Shrinkage of Genome Size in a Plant RNA Virus upon Transfer of an Essential Viral Gene into the Host Genome

Tromas, Nicolas; Zwart, Mark P.; Forment, Javier; Elena, Santiago F.

The K2269E, E1206D, I1208V, and T1210M mutations were introduced in the pMTEV infectious clone by site-directed mutagenesis. As K2269E is common among the different lineages, we also generated clones containing this mutation in combination with the other three. As a proxy for within-host fitness, we measured viral accumulation of each simple and double mutant at 2.67 dpi by RT-qPCR in three biological replicates. Data were fitted to a GLM with a log-link function and gamma distributed error structure, using replicate as factor nested within virus genotype. A significant effect of virus genotype on the observed viral accumulation was found ( 2 ¼ 134.648, 7 d.f., P < 0.001), and a Holm-Bonferroni post hoc test identified four different homogenous groups ( fig. 4C) , with a being the group with the lowest viral accumulation value and d the highest. The results are generally in agreement with our expectations based on the prevalence of residues in the Potyviridae. E1206D has the same accumulation as the ancestral virus. The other single mutations all increased accumulation, being represented in groups b and c. The double mutations resulted in the highest accumulation levels, with two being the sole members of group d and one being represented in group c.