Evolution of mutational robustness in an RNA virus

Mutational (genetic) robustness is phenotypic constancy in the face of mutational changes to the genome. Robustness is critical to the understanding of evolution because phenotypically expressed genetic variation is the fuel of natural selection. Nonetheless, the evidence for adaptive evolution of mutational robustness in biological populations is controversial. Robustness should be selectively favored when mutation rates are high, a common feature of RNA viruses. However, selection for robustness may be relaxed under virus co-infection because complementation between virus genotypes can buffer mutational effects. We therefore hypothesized that selection for genetic robustness in viruses will be weakened with increasing frequency of co-infection. To test this idea, we used populations of RNA phage φ6 that were experimentally evolved at low and high levels of co-infection and subjected lineages of these viruses to mutation accumulation through population bottlenecking. The data demonstrate that viruses evolved under high co-infection show relatively greater mean magnitude and variance in the fitness changes generated by addition of random mutations, confirming our hypothesis that they experience weakened selection for robustness. Our study further suggests that co-infection of host cells may be advantageous to RNA viruses only in the short term. In addition, we observed higher mutation frequencies in the more robust viruses, indicating that evolution of robustness might foster less-accurate genome replication in RNA viruses.     

Epistasis and its relationship to canalization in the RNA virus phi 6

Although deleterious mutations are believed to play a critical role in evolution, assessing their realized effect has been difficult. A key parameter governing the effect of deleterious mutations is the nature of epistasis, the interaction between the mutations. RNA viruses should provide one of the best systems for investigating the nature of epistasis because the high mutation rate allows a thorough investigation of mutational effects and interactions. Nonetheless, previous investigations of RNA viruses by S. Crotty and co-workers and by S. F. Elena have been unable to detect a significant effect of epistasis. Here we provide evidence that positive epistasis is characteristic of deleterious mutations in the RNA bacteriophage phi 6. We estimated the effects of deleterious mutations by performing mutation-accumulation experiments on five viral genotypes of decreasing fitness. We inferred positive epistasis because viral genotypes with low fitness were found to be less sensitive to deleterious mutations. We further examined environmental sensitivity in these genotypes and found that low-fitness genotypes were also less sensitive to environmental perturbations. Our results suggest that even random mutations impact the degree of canalization, the buffering of a phenotype against genetic and environmental perturbations. In addition, our results suggest that genetic and environmental canalization have the same developmental basis and finally that an understanding of the nature of epistasis may first require an understanding of the nature of canalization.     

Sex and the evolution of intrahost competition in RNA virus phi6.

Sex allows beneficial mutations that occur in separate lineages to be fixed in the same genome. For this reason, the Fisher-Muller model predicts that adaptation to the environment is more rapid in a large sexual population than in an equally large asexual population. Sexual reproduction occurs in populations of the RNA virus phi6 when multiple bacteriophages coinfect the same host cell. Here, we tested the model''s predictions by determining whether sex favors more rapid adaptation of phi6 to a bacterial host, Pseudomonas phaseolicola. Replicate populations of phi6 were allowed to evolve in either the presence or absence of sex for 250 generations. All experimental populations showed a significant increase in fitness relative to the ancestor, but sex did not increase the rate of adaptation. Rather, we found that the sexual and asexual treatments also differ because intense intrahost competition between viruses occurs during coinfection. Results showed that the derived sexual viruses were selectively favored only when coinfection is common, indicating that within-host competition detracts from the ability of viruses to exploit the host. Thus, sex was not advantageous because the cost created by intrahost competition was too strong. Our findings indicate that high levels of coinfection exceed an optimum where sex may be beneficial to populations of phi6, and suggest that genetic conflicts can evolve in RNA viruses.     

Antagonistic coevolution with parasites increases the cost of host deleterious mutations

The fitness consequences of deleterious mutations are sometimes greater when individuals are parasitized, hence parasites may result in the more rapid purging of deleterious mutations from host populations. The significance of host deleterious mutations when hosts and parasites antagonistically coevolve (reciprocal evolution of host resistance and parasite infectivity) has not previously been experimentally investigated. We addressed this by coevolving the bacterium Pseudomonas fluorescens and a parasitic bacteriophage in laboratory microcosms, using bacteria with high and low mutation loads. Directional coevolution between bacterial resistance and phage infectivity occurred in all populations. Bacterial population fitness, as measured by competition experiments with ancestral genotypes in the absence of phage, declined with time spent coevolving. However, this decline was significantly more rapid in bacteria with high mutation loads, suggesting the cost of bacterial resistance to phage was greater in the presence of deleterious mutations (synergistic epistasis). As such, resistance to phage was more costly to evolve in the presence of a high mutation load. Consistent with these data, bacteria with high mutation loads underwent less rapid directional coevolution with their phage populations, and showed lower levels of resistance to their coevolving phage populations. These data suggest that coevolution with parasites increases the rate at which deleterious mutations are purged from host populations.     

