RNA virus attenuation by codon pair deoptimisation is an artefact of increases in CpG/UpA dinucleotide frequencies.

Fiona Tulloch,Peter Simmonds,David J Evans,Martin D Ryan,Nicky J Atkinson

doi:10.7554/elife.04531

Fiona Tulloch, Peter Simmonds + Show 3 more

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https://doi.org/10.7554/elife.04531

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Abstract

Mutating RNA virus genomes to alter codon pair (CP) frequencies and reduce translation efficiency has been advocated as a method to generate safe, attenuated virus vaccines. However, selection for disfavoured CPs leads to unintended increases in CpG and UpA dinucleotide frequencies that also attenuate replication. We designed and phenotypically characterised mutants of the picornavirus, echovirus 7, in which these parameters were independently varied to determine which most influenced virus replication. CpG and UpA dinucleotide frequencies primarily influenced virus replication ability while no fitness differences were observed between mutants with different CP usage where dinucleotide frequencies were kept constant. Contrastingly, translation efficiency was unaffected by either CP usage or dinucleotide frequencies. This mechanistic insight is critical for future rational design of live virus vaccines and their safety evaluation; attenuation is mediated through enhanced innate immune responses to viruses with elevated CpG/UpA dinucleotide frequencies rather the viruses themselves being intrinsically defective.

Highlights

Protein encoding regions of all organisms, eukaryotic, bacterial and viral, are subject to a number of functional constraints in addition to coding capacity, many of which contribute to regulation of translation
This study sought to disentangle effects of codon pair usage and nucleotide frequencies in a re-examination of their effects on the replication of an RNA virus, echovirus 7 (E7)
Studies have documented effects of codon pair (CP) de-optimisation on virus attenuation without reference to effects of this procedure on dinucleotide frequencies (Coleman et al, 2008; Mueller et al, 2010; Martrus et al, 2013; Yang et al, 2013; Le Nouen et al, 2014; Ni et al, 2014). While frequencies of both of these dinucleotides are suppressed in most classes of mammalian RNA viruses (Rima and McFerran, 1997), all sequences modified to select disfavoured CPs (Coleman et al, 2008; Mueller et al, 2010; Martrus et al, 2013; Yang et al, 2013; Ni et al, 2014) consistently elevated frequencies of CpG and UpA dinucleotides to levels to 2.5–threefold higher levels than the original native sequences (Table 1)

Summary

Introduction

Protein encoding regions of all organisms, eukaryotic, bacterial and viral, are subject to a number of functional constraints in addition to coding capacity, many of which contribute to regulation of translation. Because of its potential effect on gene expression, altering CP frequencies towards those that are disfavoured in their hosts has recently been advocated as a novel strategy to reduce RNA virus replication (Coleman et al, 2008; Wimmer et al, 2009; Mueller et al, 2010; Martrus et al, 2013; Yang et al, 2013; Le Nouen et al, 2014; Ni et al, 2014) This procedure potentially provides the means to produce a new generation of safer, non-reverting, live attenuated vaccines. Novel strategies by which synonymous coding changes are introduced to modify codon usage (Mueller et al, 2010) (Martrus et al, 2013; Yang et al, 2013; Le Nouen et al, 2014; Ni et al, 2014) have the advantage

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