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

描述（由申请人提供）：RNA干扰（RNAi）或RNA沉默是真菌、植物和无脊椎动物中保守的一种新型抗病毒机制。RNAi介导的病毒免疫（RDVI）是一个逐步的过程，其开始于通过RNase III酶将病毒复制中间体（以双链RNA（dsRNA）的形式）加工成小干扰RNA（siRNA）双链体。然后，这些病毒衍生的siRNA（viRNA）将被掺入RNA诱导的沉默复合物（RISC）中，并作为靶病毒RNA选择的序列指导。在RISC内，Argonaut（AGO）蛋白切割由viRNA选择的病毒RNA。越来越多的证据表明，假定的RNA解旋酶也发挥重要作用，跨王国的RDVI。然而，这些RNA解旋酶如何促进RDVI仍不清楚。 DRH-1是一种推定的RNA解旋酶，在秀丽隐杆线虫（C .elegans）线虫和哺乳动物中均保守。以前，我们的研究已经证明，DRH-1是RDVI所需的，但当RNAi被触发靶向细胞转录本时，DRH-1就变成了RNA。由于已知DRH-1在siRNA生物发生的下游发挥作用，因此如果我们能够提供进一步的证据证明细胞基因沉默中的siRNA在DRH-1存在下可以介导有效的病毒沉默，则这一发现表明DRH-1在介导RNA沉默中将入侵的病毒RNA与细胞转录本区分开来。有趣的是，DRH-1在真菌、植物或昆虫中不保守，但与哺乳动物胞质病毒传感器RIG-1具有显著的序列同一性。RIG-I区分入侵的病毒RNA和细胞转录物，从而在病毒检测时诱导干扰素介导的抗病毒先天免疫，因此我们的研究支持DRH-1是RIG-I功能类似物的假设，其区分入侵的病毒RNA和细胞转录物，从而选择性地介导病毒沉默，而不管沉默siRNA的来源。为了验证这一假设，我们提出：（1）确定DRH-1是否在介导RNA沉默中区分复制型病毒和细胞转录物，而不管沉默siRNA的来源（具体目标1，研究策略部分，第4页）;（2）确定DRH-1是否在介导RNA沉默中区分复制型和非复制型病毒（具体目标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，还有助于建立C.线虫作为RIG-I样RNA解旋酶的病毒传感机制研究的遗传模型。从拟议的研究中获得的新发现也可能揭示RDVI的一些线虫特异性特征，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.