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

Valeria Lulla; Andrew E. Firth

Mammalian astrovirus nucleotide sequences were downloaded from the National Center for Biotechnology Information (NCBI) on 26 July 2018. Patent sequence records and sequences with ≥20 ambiguous nucleotide codes (e.g. "N"s) were removed. For the full-genome analyses, only sequences covering all or nearly all of ORF1a, ORF1b and ORF2 were retained, giving 221 sequences (listed in Fig. S1 ). For the ORF2 analyses, only sequences covering all or nearly all of ORF2 were retained, giving 415 sequences (listed in Supplementary Dataset 1) . To identify the correct 5′ end of ORF1b, we identified the AAAAAAC frameshift site. To identify the correct initiation site of ORF2, we identified the highly conserved sgRNA promoter nucleotides 29 and selected the next ORF2-frame AUG codon as the ORF2 start site in representative reference sequences; for the other sequences, the ORF2 start site was identified by amino acid alignment to one of the reference sequences. ORF1b and ORF2 sequences were extracted, translated to amino acid sequences, aligned with MUSCLE 30 The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/661579 doi: bioRxiv preprint acid rate matrices, with 1,000,000 (221-sequence trees; Fig. S1 and Fig. S2 ) or 5,000,000 (415sequence tree; Fig. S4 ) generations, discarding the first 25% as burn-in (other parameters were left at defaults). Trees were visualized with FigTree (http://tree.bio.ed.ac.uk/software/figtree/).