Persistence of Ambigrammatic Narnaviruses Requires Translation of the Reverse Open Reading FrameHanna Retallack, Katerina D. Popova, Matthew T. Laurie, Sara Sunshine, Joseph L. DeRisi
Journal of Virology, 2021Abstract: Narnaviruses are RNA viruses detected in diverse fungi, plants, protists, arthropods, and nematodes. Though initially described as simple single-gene nonsegmented viruses encoding RNA-dependent RNA polymerase (RdRp), a subset of narnaviruses referred to as “ambigrammatic” harbor a unique genomic con!guration consisting of overlapping open reading frames (ORFs) encoded on opposite strands. Phylogenetic analysis supports selection to maintain this unusual genome organization, but functional investigations are lacking. Here, we establish the mosquitoinfecting Culex narnavirus 1 (CxNV1) as a model to investigate the functional role of overlapping ORFs in narnavirus replication. In CxNV1, a reverse ORF without homology to known proteins covers nearly the entire 3.2-kb segment encoding the RdRp. Additionally, two opposing and nearly completely overlapping novel ORFs are found on the second putative CxNV1 segment, the 0.8-kb “Robin” RNA. We developed a system to launch CxNV1 in a naive mosquito cell line and then showed that functional RdRp is required for persistence of both segments, and an intact reverse ORF is required on the RdRp segment for persistence. Mass spectrometry of persistently CxNV1-infected cells provided evidence for translation of this reverse ORF. Finally, ribosome pro!ling yielded a striking pattern of footprints for all four CxNV1 RNA strands that was distinct from actively translating ribosomes on host mRNA or coinfecting RNA viruses. Taken together, these data raise the possibility that the process of translation itself is important for persistence of ambigrammatic narnaviruses,
potentially by protecting viral RNA with ribosomes, thus suggesting a heretofore
undescribed viral tactic for replication and transmission.
