P-260 - The role of the upstream AUGs in Triticum mosaic virus 5’ leader sequence

Upstream AUGs (uAUGs) are common in eukaryotic and viral mRNAs but are rare among Potyviridae members. Triticum mosaic virus (TriMV, Poacevirus titrici) is an unconventional potyvirus with a long 5′ UTR (739 nt) containing 13 upstream AUGs. Similar to animal picornavirus, TriMV employs an internal ribosome entry site (IRES) in its 5’UTR for cap-independent translation. Like other potyviruses, it encodes a single polyprotein that is post-translationally cleaved by the viral protease. All known potyviral proteins are thought to originate from the coding region.
Here, we investigate the role of the TriMV uAUGs and their potential coding capacity. Using ribosome profiling sequencing (Ribo-seq), we identified two strong ribosome footprint signals at uAUG5 (position 281) and uAUG10 (position 525), suggesting active translation of a 33 aa peptide and a 77 aa peptide, respectively. Mutation of uAUG5 into an AUC abolished translation, supporting a key role in IRES activity. Interestingly, the mutation of AUG10 had no effect on translation. To assess the function of the putative uORF10 in viral life cycle, we generated an AUG10-mutant TriMV infectious clone to test viral replication and systemic movement. Our findings will provide new insights into TriMV translation strategies and may have a broader impact on potyviral pathogenesis.