Upstream translation initiation expands the coding capacity of segmented negative-strand RNA viruses

Abstract

Segmented negative-strand RNA viruses (sNSVs) include the influenza viruses, the bunyaviruses, and other major pathogens of humans, other animals and plants. The genomes of these viruses are extremely short. In response to this severe genetic constraint, sNSVs use a variety of strategies to maximise their coding potential. Because the eukaryotic hosts parasitized by sNSVs can regulate gene expression through low levels of translation initiation upstream of their canonical open reading frames (ORFs), we asked whether sNSVs could use upstream translation initiation to expand their own genetic repertoires. Consistent with this hypothesis, we showed that influenza A viruses (IAVs) and bunyaviruses were capable of upstream translation initiation. Using a combination of reporter assays and viral infections, we found that upstream translation in IAVs can initiate in two unusual ways: through non-AUG initiation in virally encoded ‘untranslated’ regions, and through the appropriation of an AUG-containing leader sequence from host mRNAs through viral cap-snatching, a process we termed ‘start-snatching.’ Finally, while upstream translation of cellular genes is mainly regulatory, for sNSVs it also has the potential to create novel viral gene products. If in frame with a viral ORF, this creates N-extensions of canonical viral proteins. If not, it allows the expression of cryptic overlapping ORFs, which we found were highly conserved in IAV and widely distributed in peribunyaviruses. Thus, by exploiting their host’s capacity for upstream translation initiation, sNSVs can expand still further the coding potential of their extremely compact RNA genomes.

Accessory Gene Products of Influenza A Virus

“…A final layer of complexity in IAV gene expression comes from the demonstration that the cap-snatching mechanism itself can generate novel translational start sites on viral mRNAs, when the leader sequence generated from the host cell pre-mRNA contains an AUG codon or non-AUG initiation site (Sloan et al 2019;Ho et al 2020). The evidence for this comes from a variety of methods, including deep sequencing of the capped leader sequences, ribosomal profiling, T-cell epitope assays, and mass-spectrometric detection of novel peptides.…”