Transgene-Assisted Genetic Screen Identifies rsd-6 and Novel Genes as Key Components of Antiviral RNA Interference in Caenorhabditis elegans

RNA interference (RNAi) is a widespread antiviral mechanism triggered by virus-produced double-stranded RNAs (dsRNAs). In Caenorhabditis elegans, antiviral RNAi involves a RIG-I-like RNA helicase, termed DRH-1 (dicer related RNA helicase 1), that is not required for classical RNAi triggered by artificial dsRNA. Currently, whether antiviral RNAi in C. elegans involves novel factors that are dispensable for classical RNAi remains an open question. To address this question, we designed and carried out a genetic screen that aims to identify novel genes involved in worm antiviral RNAi. By introducing extra copies of known antiviral RNAi genes into the reporter worms, we managed to reject alleles derived from 4 known antiviral RNAi genes, including the DRH-1 coding gene, during the screen. Our genetic screen altogether identified 25 alleles, which were assigned to 11 candidate genes and 2 known antiviral RNAi genes through genetic complementation tests. Using a mapping-by-sequencing strategy, we identified one of the candidate genes as rsd-6, a gene that helps maintain genome integrity through an endogenous gene-silencing pathway but was not known to be required for antiviral RNAi. More importantly, we found that two of the candidate genes are required for antiviral RNAi targeting Orsay virus, a natural viral pathogen of C. elegans, but dispensable for classical RNAi. Since drh-1 is so far the only antiviral RNAi gene not required for classical RNAi, we believe that our genetic screen led to identification of novel worm genes that may target virus-specific features to function in RNAi. IMPORTANCE In nematode worms, drh-1 detects virus-produced double-stranded RNA (dsRNA), thereby specifically contributing to antiviral RNA silencing. To identify drh-1-like genes with dedicated function in antiviral RNAi, we recently carried out a genetic screen that was designed to automatically reject all alleles derived from 4 known antiviral silencing genes, including drh-1. Of the 11 candidate genes identified, we found two of them to be required for antiviral silencing targeting a natural viral pathogen of C. elegans but not for classical RNA silencing triggered by artificial dsRNA. We believe that these two genes are novel components of worm antiviral RNAi, considering the fact that drh-1 is the only known antiviral RNAi gene that is dispensable for classical RNAi. This genetic screen also identified rsd-6, a gene that maintains genome integrity under unfavorable conditions, as a key regulator of worm antiviral silencing, demonstrating an interplay between antiviral immunity and genome integrity maintenance.