Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

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 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.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

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 ( under accession number E-MTAB-6180.


  1. 1.

    Suresh, S., Forgie, S. & Robinson, J. Non-polio Enterovirus detection with acute flaccid paralysis: a systematic review. J. Med. Virol. 90, 3–7 (2017).

    Article  Google Scholar 

  2. 2.

    Bedard, K. M. & Semler, B. L. Regulation of picornavirus gene expression. Microbes Infect. 6, 702–713 (2004).

    CAS  Article  Google Scholar 

  3. 3.

    Sweeney, T. R., Abaeva, I. S., Pestova, T. V. & Hellen, C. U. T. The mechanism of translation initiation on Type 1 picornavirus IRESs. EMBO J. 33, 76–92 (2014).

    CAS  Article  Google Scholar 

  4. 4.

    Pelletier, J., Flynn, M. E., Kaplan, G., Racaniello, V. & Sonenberg, N. Mutational analysis of upstream AUG codons of poliovirus RNA. J. Virol. 62, 4486–4492 (1988).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. 5.

    Hellen, C. U., Pestova, T. V. & Wimmer, E. Effect of mutations downstream of the internal ribosome entry site on initiation of poliovirus protein synthesis. J. Virol. 68, 6312–6322 (1994).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. 6.

    Kaminski, A., Poyry, T. A. A., Skene, P. J. & Jackson, R. J. Mechanism of Initiation site selection promoted by the human rhinovirus 2 internal ribosome entry site. J. Virol. 84, 6578–6589 (2010).

    CAS  Article  Google Scholar 

  7. 7.

    Pestova, T. V., Hellen, C. U. T. & Wimmer, E. A conserved AUG triplet in the 5′ nontranslated region of poliovirus can function as an initiation codon in vitro and in vivo. Virology 204, 729–737 (1994).

    CAS  Article  Google Scholar 

  8. 8.

    Meerovitch, K., Nicholson, R. & Sonenberg, N. In vitro mutational analysis of cis-acting RNA translational elements within the poliovirus type 2 5′ untranslated region. J. Virol. 65, 5895–5901 (1991).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. 9.

    Firth, A. & Brown, C. Detecting overlapping coding sequences in virus genomes. BMC Bioinform. 7, 75 (2006).

    Article  Google Scholar 

  10. 10.

    Yang, Z. PAML 4: phylogenetic analysis by maximum likelihood. Mol. Biol. Evol. 24, 1586–1591 (2007).

    CAS  Article  Google Scholar 

  11. 11.

    Irigoyen, N. et al. High-resolution analysis of coronavirus gene expression by RNA sequencing and ribosome profiling. PLoS Pathog. 12, e1005473 (2016).

    Article  Google Scholar 

  12. 12.

    Slobodskaya, O. R. et al. Poliovirus neurovirulence correlates with the presence of a cryptic AUG upstream of the initiator codon. Virology 221, 141–150 (1996).

    CAS  Article  Google Scholar 

  13. 13.

    Drummond, C. G. et al. Enteroviruses infect human enteroids and induce antiviral signaling in a cell lineage-specific manner. Proc. Natl Acad. Sci. USA 114, 1672–1677 (2017).

    CAS  Article  Google Scholar 

  14. 14.

    Ettayebi, K. et al. Replication of human noroviruses in stem cell-derived human enteroids. Science 353, 1387–1393 (2016).

    Article  Google Scholar 

  15. 15.

    Sato, T. et al. Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and barrett’s epithelium. Gastroenterology 141, 1762–1772 (2011).

    CAS  Article  Google Scholar 

  16. 16.

    Kraiczy, J. et al. DNA methylation defines regional identity of human intestinal epithelial organoids and undergoes dynamic changes during development. Gut (2017).

  17. 17.

    Ward, T. et al. Decay-accelerating factor CD55 is identified as the receptor for echovirus 7 using CELICS, a rapid immuno-focal cloning method. EMBO J. 13, 5070–5074 (1994).

    CAS  Article  Google Scholar 

  18. 18.

    Racaniello, V. R. One hundred years of poliovirus pathogenesis. Virology 344, 9–16 (2006).

    CAS  Article  Google Scholar 

  19. 19.

    Kitamura, N. et al. Primary structure, gene organization and polypeptide expression of poliovirus RNA. Nature 291, 547–553 (1981).

    CAS  Article  Google Scholar 

  20. 20.

    Racaniello, V. R. & Baltimore, D. Molecular cloning of poliovirus cDNA and determination of the complete nucleotide sequence of the viral genome. Proc. Natl Acad. Sci. USA 78, 4887–4891 (1981).

    CAS  Article  Google Scholar 

  21. 21.

    Jacobson, M. F. & Baltimore, D. Polypeptide cleavages in the formation of poliovirus proteins. Proc. Natl Acad. Sci. USA 61, 77–84 (1968).

    CAS  Article  Google Scholar 

  22. 22.

    Royston, L. & Tapparel, C. Rhinoviruses and respiratory enteroviruses: not as simple as ABC. Viruses 8, 16 (2016).

    Article  Google Scholar 

  23. 23.