Complementation and Epistasis in Viral Coinfection Dynamics

Coinfection in RNA virus populations results in two important phenomena, complementation and recombination. Of the two, complementation has a strong effect on selection against deleterious mutations, as has been confirmed in earlier studies. As complementation delays the purging of less-fit mutations, coinfection may be detrimental to the evolution of a virus population. Here we employ both deterministic modeling and stochastic simulation to explore the mechanisms underlying the interactions between complementation and other evolutionary factors, namely, mutation, selection, and epistasis. We find that strong complementation reduces slightly the overall fitness of a virus population but substantially enhances its diversity and robustness, especially when interacting with selection and epistasis.     

Reduced fecundity is the cost of cheating in RNA virus phi6

Co-infection by multiple viruses affords opportunities for the evolution of cheating strategies to use intracellular resources. Cheating may be costly, however, when viruses infect cells alone. We previously allowed the RNA bacteriophage phi6 to evolve for 250 generations in replicated environments allowing co-infection of Pseudomonas phaseolicola bacteria. Derived genotypes showed great capacity to compete during co-infection, but suffered reduced performance in solo infections. Thus, the evolved viruses appear to be cheaters that sacrifice between-host fitness for within-host fitness. It is unknown, however, which stage of the lytic growth cycle is linked to the cost of cheating. Here, we examine the cost through burst assays, where lytic infection can be separated into three discrete phases (analogous to phage life history): dispersal stage, latent period (juvenile stage), and burst (adult stage). We compared growth of a representative cheater and its ancestor in environments where the cost occurs. The cost of cheating was shown to be reduced fecundity, because cheaters feature a significantly smaller burst size (progeny produced per infected cell) when infecting on their own. Interestingly, latent period (average burst time) of the evolved virus was much longer than that of the ancestor, indicating the cost does not follow a life history trade-off between timing of reproduction and lifetime fecundity. Our data suggest that interference competition allows high fitness of derived cheaters in mixed infections, and we discuss preferential encapsidation as one possible mechanism.     

Parasites and mutational load: an experimental test of a pluralistic theory for the evolution of sex

Ecological and mutational explanations for the evolution of sexual reproduction have usually been considered independently. Although many of these explanations have yielded promising theoretical results,experimental support for their ability to overcome a twofold cost of sex has been limited. For this reason, it has recently been argued that a pluralistic approach, combining effects from multiple models, may be necessary to explain the apparent advantage of sex. One such pluralistic model proposes that parasite load and synergistic epistasis between deleterious mutations might interact to create an advantage for recombination.Here, we test this proposal by comparing the fitness functions of parasitized and parasite-free genotypes of Escherichia coli bearing known numbers of transposon-insertion mutations. In both classes, we failed to detect any evidence for synergistic epistasis. However, the average effect of deleterious mutations was greater in parasitized than parasite-free genotypes. This effect might broaden the conditions under which another proposed model combining parasite-host coevolutionary dynamics and mutation accumulation can explain the maintenance of sex. These results suggest that, on average, deleterious mutations act multiplicatively with each other but in synergy with infection in determining fitness.     

Evolution can favor antagonistic epistasis

The accumulation of deleterious mutations plays a major role in evolution, and key to this are the interactions between their fitness effects, known as epistasis. Whether mutations tend to interact synergistically (with multiple mutations being more deleterious than would be expected from their individual fitness effects) or antagonistically is important for a variety of evolutionary questions, particularly the evolution of sex. Unfortunately, the experimental evidence on the prevalence and strength of epistasis is mixed and inconclusive. Here we study theoretically whether synergistic or antagonistic epistasis is likely to be favored by evolution and by how much. We find that in the presence of recombination, evolution favors less synergistic or more antagonistic epistasis whenever mutations that change the epistasis in this direction are possible. This is because evolution favors increased buffering against the effects of deleterious mutations. This suggests that we should not expect synergistic epistasis to be widespread in nature and hence that the mutational deterministic hypothesis for the advantage of sex may not apply widely.     

Human immunodeficiency virus evolution towards reduced replicative fitness in vivo and the development of AIDS

Human immunodeficiency virus (HIV) infection progresses to AIDS following an asymptomatic period during which the virus is thought to evolve towards increased fitness and pathogenicity. We show mathematically that progression to the strongest HIV-induced pathology requires evolution of the virus towards reduced replicative fitness in vivo. This counter-intuitive outcome can happen if multiple viruses co-infect the same cell frequently, which has been shown to occur in recent experiments. According to our model, in the absence of frequent co-infection, the less fit AIDS-inducing strains might never emerge. The frequency of co-infection can correlate with virus load, which in turn is determined by immune responses. Thus, at the beginning of infection when immunity is strong and virus load is low, co-infection is rare and pathogenic virus variants with reduced replicative fitness go extinct. At later stages of infection when immunity is less efficient and virus load is higher, co-infection occurs more frequently and pathogenic virus variants with reduced replicative fitness can emerge, resulting in T-cell depletion. In support of these notions, recent data indicate that pathogenic simian immunodeficiency virus (SIV) strains occurring late in the infection are less fit in specific in vitro experiments than those isolated at earlier stages. If co-infection is blocked, the model predicts the absence of any disease even if virus loads are high. We hypothesize that non-pathogenic SIV infection within its natural hosts, which is characterized by the absence of disease even in the presence of high virus loads, could be explained by a reduced occurrence of co-infection in this system.     

Testing for epistasis between deleterious mutations in a parasitoid wasp

Abstract.— Determining the way in which deleterious mutations interact to effect fitness is crucial to numerous areas in evolutionary biology. For example, if each additional mutation leads to a greater decrease in log fitness than the last, termed synergistic epistasis, then sex and recombination provide an advantage because they enable deleterious mutations to be eliminated more efficiently. However, there is a severe shortage of relevant empirical data, especially of the form that can help test mutational explanations for the widespread occurrence of sex. Here, we test for epistasis in the parasitic wasp Nasonia vitripennis , examining the fitness consequences of chemically induced deleterious mutations. We examine two components of fitness, both of which are thought to be important in natural populations of parasitic wasps: longevity and egg production. Our results show synergistic epistasis for longevity, but not for egg production.     

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.     

Evolution of sex in RNA viruses

The distribution of deleterious mutations in a population of organisms is determined by the opposing effects of two forces, mutation pressure and selection. If mutation rates are high, the resulting mutation-selection balance can generate a substantial mutational load in the population. Sex can be advantageous to organisms experiencing high mutation rates because it can either buffer the mutation-selection balance from genetic drift, thus preventing any increases in the mutational load (Muller, 1964: Mut. Res. 1, 2), or decrease the mutational load by increasing the efficiency of selection (Crow, 1970: Biomathematics 1, 128). Muller's hypothesis assumes that deleterious mutations act independently, whereas Crow's hypothesis assumes that deleterious mutations interact synergistically, i.e., the acquisition of a deleterious mutation is proportionately more harmful to a genome with many mutations than it is to a genome with a few mutations. RNA viruses provide a test for these two hypotheses because they have extremely high mutation rates and appear to have evolved specific adaptations to reproduce sexually. Population genetic models for RNA viruses show that Muller's and Crow's hypotheses are also possible explanations for why sex is advantageous to these viruses. A re-analysis of published data on RNA viruses that are cultured by undiluted passage suggests that deleterious mutations in such viruses interact synergistically and that sex evolved there as a mechanism to reduce the mutational load.     

Evaluating the within-host fitness effects of mutations fixed during virus adaptation to different ecotypes of a new host

The existence of genetic variation for resistance in host populations is assumed to be essential to the spread of an emerging virus. Models predict that the rate of spread slows down with the increasing frequency and higher diversity of resistance alleles in the host population. We have been using the experimental pathosystem Arabidopsis thaliana—tobacco etch potyvirus (TEV) to explore the interplay between genetic variation in host''s susceptibility and virus diversity. We have recently shown that TEV populations evolving in A. thaliana ecotypes that differ in susceptibility to infection gained within-host fitness, virulence and infectivity in a manner compatible with a gene-for-gene model of host–parasite interactions: hard-to-infect ecotypes were infected by generalist viruses, whereas easy-to-infect ecotypes were infected by every virus. We characterized the genomes of the evolved viruses and found cases of host-driven convergent mutations. To gain further insights in the mechanistic basis of this gene-for-gene model, we have generated all viral mutations individually as well as in specific combinations and tested their within-host fitness effects across ecotypes. Most of these mutations were deleterious or neutral in their local ecotype and only a very reduced number had a host-specific beneficial effect. We conclude that most of the mutations fixed during the evolution experiment were so by drift or by selective sweeps along with the selected driver mutation. In addition, we evaluated the ruggedness of the underlying adaptive fitness landscape and found that mutational effects were mostly multiplicative, with few cases of significant epistasis.     

