Biased Genetic Screen Identifies Novel Genes Involved in Antiviral Defense

Abstract

RNA interference (RNAi) mediates potent antiviral response across kingdoms. In Caenorhabditis elegans nematodes, antiviral RNAi requires a virus sensor that is conserved in mammals and is amplified by secondary small interfering RNAs that are produced in a Dicer-independent manner. To better understand worm antiviral RNAi, I carried out a biased genetic screen, aiming to identify novel antiviral RNAi genes. To speed up the gene discovery process, the reporter worms used for this genetic screen were engineered to contain extra copies of 4 known antiviral RNAi genes. Therefore, genetic alleles derived from these 4 genes will be automatically rejected during screening process. Since a viral replicon transgene was used as reporter for loss of antiviral RNAi my genetic screen was expected to identify genes required for antiviral RNAi but not artificial double-stranded RNA (dsRNA) triggered RNAi. My genetic screen identified altogether 25 candidate alleles that appear to have derived from 13 candidate genes. Through genetic complementation tests and coding region sequencing I confirmed that 2 of the candidate genes are known antiviral RNAi genes rde-3 and rrf-1. Of the rest 11 candidate genes, 9 were found not to be required for classical RNAi. Interestingly, Orsay virus infection assay further suggested that 7 of these 9 candidate genes are dispensable for antiviral RNAi against Orsay virus. Since Orsay virus specifically infects intestine cells which have much weaker antiviral activity I believe that these 7 genes mainly function in non-intestine cells. Through whole genome sequencing I identified 2 of the candidate genes as rsd-6 and mut-16, 2 known genes required for artificial dsRNA triggered RNAi. Currently, rsd-6 and mut-16 are not known to play a role in antiviral RNAi. Thus, my genetic screen, for the first time, identified both rsd-6 and mut-16 as key components for antiviral RNAi.