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

Valeria Lulla; Andrew E. Firth

All virus genome mutations (Fig. S11) were introduced using site-directed mutagenesis of pAVIC1 and confirmed by sequencing. The resulting plasmids were linearized with XhoI prior to T7 RNA transcription. To create the HAstV1 replicon system, the pAVIC1 infectious clone was left intact up to the end of ORFX, then followed by a foot and mouth disease virus 2A sequence and a Renilla luciferase (RLuc) sequence fused in either the ORF2 (pO2RL) or ORFX (pXRL) reading frame, followed by the last 624 nt of the virus genome and a poly-A tail (Fig. 4A ). All mutations were introduced from the corresponding pAVIC1 mutants using available restriction sites and all constructs were confirmed by sequencing. The resulting plasmids were linearized with XhoI prior to T7 RNA transcription. Each XP coding sequence was inserted into the pOPT plasmid with a C-or N-terminal Streptag as indicated (Fig. 6I) . For the permeabilization assay, Sindbis virus derived replicons (SINV . CC-BY 4.0 International license author/funder. It is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/661579 doi: bioRxiv preprint repC) expressing XP, mCherry or Strep-tagged enterovirus (echovirus 7) 2B were created exactly as previously described 23 .