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International audience; 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.

This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License. The images in this article are included in the article's Creative Commons license, unless indicated otherwise in the image credit; if the image is not included under the Creative Commons license, users will need to obtain permission from the license holder in order to reproduce the image., The distribution of mutational fitness effects (DMFE) is crucial to the evolutionary fate of quasispecies. In this article we analyze the effect of the DMFE on the dynamics of a large quasispecies by means of a phenotypic version of the classic Eigen's model that incorporates beneficial, neutral, deleterious, and lethal mutations. By parameterizing the model with available experimental data on the DMFE of Vesicular stomatitis virus (VSV) and Tobacco etch virus (TEV), we found that increasing mutation does not totally push the entire viral quasispecies towards deleterious or lethal regions of the phenotypic sequence space. The probability of finding regions in the parameter space of the general model that results in a quasispecies only composed by lethal phenotypes is extremely small at equilibrium and in transient times. The implications of our findings can be extended to other scenarios, such as lethal mutagenesis or genomically unstable cancer, where increased mutagenesis has been suggested as a potential therapy., This work was partially funded by the Botin Foundation (JS, RVS), by the Spanish Secretaria de Estado de Investigacion, Desarrollo e Innovacion grants MTM2010-16425 (CS, RM) and BFU2012-30805 (SFE), by grant 2009-SGR-67 from the Catalan government (CS, RM), by grant NSF PHY05-51164 (JS, SFE), and by the Santa Fe Institute (RVS, SFE).

Measuring fitness with precision is a key issue in evolutionary biology, particularly in studying mutations of small effects. It is usually thought that sampling error and drift prevent precise measurement of very small fitness effects. We circumvented these limits by using a new combined approach to measuring and analyzing fitness. We estimated the mutational fitness effect (MFE) of three independent mini-Tn10 transposon insertion mutations by conducting competition experiments in large populations of Escherichia coli under controlled laboratory conditions. Using flow cytometry to assess genotype frequencies from very large samples alleviated the problem of sampling error, while the effect of drift was controlled by using large populations and massive replication of fitness measures. Furthermore, with a set of four competition experiments between ancestral and mutant genotypes, we were able to decompose fitness measures into four estimated parameters that account for fitness effects of our fluorescent marker (α), the mutation (β), epistasis between the mutation and the marker (γ), and departure from transitivity (τ). Our method allowed us to estimate mean selection coefficients to a precision of 2 × 10−4. We also found small, but significant, epistatic interactions between the allelic effects of mutations and markers and confirmed that fitness effects were transitive in most cases. Unexpectedly, we also detected variation in measures of s that were significantly bigger than expected due to drift alone, indicating the existence of cryptic variation, even in fully controlled experiments. Overall our results indicate that selection coefficients are best understood as being distributed, representing a limit on the precision with which selection can be measured, even under controlled laboratory conditions., This work was supported by the ERC Starting Grant ‘Quantevol’ to T.L. and NSF (IOS-­‐1022373) to T.F.C.

6 pages, 3 figures.-- PMID: 15159545 [PubMed].-- PMCID: PMC420405.-- Supporting information (Table 3: Relevant information about each single-nucleotide substation mutant created) available at: http://www.pnas.org/content/101/22/8396/suppl/DC1, Printed version published on Jun 1, 2004., Full-text paper available Open Access via PubMed Central: http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=15159545, Little is known about the mutational fitness effects associated with single-nucleotide substitutions on RNA viral genomes. Here, we used site-directed mutagenesis to create 91 single mutant clones of vesicular stomatitis virus derived from a common ancestral cDNA and performed competition experiments to measure the relative fitness of each mutant. The distribution of nonlethal deleterious effects was highly skewed and had a long, flat tail. As expected, fitness effects depended on whether mutations were chosen at random or reproduced previously described ones. The effect of random deleterious mutations was well described by a log-normal distribution, with -19% reduction of average fitness; the effects distribution of preobserved deleterious mutations was better explained by a β model. The fit of both models was improved when combined with a uniform distribution. Up to 40% of random mutations were lethal. The proportion of beneficial mutations was unexpectedly high. Beneficial effects followed a γ distribution, with expected fitness increases of 1% for random mutations and 5% for preobserved mutations., This study was supported by Spanish Ministerio de Ciencia y Tecnología Grant BMC2001-3096 (to A.M.) and Generalitat Valenciana Grant GV01-65 (to S.F.E.). R.S. enjoyed a predoctoral fellowship from the Ministerio de Educación, Cultura y Deporte.