From genotypes to organisms: State-of-the-art and perspectives of a cornerstone in evolutionary dynamics

Understanding how genotypes map onto phenotypes, fitness, and eventually organisms is arguably the next major missing piece in a fully predictive theory of evolution. We refer to this generally as the problem of the genotype-phenotype map. Though we are still far from achieving a complete picture of these relationships, our current understanding of simpler questions, such as the structure induced in the space of genotypes by sequences mapped to molecular structures, has revealed important facts that deeply affect the dynamical description of evolutionary processes. Empirical evidence supporting the fundamental relevance of features such as phenotypic bias is mounting as well, while the synthesis of conceptual and experimental progress leads to questioning current assumptions on the nature of evolutionary dynamics-cancer progression models or synthetic biology approaches being notable examples. This work delves into a critical and constructive attitude in our current knowledge of how genotypes map onto molecular phenotypes and organismal functions, and discusses theoretical and empirical avenues to broaden and improve this comprehension. As a final goal, this community should aim at deriving an updated picture of evolutionary processes soundly relying on the structural properties of genotype spaces, as revealed by modern techniques of molecular and functional analysis.Comment: 111 pages, 11 figures uses elsarticle latex clas

RNA structures and their molecular evolution in HIV: evolution of robustness in RNA structures and theoretical systems

The known functions of RNA structures have expanded of late, such that RNA is considered a more active player in molecular biology. The presence of RNA secondary structure in a sequence should constrain evolution of its constituent nucleotides because of the requirement to maintain the base-pairing regions in the structure. In a previous work, we found support for this hypothesis in nine molecules from various organisms, the exception being a structure found in a protein-coding region of the HIV-1 genome. In this work, I examine the interaction of constraints imposed by RNA structures and host-induced hypermutation on molecular evolution in HIV-1. I conclude that RNA structures in HIV do evolve via compensatory evolution, but that hypermutation can obscure the expected signal. Since RNA's known roles have increased, so have the methods for identification and prediction of RNA structures in genetic sequence. I use a method adapted for searching in multiple coding regions to identify conserved RNA structures throughout the HIV-1 and HIV-2 genomes. I find evidence for several new, small structures in HIV-1, but evidence is less strong for HIV-2. Finally, I consider the evolution of robustness, the property of phenotypic constancy, using RNA structures and two other theoretical model systems. I find that pervasive environmental variation can select for environmental and genetic robustness in all three systems, and conclude that it may be a generic mechanism for the evolution of robustness

Robustness promotes evolvability of thermotolerance in an RNA virus

<p>Abstract</p> <p>Background</p> <p>The ability for an evolving population to adapt to a novel environment is achieved through a balance of robustness and evolvability. Robustness is the invariance of phenotype in the face of perturbation and evolvability is the capacity to adapt in response to selection. Genetic robustness has been posited, depending on the underlying mechanism, to either decrease the efficacy of selection, or increase the possibility of future adaptation. However, the true effect of genetic robustness on evolvability in biological systems remains uncertain.</p> <p>Results</p> <p>Here we demonstrate that genetic robustness increases evolvability of thermotolerance in laboratory populations of the RNA virus φ6. We observed that populations founded by robust clones evolved greater resistance to heat shock, relative to populations founded by brittle (less-robust) clones. Thus, we provide empirical evidence for the idea that robustness can promote evolvability in this environment, and further suggest that evolvability can arise indirectly via selection for robustness, rather than through direct selective action.</p> <p>Conclusion</p> <p>Our data imply that greater tolerance of mutational change is associated with virus adaptability in a new niche, a finding generally relevant to evolutionary biology, and informative for elucidating how viruses might evolve to emerge in new habitats and/or overcome novel therapies.</p

The influence of CpG and UpA dinucleotide frequencies on RNA virus replication and characterization of the innate cellular pathways underlying virus attenuation and enhanced replication

