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

Valeria Lulla; Andrew E. Firth

To further investigate the function of XP, we studied its intracellular localization. To make the small XP protein more stable and enable visualization in transfected cells, we fused it either Nor C-terminally with mCherry in the context of a mammalian expression vector. The diffuse cytoplasmic localization of mCherry was drastically affected in both fusions, where it was relocalized to plasma and perinuclear membranes (Fig. 5A ). This strongly suggests the presence of a membrane-interacting domain in XP, which was not predicted by TM domain prediction software ( Fig. S8 ; see Methods). Since ORFX is completely embedded within ORF2, it may have greatly decreased evolutionary flexibility, perhaps resulting in the evolution of a non-. 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 It is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the .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 canonical TM region. Some of the putative XP proteins encoded by other astroviruses do in fact have predicted TM regions (Fig. S9 ). We also confirmed membrane and nuclear association of the XP-fused mCherry proteins by subcellular fractionation of transfected HeLa cells and subsequent analysis of the fractions (Fig. 5B ). To investigate the potential topology of XP within the plasma membrane, we probed live electroporated HeLa cells with anti-mCherry antibody, fixed the cells and analyzed them by confocal microscopy. We observed punctate staining across the plasma membrane when mCherry was fused to the N-terminus but not when it was fused to the C-terminus, suggesting an extracellular N-terminal topology and potential multimerization ( Fig. 5C-D) .