Fitness ranking of individual mutants drives patterns of epistatic interactions in HIV-1

Fitness interactions between mutations, referred to as epistasis, can strongly impact evolution. For RNA viruses and retroviruses with their high mutation rates, epistasis may be particularly important to overcome fitness losses due to the accumulation of deleterious mutations and thus could influence the frequency of mutants in a viral population. As human immunodeficiency virus type 1 (HIV-1) resistance to azidothymidine (AZT) requires selection of sequential mutations, it is a good system to study the impact of epistasis. Here we present a thorough analysis of a classical AZT-resistance pathway (the 41-215 cluster) of HIV-1 variants by fitness measurements in single round infection assays covering physiological drug concentrations ex vivo. The sign and value of epistasis varied and did not predict the epistatic effect on the mutant frequency. This complex behavior is explained by the fitness ranking of the variants that strongly depends on environmental factors, i.e., the presence and absence of drugs and the host cells used. Although some interactions compensate fitness losses, the observed small effect on the relative mutant frequencies suggests that epistasis might be inefficient as a buffering mechanism for fitness losses in vivo. While the use of epistasis-based hypotheses to make general assumptions on the evolutionary dynamics of viral populations is appealing, our data caution their interpretation without further knowledge on the characteristics of the viral mutant spectrum under different environmental conditions.     

MUTATION AND EXTINCTION: THE ROLE OF VARIABLE MUTATIONAL EFFECTS,SYNERGISTIC EPISTASIS,BENEFICIAL MUTATIONS,AND DEGREE OF OUTCROSSING

Recent theoretical studies have illustrated the potential role of spontaneous deleterious mutation as a cause of extinction in small populations. However, these studies have not addressed several genetic issues, which can in principle have a substantial influence on the risk of extinction. These include the presence of synergistic epistasis, which can reduce the rate of mutation accumulation by progressively magnifying the selective effects of mutations, and the occurrence of beneficial mutations, which can offset the effects of previous deleterious mutations. In stochastic simulations of small populations (effective sizes on the order of 100 or less), we show that both synergistic epistasis and the rate of beneficial mutation must be unrealistically high to substantially reduce the risk of extinction due to random fixation of deleterious mutations. However, in analytical calculations based on diffusion theory, we show that in large, outcrossing populations (effective sizes greater than a few hundred), very low levels of beneficial mutation are sufficient to prevent mutational decay. Further simulation results indicate that in populations small enough to be highly vulnerable to mutational decay, variance in deleterious mutational effects reduces the risk of extinction, assuming that the mean deleterious mutational effect is on the order of a few percent or less. We also examine the magnitude of outcrossing that is necessary to liberate a predominantly selfing population from the threat of long-term mutational deterioration. The critical amount of outcrossing appears to be greater than is common in near-obligately selfing plant species, supporting the contention that such species are generally doomed to extinction via random drift of new mutations. Our results support the hypothesis that a long-term effective population size in the neighborhood of a few hundred individuals defines an approximate threshold, below which outcrossing populations are vulnerable to extinction via fixation of deleterious mutations, and above which immunity is acquired.     

Epistasis among Deleterious Mutations in the HIV-1 Protease

A central goal in molecular evolution is to understand how genetic interactions between protein mutations shape protein function and fitness. While intergenic epistasis has been extensively explored in eukaryotes, bacteria, and viruses, intragenic epistatic interactions have been insufficiently studied. Here, we employ a model system in which lambda phage fitness correlates with the enzymatic activity of human immunodeficiency virus type 1 (HIV-1) protease to systematically determine the epistatic interactions between intragenic pairs of deleterious protein substitutions. We generated 114 genotypes of the HIV-1 protease, each carrying pairs of nucleotide substitution mutations whose separated and combined deleterious effects on fitness were then determined. A high proportion (39%) of pairs displayed lethality. Several pairs exhibited significant interactions for fitness, including positive and negative epistasis. Significant negative epistatic interactions predominated (15%) over positive interactions (2%). However, the average ± SD epistatic effect, ē = 0.0025 ± 0.1334, was not significantly different from zero (p = 0.8368). Notably, epistatic interactions, regardless of epistatic direction, tend to be more frequent in the context of less deleterious mutations. In the present study, the high frequencies of lethality and negative epistasis indicate that the HIV-1 protease is highly sensitive to the effects of deleterious mutations. Therefore, proteins may not be as robust to mutational change as is usually expected.     

