a lack of sufficiently precise and powerful tools. This Exploratory and Developmental Research Proposal explores a new strategy to identify virus-host cell interaction by employing a large collection of bacterial effector proteins as cell biological probes, based on the observation that these proteins frequently target the same cellular pathways used by (+) RNA viruses. Because bacterial effector proteins are genetically encoded enzymes that have evolved to manipulate host cell biology with high specificity and activity, they have distinct advantages over other reagents for probing virus-host interaction, such as RNAi or pharmacological agents. By using a high-throughput screening approach, we will express a panel of 435 bacterial effector proteins to manipulate host cell biology, and examine how these perturbations affect the replication of hepatitis C virus, dengue virus, and yellow fever virus, three representative members of the Flaviviridae. Hits will then be prioritized for further mechanistic characterization and follow up studies. Our preliminary data demonstrate feasibility of this approach and provide interesting leads for future experiments. The outcome of these studies will provide proof of concept for our strategy, will generate a set of unique tools to study an important group of (+) RNA viruses, and will collaboratively synergize with ongoing research into bacterial effector mechanisms. We envision a future experimental niche wherein virologists and cell biologists will be able to utiliz a toolkit of bacterial effector proteins to probe many different biological questions.