Detection of Orsay viral replication intermediates reveals spatial and regulatory links to Caenorhabditis elegans innate immune responses
Discuss this preprint
Start a discussion What are Sciety discussions?Listed in
This article is not in any list yet, why not save it to one of your lists.Abstract
For a positive-strand RNA virus, the encoded viral RNA-dependent RNA polymerase (oRdRP) synthesizes complementary antigenome strand and uses it as a template for amplifying the viral genome, generating various replication intermediates. Structural proteins and viral genome are packaged into virions, but the fate of replication intermediates is underexplored. Here, we investigate Orsay Virus (OV) replication intermediates, including antigenome, oRdRP and double stranded RNA (dsRNA), using PCR and fluorescence-based imaging in C. elegans intestines. As for other positive-strand RNA viruses, we find that genome is in vast excess of antigenome. Antigenome is only visualized in cells when using denaturation protocols, indicating basepaired intermediates. OV antigenome is observed with distinct cytoplasmic and perinuclear localization patterns that depend on factors required for generation of primary, but not secondary, siRNAs. In both wildtype and RNA interference (RNAi) mutants, viral dsRNA is observed in the cytoplasm associated with oRdRP, suggesting cytoplasmic virus replication hubs. Additionally, using antibodies to oRdRP, we observed spherical structures of ~1μm in diameter defined by oRdRP at their surface; over 75% of infected wildtype animals show these structures, which associate with mitochondria and autophagosomes in an antiviral RNAi- and autophagy-dependent manner, respectively. Our study defines new features of OV replication intermediates in wildtype animals, setting the stage for understanding their connection to the viral life cycle and host antiviral pathways.
Author Summary
Viruses infect and cause disease in all organisms. Once they enter an organism, or host, viruses make copies of themselves in a process called replication, and this process generates byproducts that are recognized as foreign by the host. This signals the host to begin protecting itself using pathways such as the innate immune response, underscoring the importance of understanding where and when replication byproducts are generated. Here we utilize Caenorhabditis elegans , a microscopic roundworm, to detect the viral replication intermediates of its naturally infecting pathogen, Orsay Virus (OV). For the first time, we detect OV antigenome, a byproduct that is used as a template for viral genome replication. We report a previously uncharacterized antigenome-containing perinuclear granule that is partially dependent on the antiviral RNA interference (RNAi) pathway, an invertebrate immune response. We also identify regions of cytoplasmic viral replication and unusually large (~1 micron) spherical structures lined with the viral RNA-dependent RNA polymerase, an enzyme made by the virus for its replication. By characterizing important viral replication byproducts, our studies set the stage for understanding mechanistic connections between viral infection and the host response.
