Homologous RIG-I–like helicase proteins direct RNAi-mediated antiviral immunity in C. elegans by distinct mechanisms

Abstract

RNAi-mediated antiviral immunity in Caenorhabditis elegans requires Dicer-related helicase 1 (DRH-1), which encodes the helicase and C-terminal domains homologous to the mammalian retinoic acid inducible gene I (RIG-I)-like helicase (RLH) family of cytosolic immune receptors. Here we show that the antiviral function of DRH-1 requires the RIG-I homologous domains as well as its wormspecific N-terminal domain. We also demonstrate that the helicase and C-terminal domains encoded by either worm DRH-2 or human RIG-I can functionally replace the corresponding domains of DRH-1 to mediate antiviral RNAi in C. elegans. Notably, substitutions in a three-residue motif of the C-terminal regulatory domain of RIG-I that physically interacts with viral double-stranded RNA abolish the antiviral activity of C-terminal regulatory domains of both RIG-I and DRH-1 in C. elegans. Genetic analysis revealed an essential role for both DRH-1 and DRH-3 in C. elegans antiviral RNAi targeting a natural viral pathogen. However, Northern blot and small RNA deep sequencing analyses indicate that DRH-1 acts to enhance production of viral primary siRNAs, whereas DRH-3 regulates antiviral RNAi by participating in the biogenesis of secondary siRNAs after Dicer-dependent production of primary siRNAs. We propose that DRH-1 facilitates the acquisition of viral double-stranded RNA by the worm dicing complex for the subsequent processing into primary siRNAs. The strong parallel for the antiviral function of RLHs in worms and mammals suggests that detection of viral doublestranded RNA may activate completely unrelated effector mechanisms or, alternatively, that the mammalian RLHs have a conserved activity to stimulate production of viral siRNAs for antiviral immunity by an RNAi effector mechanism.I nnate immunity refers to ancient host defense systems that act immediately after pathogen attack and are under the control of germline-encoded immune receptors. Pathogen sensing by several classes of immune receptors such as retinoic acid inducible gene I (RIG-I)-like cytosolic sensors in mammals leads to the transcriptional activation of effector genes in the nucleus (1). In contrast, detection of viral double-stranded RNA (dsRNA) by Dicer endonucleases is associated with the production of small interfering RNAs (siRNAs), which subsequently direct sequencespecific antiviral immunity through RNA interference (RNAi) (2-5). Antiviral RNAi is a major antiviral defense mechanism in fungi, plants, insects, and nematodes, so that suppression of the defense by a virus-encoded suppressor of RNAi is essential to establish infection (6-8). Although the RNAi machinery is highly conserved in mammals, conclusive evidence for a natural antiviral role of RNAi in mammals is not available (9).The nematode Caenorhabditis elegans has recently emerged as a small-animal model for innate immunity studies (10, 11). Genetic studies indicate that antiviral RNAi in C. elegans requires the genes essential for RNAi induced by exogenous dsRNA (12-17). These include Dicer-1 and dsRNA-bind...

The RIG‐I‐like receptor LGP2 inhibits Dicer‐dependent processing of long double‐stranded RNA and blocks RNA interference in mammalian cells

“…DRH‐1 and DRH‐3 additionally contain a worm‐specific N‐terminal domain, whilst DRH‐2 lacks this domain and is solely composed of the helicase domain and the CTD, thereby exhibiting a remarkable resemblance with LGP2 (Paro et al , 2015). Whilst DRH‐1 is essential in antiviral, but not exogenous, RNAi in worms, DRH‐2 mutants show enhanced RNAi upon exposure to either exogenous or viral dsRNA (Lu et al , 2009; Ashe et al , 2013; Guo et al , 2013). This suggests that DRH‐2, through an unknown mechanism, functions as a negative regulator that inhibits the RNAi response in nematodes, resembling the observations made for its mammalian counterpart LGP2.…”

Caenorhabditis elegans as an Emerging Model for Virus-Host Interactions

“…DRH-1 physically interacts with DCR-1/RDE-4 complexes (23) and is required for fulminate antiviral RNAi responses but is dispensable for exo-RNAi (20,24). Interest- ingly, DRH-1 shares homology with mammalian retinoic acid-inducible gene-I (RIG-I)-like helicases (20,24), which act as cytosolic sensors of viral RNA that activate the vertebrate interferon response (25).…”

“…Interest- ingly, DRH-1 shares homology with mammalian retinoic acid-inducible gene-I (RIG-I)-like helicases (20,24), which act as cytosolic sensors of viral RNA that activate the vertebrate interferon response (25). Remarkably, human RIG-I domains can substitute for the corresponding DRH-1 domains to promote antiviral RNAi in C. elegans (24). These observations suggest that DRH-1 might function as a sensor of viral RNA, analogous to RIG-I, but instead trigger an RNAi response (26).…”

Sensing viral RNAs by Dicer/RIG-I like ATPases across species

“…The identification of a natural viral pathogen infecting C. elegans has opened the way for the genetic characterization of antiviral resistance [21]. One major locus associated with sensitivity to virus infection is the sDRA Dicer-related helicase-1 (DRH-1), which senses viral RNAs and recruits Dicer-1 to the antiviral RNAi pathway [22,23]. Interestingly, DRH-1 is not required for processing exogenous dsRNAs into siRNAs, indicating that it discriminates something more than the double-strandedness of viral RNAs.…”