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

Valeria Lulla; Andrew E. Firth

Comparative genomic analysis suggests that the presence of a protein-coding ORF overlapping ORF2 is widespread in mammalian astroviruses. It appears to be almost ubiquitous in genogroups I, III and IV and is probably present in the single genogroup V sequence. It also appears to be present in various unassigned sequences and clades. ORFX frequently has a hydrophobic stretch that is often predicted to be a TM domain and in other cases may represent a non-canonical TM domain refractory to detection with standard TM-predicting software (as appears to be the case for HAstV1). While normally absent from genogroup II astroviruses, an ORFX appears to be present in one clade (herein referred to as IId), whereas in the clade referred to herein as IIc we predicted an ORFY in the −1 instead of +1 frame, and accessed via ribosomal frameshifting instead of leaky scanning. Given the sporadic appearance of an overlapping ORF across the genogroup II phylogeny, it seems likely that the genogroup II ORFX and ORFY evolved independently from ORFX in genogroups I, III and IV, and may also therefore have different functions. In contrast, these other ORFXs may or may not have a common ancestor and/or common function (it is unclear to what extent genogroups I, III and IV form a monophyletic group; Fig. S1, Fig. S2 ). The N-terminal ~70 aa of CP are dispensable for particle assembly and likely structurally disordered 20 and may therefore be evolutionarily fairly flexible. Thus, this region of ORF2 may be unusually tolerant to the coding constraints imposed by overlapping genes. Together with the high translation level of sgRNAs at later timepoints ( Fig. 2A) , and the ease with which 5′ proximal ORFs can be expressed (requiring only leaky scanning rather than more complex expression mechanisms such as internal ribosome entry or ribosomal frameshifting 10 ), the 5′ region of the sgRNA may be particularly well-suited to the evolution of an overlapping gene, consistent with multiple independent origins of ORFXs.