Unusual dicistronic expression from closely spaced initiation codons in an umbravirus subgenomic RNA

Translation commencing at closely spaced initiation codons is common in RNA viruses with limited genome space. In the subgenomic RNA (sgRNA) of Pea enation mosaic virus 2, two closely spaced, out-of-frame start codons direct synthesis of movement/stability proteins p26 and p27. Efficient translation from AUG(26)/AUG(27) is dependent on three 3'-proximal cap-independent translation enhancers (3'CITEs), whereas translation of the genomic (gRNA) requires only two. Contrary to strictly scanning-dependent initiation at the gRNA, sequence context of AUG(26)/AUG(27) does not conform with Kozak requirements and insertion of efficient upstream AUGs had pronounced effects for AUG(26) but only moderate effects for AUG27. Insertion of a hairpin within an extended 5'UTR did not significantly impact translation from AUG(26)/AUG(27). Furthermore, AUG(27) repressed translation from upstream AUG(26) and this effect was mitigated when inter-codon spacing was reduced. Addition of a stable hairpin to the very 5' end of the sgRNA severely restricted translation, testifying that this 3'CITE-driven initiation is 5' end-dependent. Similar to gRNA, sgRNA reporter transcripts were nearly exclusively associated with light polysomes and 3'CITE-promoted long-distance interaction connecting the sgRNA ends affected the number of templates translated and not the initiation rate. We propose a non-canonical, 3' CITE-driven mechanism for efficient dicistronic expression from umbravirus sgRNAs.