Characterization of Orsay virus replication intermediates in Caenorhabditis elegans reveals links to antiviral RNA interference

Orsay Virus (OV) is a positive-sense, single-stranded RNA (+ssRNA) virus that naturally infects C. elegans intestines. Like other +ssRNA viruses, the OV-encoded RNA-dependent RNA polymerase (oRdRP) synthesizes complementary antigenome for use as template for amplifying viral genome, but OV replication intermediates are underexplored. Using PCR, we observed viral genome in vast excess of antigenome, as for other +ssRNA viruses. Unlike interferon-based antiviral defense, C. elegans utilizes RNA interference (RNAi) for antiviral defense, producing sense and antisense small interfering RNAs (siRNAs) that cannot be distinguished from genome and antigenome with conventional hybridization protocols. Fluorescence-based imaging in C. elegans intestines using probes to antigenomic sequences revealed cytoplasmic as well as perinuclear localization patterns. The latter depended on factors required for generation of primary, but not secondary, siRNAs, connecting the antigenomic hybridization pattern to RNAi. We also observed cytoplasmic double-stranded RNA (dsRNA) associated with oRdRP, suggesting viral replication hubs, as well as infection-induced nuclear dsRNA, likely from endogenous dsRNA. Finally, using antibodies to oRdRP, we observed spherical structures of [~]1{micro}m in diameter with oRdRP at their surface, which decrease in animals lacking RDE-1. Our study defines features of OV replication intermediates, setting the stage for understanding their connection to host antiviral pathways. Significance StatementO_LIOrsay virus is a +ssRNA virus that infects C. elegans intestines. We advance understanding of viral replication intermediates and address the issue that for animals that use antiviral RNA interference, hybridization of probes occurs with both genome and antigenome and small interfering RNAs. C_LIO_LISingle-molecule fluorescence in-situ hybridization using antigenomic probes revealed cytoplasmic and perinuclear puncta, only upon denaturation, with perinuclear signal dependent on primary, but not secondary, siRNA biogenesis. Viral RNA-dependent RNA polymerase lined the perimeter of spherical structures of [~]1{micro}m diameter. C_LIO_LIThis study sets the stage for understanding the relationship between viral replication dynamics and antiviral RNA interference. C_LI