A targeted genetic screen identifies Caenorhabditis elegans genes involved in RNAi-independent antiviral defense

Cellular organisms are constantly challenged by potentially lethal viral infections and rely on diverse antiviral mechanisms for survival. In several systems, including plants and insects, parallel antiviral pathways provide redundancy, ensuring host protection even if one pathway is compromised by a virus. However, whether such alternative antiviral mechanisms exist in nematodes beyond RNA interference (RNAi) remains largely unexplored. To address this question and as proof of principle, we conducted a small-scale genetic screen to identify C. elegans genes involved in RNAi-independent antiviral defense (RiAD). The reporter system for this screen was a combination of a GFP-tagged flock house virus replicon, as the readout of efficient viral replication, and an RNAi-deficient triple mutant. The reporter C. elegans strain also carried a recessive E3 allele, which suppresses the replication of both flock house virus and Orsay virus. Because this reporter strain lacks a functional RNAi response, any restoration of viral replication in the screen is unlikely due to RNAi disruption, thereby enriching for mutants with defects in RiAD. Upon completing this biased genetic screen we identified ten recessive alleles which were assigned to eight candidate genes. Notably, five of these genes also contributed to RiAD against Orsay virus, indicating a role in broad-spectrum natural antiviral defense. Strikingly, removal of the E3 allele in one of the mutant backgrounds resulted in lethal Orsay virus infection, suggesting that RiAD plays a critical role in protecting C. elegans from fatal viral infection in the absence of RNAi under natural conditions.