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

Valeria Lulla; Andrew E. Firth

To compare phasing in the region of ORF2 overlapped by ORFX with the region of ORF2 not overlapped by ORFX (Fig. 2C) we counted RPFs whose 5′ end (+12 nt offset) mapped within the regions 4388-4693 or 4727-6673, respectively. We then compared the fraction of RPFs in The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/661579 doi: bioRxiv preprint media (VP-SFM, Gibco) containing 0.6 µg/ml trypsin. At 48 hpe media samples were collected and centrifuged at 9,600 g for 5 min. The released virus samples were titrated on Caco2 cells as described above. For RT-qPCR analysis, a 150 µl aliquot of each sample was mixed with 4 × 10 6 Lysates from the above mentioned assays were analyzed by SDS-PAGE, using standard 12% SDS-PAGE to resolve mCherry and its XP-fusion variants, and precast Novex™ 10-20% tricine protein gels (Thermo Fisher) to resolve XPs and enterovirus 2B. Proteins were then transferred to 0.2 µm nitrocellulose membranes and blocked with 4% Marvel milk powder in phosphate-buffered saline (PBS). Immunoblotting of mCherry was performed using anti-mCherry antibody (Abcam, ab167453). A custom rabbit polyclonal antibody raised against XP peptide SNSGNRVSQDQNLQ (GenScript; only able to detect strongly overexpressed XP) and an anti-Strep mouse antibody (Abcam, ab184224) were used for detecting HAstV1 XP and Strep-tagged proteins, respectively. The following antibodies were used for cellular targets: