Compensatory molecular evolution of HC-Pro, an RNA-silencing suppressor from a plant RNA virus

RNA silencing is a eukaryotic mechanism involved in several cellular processes, one example being a sequence-specific antiviral defense. Many plant viruses have developed counterdefensive proteins that in many instances are multifunctional, such as helper component protease (HC-Pro) of Tobacco etch virus (TEV). In a previous work, a collection of mutants with amino acid replacements in TEV HC-Pro was generated, and their effects in the capacity of suppressing RNA silencing were quantified in a transient expression assay. In this study, three mutations that caused a reduction in suppression activity and three that increased it were used to create replicate experimental lineages that were evolved through serial passages. We have evaluated the number of genotypic changes that occurred during evolution in HC-Pro and their phenotypic effects on virus viability, virulence, and suppression of RNA silencing. In no instance did the original mutation revert to the wildtype (WT) sequence. In several cases, fixed mutations were canonical compensatory changes, returning the suppressor activity to the WT HC-Pro value, pointing to the existence of stabilizing selection pressures and pleiotropic effects of the introduced original mutations. However, in other instances, the fixed mutations were overcompensatory, driving the activity of the mutant beyond the optimal value. Negative epistatic effects among beneficial mutations as well as decompensatory epistasis also play an important role during compensatory evolution of RNA-silencing suppression.