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

Valeria Lulla; Andrew E. Firth

To evaluate the significance of ORFX in the context of virus infection, a set of mutant virus genomes was created based on the pAVIC1 infectious clone 16 by introducing mutations that knock out ORFX without affecting the CP amino acid sequence (Fig. 3A) . Four independent mutations were introduced to guard against the possibility of affecting potential RNA secondary structures overlapping with this region, resulting in pAVIC1-AUGm (AUG to ACG), pAVIC1-PTC1 (stop codon after 20 amino acids), pAVIC1-2×PTC (double stop codon after 20 amino acids) and pAVIC1-PTC2 (stop codon after 73 amino acids) ( Fig. 3B; Fig. S11 ). Corresponding T7 RNA transcripts were used for virus rescue in Huh7.5.1 cells followed by infection in Caco2 cells. All four ORFX knockout viruses were strongly attenuated compared to wt virus (Fig. 3C ).