Translation of Overlapping Open Reading Frames Promoted by Type 2 IRESs in Avian Calicivirus Genomes

Caliciviruses have positive-sense RNA genomes, typically with short 5'-untranslated regions (5'UTRs) that precede the long open reading frame 1 (ORF1). Exceptionally, some avian caliciviruses have long 5'UTRs containing a picornavirus-like internal ribosomal entry site (IRES), which was likely acquired by horizontal gene transfer. Here, we identified numerous additional avian calicivirus genomes with IRESs, predominantly type 2, and determined that many of these genomes contain a ~200-300 codon-long ORF (designated ORF1*) that overlaps the 5'-terminal region of ORF1. The activity of representative type 2 IRESs from grey teal calicivirus (GTCV) and Caliciviridae sp. isolate yc-13 (RaCV1) was confirmed by in vitro translation. Toeprinting showed that in cell-free extracts and in vitro reconstituted reactions, ribosomal initiation complexes assembled on the ORF1* initiation codon and at one or two AUG codons in ORF1 at the 3'-border and/or downstream of the IRES. Initiation at all three sites required eIF4A and eIF4G, which bound to a conserved region of the IRES; initiation on the ORF1* and principal ORF1 initiation codons involved eIF1/eIF1A-dependent scanning from the IRES's 3'-border. Initiation on these IRESs was enhanced by the IRES trans-acting factors (ITAFs) Ebp1/ITAF₄₅, which bound to the apical subdomain Id of the IRES, and PTB (GTCV) or PCBP2 (RaCV1).