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

Abstract

ABSTRACTHuman astroviruses are small nonenveloped viruses with positive-sense single-stranded RNA genomes that contain three main open reading frames: ORF1a, ORF1b and ORF2. Astroviruses cause acute gastroenteritis in children worldwide and have been associated with encephalitis and meningitis in immunocompromised individuals. Through comparative genomic analysis of >400 astrovirus sequences, we identified a conserved “ORFX” overlapping the capsid-encoding ORF2 in genogroup I, III and IV astroviruses. ORFX appears to be subject to purifying selection, consistent with it encoding a functional protein product, termed XP. Using ribosome profiling of cells infected with human astrovirus 1, we confirm initiation at the ORFX AUG. XP-knockout astroviruses are strongly attenuated and after passaging can partly restore viral titer via pseudo-reversions, thus demonstrating that XP plays an important role in virus growth. To further investigate XP, we developed an astrovirus replicon system. We demonstrate that XP has only minor effects on RNA replication and structural protein production. Instead, XP associates with the plasma membrane with an extracellular N-terminus topology and promotes efficient virus release. Using two different assays, we show that expression of human or related astrovirus XPs leads to cell permeabilization, suggesting a viroporin-like activity. The discovery of XP advances our knowledge of these important human viruses and opens a new direction of research into astrovirus replication and pathogenesis.