Experimental evolution of Tobacco etch virus carrying eGFP

Viruses have evolved highly streamlined genomes and a variety of mechanisms to compress them, suggesting that genome size is under strong selection. Horizontal gene transfer has, on the other hand, played an important role in virus evolution. However, evolution cannot integrate initially nonfunctional sequences into the viral genome if they are rapidly purged by selection. Here we explore the process of pseudogenization in virusgenomes by means of experimental evolution. As a model system, we consider a plant RNA virus expressing a nontoxic heterologous gene, whose expression has been engineered to minimally disrupt the viral genome. We first looked for conditions under which the heterologous gene would be maintained in the genome. We then consider whether the deletion of the heterologous gene was adaptive and what virus characteristics it modifies. Finally, we consider whether there are conditions that fulfill two requirements: (i) the heterologous gene has a high probability of being maintained in the evolved virus population and (ii) there isevidence that the virus population is under positive selection and experiences increases in fitness. We found that that passage duration is crucial to the outcome of plant virus evolution and that adaptive and convergent evolution can both be observed in real time during long passages. Moreover, the results suggest a demographically determined sweet spot might exist, where heterologous insertions are not immediately lost while evolution can act to integrate them into the viral genome.