Most RNA viruses infecting mammals and other vertebrates show profound suppression of CpG and UpA dinucleotide frequencies. To investigate this functionally, mutants of the picornavirus, echovirus 7 (E7), were constructed with altered CpG and UpA compositions in two 1.1–1.3 Kbase regions. Those with increased frequencies of CpG and UpA showed impaired replication kinetics and higher RNA/infectivity ratios compared with wild-type virus. Remarkably, mutants with CpGs and UpAs removed showed enhanced replication, larger plaques and rapidly outcompeted wild-type virus on co-infections. Luciferase-expressing E7 sub-genomic replicons with CpGs and UpAs removed from the reporter gene showed 100-fold greater luminescence. E7 and mutants were equivalently sensitive to exogenously added interferon-β, showed no evidence for differential recognition by ADAR1 or pattern recognition receptors RIG-I, MDA5 or PKR. However, kinase inhibitors roscovitine and C16 partially or entirely reversed the attenuated phenotype of high CpG and UpA mutants, potentially through inhibition of currently uncharacterized pattern recognition receptors that respond to RNA composition. Generating viruses with enhanced replication kinetics has applications in vaccine production and reporter gene construction. More fundamentally, the findings introduce a new evolutionary paradigm where dinucleotide composition of viral genomes is subjected to selection pressures independently of coding capacity and profoundly influences host–pathogen interactions

Systematic Experimental Determination of Functional Constraints on Proteins and Adaptive Potential of Mutations: A Dissertation

Sequence-function relationship is a fundamental question for many branches of modern biomedical research. It connects the primary sequence of proteins to the function of proteins and fitness of organisms, holding answers for critical questions such as functional consequences of mutations identified in whole genome sequencing and adaptive potential of fast evolving pathogenic viruses and microbes. Many different approaches have been developed to delineate the genotype-phenotype map for different proteins, but are generally limited by their throughput or precision. To systematically quantify the fitness of large numbers of mutations, I modified a novel high throughput mutational scanning approach (EMPIRIC) to investigate the fitness landscape of mutations in important regions of essential proteins from the yeast or RNA viruses. Using EMPIRIC, I analyzed the interplay of the expression level and sequence of Hsp90 on the yeast growth and revealed latent effect of mutations at reduced expression levels of Hsp90. I also examined the functional constraint on the receptor binding site of the Env of Human Immunodeficiency Virus (HIV) and uncovered enhanced receptor binding capacity as a common pathway for adaptation of HIV to laboratory conditions. Moreover, I explored the adaptive potential of neuraminidase (NA) of influenza A virus to a NA inhibitor, oseltamivir, and identified novel oseltamivir resistance mutations with distinct molecular mechanisms. In summary, I applied a high throughput functional genomics approach to map the sequence-function relationship in various systems and examined the evolutionary constraints and adaptive potential of essential proteins ranging from molecular chaperones to drug-targetable viral proteins

Relationship between mRNA secondary structure and sequence variability in Chloroplast genes: possible life history implications

<p>Abstract</p> <p>Background</p> <p>Synonymous sites are freer to vary because of redundancy in genetic code. Messenger RNA secondary structure restricts this freedom, as revealed by previous findings in mitochondrial genes that mutations at third codon position nucleotides in helices are more selected against than those in loops. This motivated us to explore the constraints imposed by mRNA secondary structure on evolutionary variability at all codon positions in general, in chloroplast systems.</p> <p>Results</p> <p>We found that the evolutionary variability and intrinsic secondary structure stability of these sequences share an inverse relationship. Simulations of most likely single nucleotide evolution in <it>Psilotum nudum </it>and <it>Nephroselmis olivacea </it>mRNAs, indicate that helix-forming propensities of mutated mRNAs are greater than those of the natural mRNAs for short sequences and vice-versa for long sequences. Moreover, helix-forming propensity estimated by the percentage of total mRNA in helices increases gradually with mRNA length, saturating beyond 1000 nucleotides. Protection levels of functionally important sites vary across plants and proteins: <it>r</it>-strategists minimize mutation costs in large genes; <it>K</it>-strategists do the opposite.</p> <p>Conclusion</p> <p>Mrna length presumably predisposes shorter mRNAs to evolve under different constraints than longer mRNAs. The positive correlation between secondary structure protection and functional importance of sites suggests that some sites might be conserved due to packing-protection constraints at the nucleic acid level in addition to protein level constraints. Consequently, nucleic acid secondary structure <it>a priori </it>biases mutations. The converse (exposure of conserved sites) apparently occurs in a smaller number of cases, indicating a different evolutionary adaptive strategy in these plants. The differences between the protection levels of functionally important sites for <it>r</it>- and <it>K-</it>strategists reflect their respective molecular adaptive strategies. These converge with increasing domestication levels of <it>K</it>-strategists, perhaps because domestication increases reproductive output.</p

