The goal of this project is to identify novel mechanisms of antiviral immunity in eukaryotic organisms. This proposal takes an innovative approach that capitalizes upon a unique virus infection system in the model organism C. elegans. The success of C. elegans as a model multicellular organism stems from it relative simplicity, genetic tractability, and extensive evolutionary conservation with humans. With the recent discovery of Orsay virus, the first and only natural virus capable of bona fide infection of C. elegans, it is now possible to define genes that function in the defense against viral infection in C. elegans. Because C. elegans possesses neither adaptive immunity nor professional immune cells, it provides a robust platform to identify novel innate immunity mechanisms. Currently, RNA interference (RNAi) is the only known antiviral pathway in C. elegans that functions against natural virus infection. In preliminary experiments, high-resolution RNA-seq time courses of the C. elegans transcriptional response to Orsay virus infection identified a set of ~200 genes that are differentially regulated in the first 12 hours post-infection. Using RNAi knockdown, multiple candidate genes have been identified that, upon depletion, yield increased levels of viral RNA and protein expression. This proposal seeks to characterize the most compelling candidate genes from the preliminary studies in order to generate additional insights into mechanisms of metazoan antiviral immunity. Specifically, the proposal will focus upon one gene, C01G10.6, and a gene family, the C17H1.3 family of paralogs. Consistent with the goal of identifying novel mechanisms of antiviral immunity, these genes have no known function and have not been previously linked to antimicrobial immunity. Aim 1 focuses on characterization of C01G10.6, a gene that is transcriptionally induced at early time points post infection and yields a robust 20-fold increase in Orsay virus RNA levels when depleted by RNAi. Aim 2 will focus upon the C17H1.3 gene family, a family of 34 paralogs of which 24 members are highly induced 6-12 hours post Orsay virus infection. Individual RNAi knockdown of several paralogs in the family (C17H1.3, C54D10.12 and C31B8.4) in C. elegans leads to modest increases in Orsay virus RNA and protein levels. Together, these data suggest that this paralog family plays an important role in the response to viral infection. Critically, C17H1.3 family members are orthologous to a human gene, Amyotropic lateral sclerosis candidate region 12, which encodes a protein of unknown function. Thus, characterizing the mechanism of antiviral defense afforded by the C17H1.3 family members may have direct relevance to understanding human antiviral defenses.