Genetic dissection of the RNAi-mediated antiviral immunity in C. elegans

描述（由申请人提供）：具有RNA基因组的病毒通过不同于DNA基因组的机制复制和进化，是重要的人类、动物和植物病原体。哺乳动物防御RNA病毒的免疫机制尚不清楚。本研究将从遗传学角度剖析由禽舍病毒（Flock house virus， FHV）引发的秀丽隐杆线虫（Caenorhabditis elegans）动物的免疫途径。我们最近已经证明，在秀丽隐杆线虫基因组中整合的可诱导转基因启动FHV复制，触发病毒衍生的sirna的产生，这些sirna通过RNA沉默或RNA干扰（RNAi）引导病毒RNA清除。进一步的遗传分析为秀丽隐杆线虫对FHV或另一种不同RNA病毒的反应提供了保守的RNA沉默病毒免疫（RSI）途径的证据。我们已经使用秀丽隐杆线虫/FHV系统进行了RNAi喂养和化学诱变筛选试点。我们的喂养RNAi筛选已经鉴定出35个RSI所需的基因，包括drh-1，它是外源dsRNA诱导的RNAi所必需的，并且与最近发现的哺乳动物RNA病毒免疫受体高度同源。从化学筛选中恢复的存活的rsi缺陷蠕虫突变体包括rnai缺陷突变体和rnai敏感突变体，并且没有与drh-1或其他常见的rnai缺陷等位基因等位。因此，我们的研究结果表明，秀丽隐杆线虫的RSI需要与外源RNAi共享且不可缺少的成分，以及那些与哺乳动物病毒免疫成分同源的成分。我们计划进一步表征病毒sirna引导下的病毒RNA清除机制，次级病毒sirna的特征和作用，以及外源RNAi在蠕虫中缺失的RSI组分的特异性机制。此外，我们将利用实验室已经建立的方法，通过全基因组喂养RNAi和化学诱变筛选，分离动物RSI通路的其他成分。该实验将提供全动物病毒免疫途径的第一个基因组视图，鉴定在以前使用非病毒RNAi触发器的筛选中无法获得的新的RNAi成分，并揭示针对RNA病毒的免疫机制，从而促进理解哺乳动物病毒免疫。

DESCRIPTION (provided by applicant): Viruses with an RNA genome replicate and evolve via mechanisms distinct from DNA genomes and are important human, animal and plant pathogens. The mammalian immune mechanisms that defend against RNA viruses are poorly characterized. The proposed research will genetically dissect the immune pathway of Caenorhabditis elegans animals triggered by Flock house virus (FHV), one of the best characterized RNA viruses. We have shown recently that FHV replication launched from an inducible transgene integrated in the C. elegans genome triggers production of virus-derived siRNAs, which guide viral RNA clearance by RNA silencing or RNA interference (RNAi). Further genetic analyses have provided evidence for a conserved RNA silencing virus immunity (RSI) pathway in C. elegans in response to either FHV or another distinct RNA virus. We have carried out pilot feeding RNAi and chemical mutagenesis screens using the C. elegans/FHV system. Our feeding RNAi screen has identified 35 genes required for RSI including drh-1, which is dispensable for RNAi induced by exogenous dsRNA and highly homologous to the recently identified mammalian immune receptor for RNA viruses. Viable RSI-defective worm mutants recovered from the chemical screen include both RNAi-defective and RNAi-sensitive mutants and none are allelic with drh-1 or another frequent occurring RNAi-defective allele. Hence, our results have shown that C. elegans RSI requires components that are shared with, and dispensable for, exogenous RNAi, as well as those homologous to mammalian viral immunity components. We plan to further characterize the mechanism of viral RNA clearance guided by viral siRNAs, the features and roles of secondary viral siRNAs, and the specificity mechanism of RSI components dispensable for exogenous RNAi in worms. Further, we will isolate additional components of the animal RSI pathway through genome-wide feeding RNAi and chemical mutagenesis screens using the methodology already established in the lab. The proposed experiments will provide the first genome-view of the viral immunity pathway in whole animals, identify novel RNAi components that cannot be obtained in previous screens using non-viral RNAi triggers, and reveal immune mechanisms against RNA viruses that facilitate understanding mammalian viral immunity. PUBLIC HEALTH RELEVANCE: The nematode C. elegans has been a powerful model for elucidating the molecular mechanisms of both RNA interference (RNAi) and programmed cell death in humans. Many RNA viruses such as influenza, hepatitis C virus, poliovirus, dengue virus and West Nile virus are important human pathogens. Thus, it is likely that the proposed studies on the RNAi-mediated viral immunity pathway of C. elegans to RNA viruses will provide mechanistic insights into mammalian viral immunity and RNAi.