An Upstream Protein-Coding Region in Enteroviruses Modulates Virus Infection in Gut Epithelial Cells

Enteroviruses comprise a large group of mammalian pathogens that includes poliovirus. Pathology in humans ranges from sub-clinical to acute flaccid paralysis, myocarditis and meningitis. Until now, all the enteroviral proteins were thought to derive from proteolytic processing of a polyprotein encoded in a single open reading frame (ORF). We report that many enterovirus genomes also harbor an upstream ORF (uORF) that is subject to strong purifying selection. Using echovirus 7 and poliovirus 1, we confirmed expression of uORF protein (UP) in infected cells. Using ribosome profiling (a technique for global footprinting of translating ribosomes), we also demonstrated translation of the uORF in representative members of the predominant human enterovirus species, namely Enterovirus A, B, and C. In differentiated human intestinal organoids, UP-knockout echoviruses are attenuated compared to wild-type virus at late stages of infection where membrane-associated UP facilitates virus release. Thus we have identified a previously unknown enterovirus protein that facilitates virus growth in gut epithelial cells – the site of initial viral invasion into susceptible hosts. These findings overturn the 50-year-old dogma that enteroviruses use a single-polyprotein gene expression strategy, and have important implications for understanding enterovirus pathogenesis.