An RIG-I-Like RNA helicase mediates antiviral RNAi downstream of viral siRNA biogenesis in Caenorhabditis elegans.
An RIG-I-Like RNA helicase mediates antiviral RNAi downstream of viral siRNA biogenesis in Caenorhabditis elegans.
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DOI：
10.1371/journal.ppat.1000286
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发表时间：
2009-02
期刊：
影响因子：
6.7
通讯作者：
Ding SW
中科院分区：
文献类型：
--
作者：
Lu R;Yigit E;Li WX;Ding SW
植物和无脊椎动物包括秀丽隐杆线虫的Dicer核糖核酸酶识别病毒RNA触发物并将其加工成病毒衍生的小干扰RNA（siRNA），以通过Argonaute-dependent RNA干扰（RNAi）引导特异性病毒免疫。C.秀丽线虫还编码三种与RIG-1样RNA解旋酶受体密切相关的Dicer相关解旋酶（drh）基因，所述RIG-1样RNA解旋酶受体在哺乳动物中启动针对RNA病毒的广谱先天免疫。在这里，我们开发了一个转基因C。仅在敲低或敲除抗病毒RNAi所需的基因后，从染色体整合的鸡舍病毒复制子表达强烈的绿色荧光的线虫菌株。在摄食RNAi筛选中使用报告线虫菌株鉴定了drh-1作为抗病毒RNAi途径的重要组分。然而，由外源dsRNA或病毒复制子诱导的RNAi在drh-2突变体线虫中增强，而外源RNAi在drh-1突变体线虫中基本上未改变，表明外源和抗病毒RNAi途径在遗传上是不同的。遗传上位性分析表明，drh-1作用于病毒传感和病毒siRNA生物合成的下游，介导特异性抗病毒RNAi。值得注意的是，我们发现，两个成员的大幅扩大亚家族的Argonautes具体到C。线虫控制着平行的抗病毒RNAi途径。这些发现证明了C. elegans在抗病毒防御中的作用秀丽隐杆线虫基因组编码三种Dicer相关解旋酶（DRH），它们与DexD/H盒解旋酶结构域高度同源，Dicer核糖核酸酶和RIG-I样解旋酶（RLR）是两种不同的病毒感受器家族。Dicer通过产生病毒衍生的小干扰RNA（siRNA）在植物、真菌和无脊椎动物中启动特异性RNAi介导的病毒免疫。相比之下，哺乳动物的RLR触发干扰素的生产和广谱病毒免疫，虽然三个RLR之一可以作为一个负和正调节病毒免疫。在本研究中，我们开发了一个转基因C。elegans菌株进行高通量遗传筛选，并鉴定了包括drh-1在内的35个RNAi介导的病毒免疫所需的基因。遗传上位性分析表明，drh-1介导的RNAi免疫下游的病毒siRNA的生产。值得注意的是，我们发现drh-2作为病毒免疫的负调节剂发挥作用。因此，线虫DRH和哺乳动物RLR都参与抗病毒免疫应答。然而，与哺乳动物RLR不同，线虫DRH-1采用RNAi效应器机制，不太可能参与直接病毒感测。
Dicer ribonucleases of plants and invertebrate animals including Caenorhabditis elegans recognize and process a viral RNA trigger into virus-derived small interfering RNAs (siRNAs) to guide specific viral immunity by Argonaute-dependent RNA interference (RNAi). C. elegans also encodes three Dicer-related helicase (drh) genes closely related to the RIG-I-like RNA helicase receptors which initiate broad-spectrum innate immunity against RNA viruses in mammals. Here we developed a transgenic C. elegans strain that expressed intense green fluorescence from a chromosomally integrated flock house virus replicon only after knockdown or knockout of a gene required for antiviral RNAi. Use of the reporter nematode strain in a feeding RNAi screen identified drh-1 as an essential component of the antiviral RNAi pathway. However, RNAi induced by either exogenous dsRNA or the viral replicon was enhanced in drh-2 mutant nematodes, whereas exogenous RNAi was essentially unaltered in drh-1 mutant nematodes, indicating that exogenous and antiviral RNAi pathways are genetically distinct. Genetic epistatic analysis shows that drh-1 acts downstream of virus sensing and viral siRNA biogenesis to mediate specific antiviral RNAi. Notably, we found that two members of the substantially expanded subfamily of Argonautes specific to C. elegans control parallel antiviral RNAi pathways. These findings demonstrate both conserved and unique strategies of C. elegans in antiviral defense. The genome of Caenorhabditis elegans encodes three Dicer-related helicases (DRHs) highly homologous to the DExD/H box helicase domain found in two distinct families of virus sensors, Dicer ribonucleases and RIG-I-like helicases (RLRs). Dicer initiates the specific, RNAi-mediated viral immunity in plants, fungi and invertebrates by producing virus-derived small interfering RNAs (siRNAs). By contrast, mammalian RLRs trigger interferon production and broad-spectrum viral immunity, although one of the three RLRs may act as both a negative and positive regulator of viral immunity. In this study we developed a transgenic C. elegans strain for high-throughput genetic screens and identified 35 genes including drh-1 that are required for RNAi-mediated viral immunity. Genetic epistatic analyses demonstrate that drh-1 mediates RNAi immunity downstream of the production of viral siRNAs. Notably, we found that drh-2 functions as a negative regulator of the viral immunity. Thus, both nematode DRHs and mammalian RLRs participate in antiviral immune responses. Unlike mammalian RLRs, however, nematode DRH-1 employs an RNAi effector mechanism and is unlikely to be involved in direct virus sensing.
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