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      Distribution of mutational fitness effects and of epistasis in the 5’ untranslated region of a plant RNA virus


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          Understanding the causes and consequences of phenotypic variability is a central topic of evolutionary biology. Mutations within non-coding cis-regulatory regions are thought to be of major effect since they affect the expression of downstream genes. To address the evolutionary potential of mutations affecting such regions in RNA viruses, we explored the fitness properties of mutations affecting the 5’-untranslated region (UTR) of a prototypical member of the picorna-like superfamily, Tobacco etch virus (TEV). This 5’ UTR acts as an internal ribosomal entry site (IRES) and is essential for expression of all viral genes.


          We determined in vitro the folding of 5’ UTR using the selective 2’-hydroxyl acylation analyzed by primer extension (SHAPE) technique. Then, we created a collection of single-nucleotide substitutions on this region and evaluated the statistical properties of their fitness effects in vivo. We found that, compared to random mutations affecting coding sequences, mutations at the 5’ UTR were of weaker effect. We also created double mutants by combining pairs of these single mutations and found variation in the magnitude and sign of epistatic interactions, with an enrichment of cases of positive epistasis. A correlation exists between the magnitude of fitness effects and the size of the perturbation made in the RNA folding structure, suggesting that the larger the departure from the predicted fold, the more negative impact in viral fitness.


          Evidence that mutational fitness effects on the short 5’ UTR regulatory sequence of TEV are weaker than those affecting its coding sequences have been found. Epistasis among pairs of mutations on the 5’ UTR ranged between the extreme cases of synthetic lethal and compensatory. A plausible hypothesis to explain all these observations is that the interaction between the 5’ UTR and the host translational machinery was shaped by natural selection to be robust to mutations, thus ensuring the homeostatic expression of viral genes even at high mutation rates.

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          Most cited references53

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          Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE): quantitative RNA structure analysis at single nucleotide resolution.

          Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) interrogates local backbone flexibility in RNA at single-nucleotide resolution under diverse solution environments. Flexible RNA nucleotides preferentially sample local conformations that enhance the nucleophilic reactivity of 2'-hydroxyl groups toward electrophiles, such as N-methylisatoic anhydride (NMIA). Modified sites are detected as stops in an optimized primer extension reaction, followed by electrophoretic fragment separation. SHAPE chemistry scores local nucleotide flexibility at all four ribonucleotides in a single experiment and discriminates between base-paired versus unconstrained or flexible residues with a dynamic range of 20-fold or greater. Quantitative SHAPE reactivity information can be used to establish the secondary structure of an RNA, to improve the accuracy of structure prediction algorithms, to monitor structural differences between related RNAs or a single RNA in different states, and to detect ligand binding sites. SHAPE chemistry rarely needs significant optimization and requires two days to complete for an RNA of 100-200 nucleotides.
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            The distribution of fitness effects caused by single-nucleotide substitutions in an RNA virus.

            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 beta 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 gamma distribution, with expected fitness increases of 1% for random mutations and 5% for preobserved mutations.
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              Comparative studies of gene expression and the evolution of gene regulation.

              The hypothesis that differences in gene regulation have an important role in speciation and adaptation is more than 40 years old. With the advent of new sequencing technologies, we are able to characterize and study gene expression levels and associated regulatory mechanisms in a large number of individuals and species at an unprecedented resolution and scale. We have thus gained new insights into the evolutionary pressures that shape gene expression levels and have developed an appreciation for the relative importance of evolutionary changes in different regulatory genetic and epigenetic mechanisms. The current challenge is to link gene regulatory changes to adaptive evolution of complex phenotypes. Here we mainly focus on comparative studies in primates and how they are complemented by studies in model organisms.

                Author and article information

                BMC Evol Biol
                BMC Evol. Biol
                BMC Evolutionary Biology
                BioMed Central (London )
                7 December 2015
                7 December 2015
                : 15
                : 274
                [ ]Instituto de Biología Molecular y Celular de Plantas, Consejo Superior de Investigaciones Científicas-UPV, Campus UPV CPI 8E, Ingeniero Fausto Elio s/n, 46022 València, Spain
                [ ]The Santa Fe Institute, 1399 Hyde Park Road, Santa Fe, NM 87501 USA
                Author information
                © Bernet and Elena. 2015

                Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

                : 30 September 2015
                : 2 December 2015
                Funded by: FundRef http://dx.doi.org/10.13039/501100003329, Ministerio de Economía y Competitividad (ES);
                Award ID: BFU2012-30805
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/501100003359, Generalitat Valenciana (ES);
                Award ID: PROMETEOII/201/021
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/501100000780, European Commission;
                Award ID: EvoEvo (ICT610427)
                Award Recipient :
                Research Article
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                © The Author(s) 2015

                Evolutionary Biology
                distribution of mutational fitness effects,plant virus,potyvirus,rna regulatory sequences,rna folding,virus evolution


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