On the stability of sequences inserted into viral genomes

Willemsen, Anouk; Zwart, Mark P

In a previous study we reported on experimental evolution of pseudogenization in virus genomes using Tobacco etch virus (TEV) expressing eGFP (Zwart et al. 2014 ), a gene known to be toxic in many expression systems. In this case eGFP can be considered a non-functional sequence, as it does not add any function to the viral genome. We showed that eGFP has a high fitness cost in TEV, and the loss of eGFP depended on the passage length, where longer passages led to a faster and assured loss. Similarly, prolonged propagation of TEV and plum pox potyvirus expressing GUS (Dolja, McBride, and Carrington 1992; Dolja et al. 1993; Guo, Ló pez-Moya, and García 1998) , and TMV expressing GFP (Rabindran and Dawson 2001) , led to the appearance of spontaneous deletion variants. Due to the increase in genome size, viruses that carry an insert are unlikely to be as fit as the parental (ancestral) virus, even if they accumulate initially to similar levels. The TEV-eGFP genomes that had lost the insert had a within-host competitive fitness advantage, where the smaller the genome the higher the within-host competitive fitness. Interestingly, although the size of the deletions varied, convergent evolution did occur in terms of fixed point mutations (Zwart et al. 2014) . This result also suggests that a demographic 'sweet spot' exists, where heterologous insertions are not immediately lost while evolution can act to integrate them into the viral genome. In summary, in several studies passage duration has an effect on insert stability, with inserts being more stable in shorter passages. We explore these effects in the conceptual section presented at the end of this paper (see also Box 1).