A hidden gene in astroviruses encodes a cell-permeabilizing protein involved in virus release

Valeria Lulla; Andrew E. Firth

To test for XP expression in infected cells, we first raised various antibodies against XP peptides and we also tested the viability of tagged-XP viruses. However, neither antibody nor tagged virus approaches were successful. Thus we turned to ribosome profiling (Ribo-Seq). Ribo-Seq is a high throughput sequencing technique that globally maps the footprints of initiating or elongating 80S ribosomes on mRNAs 12, 13 . We infected Caco2 cells and performed Ribo-Seq at 12 hours post infection (hpi). Ribo-Seq quality was assessed as previously described 14 (Fig. S10 ). Using flash-freezing with no drug pre-treatment (NT), we mapped the translational landscape of the HAstV1 genome ( Fig. 2A) . ORF2 is translated at ~9ï‚´ the level of ORF1a, whereas ORF1b is translated at ~25% the level of ORF1a, indicating a ribosomal frameshifting efficiency of ~25%. The higher expression of ORF2 is likely a result of higher levels of sgRNA than gRNA in the translation pool. To identify translation initiation sites, we utilized the translation inhibitor lactimidomycin (LTM), which acts preferentially on the initiating ribosome but not on the elongating ribosome 15 . LTM binds to the 80S ribosome already assembled at the initiation codon and occupies the empty exit (E-site) of initiating ribosomes, thus completely blocking translocation. Using this approach, we confirm the two previously known initiation . CC-BY 4.0 International license author/funder. It is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/661579 doi: bioRxiv preprint sites, i.e. for ORF1a and ORF2, and also identify substantial initiation at the ORFX start codon ( Fig. 2B) , this being the third largest peak in the LTM virus profile.