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

Valeria Lulla; Andrew E. Firth

For the ORF2-only SYNPLOT2 analyses, any duplicate sequences were removed and the remaining ORF2 sequences in each clade were translated, aligned using MUSCLE as amino acid sequences, back-translated to codon-respecting nucleotide alignments, and the alignment analyzed with SYNPLOT2 as above. In contrast to the full-genome alignments, all alignment gaps were retained instead of mapping to a specific reference sequence coordinate system. The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/661579 doi: bioRxiv preprint Calculation of the pI and molecular mass of XP peptides and other sequence processing were performed with pepstats and other programs from the EMBOSS package 33 . TM domains were predicted with Phobius (EMBL-EBI) 34 . The XP proteins of HAstVs 1-8 were additionally queried with TMHMM (http://www.cbs.dtu.dk/services/TMHMM/; weak TM prediction for HAstV3, no TM predicted for other HAstVs) and SOSUI (http://harrier.nagahama-ibio.ac.jp/sosui/sosui_submit.html; no TMs predicted). XP secondary structures were predicted with RaptorX 35 . To search for potential homologues of XP, XPs from Fig. S6D, Fig. S7 and The infectious clone of HAstV1 (pAVIC1, GenBank accession number L23513.1) was described previously 16 . The reverse genetics procedure was compiled from several previously published approaches 16, 17 . Initial virus was recovered from T7 transcribed RNA using reverse transfection of Huh7.5.1 cells by Lipofectamine® 2000 (Invitrogen) (for virus rescue, see Fig. 3) or electroporation of BSR cells in PBS at 800 V and 25 µF using a Bio-Rad Gene Pulser Xcell TM electroporation system (see Fig. 4 for an analysis of virus RNA in cells versus released particles). For virus passaging, the collected supernatant was treated with 10 µg/ml trypsin (Type IX, Sigma, #T0303) for 30 min at 37 °C, diluted 5 times with serum-free media, and used for infection of Caco2 cells. After 3 h of incubation, the virus containing media was replaced with serum free media containing 0.6 µg/ml trypsin, and cells were incubated for 48-72 h until appearance of CPE. After 3 freeze-thaw cycles, the viral stocks were aliquoted, frozen and stored at −70 °C. Viral stocks were titrated as previously described 35 , but using infrared detection readout, combined with automated LI-COR software-based quantification.