An RIG-I-Like RNA Helicase Mediates Antiviral RNAi Downstream of Viral siRNA Biogenesis in Caenorhabditis elegans

Rui Lu,Shou-Wei Ding,Erbay Yigit,Wan-Xiang Li,David S Schneider

doi:10.1371/journal.ppat.1000286

Abstract

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.

Highlights

Innate immunity is active immediately upon pathogen attack and represents an ancient defense mechanism conserved in diverse multicellular organisms
We found that no green fluorescence or only a tiny green spot in the pharynx area was observed in FR1gfp worms after heat induction of the Flock house virus (FHV) replicon transgene (Figure 1B, top left)
Recent studies demonstrate that viruses can infect, replicate, and assemble within C. elegans cells and that RNA viruses are targeted for silencing by the canonical RNA interference (RNAi) pathway in C. elegans [46,47,48,49]

Summary

Introduction

Innate immunity is active immediately upon pathogen attack and represents an ancient defense mechanism conserved in diverse multicellular organisms. Innate immunity is initiated by pattern recognition receptors (PRRs) that recognize conserved molecular patterns associated with microbes. Well-characterized PRR families include the transmembrane Toll-like receptors (TLRs) and the cytosolic NOD-like receptors (NLRs) and RIG-I-like RNA helicase receptors (RLRs), all of which contain members in vertebrates that recognize viral single- and/or double-stranded RNAs as the pathogen signatures [1,2,3]. The Dicer family of ribonucleases recognizes viral RNA like these PRRs to initiate the viral immunity in plants and invertebrates that is mechanistically related to RNA silencing or RNA interference (RNAi). Unlike TLR and RLRs, Dicer further processes the viral RNA trigger into small RNAs of 21–24 nucleotides to guide specific antiviral silencing [4]

Methods

Results

Conclusion