    Ohlmann, T. & Jackson, R. J. The properties of chimeric picornavirus IRESes show that discrimination between internal translation initiation sites is influenced by the identity of the IRES and not just the context of the AUG codon. RNA 5, 764–778 (1999).

    CAS  Article  Google Scholar 

  24. 24.

    Andreev, D. E. et al. Glycyl-tRNA synthetase specifically binds to the poliovirus IRES to activate translation initiation. Nucleic Acids Res. 40, 5602–5614 (2012).

    CAS  Article  Google Scholar 

  25. 25.

    Feng, Z. et al. A pathogenic picornavirus acquires an envelope by hijacking cellular membranes. Nature 496, 367–371 (2013).

    CAS  Article  Google Scholar 

  26. 26.

    Chen, Y.-H. et al. Phosphatidylserine vesicles enable efficient en bloc transmission of enteroviruses. Cell 160, 619–630 (2015).

    CAS  Article  Google Scholar 

  27. 27.

    Richards, A. L. & Jackson, W. T. Behind closed membranes: the secret lives of picornaviruses? PLoS Pathog. 9, e1003262 (2013).

    CAS  Article  Google Scholar 

  28. 28.

    Sin, J., McIntyre, L., Stotland, A., Feuer, R. & Gottlieb, R. A. Coxsackievirus B escapes the infected cell in ejected mitophagosomes. J. Virol. 91, e01347-17 (2017).

    Article  Google Scholar 

  29. 29.

    Cornell, C. T., Perera, R., Brunner, J. E. & Semler, B. L. Strand-specific RNA synthesis determinants in the RNA-dependent RNA polymerase of poliovirus. J. Virol. 78, 4397–4407 (2004).

    CAS  Article  Google Scholar 

  30. 30.

    Lulla, V. et al. Assembly of replication-incompetent African horse sickness virus particles: rational design of vaccines for all serotypes. J. Virol. 90, 7405–7414 (2016).

    CAS  Article  Google Scholar 

  31. 31.

    Loughran, G., Howard, M. T., Firth, A. E. & Atkins, J. F. Avoidance of reporter assay distortions from fused dual reporters. RNA 23, 1285–1289 (2017).

    CAS  Article  Google Scholar 

  32. 32.

    Vogt, D. A. & Ott, M. Membrane flotation assay. Bio-protocol 5, e1435 (2015).

    Article  Google Scholar 

  33. 33.

    Goodfellow, I. G. et al. Inhibition of coxsackie B virus infection by soluble forms of its receptors: binding affinities, altered particle formation, and competition with cellular receptors. J. Virol. 79, 12016–12024 (2005).

    CAS  Article  Google Scholar 

  34. 34.

    Ingolia, N. T., Brar, G. A., Rouskin, S., McGeachy, A. M. & Weissman, J. S. The ribosome profiling strategy for monitoring translation in vivo by deep sequencing of ribosome-protected mRNA fragments. Nat. Protoc. 7, 1534–1550 (2012).

    CAS  Article  Google Scholar 

  35. 35.

    Langmead, B., Trapnell, C., Pop, M. & Salzberg, S. L. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 10, R25 (2009).

    Article  Google Scholar 

  36. 36.

    Simmonds, P. & Welch, J. Frequency and dynamics of recombination within different species of human enteroviruses. J. Virol. 80, 483–493 (2006).

    CAS  Article  Google Scholar 

  37. 37.

    Edgar, R. C. MUSCLE: a multiple sequence alignment method with reduced time and space complexity. BMC Bioinform. 5, 113 (2004).

    Article  Google Scholar 

  38. 38.

    Castresana, J. Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis. Mol. Biol. Evol. 17, 540–552 (2000).

    CAS  Article  Google Scholar 

  39. 39.

    Ronquist, F. et al. MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Syst. Biol. 61, 539–542 (2012).

    Article  Google Scholar 

  40. 40.

    Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. Basic local alignment search tool. J. Mol. Biol. 215, 403–410 (1990).

    CAS  Article  Google Scholar 

  41. 41.

    Rice, P., Longden, I. & Bleasby, A. EMBOSS: the European molecular biology open software suite. Trends Genet. 16, 276–277 (2000).

    CAS  Article  Google Scholar 

  42. 42.

    Guindon, S. & Gascuel, O. A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst. Biol. 52, 696–704 (2003).

    Article  Google Scholar 

  43. 43.

    Stocsits, R. R., Hofacker, I. L., Fried, C. & Stadler, P. F. Multiple sequence alignments of partially coding nucleic acid sequences. BMC Bioinform. 6, 160 (2005).

    Article  Google Scholar 

  44. 44.

    Firth, A. E. Mapping overlapping functional elements embedded within the protein-coding regions of RNA viruses. Nucleic Acids Res. 42, 12425–12439 (2014).

    CAS  Article  Google Scholar 

  45. 45.

    McWilliam, H. et al. Analysis tool web services from the EMBL-EBI. Nucleic Acids Res. 41, W597–W600 (2013).

    Article  Google Scholar 

Download references


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.

Author information




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.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

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

Reporting Summary

Supplementary Table 3

Statistical analysis of data from organoid-derived samples.

Supplementary Table 4

Statistical analysis of dual luciferase data.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

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).

Download citation

Further reading


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing