Mutational fitness effects in RNA and single-stranded DNA viruses: common patterns revealed by site-directed mutagenesis studies

The fitness effects of mutations are central to evolution, yet have begun to be characterized in detail only recently. Site-directed mutagenesis is a powerful tool for achieving this goal, which is particularly suited for viruses because of their small genomes. Here, I discuss the evolutionary relevance of mutational fitness effects and critically review previous site-directed mutagenesis studies. The effects of single-nucleotide substitutions are standardized and compared for five RNA or single-stranded DNA viruses infecting bacteria, plants or animals. All viruses examined show very low tolerance to mutation when compared with cellular organisms. Moreover, for non-lethal mutations, the mean fitness reduction caused by single mutations is remarkably constant (0.10–0.13), whereas the fraction of lethals varies only modestly (0.20–0.41). Other summary statistics are provided. These generalizations about the distribution of mutational fitness effects can help us to better understand the evolution of RNA and single-stranded DNA viruses.     

Conservation of mRNA secondary structures may filter out mutations in Escherichia coli evolution

Recent reports indicate that mutations in viral genomes tend to preserve RNA secondary structure, and those mutations that disrupt secondary structural elements may reduce gene expression levels, thereby serving as a functional knockout. In this article, we explore the conservation of secondary structures of mRNA coding regions, a previously unknown factor in bacterial evolution, by comparing the structural consequences of mutations in essential and nonessential Escherichia coli genes accumulated over 40 000 generations in the course of the ‘long-term evolution experiment’. We monitored the extent to which mutations influence minimum free energy (MFE) values, assuming that a substantial change in MFE is indicative of structural perturbation. Our principal finding is that purifying selection tends to eliminate those mutations in essential genes that lead to greater changes of MFE values and, therefore, may be more disruptive for the corresponding mRNA secondary structures. This effect implies that synonymous mutations disrupting mRNA secondary structures may directly affect the fitness of the organism. These results demonstrate that the need to maintain intact mRNA structures imposes additional evolutionary constraints on bacterial genomes, which go beyond preservation of structure and function of the encoded proteins.     

Patterns of epistasis in RNA viruses: a review of the evidence from vaccine design

Epistasis results when the fitness effects of a mutation change depending on the presence or absence of other mutations in the genome. The predictions of many influential evolutionary hypotheses are determined by the existence and form of epistasis. One rich source of data on the interactions among deleterious mutations that has gone untapped by evolutionary biologists is the literature on the design of live, attenuated vaccine viruses. Rational vaccine design depends upon the measurement of individual and combined effects of deleterious mutations. In the current study, we have reviewed data from 29 vaccine-oriented studies using 14 different RNA viruses. Our analyses indicate that (1) no consistent tendency towards a particular form of epistasis exists across RNA viruses and (2) significant interactions among groups of mutations within individual viruses occur but are not common. RNA viruses are significant pathogens of human disease, and are tractable model systems for evolutionary studies--we discuss the relevance of our findings in both contexts.     

Positive selection of synonymous mutations in vesicular stomatitis virus

Prevailing evolutionary forces are typically deduced from the pattern of differences in synonymous and non-synonymous mutations, under the assumption of neutrality in the absence of amino acid change. We determined the complete sequence of ten vesicular stomatitis virus populations evolving under positive selection. A significant number of the mutations occurred independently in two or more strains, a process known as parallel evolution, and a substantial fraction of the parallel mutations were silent. Parallel evolution was also identified in non-coding regions. These results indicate that silent mutations can significantly contribute to adaptation in RNA viruses, and relative frequencies of synonymous and non-synonymous substitutions may not be useful to resolve their evolutionary history.     

Quantifying the adaptive potential of an antibiotic resistance enzyme

For a quantitative understanding of the process of adaptation, we need to understand its "raw material," that is, the frequency and fitness effects of beneficial mutations. At present, most empirical evidence suggests an exponential distribution of fitness effects of beneficial mutations, as predicted for Gumbel-domain distributions by extreme value theory. Here, we study the distribution of mutation effects on cefotaxime (Ctx) resistance and fitness of 48 unique beneficial mutations in the bacterial enzyme TEM-1 β-lactamase, which were obtained by screening the products of random mutagenesis for increased Ctx resistance. Our contributions are threefold. First, based on the frequency of unique mutations among more than 300 sequenced isolates and correcting for mutation bias, we conservatively estimate that the total number of first-step mutations that increase Ctx resistance in this enzyme is 87 [95% CI 75-189], or 3.4% of all 2,583 possible base-pair substitutions. Of the 48 mutations, 10 are synonymous and the majority of the 38 non-synonymous mutations occur in the pocket surrounding the catalytic site. Second, we estimate the effects of the mutations on Ctx resistance by determining survival at various Ctx concentrations, and we derive their fitness effects by modeling reproduction and survival as a branching process. Third, we find that the distribution of both measures follows a Fréchet-type distribution characterized by a broad tail of a few exceptionally fit mutants. Such distributions have fundamental evolutionary implications, including an increased predictability of evolution, and may provide a partial explanation for recent observations of striking parallel evolution of antibiotic resistance.     

Directionality in the evolution of influenza A haemagglutinin

The evolution of haemagglutinin (HA), an important influenza virus antigen, has been the subject of intensive research for more than two decades. Many characteristics of HA's sequence evolution are captured by standard Markov chain substitution models. Such models assign equal fitness to all accessible amino acids at a site. We show, however, that such models strongly underestimate the number of homoplastic amino acid substitutions during the course of HA's evolution, i.e. substitutions that repeatedly give rise to the same amino acid at a site. We develop statistics to detect individual homoplastic events and find that they preferentially occur at positively selected epitopic sites. Our results suggest that the evolution of the influenza A HA, including evolution by positive selection, is strongly affected by the long-term site-specific preferences for individual amino acids.     

Phylogenetic evidence for deleterious mutation load in RNA viruses and its contribution to viral evolution

Populations of RNA viruses are often characterized by abundant genetic variation. However, the relative fitness of these mutations is largely unknown, although this information is central to our understanding of viral emergence, immune evasion, and drug resistance. Here we develop a phylogenetic method, based on the distribution of nonsynonymous and synonymous changes, to assess the relative fitness of polymorphisms in the structural genes of 143 RNA viruses. This reveals that a substantial proportion of the amino acid variation observed in natural populations of RNA viruses comprises transient deleterious mutations that are later purged by purifying selection, potentially limiting virus adaptability. We also demonstrate, for the first time, the existence of a relationship between amino acid variability and the phylogenetic distribution of polymorphisms. From this relationship, we propose an empirical threshold for the maximum viable deleterious mutation load in RNA viruses.     

Molecular evolution of hemagglutinin genes of H1N1 swine and human influenza A viruses

Summary The hemagglutinin (HA) genes of influenza type A (H1N1) viruses isolated from swine were cloned into plasmid vectors and their nucleotide sequences were determined. A phylogenetic tree for the HA genes of swine and human influenza viruses was constructed by the neighbor-joining method. It showed that the divergence between swine and human HA genes might have occurred around 1905. The estimated rates of synonymous (silent) substitutions for swine and human influenza viruses were almost the same. For both viruses, the rate of synonymous substitution was much higher than that of nonsynonymous (amino acid altering) substitution. It is the case even for only the antigenic sites of the HA. This feature is consistent with the neutral theory of molecular evolution. The rate of nonsynonymous substitution for human influenza viruses was three times the rate for swine influenza viruses. In particular, nonsynonymous substitutions at antigenic sites occurred less frequently in swine than in humans. The difference in the rate of nonsynonymous substitution between swine and human influenza viruses can be explained by the different degrees of functional constraint operating on the amino acid sequence of the HA in both hosts.     

Evolutionary genomics of host-use in bifurcating demes of RNA virus phi-6

ABSTRACT: BACKGROUND: Viruses are exceedingly diverse in their evolved strategies to manipulate hosts for viral replication. However, despite these differences, most virus populations will occasionally experience two commonly-encountered challenges: growth in variable host environments, and growth under fluctuating population sizes. We used the segmented RNA bacteriophage [GREEK PHI SYMBOL]6 as a model for studying the evolutionary genomics of virus adaptation in the face of host switches and parametrically varying population sizes. To do so, we created a bifurcating deme structure that reflected lineage splitting in natural populations, allowing us to test whether phylogenetic algorithms could accurately resolve this 'known phylogeny'. The resulting tree yielded 32 clones at the tips and internal nodes; these strains were fully sequenced and measured for phenotypic changes in selected traits (fitness on original and novel hosts). RESULTS: We observed that RNA segment size was negatively correlated with the extent of molecular change in the imposed treatments; molecular substitutions tended to cluster on the Small and Medium RNA chromosomes of the virus, and not on the Large segment. Our study yielded a very large molecular and phenotypic dataset, fostering possible inferences on genotype-phenotype associations. Using further experimental evolution, we confirmed an inference on the unanticipated role of an allelic switch in a viral assembly protein, which governed viral performance across host environments. CONCLUSIONS: Our study demonstrated that varying complexities can be simultaneously incorporated into experimental evolution, to examine the combined effects of population size, and adaptation in novel environments. The imposed bifurcating structure revealed that some methods for phylogenetic reconstruction failed to resolve the true phylogeny, owing to a paucity of molecular substitutions separating the RNA viruses that evolved in our study.     

Synonymous substitutions substantially improve evolutionary inference from highly diverged proteins

Codon-and amino acid-substitution models are widely used for the evolutionary analysis of protein-coding DNA sequences. Using codon models, the amounts of both nonsynonymous and synonymous DNA substitutions can be estimated. The ratio of these amounts represents the strength of selective pressure. Using amino acid models, the amount of nonsynonymous substitutions is estimated, but that of synonymous substitutions is ignored. Although amino acid models lose any information regarding synonymous substitutions, they explicitly incorporate the information for amino acid replacement, which is empirically derived from databases. It is often presumed that when the protein-coding sequences are highly divergent, synonymous substitutions might be saturated and the evolutionary analysis may be hampered by synonymous noise. However, there exists no quantitative procedure to verify whether synonymous substitutions can be ignored; therefore, amino acid models have been arbitrarily selected. In this study, we investigate the issue of a statistical comparison between codon-and amino acid-substitution models. For this purpose, we propose a new procedure to transform a 20-dimensional amino acid model to a 61-dimensional codon model. This transformation reveals that amino acid models belong to a subset of the codon models and enables us to test whether synonymous substitutions can be ignored by using the likelihood ratio. Our theoretical results and analyses of real data indicate that synonymous substitutions are very informative and substantially improve evolutionary inference, even when the sequences are highly divergent. Therefore, we note that amino acid models should be adopted only after carefully investigating and discarding the possibility that synonymous substitutions can reveal important evolutionary information.     

Assessing in vivo mutation frequencies and creating a high-resolution genome-wide map of fitness costs of Hepatitis C virus

Like many viruses, Hepatitis C Virus (HCV) has a high mutation rate, which helps the virus adapt quickly, but mutations come with fitness costs. Fitness costs can be studied by different approaches, such as experimental or frequency-based approaches. The frequency-based approach is particularly useful to estimate in vivo fitness costs, but this approach works best with deep sequencing data from many hosts are. In this study, we applied the frequency-based approach to a large dataset of 195 patients and estimated the fitness costs of mutations at 7957 sites along the HCV genome. We used beta regression and random forest models to better understand how different factors influenced fitness costs. Our results revealed that costs of nonsynonymous mutations were three times higher than those of synonymous mutations, and mutations at nucleotides A or T had higher costs than those at C or G. Genome location had a modest effect, with lower costs for mutations in HVR1 and higher costs for mutations in Core and NS5B. Resistance mutations were, on average, costlier than other mutations. Our results show that in vivo fitness costs of mutations can be site and virus specific, reinforcing the utility of constructing in vivo fitness cost maps of viral genomes.     

Evolutionary models accounting for layers of selection in protein-coding genes and their impact on the inference of positive selection

The selective forces acting on a protein-coding gene are commonly inferred using evolutionary codon models by contrasting the rate of nonsynonymous substitutions to the rate of synonymous substitutions. These models usually assume that the synonymous substitution rate, Ks, is homogenous across all sites, which is justified if synonymous sites are free from selection. However, a growing body of evidence indicates that the DNA and RNA levels of protein-coding genes are subject to varying degrees of selective constraints due to various biological functions encoded at these levels. In this paper, we develop evolutionary models that account for these layers of selection by allowing for both among-site variability of substitution rates at the DNA/RNA level (which leads to Ks variability among protein-coding sites) and among-site variability of substitution rates at the protein level (Ka variability). These models are constructed so that positive selection is either allowed or not. This enables statistical testing of positive selection when variability at the DNA/RNA substitution rate is accounted for. Using this methodology, we show that variability of the baseline DNA/RNA substitution rate is a widespread phenomenon in coding sequence data of mammalian genomes, most likely reflecting varying degrees of selection at the DNA and RNA levels. Additionally, we use simulations to examine the impact that accounting for the variability of the baseline DNA/RNA substitution rate has on the inference of positive selection. Our results show that ignoring this variability results in a high rate of erroneous positive-selection inference. Our newly developed model, which accounts for this variability, does not suffer from this problem and hence provides a likelihood framework for the inference of positive selection on a background of variability in the baseline DNA/RNA substitution rate.     

The impact of high‐order epistasis in the within‐host fitness of a positive‐sense plant RNA virus

RNA viruses are the main source of emerging infectious diseases because of the evolutionary potential bestowed by their fast replication, large population sizes and high mutation and recombination rates. However, an equally important property, which is usually neglected, is the topography of the fitness landscape. How many fitness maxima exist and how well they are connected is especially interesting, as this determines the number of accessible evolutionary pathways. To address this question, we have reconstructed a region of the fitness landscape of tobacco etch potyvirus constituted by mutations observed during the experimental adaptation of the virus to the novel host Arabidopsis thaliana. Fitness was measured for many genotypes and showed the existence of multiple peaks and holes in the landscape. We found prevailing epistatic effects between mutations, with cases of reciprocal sign epistasis being common among pairs of mutations. We also found that high‐order epistasis was as important as pairwise epistasis in their contribution to fitness. Therefore, results suggest that the landscape was rugged due to the existence of holes caused by lethal genotypes, that a very limited number of potential neutral paths exist and that it contained a single adaptive peak.     

The fitness effects of synonymous mutations in DNA and RNA viruses

Despite being silent with respect to protein sequence, synonymous nucleotide substitutions can be targeted by natural selection directly at the DNA or RNA level. However, there has been no systematic assessment of how frequent this type of selection is. Here, we have constructed 53 single random synonymous substitution mutants of the bacteriophages Qβ and ΦX174 by site-directed mutagenesis and assayed their fitness. Analysis of this mutant collection and of previous studies undertaken with a variety of single-stranded (ss) viruses demonstrates that selection at synonymous sites is stronger in RNA viruses than in DNA viruses. We estimate that this type of selection contributes approximately 18% of the overall mutational fitness effects in ssRNA viruses under our assay conditions and that random synonymous substitutions have a 5% chance of being lethal to the virus, whereas in ssDNA viruses, these figures drop to 1.4% and 0%, respectively. In contrast, the effects of nonsynonymous substitutions appear to be similar in ssRNA and ssDNA viruses.     

Synonymous and nonsynonymous substitutions in mammalian genes and the nearly neutral theory

The nearly neutral theory of molecular evolution predicts larger generation-time effects for synonymous than for nonsynonymous substitutions. This prediction is tested using the sequences of 49 single-copy genes by calculating the average and variance of synonymous and nonsynonymous substitutions in mammalian star phylogenies (rodentia, artiodactyla, and primates). The average pattern of the 49 genes supports the prediction of the nearly neutral theory, with some notable exceptions.The nearly neutral theory also predicts that the variance of the evolutionary rate is larger than the value predicted by the completely neutral theory. This prediction is tested by examining the dispersion index (ratio of the variance to the mean), which is positively correlated with the average substitution number. After weighting by the lineage effects, this correlation almost disappears for nonsynonymous substitutions, but not quite so for synonymous substitutions. After weighting, the dispersion indices of both synonymous and nonsynonymous substitutions still exceed values expected under the simple Poisson process. The results indicate that both the systematic bias in evolutionary rate among the lineages and the episodic type of rate variation are contributing to the large variance. The former is more significant to synonymous substitutions than to nonsynonymous substitutions. Isochore evolution may be similar to synonymous substitutions. The rate and pattern found here are consistent with the nearly neutral theory, such that the relative contributions of drift and selection differ between the two types of substitutions. The results are also consistent with Gillespie's episodic selection theory.     

Delayed transmission selects for increased survival of vesicular stomatitis virus

Life-history theory predicts that traits for survival and reproduction cannot be simultaneously maximized in evolving populations. For this reason, in obligate parasites such as infectious viruses, selection for improved between-host survival during transmission may lead to evolution of decreased within-host reproduction. We tested this idea using experimental evolution of RNA virus populations, passaged under differing transmission times in the laboratory. A single ancestral genotype of vesicular stomatitis virus (VSV), a negative-sense RNA Rhabdovirus, was used to found multiple virus lineages evolved in either ordinary 24-h cell-culture passage, or in delayed passages of 48 h. After 30 passages (120 generations of viral evolution), we observed that delayed transmission selected for improved extracellular survival, which traded-off with lowered viral fecundity (slower exponential population growth and smaller mean plaque size). To further examine the confirmed evolutionary trade-off, we obtained consensus whole-genome sequences of evolved virus populations, to infer phenotype–genotype associations. Results implied that increased virus survival did not occur via convergence; rather, improved virion stability was gained via independent mutations in various VSV structural proteins. Our study suggests that RNA viruses can evolve different molecular solutions for enhanced survival despite their limited genetic architecture, but suffer generalized reproductive trade-offs that limit overall fitness gains.     

抗体选择压作用下H9N2亚型禽流感病毒HA基因的变异

摘要：【目的】了解H9N2亚型禽流感病毒(AIV)在抗体选择压作用下的遗传变异。【方法】将制备疫苗用的LG1株H9N2亚型AIV分别接种含有母源抗体鸡胚(A组)和不含有母源抗体的SPF鸡胚(B组),并连续传代。其中A组再分为4 个独立的传代系列A1-4,B组再分为2 个独立传代系列B1-2。在每个传代系列,分别对第10,20,30,40,50 代病毒的HA基因进行扩增克隆测序,并与原始病毒的序列比较。【结果】LG1株H9N2在没有抗体的鸡胚的传代过程中,仅发生少数碱基的不稳定随机变异,且多为无义突变。在2 个传代系列的10 个代次病毒,共出现了29 个位点变异,有义突变(NS)与无义突变(S)比值NS/S为1.42 。但在有抗体的鸡胚的传代过程中,发生了多个呈现稳定遗传的有义突变。在4 个传代系列的20 个代次病毒,共出现了45 个位点变异,有义突变(NS)与无义突变(S)比值NS/S为3.46。【结论】在鸡胚传代过程中母源抗体提供的免疫选择压确实能影响H9N2的HA基因的变异。同时表明,带有母源抗体的鸡胚是实验室条件下研究免疫选择压对病毒抗原性变异影响的一种有效的实验模型。     

The Structural Determinants of Intra-Protein Compensatory Substitutions

Compensatory substitutions happen when one mutation is advantageously selected because it restores the loss of fitness induced by a previous deleterious mutation. How frequent such mutations occur in evolution and what is the structural and functional context permitting their emergence remain open questions. We built an atlas of intra-protein compensatory substitutions using a phylogenetic approach and a dataset of 1,630 bacterial protein families for which high-quality sequence alignments and experimentally derived protein structures were available. We identified more than 51,000 positions coevolving by the mean of predicted compensatory mutations. Using the evolutionary and structural properties of the analyzed positions, we demonstrate that compensatory mutations are scarce (typically only a few in the protein history) but widespread (the majority of proteins experienced at least one). Typical coevolving residues are evolving slowly, are located in the protein core outside secondary structure motifs, and are more often in contact than expected by chance, even after accounting for their evolutionary rate and solvent exposure. An exception to this general scheme is residues coevolving for charge compensation, which are evolving faster than noncoevolving sites, in contradiction with predictions from simple coevolutionary models, but similar to stem pairs in RNA. While sites with a significant pattern of coevolution by compensatory mutations are rare, the comparative analysis of hundreds of structures ultimately permits a better understanding of the link between the three-dimensional structure of a protein and its fitness landscape.     

A likelihood-based index of protein protein binding affinities with application to influenza HA escape from antibodies

In many biological systems, proteins interact with other organic molecules to produce indispensable functions, in which molecular recognition phenomena are essential. Proteins have kept or gained their functions during molecular evolution. Their functions seem to be flexible, and a few amino acid substitutions sometimes cause drastic changes in function. In order to monitor and predict such drastic changes in the early stages in target populations, we need to identify patterns of structural changes during molecular evolution causing decreases or increases in the binding affinity of protein complexes. In previous work, we developed a likelihood-based index to quantify the degree to which a sequence fits a given structure. This index was named the sequence-structure fitness (SSF) and is calculated empirically based on amino acid preferences and pairwise interactions in the structural environment present in template structures. In the present work, we used the SSF to develop an index to measure the binding affinity of protein-protein complexes defined as the log likelihood ratio, contrasting the fitness of the sequences to the structure of the complex and that of the uncomplexed proteins. We applied the developed index to the complexes formed between influenza A hemagglutinin (HA) and four antibodies. The antibody-antigen binding region of HA is under strong selection pressure by the host immune system. Hence, examination of the long-term adaptation of HA to the four antibodies could reveal the strategy of the molecular evolution of HA. Two antibodies cover the HA receptor-binding region, while the other two bind away from the receptor-binding region. By focusing on branches with a significant decline in binding ability, we could detect key amino acid replacements and investigate the mechanism via conditional probabilities. The contrast between the adaptations to the two types of antibodies suggests that the virus adapts to the immune system at the cost of structural change.     

Slow fitness recovery in a codon-modified viral genome

Extensive synonymous codon modification of viral genomes appears to be an effective way of attenuating strains for use as live vaccines. An assumption of this method is that codon changes have individually small effects, such that codon-attenuated viruses will be slow to evolve back to high fitness (and thus to high virulence). The major capsid gene of the bacterial virus T7 was modified to have varying levels of suboptimal synonymous codons in different constructs, and fitnesses declined linearly with the number of changes. Adaptation of the most extreme design, with 182 codon changes, resulted in a slow fitness recovery by standards of previous experimental evolution with this virus, although fitness effects of substitutions were higher than expected from the average effect of an engineered codon modification. Molecular evolution during recovery was modest, and changes evolved both within the modified gene and outside it. Some changes within the modified gene evolved in parallel across replicates, but with no obvious explanation. Overall, the study supports the premise that codon-modified viruses recover fitness slowly, although the evolution is substantially more rapid than expected from the design principle.