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

DRH-1 在 RNAi 定向病毒免疫中的功能作用

• 批准号: 8242983
• 负责人: Rui Lu
• 金额: $ 7.4万
• 依托单位: LOUISIANA STATE UNIV A&M COL BATON ROUGE
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-02-01 至 2014-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8242983
• 关键词: Antiviral Agents; Biogenesis; Biological Models; Biological Process; Biomedical Research; Boxing; Caenorhabditis elegans; Cleaved cell; Double-Stranded RNA; Enzymes; Gene Silencing; Genetic; Genetic Models; Goals; Homologous Gene; Housing; Human; Immunity; Insecta; Interferons; Invaded; Invertebrates; Lead; Light; Mammals; Mediating; Natural Immunity; Nematoda; Play; Process; Proteins; RNA Helicase; RNA I; RNA Interference; RNA-Induced Silencing Complex; Replicon; Research; Ribonuclease III; Role; Signal Pathway; Small Interfering RNA; Testing; Transcript; Transgenes; Viral; Virus; analog; cellular targeting; mutant; novel; plant fungi; sensor; success; viral RNA; viral detection

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. PUBLIC HEALTH RELEVANCE: In mammals, including humans, an RNA helicase, termed RIG-I, functions as a virus sensor in antiviral innate immunity. Here we propose to delineate a functional role for DRH-1, a RIG-I homologue found in Caenorhabditis elegans (C. elegans). Because C. elegans represents one of the best genetic models for modern biomedical research the proposed studies have the potential to establish C. elegans as a genetic model for mechanistic study of virus sensing by RIG-I.

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新型抗病毒机制。 RNAi 定向病毒免疫 (RDVI) 是一个逐步过程，首先通过 RNase III 酶将病毒复制中间体（以双链 RNA (dsRNA) 形式）加工成小干扰 RNA (siRNA) 双链体。然后，这些病毒衍生的 siRNA (viRNA) 将被整合到 RNA 诱导沉默复合物 (RISC) 中，并作为目标病毒 RNA 选择的序列指南。在 RISC 内，Argonaut (AGO) 蛋白会裂解 viRNA 选择的病毒 RNA。越来越多的证据表明，假定的 RNA 解旋酶在各个王国的 RDVI 中也发挥着重要作用。然而，这些 RNA 解旋酶如何促进 RDVI 仍不清楚。 DRH-1 是一种假定的 RNA 解旋酶，在秀丽隐杆线虫 (C .elegans) 线虫和哺乳动物中均保守。此前，我们的研究表明，DRH-1 是 RDVI 所必需的，但当 RNAi 被触发以靶向细胞转录本时，DRH-1 就变得可有可无。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 存在的情况下可以介导有效的病毒沉默，那么这一发现表明，DRH-1 在介导 RNA 沉默时能够区分细胞转录物中的入侵病毒 RNA。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 具有显着的序列同一性。 RIG-I区分入侵的病毒RNA和细胞转录本，从而在检测到病毒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这样的假设：DRH-1是RIG-I功能类似物，区分入侵的病毒RNA和细胞转录本，从而选择性介导病毒沉默，无论沉默siRNA的来源如何。为了在这里检验这一假设，我们建议：（1）确定 DRH-1 在介导 RNA 沉默时是否区分复制病毒和细胞转录本，无论沉默 siRNA 的来源如何（具体目标 1，研究策略部分，第 4 页）； (2) 确定 DRH-1 在介导 RNA 沉默时是否区分复制病毒和非复制病毒（具体目标 2，研究策略部分，第 6 页）。拟议的研究预计不仅有助于确定 DRH-1 在蠕虫 RDVI 中的功能作用，而且还将建立秀丽隐杆线虫作为遗传模型，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 的一些线虫特异性特征，因为 DRH-1 在真菌、植物或昆虫中并不保守。 公共卫生相关性：在包括人类在内的哺乳动物中，一种称为 RIG-I 的 RNA 解旋酶在抗病毒先天免疫中充当病毒传感器。在这里，我们建议描述 DRH-1 的功能作用，DRH-1 是一种在秀丽隐杆线虫 (C. elegans) 中发现的 RIG-I 同源物。由于线虫代表了现代生物医学研究的最佳遗传模型之一，因此拟议的研究有可能将线虫建立为 RIG-I 病毒传感机制研究的遗传模型。