Epistasis and the adaptability of an RNA virus

We have explored the patterns of fitness recovery in the vesicular stomatitis RNA virus. We show that, in our experimental setting, reversions to the wild-type genotype were rare and fitness recovery was at least partially driven by compensatory mutations. We compared compensatory adaptation for genotypes carrying (1) mutations with varying deleterious fitness effects, (2) one or two deleterious mutations, and (3) pairs of mutations showing differences in the strength and sign of epistasis. In all cases, we found that the rate of fitness recovery and the proportion of reversions were positively affected by population size. Additionally, we observed that mutations with large fitness effect were always compensated faster than mutations with small fitness effect. Similarly, compensatory evolution was faster for genotypes carrying a single deleterious mutation than for those carrying pairs of mutations. Finally, for genotypes carrying two deleterious mutations, we found evidence of a negative correlation between the epistastic effect and the rate of compensatory evolution.     

A network model for the correlation between epistasis and genomic complexity

The study of genetic interactions (epistasis) is central to the understanding of genome organization and evolution. A general correlation between epistasis and genomic complexity has been recently shown, such that in simpler genomes epistasis is antagonistic on average (mutational effects tend to cancel each other out), whereas a transition towards synergistic epistasis occurs in more complex genomes (mutational effects strengthen each other). Here, we use a simple network model to identify basic features explaining this correlation. We show that, in small networks with multifunctional nodes, lack of redundancy, and absence of alternative pathways, epistasis is antagonistic on average. In contrast, lack of multi-functionality, high connectivity, and redundancy favor synergistic epistasis. Moreover, we confirm the previous finding that epistasis is a covariate of mutational robustness: in less robust networks it tends to be antagonistic whereas in more robust networks it tends to be synergistic. We argue that network features associated with antagonistic epistasis are typically found in simple genomes, such as those of viruses and bacteria, whereas the features associated with synergistic epistasis are more extensively exploited by higher eukaryotes.     

Sexual selection and its effect on the fixation of an asexual clone

Sexual selection is a powerful and ubiquitous force in sexual populations. It has recently been argued that sexual selection can eliminate the twofold cost of sex even with low genomic mutation rates. By means of differential male mating success, deleterious mutations in males become more deleterious than in females, and it has been shown that sexual selection can drastically reduce the mutational load in a sexual population, with or without any form of epistasis. However, any mechanism that claims to maintain sexual reproduction must be able to prevent the fixation of an asexual mutant clone with a twofold fitness advantage. Here, I show that despite very strong sexual selection, the fixation of an asexual mutant cannot be prevented under reasonable genomic mutation rates. Sexual selection can have a strong effect on the average mutational load in a sexual population, but as it cannot prevent the fixation of an asexual mutant, it is unlikely to play a key role on the maintenance of sexual reproduction.     

A phylogenetic method for detecting positive epistasis in gene sequences and its application to RNA virus evolution

RNA virus genomes are compact, often containing multiple overlapping reading frames and functional secondary structure. Consequently, it is thought that evolutionary interactions between nucleotide sites are commonplace in the genomes of these infectious agents. However, the role of epistasis in natural populations of RNA viruses remains unclear. To investigate the pervasiveness of epistasis in RNA viruses, we used a parsimony-based computational method to identify pairs of co-occurring mutations along phylogenies of 177 RNA virus genes. This analysis revealed widespread evidence for positive epistatic interactions at both synonymous and nonsynonymous nucleotide sites and in both clonal and recombining viruses, with the majority of these interactions spanning very short sequence regions. These findings have important implications for understanding the key aspects of RNA virus evolution, including the dynamics of adaptation. Additionally, many comparative analyses that utilize the phylogenetic relationships among gene sequences assume that mutations represent independent, uncorrelated events. Our results show that this assumption may often be invalid.