Flexibility and modulation of translation initiation in enterovirus genomes

  1. Rhian L. O’Connor
  2. Georgia M. Cook
  3. Jacqueline Hankinson
  4. Ksenia Fominykh
  5. Samantha H. Cheng
  6. Daniel A. Nash
  7. Aurélie Cenier
  8. Komal M. Nayak
  9. Stephen C. Graham
  10. Janet E. Deane
  11. Matthias Zilbauer
  12. Andrew E. Firth
  13. Valeria Lulla

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Abstract

Enteroviruses comprise a large group of mammalian pathogens that often utilize two open reading frames (ORFs) to encode their proteins: the upstream protein (UP) and the main polyprotein. In some enteroviruses, in addition to the canonical upstream AUG (uAUG), there is another AUG that may represent an alternative upstream initiation site. An analysis of enterovirus sequences containing additional upstream AUGs identified several clusters, including strains of pathogenic Enterovirus alphacoxsackie and E. coxsackiepol . Using ribosome profiling on coxsackievirus CVA-13 ( E. coxsackiepol ), we demonstrate that both upstream AUG codons can be used for translation initiation in infected cells. Moreover, we confirm translation from both upstream AUGs using a reporter system. Mutating the additional upstream AUG in the context of CVA-13 did not result in phenotypic changes in immortalized cell lines. However, the wild-type virus outcompeted this mutant in human intestinal organoids and differentiated neuronal systems, representing an advantage in physiologically relevant infection sites. Mutation of the stop codon of the shorter upstream ORF led to dysregulated translation of the other ORFs in the reporter system, suggesting a potential role for the additional uORF in modulating the expression level of the other ORFs. These findings demonstrate the remarkable plasticity of enterovirus IRES-mediated initiation and the competitive advantage of double-upstream-AUG-containing viruses in terminally differentiated intestinal organoids and neuronal systems.

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    I thank the Referees for their...

    Referee #1

    1. The authors should provide more information when...

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    1. Figure 6: Why has only...

    Response: We expanded the comparison

    Minor comments:

    1. The text contains several...

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    Referee #2

    Evidence, reproducibility and clarity

    Enterovirus genomes contain an AUG triplet at the 3'-border of the IRES, often far upstream of the initiation codon for the principal ORF that encodes the viral polyprotein. Prior in vitro and in vivo studies have shown that this upstream AUG triplet (uAUG) initiates translation of a short polypeptide ("UP") encoded by an upstream ORF (uORF) that promotes viral infection in gut epithelial cells (Refs. 5, 6). In the present thorough and rigorously controlled study, O'Connor et al. extend these observations, thereby providing further insights into the regulatory and coding potential of translation of in alternate reading frames in viral mRNAs.

    They first undertook detailed analyses of almost 10000 enterovirus genomic sequences and determined that one third contained additional AUG triplets in the vicinity of the uAUG, collectively designated upstream uAUGs (uuAUGs), that could potentially initiate translation of uuORFs that are mostly very short but that in a few instances encode UP-related polypeptides.

    Systematic studies involving (a) ribosomal profiling and (b) the use of a dual luciferase reporter system showed that uuAUG triplets are recognized by ribosomes in infected cells and are functional albeit inefficient initiation codons. The uuAUG triplet in the enterovirus CVA-13 (Flores strain) initiates translation of an 8aa-long non-UP-like peptide, and the functional importance of this uuAUG was assayed by substituting it by a GUG triplet to downregulate uuORF translation. This mutation had no effect of infection in HeLa cells, but the uuAUG-containing (wt) virus had a competitive advantage over the mutant in mixed mutant/wt infections in terminally differentiated neuronal cells and in differentiated human intestinal organoids. This differential effect was similar to the previously reported competitive advantage conferred by UP expression during enterovirus infection in differentiated cells (Ref. 5). The function of non-UP-like proteins initiating at uuAUG codons remains unknown. However, elimination of stop codons that modify their length of uORFs modulated upstream ORF expression, although the mechanism responsible for this effect remains unknown. These results suggest that the interplay between initiation, termination and recycling steps on the 5'UTR of enteroviruses has the potential to affect viral pathogenicity.

