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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 of the enteroviral proteins were thought to derive from the proteolytic processing of a polyprotein encoded in a single open reading frame. Here we report that many enterovirus genomes also harbour an upstream open reading frame (uORF) that is subject to strong purifying selection. Using echovirus 7 and poliovirus 1, we confirmed the expression of uORF protein in infected cells. Through ribosome profiling (a technique for the 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, uORF protein-knockout echoviruses are attenuated compared to the wild-type at late stages of infection where membrane-associated uORF protein 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 the understanding of enterovirus pathogenesis.

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Fig. 1: Comparative genomic analysis of the Enterovirus genus.
Fig. 2: Analysis of WT and mutant EV7 viruses.
Fig. 3: Timecourse of UP expression in EV7-infected cells.
Fig. 4: Translation of the uORF in PV1 and EV-A71.
Fig. 5: Analysis of EV7 infection in differentiated human intestinal organoids.
Fig. 6: Membrane association of UP and temporal analysis of uORF translation.

Data availability

The sequencing data reported in this paper have been deposited in ArrayExpress (http://www.ebi.ac.uk/arrayexpress) under accession number E-MTAB-6180.

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Acknowledgements

We thank the Cambridge NIHR BRC Cell Phenotyping Hub for assistance with confocal microscopy. We thank T. Sweeney, I. Brierley and E. Jan for stimulating discussions. This work was supported by Wellcome Trust grant no. 106207 and European Research Council grant no. 646891 to A.E.F and Wellcome Trust grant nos 097997/Z/11/Z and 207498/Z/17/Z to I.G.

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Contributions

A.E.F. and V.L. conceived the project. V.L. performed the experiments. M.Z., K.M.N., M.H., Y.C. and I.G. established the organoid system, prepared and maintained the organoids and assisted with the organoid experiments. L.S. and N.J.S. established the poliovirus system and helped prepare poliovirus samples. N.I. advised and assisted with the Ribo-Seq experiments. A.E.F. performed the comparative genomic analyses. A.M.D. analysed the Ribo-Seq data. V.L. and A.E.F. wrote the manuscript. All authors edited the manuscript.

Corresponding authors

Correspondence to Valeria Lulla or Andrew E. Firth.

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Supplementary information

Supplementary Information

Supplementary Figures 1–23, Supplementary Tables 1–4 and Supplementary References.

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Supplementary Table 3

Statistical analysis of data from organoid-derived samples.

Supplementary Table 4

Statistical analysis of dual luciferase data.

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Lulla, V., Dinan, A.M., Hosmillo, M. et al. An upstream protein-coding region in enteroviruses modulates virus infection in gut epithelial cells. Nat Microbiol 4, 280–292 (2019). https://doi.org/10.1038/s41564-018-0297-1

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