On the stability of sequences inserted into viral genomes

Willemsen, Anouk; Zwart, Mark P

These different mutation classes are likely to have different mutation rates, and mutation bias might therefore drive the evolutionary route that is followed (Stoltzfus and McCandlish 2017) . For example, consider that recombination rates are high for many viruses (Tromas and Elena 2010) , and there are many Figure 3 . In panel A, we illustrate how the cellular MOI can have a direct effect on selection strength. Consider a virus that expresses a product that is toxic and acts in trans within cells to lower replication levels, but deletions can remove the gene coding this gene. If there is a mixed virus population with variants with the insertion intact and deleted, at high MOI all cells will be infected with both variants and the toxin will lower replication. The ubiquitousness of the toxin will limit selection against the virus variant with the deletion. When MOI is low, due to genetic drift at the cellular not all cells will contain both variants, and virus variant with the deletion is selected because those cells infected only with this variant have higher replication. In panel B, the relationship between the cellular MOI (ordinate) and the frequency of single-genotype infection (abscissa) for a virus population with genotypes a and b is given, for different frequencies of the two virus genotypes in the population (f a shown, f b ¼ 1 -f a ). Note that the frequency of single-genotype infections is given as the proportion of infected cells in which only virus genotypes a or b are present. As the MOI increases, the frequency of single-genotype infections decreases, although it depends on the frequency of the two virus genotypes in the population. If genotype a expresses a gene that has fitness costs that act in trans (e.g. toxicity), then selection can only act against this genotype when there is an appreciable number of singlegenotype infections. possible recombination events that partially remove an insertion. In contrast, probably only a small fraction of point mutations will be beneficial (Sanjuá n, Moya, and Elena 2004; Carrasco, de la Iglesia, and Elena 2007) , e.g. in this case by lowering expression of the inserted gene or leading to more favorable codon usage. We therefore conjecture that mutation supply is likely to favor the evolution of deletions in the transgene over beneficial point mutations that affect fitness cost. Consider the 'genomic accordion' observed in poxviruses (Elde et al. 2012) : beneficial point mutations typically occur long after gene amplification by copy number variation. Likewise we expect deletions that remove an insertion to be fixed before point mutations that also lessen its impact occur. Nevertheless, the occurrence of alternative evolutionary trajectories could, depending on the exact mutation supply and effect sizes for different classes of mutations, contribute to making stability of genomic inserts less repeatable and predictable in some cases (De Visser and Krug 2014; Bolnick et al. 2018 ).