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Identification and Characterization of Novel Alleles Required for Antiviral RNA Interference in Caenorhabditis elegans


Antiviral RNAi is a conserved antiviral pathway in plants and invertebrates that mediates clearance of viral RNA. Antiviral RNAi has been extensively studied in plants and insects, but Caenorhabditis elegans has only recently emerged as a model to study antiviral immunity. C. elegans is an ideal model to study antiviral RNAi, because it shares key commonalities with mammals, including a single Dicer and an expanded class of duplex RNA activated ATPases (DRAs). While several genetic screens have been done to identify the genes required for RNAi induced by exogenous long dsRNA, this unbiased forward genetic screen is the first with the ability to reveal gene required uniquely for antiviral RNAi or novel antiviral pathways. This dissertation develops an EMS screen based on a viral replicon that is defective in RNAi suppression. Viral replication is rescued in animals defective in RNAi and visualized by GFP. Thirteen worm mutants were identified that are defective in antiviral RNAi and four novel alleles of drh-1, a DRA homologous to the mammalian gene RIG-I, were revealed.

Both RIG-I and DRH-1 function in antiviral immunity through the activation of type-I interferon signaling in mammals and antiviral RNAi in C. elegans, respectively. This work investigates the function of DRH-1 in the production of virus-derived siRNAs (vsiRNAs). Deep sequencing of wild-type and drh-1 mutants challenged with Orsay Virus demonstrated that drh-1 mutants produced primary vsiRNAs that are dependent on DCR-1 and the dsRNA-binding protein RDE-4. However, these DRH-1 independent vsiRNAs are predominantly derived from the 5’ terminal regions of both OrV RNA1 and RNA2 and were depleted from the internal regions of viral RNA. In contrast, wild-type and other mutant animals did not exhibit a bias for primary vsiRNA biogenesis from the 5’ terminal regions. Mammalian RIG-I acts as an ATP-powered translocase, though the biological function of this activity is unknown. Therefore, DRH-1 may function to promote the production of primary vsiRNAs spreading from the 5’ terminal regions to the internal and 3’ regions of the viral RNA using translocase activity.

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