    The data in the manuscript are strong, well controlled and validated. Elements of the manuscript could be presented more clearly.

    Minor comments

    1. Line 56. Domain 1 is a cloverleaf i.e. not just a stemloop.
    2. Fig. 4A, 5B, 8C. It would be informative to add an additional 5'-terminal nucleotide to the structure of the SL-VI region to show the Kozak context of the uuAUG codon.
    3. Figs. 8C, 8E, 8F. It might be more reader-friendly to replace structural models of sections of enterovirus 5'UTRs by a schematic representations to show uuORFs, uORFs, ppORFs etc and how altered stop codons affect their overlap. The corresponding section of the manuscript could also be presented in a more straightforward manner.
    4. Lines 473-4. This statement is incorrect, because eIF4G is required for IRES-dependent initiation. 2A-mediated cleavage of eIF4G does not abrogate IRES function because it splits off the non-essential N-terminal (eIF4E-binding) region from the critical C-terminal region that binds directly to enterovirus IRESs and recruits eIF4A (Ref. 7; PMID: 19470487).
    5. Ref. 27 is annotated incorrectly

    Significance

    The study reinforces and extends the authors' previous conclusions (Ref. 5, 13, 28, 31) that the genomes of positive-sense RNA viruses can and do have coding properties that are more complex than simply encoding a single open reading frame. Careful examination of a large panel of enterovirus genomes revealed a great diversity in coding potential, and the authors are right to suggest that further correlation of coding potential (particularly alternate ORFs/alternate reading frames) with pathogenic phenotypes is merited, particularly for variants of a single virus.

    This study also provides insights into the influence of alternative upstream open reading frames on viral fitness using strong experimental models (viral infection of differentiated cells and organoids in addition to HeLa cells), and appropriate methods (e.g. an innovative competition assay to compare the competitive advantage of co-infecting variants of a virus, sophisticated reporter assays). Although the mechanistic basis for the influence of uuORFs on enterovirus infection of cells remains to be fully elucidated, these studies indicate that the topic strongly merits further study. In consequence, this report will be of interest both to molecular virologists and to scientists with an interest in gene expression mechanisms.

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    Referee #1

    Evidence, reproducibility and clarity

    This study provides a detailed analysis of upstream and "upstream upstream" open reading frames in enteroviruses from the Cosackiepol and Alphacoxsackie species. The work includes a comprehensive bioinformatic analysis of uORF and uuORF diversity and conservation across the EVs, along with characterization of these ORFs by Riboseq and reporter assays. The authors also include a characterization of the uORFs through the mutation of these ORFs in both cell lines and iPSC derived cells.

    The manuscript is detailed, the experiments rigorous, and their description clear. The work provides a number of orthogonal experiments to support the claims of the study.

    Significance

    General assessment: This work is of high quality, and an important addition to the literature on uORFs, though it doesn't provide much mechanistic insight into the function of these ORFs. It falls short in pushing our understanding of uORF function forward.

    • I wonder if the authors can expand their studies to address the potential mechanisms by which these ORFs function, whether through their translation or translation products.
    • Have the authors considered exploring the possible functions similar to cellular transcripts, e.g. what they reference in the discussion regarding ATF4, by modulating stress responses and assessing expression of uORF and ppORF? These studies would greatly enhance the additional insights the manuscript provides.
    • A more comprehensive accounting of the ORF diversity across the EV's would be a valuable analysis. Are there ORFs in the negative strand (as have been characterized in influenza), or elsewhere in the positive strand, that may have functions?

    In Fig. 7A: "iPCS" -> "iPSC"

    Advance: This work builds on the authors' previous characterization of the uORFs of related EV's. It provides further support consistent with their previous findings that these uORFs are of importance of these regions in the replication of the virus, especially in differentiated target tissues, suggesting they contribute to the pathogenesis of the virus as part of the known important role the IRES regions are known to play. How the translation, or the products of translation, function to confer this phenotype remains elusive.

    Audience: This will be of interest to virologists working on cryptic translational elements in viruses, which are found in many viruses, and sure to be discovered in more as we begin to appreciate their important role, however the findings are not likely to be especially relevant to very broad audience.

  4. Version published to 10.1101/2025.03.24.645098v1 on bioRxiv