The functional role of DRH-1 in RNAi directed viral immunity

描述(申请人提供)：RNA干扰(RNAi)，或RNA沉默，是一种新的抗病毒机制，保存在真菌、植物和无脊椎动物中。RNAi定向病毒免疫(RDVI)是一个逐步的过程，首先由RNaseIII酶将病毒复制中间产物(以双链RNA(DsRNA)的形式)加工成小干扰RNA(SiRNA)双链。这些病毒来源的siRNAs(ViRNAs)将被整合到RNA诱导沉默复合体(RISC)中，作为靶向病毒RNA选择的序列指南。在RISC内，ArgAert(AGO)蛋白裂解由病毒RNA选择的病毒RNA。越来越多的证据表明，假定的RNA解旋酶在跨王国的RDVI中也发挥着重要作用。然而，这些RNA解旋酶如何促进RDVI在很大程度上仍不清楚。DRH-1是一种可能存在于线虫和哺乳动物体内的RNA解旋酶。此前，我们的研究已经证明，DRH-1是RDVI所必需的，但当RNAi被触发靶向细胞转录时，DRH-1就变得不可或缺。由于已知DRH-1在siRNA生物发生的下游发挥作用，这一发现表明，如果我们能够提供进一步的证据，证明在DRH-1存在的情况下，细胞基因沉默中的siRNAs可以介导有效的病毒沉默，那么DRH-1在介导RNA沉默方面将入侵的病毒RNA与细胞转录产物区分开来。有趣的是，DRH-1在真菌、植物或昆虫中并不保守，但与哺乳动物胞质病毒传感器RIG-I有显著的序列同源性。RIG-I区分入侵的病毒RNA和细胞转录产物，从而诱导干扰素介导的抗病毒天然免疫。因此，我们的研究支持这样一种假设，即DRH-1是一种RIG-I功能类似物，它区分入侵的病毒RNA和细胞转录产物，从而选择性地介导病毒沉默，而与沉默siRNA的来源无关。为了验证这一假设，我们建议：(1)确定DRH-1在介导RNA沉默方面是否区分复制病毒和细胞转录产物，而不管沉默siRNA的来源如何(“特定目的1，研究策略”部分，第4页)；(2)确定DRH-1在介导RNA沉默方面是否区分复制病毒和非复制病毒(“特定目标2”，“研究策略”部分，第6页)。这些研究不仅有助于确定DRH-1在蠕虫RDVI中的功能作用，还将建立线虫作为一种遗传模型，用于RIG-I样RNA解旋酶感知病毒的机制研究。拟议研究的新发现也可能揭示RDVI的一些线虫特有特征，因为DRH-1在真菌、植物或昆虫中不保守。

DESCRIPTION (provided by applicant): RNA interference (RNAi), or RNA silencing, is a novel antiviral mechanism conserved in fungi, plants and invertebrates. RNAi directed viral immunity (RDVI) is a stepwise process that begins with the processing of viral replication intermediates (in the form of double stranded RNA (dsRNA)), into small interfering RNA (siRNA) duplexes by RNase III enzymes. These virus-derived siRNAs (viRNAs) will then be incorporated into an RNA induced silencing complex (RISC) and serve as sequence guide for target viral RNA selection. Within the RISC, an Argonaut (AGO) protein cleaves the viral RNAs selected by viRNAs. Increasing evidence suggests that putative RNA helicases also play important roles in RDVI across kingdoms. However, how these RNA helicases contribute to RDVI remains largely unclear. DRH-1 is a putative RNA helicase conserved in both the Caenorhabditis elegans (C .elegans) nematode and mammals. Previously, our studies have demonstrated that DRH-1 is required for RDVI but becomes dispensable when RNAi is triggered to target cellular transcripts. Because DRH-1 is known to function downstream of siRNA biogenesis, this finding suggests that DRH-1 discriminates invading viral RNAs from cellular transcripts in mediating RNA silencing if we can provide further evidence that siRNAs in cellular gene silencing can mediate efficient virus silencing in the presence of DRH-1. Interestingly, DRH-1 is not conserved in fungi, plants or insects but shares significant sequence identity with RIG-I, a mammalian cytosolic virus sensor. RIG-I discriminates invading viral RNAs from cellular transcripts thereby to induce interferon mediated antiviral innate immunity upon virus detection, our studies thus support a hypothesis that DRH-1 is a RIG-I function analog that discriminates between invading viral RNAs and cellular transcripts thereby to selectively mediate virus silencing irrespective of the origin of silencing siRNAs. To test this hypothesis here we propose to: (1) determine if DRH-1 discriminates replicating viruses from cellular transcripts in mediating RNA silencing regardless of the origin of silencing siRNAs (Specific Aim 1, Research Strategy section, page 4); (2) determine if DRH-1 discriminates between replicating and non-replicating viruses in mediating RNA silencing (Specific Aim 2, Research Strategy section, page 6). The proposed studies are expected to not only help define a functional role for DRH-1 in worm RDVI but also establish C. elegans as a genetic model for mechanistic studies of virus sensing by RIG-I like RNA helicases. Novel findings from the proposed studies may also shed light on some nematode-specific features of RDVI since DRH-1 is not conserved in fungi, plants or insects.