An upstream protein-coding region in enteroviruses modulates virus infection in gut epithelial cells

Abstract

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.

A case for a reverse-frame coding sequence in a group of positive-sense RNA viruses

“…To fully understand the molecular biology of viruses, it is crucially important to know their coding capacity. In recent years, a number of "hidden" coding open-reading frames (ORFs) have been discovered in the genomes of various (+)ssRNA viruses (Chung et al 2008;Loughran et al 2011;Fang et al 2012;Firth 2014;Smirnova et al 2015;Napthine et al 2017;Lulla et al 2019). Such "hidden" genes tend to be very short and/or to overlap previously known coding ORFs, explaining why they have escaped previous detection prior to the application of sensitive comparative genomic methods.…”

OUP accepted manuscript

“…To fully understand the molecular biology of viruses, it is crucially important to know their coding capacity. In recent years, a number of 'hidden' protein-coding open-reading frames (ORFs) have been discovered in the genomes of various (þ)ssRNA viruses (Chung et al 2008;Loughran, Firth, and Atkins 2011;Fang et al 2012;Firth 2014;Smirnova et al 2015;Napthine et al 2017;Lulla et al 2019). Such genes tend to be very short and/or to overlap previously known coding ORFs, explaining why they have escaped detection prior to the application of sensitive comparative genomic methods.…”

Upstream translation initiation expands the coding capacity of segmented negative-strand RNA viruses

“…Indeed, it is important to note that the viral UTRs we examined in this study do not contain AUG codons, meaning that translation initiation in these regions is presumably from non-AUG codons, a common if low-level effect in both host and viral genes [4,39,40]. Our data therefore add to a growing number of reports of upstream translation initiating on virally-encoded sequences, in some cases with clear regulatory effects [4,41–46]. On the basis of our results we would argue that upstream translation of viral genes should not be discounted, even in the absence of virally-encoded AUGs.…”