Persistence of ambigrammatic narnaviruses requires translation of the reverse open reading frame

Narnaviruses are RNA viruses detected in diverse fungi, plants, protists,
arthropods and nematodes. Though initially described as simple single-gene
non-segmented viruses encoding RNA-dependent RNA polymerase (RdRp), a
subset of narnaviruses referred to as “ambigrammatic” harbor a unique
genomic configuration 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 mosquito-infecting 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 naïve mosquito cell line, 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 profiling yielded a
striking pattern of footprints for all four CxNV1 RNA strands that was
distinct from actively-translating ribosomes on host mRNA or co-infecting
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.