Caenorhabditis elegans RIG-I homolog mediates antiviral RNA interference downstream of Dicer-dependent biogenesis of viral small interfering RNAs

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© 2017 Coffman et al. Dicer enzymes process virus-specific double-stranded RNA (ds RNA) into small interfering RNAs (si RNAs) to initiate specific antiviral defense by related RNA interference (RNAi) pathways in plants, insects, nematodes, and mammals. Antiviral RNAi in Caenorhabditis elegans requires Dicer-related helicase 1 (DRH-1), not found in plants and insects but highly homologous to mammalian retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs), intracellular viral RNA sensors that trigger innate immunity against RNA virus infection. However, it remains unclear if DRH-1 acts analogously to initiate antiviral RNAi in C. elegans. Here, we performed a forward genetic screen to characterize antiviral RNAi in C. elegans. Using a mapping-bysequencing strategy, we uncovered four loss-of-function alleles of drh-1, three of which caused mutations in the helicase and C-terminal domains conserved in RLRs. Deep sequencing of small RNAs revealed an abundant population of Dicer-dependent virus-derived small interfering RNAs (vsi RNAs) in drh-1 single and double mutant animals after infection with Orsay virus, a positive-strand RNA virus. These findings provide further genetic evidence for the antiviral function of DRH-1 and illustrate that DRH-1 is not essential for the sensing and Dicer-mediated processing of the viral ds RNA replicative intermediates. Interestingly, vsi RNAs produced by drh-1 mutants were mapped overwhelmingly to the terminal regions of the viral genomic RNAs, in contrast to random distribution of vsi RNA hot spots when DRH-1 is functional. As RIG-I translocates on long ds RNA and DRH-1 exists in a complex with Dicer, we propose that DRH-1 facilitates the biogenesis of vsi RNAs in nematodes by catalyzing translocation of the Dicer complex on the viral long ds RNA precursors. IMPORTANCE The helicase and C-terminal domains of mammalian RLRs sense intracellular viral RNAs to initiate the interferon-regulated innate immunity against RNA virus infection. Both of the domains from human RIG-I can substitute for the corresponding domains of DRH-1 to mediate antiviral RNAi in C. elegans, suggesting an analogous role for DRH-1 as an intracellular ds RNA sensor to initiate antiviral RNAi. Here, we developed a forward genetic screen for the identification of host factors required for antiviral RNAi in C. elegans. Characterization of four distinct drh-1 mutants obtained from the screen revealed that DRH-1 did not function to initiate antiviral RNAi. We show that DRH-1 acted in a downstream step to enhance Dicer-dependent biogenesis of viral si RNAs in C. elegans. As mammals produce Dicer-dependent viral si RNAs to target RNA viruses, our find-ings suggest a possible role for mammalian RLRs and interferon signaling in the biogenesis of viral siRNAs.

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