The Critical role of codon composition on the translation efficiency robustness of the Hepatitis A virus capsid

Hepatoviruses show an intriguing deviated codon usage, suggesting an evolutionary signature. Abundant and rare codons in the cellular genome are scarce in the human hepatitis A virus (HAV) genome, while intermediately abundant host codons are abundant in the virus. Genotype-phenotype maps, or fitness landscapes, are a means of representing a genotype position in sequence space and uncovering how genotype relates to phenotype and fitness. Using genotype-phenotype maps of the translation efficiency, we have shown the critical role of the HAV capsid codon composition in regulating translation and determining its robustness. Adaptation to an environmental perturbation such as the artificial induction of cellular shutoff not naturally occurring in HAV infection involved movements in the sequence space and dramatic changes of the translation efficiency. Capsid rare codons, including abundant and rare codons of the cellular genome, slowed down the translation efficiency in conditions of no cellular shutoff. In contrast, rare capsid codons that are abundant in the cellular genome were efficiently translated in conditions of shutoff. Capsid regions very rich in slowly translated codons adapt to shutoff through sequence space movements from positions with highly robust translation to others with diminished translation robustness. These movements paralleled decreases of the capsid physical and biological robustness, and resulted in the diversification of capsid phenotypes. The deviated codon usage of extant hepatoviruses compared with that of their hosts may suggest the occurrence of a virus ancestor with an optimized codon usage with respect to an unknown ancient host

Inferring stabilizing mutations from protein phylogenies : application to influenza hemagglutinin

One selection pressure shaping sequence evolution is the requirement that a protein fold with sufficient stability to perform its biological functions. We present a conceptual framework that explains how this requirement causes the probability that a particular amino acid mutation is fixed during evolution to depend on its effect on protein stability. We mathematically formalize this framework to develop a Bayesian approach for inferring the stability effects of individual mutations from homologous protein sequences of known phylogeny. This approach is able to predict published experimentally measured mutational stability effects (ΔΔG values) with an accuracy that exceeds both a state-of-the-art physicochemical modeling program and the sequence-based consensus approach. As a further test, we use our phylogenetic inference approach to predict stabilizing mutations to influenza hemagglutinin. We introduce these mutations into a temperature-sensitive influenza virus with a defect in its hemagglutinin gene and experimentally demonstrate that some of the mutations allow the virus to grow at higher temperatures. Our work therefore describes a powerful new approach for predicting stabilizing mutations that can be successfully applied even to large, complex proteins such as hemagglutinin. This approach also makes a mathematical link between phylogenetics and experimentally measurable protein properties, potentially paving the way for more accurate analyses of molecular evolution

Synonymous genome recoding : a tool to explore microbial biology and new therapeutic strategies

Synthetic genome recoding is a new means of generating designed organisms with altered phenotypes. Synonymous mutations introduced into the protein coding region tolerate modifications in DNA or mRNA without modifying the encoded proteins. Synonymous genome-wide recoding has allowed the synthetic generation of different small-genome viruses with modified phenotypes and biological properties. Recently, a decreased cost of chemically synthesizing DNA and improved methods for assembling DNA fragments (e.g. lambda red recombination and CRISPR-based editing) have enabled the construction of an Escherichia coli variant with a 4-Mb synthetic synonymously recoded genome with a reduced number of sense codons (n = 59) encoding the 20 canonical amino acids. Synonymous genome recoding is increasing our knowledge of microbial interactions with innate immune responses, identifying functional genome structures, and strategically ameliorating cis-inhibitory signaling sequences related to splicing, replication (in eukaryotes), and complex microbe functions, unraveling the relevance of codon usage for the temporal regulation of gene expression and the microbe mutant spectrum and adaptability. New biotechnological and therapeutic applications of this methodology can easily be envisaged. In this review, we discuss how synonymous genome recoding may impact our knowledge of microbial biology and the development of new and better therapeutic methodologies