Animal virus replication and RNAi-mediated antiviral silencing in Caenorhabditis elegans

Abstract

The worm Caenorhabditis elegans is a model system for studying many aspects of biology, including host responses to bacterial pathogens, but it is not known to support replication of any virus. Plants and insects encode multiple Dicer enzymes that recognize distinct precursors of small RNAs and may act cooperatively. However, it is not known whether the single Dicer of worms and mammals is able to initiate the small RNA-guided RNA interference (RNAi) antiviral immunity as occurs in plants and insects. Here we show complete replication of the Flock house virus (FHV) bipartite, plus-strand RNA genome in C. elegans. We show that FHV replication in C. elegans triggers potent antiviral silencing that requires RDE-1, an Argonaute protein essential for RNAi mediated by small interfering RNAs (siRNAs) but not by microRNAs. This immunity system is capable of rapid virus clearance in the absence of FHV B2 protein, which acts as a broad-spectrum RNAi inhibitor upstream of rde-1 by targeting the siRNA precursor. This work establishes a C. elegans model for genetic studies of animal virus-host interactions and indicates that mammals might use a siRNA pathway as an antiviral response.

Fig. 1

Replication and silencing of FHV in C. elegans. a, Structure, replication and expression of FHV genome. b, Structure of FHV transgenes. HIP- heat inducible promoter. The junction region between HIP (lower case) and FHV cDNA (upper case) is shown. Transcriptional initiation sites from HIP were indicated. Rz — a self-cleaving ribozyme used to reduce non-viral extensions at the 3'-termini of heat-induced transcripts. Northern blot detection of FHV RNA accumulation in C. elegans (c) and fruit fly S2 cells (d). Four μg total RNA was loaded per lane in both c and d, except for lane 8 of c.

Fig. 2

FHV RNAi suppressor is active in rde-1 worms. Total RNA was extracted from worms of either wild type or rde-1 genotype carrying an integrated FR1-3 or FR1-3ΔB2 transgene two days after transcriptional induction. Northern blot hybridizations were carried out as in upper panels of Fig. 1c/d.

Fig. 3

FHV B2 is a dsRNA-binding protein and inhibits siRNA production in vitro. a — d. GST-tagged B2 protein (GST-B2) binds both 21-nt siRNA duplex (a and d) and 44-nt dsRNA (d) in vitro. Volumes of the top siRNA/B2 band in lanes 7-10 and 15-18 were quantified by phosphoimager and plotted against the concentrations of the cold competitor RNAs (c). Note the presence of non-specific signals in lanes 1, 8, 13 and 16 of d. e. B2 inhibits in vitro processing of a labeled long dsRNA by the Dicer extracts from fruit fly S2 cells. Concentration gradients of GST or GST-B2 used were 50, 200, 800, 3,200 or 10,000 nM. A labeled 21-nt siRNA was used as a marker (M) at left.