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

Valeria Lulla; Andrew E. Firth

Another widely used assay to assess the ability of proteins to permeabilize cellular membranes is based on impaired growth of Escherichia coli upon induced overexpression of a membranepermeabilizing protein 26, 27 . Consistent with the results observed in the mammalian system, . 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 It is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the .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 induced expression of XP or the enteroviral viroporin 2B in E. coli resulted in cytotoxicity and impaired growth (Fig. 6E ). There are two features that are generally associated with viroporin membrane permeabilizing activity: (i) an amphipathic α-helix that facilitates oligomerization to form the TM pore, and (ii) adjacent positively charged residues that anchor the viroporin in the membrane 27, 28 . To test for these features in XP, two conserved RR motifs present in all eight HAstV serotypes (Fig. 1F ) were mutated to alanines (Fig. 6F) . Interestingly, only the mutation of the very C-terminal arginines resulted in loss of XP activity in this system (Fig. 6G ). Mutation of the hydrophobic leucine stretch (L95, L98, L102, L105 and L109, Fig. 6A ) to serines and threonines (Fig. 6F ) also resulted in complete loss of XP activity (Fig. 6G) . Thus, the C-terminal domain of XP is involved in membrane permeabilization and harbours key features of a viroporin.