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

Valeria Lulla; Andrew E. Firth

Although these leucines are not entirely conserved between the eight HAstV serotypes (Fig. 1F ), each XP was still predicted to contain an amphipathic α-helix in this region 22 . To test for viroporin-like activity, we utilized a previously described Sindbis virus replicon (SINV repC) 23 to overexpress XP or control proteins in mammalian cells. This replicon has been demonstrated to be an extremely useful tool for investigating viroporins from different RNA viruses via induced permeabilization of the plasma membrane in BHK cells to the translation inhibitor hygromycin B (HB). BSR cells (a clone of BHK cells) were electroporated with in vitro transcribed SINV repC RNAs, and new protein synthesis was labelled with Lazidohomoalanine (AHA) in the presence or absence of HB, followed by lysis and click chemistry-based on-gel detection 24 . Expression of XP or Strep-tagged enterovirus 2B (a wellcharacterized viroporin 25 ) led to almost total inhibition of protein synthesis (Fig. 6C-D) indicating that both proteins induced pronounced cell permeabilization to HB.