Experimental evolution of alternative gene orders in Tobacco etch virus

In this study, we used experimental evolution to better understand the dynamics of genome architecture evolution and gene-order conservation. Why has gene order been conserved within positive-strand RNA virus orders and families? Are there accessible evolutionary trajectories to alternative orders, or is a lack of accessible trajectories an important impediment in present-day viruses? Here we use the two viable reordered TEV variants to address these issues. We first explore whether there are accessible evolutionary trajectories that result in these two viable reordered viruses. To consider whether such natural trajectories exist, genomes containing duplications of the NIb gene were generated, tested for viability, and evolved in plants. We consistently found that the NIb gene at the alternative position was rapidly lost owing to the occurrence of large genomic deletions. Finally, we explored the evolutionary potential of viruses with reordered genomes by evolving viruses with a single NIb gene in an alternative position. We then measured virus accumulation and fitness and used next-generation sequencing to identify genomic changes. Although we found evidence for adaptation of these reordered viruses in terms of increasing virus accumulation, they were still less fit than the wild-type virus. This study therefore revealed multiple barriers to the evolution of alternative gene orders in TEV.