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Evolution of fast mutating replicators—RNA viruses and the RNA world
Abstract
In the last two decades, viruses have become the model system to witness evolution in the laboratory. Large population sizes, high mutation rates, and short generation times are the three features that permit to carry out in vitro experiments under controlled conditions. In this contribution we briefly review a number of recent experiments that open new prospectives in our understanding of molecular evolutionary mechanisms, in their dependence with population dynamics and quasispecies organization, and in the interaction between heterogeneous populations and the environment. One of the possible origins of RNA viruses is a hypothetical RNA world, previous to our present DNA world, where information coding and catalytic functions would be simultaneously performed by RNA molecules.
... Moreover, when genotype and phenotype are decoupled in models of evolution, it turns out that the most abundant sequence does not necessarily coincide with the master sequence, i.e. the fittest sequence, as defined in quasispecies theory. This agrees with experimental observations demonstrating that even if a population accumulates mutations steadily through bottleneck events, its fitness can achieve high values comparable to those of an optimized initial population [16,39]. That is to say, there are many different genotypes which yield comparable levels of biological performance. ...
RNA molecules, through their dual appearance as sequence and structure, represent a suitable model to study evolutionary properties of quasispecies. The essential ingredient in this model is the differentiation between genotype (molecular sequences which are affected by mutation) and phenotype (molecular structure, affected by selection). This framework allows a quantitative analysis of organizational properties of quasispecies as they adapt to different environments, such as their robustness, the effect of the degeneration of the sequence space, or the adaptation under different mutation rates and the error threshold associated.
We describe and analyze the structural properties of molecular quasispecies adapting to different environments both during the transient time before adaptation takes place and in the asymptotic state, once optimization has occurred. We observe a minimum in the adaptation time at values of the mutation rate relatively far from the phenotypic error threshold. Through the definition of a consensus structure, it is shown that the quasispecies retains relevant structural information in a distributed fashion even above the error threshold. This structural robustness depends on the precise shape of the secondary structure used as target of selection. Experimental results available for natural RNA populations are in qualitative agreement with our observations.
Adaptation time of molecular quasispecies to a given environment is optimized at values of the mutation rate well below the phenotypic error threshold. The optimal value results from a trade-off between diversity generation and fixation of advantageous mutants. The critical value of the mutation rate is a function not only of the sequence length, but also of the specific properties of the environment, in this case the selection pressure and the shape of the secondary structure used as target phenotype. Certain functional motifs of RNA secondary structure that withstand high mutation rates (as the ubiquitous hairpin motif) might appear early in evolution and be actually frozen evolutionary accidents.
Dispersal mechanisms play a main role in the dynamics of infection spread. Recent experimental results with in vitro infections of foot-and-mouth disease virus reveal that the time needed for the virus to kill a cellular monolayer depends qualitatively on the number of viral particles required to initiate infection in a susceptible cell. A two-dimensional susceptible-infected-removed (SIR) model based on the experimental setting agrees with the observations only when viral particles are subject to long-range transport. Numerical and analytical results show that this long-range transport plays a role when a single particle causes infection, while it is inefficient when complementation between two or more particles is necessary.
We explore a mean-field model for the evolution of exponentially growing populations of mutating replicators. Motivated by recent in vitro experiments devised to analyze phenotypic properties of bacterial and viral populations subjected to serial population transfers, we allow our in silico individuals to undergo unrestricted growth before applying bottleneck events. Different dynamical regimes of our model can be mapped to different experimental situations. Numerical and analytical results for fitness distributions calculated at the statistically stationary states of the dynamics compare favorably with available experimental data. Our model and results provide a common framework to better understand populations evolving under different selection pressures.
RNA viruses display high mutation rates and their populations replicate as dynamic and complex mutant distributions, termed viral quasispecies. Repeated genetic bottlenecks, which experimentally are carried out through serial plaque-to-plaque transfers of the virus, lead to fitness decrease (measured here as diminished capacity to produce infectious progeny). Here we report an analysis of fitness evolution of several low fitness foot-and-mouth disease virus clones subjected to 50 plaque-to-plaque transfers. Unexpectedly, fitness decrease, rather than being continuous and monotonic, displayed a fluctuating pattern, which was influenced by both the virus and the state of the host cell as shown by effects of recent cell passage history. The amplitude of the fluctuations increased as fitness decreased, resulting in a remarkable resistance of virus to extinction. Whereas the frequency distribution of fitness in control (independent) experiments follows a log-normal distribution, the probability of fitness values in the evolving bottlenecked populations fitted a Weibull distribution. We suggest that multiple functions of viral genomic RNA and its encoded proteins, subjected to high mutational pressure, interact with cellular components to produce this nontrivial, fluctuating pattern.
Passage of foot-and-mouth disease virus (FMDV) in cell culture resulted in the generation of defective RNAs that were infectious
by complementation. Deletions (of nucleotides 417, 999, and 1017) mapped in the L proteinase and capsid protein-coding regions.
Cell killing followed two-hit kinetics, defective genomes were encapsidated into separate viral particles, and individual
viral plaques contained defective genomes with no detectable standard FMDV RNA. Infection in the absence of standard FMDV
RNA was achieved by cotransfection of susceptible cells with transcripts produced in vitro from plasmids encoding the defective
genomes. These results document the first step of an evolutionary transition toward genome segmentation of an unsegmented
RNA virus and provide an experimental system to compare rates of RNA progeny production and resistance to enhanced mutagenesis
of a segmented genome versus its unsegmented counterpart.
RNA viruses replicate with a very high error rate and give rise to heterogeneous, highly plastic populations able to adapt very rapidly to changing environments. Viral diseases are thus difficult to control because of the appearance of drug-resistant mutants, and it becomes essential to seek mechanisms able to force the extinction of the quasispecies before adaptation emerges. An alternative to the use of conventional drugs consists in increasing the replication error rate through the use of mutagens. Here, we report about persistent infections of lymphocytic choriomeningitis virus treated with fluorouracil, where a progressive debilitation of infectivity leading to eventual extinction occurs. The transition to extinction is accompanied by the production of large amounts of RNA, indicating that the replicative ability of the quasispecies is not strongly impaired by the mutagen. By means of experimental and theoretical approaches, we propose that a fraction of the RNA molecules synthesized can behave as a defective subpopulation able to drive the viable class extinct. Our results lead to the identification of two extinction pathways, one at high amounts of mutagen, where the quasispecies completely loses its ability to infect and replicate, and a second one, at lower amounts of mutagen, where replication continues while the infective class gets extinct because of the action of defectors. The results bear on a potential application of increased mutagenesis as an antiviral strategy in that low doses of a mutagenic agent may suffice to drive persistent virus to extinction.
• error catastrophe
• mutagen
• RNA virus
• replicative ability
• mathematical model
In the last two decades, viruses have been used as model systems to study evolution in short periods of time. Due to their characteristics, virus adapt rapidly to changing conditions, thus allowing the quantification of several evolutionary features under controlled laboratory conditions. Here we review the basic biology of viruses and describe in detail a number of experiments performed with RNA viruses. Particular emphasis is devoted to the interpretation of the experiments and to the involved phenomenology. This analysis sometimes represents the basis to formulate simple evolutionary models that aim at describing the observed dynamics. In other cases, theoretical results have prompted the realization of related experiments, as we discuss. Concepts as fitness loss and fitness recovery, the error threshold, increased mutagenesis, viral sex, or viral competition and interference, are discussed in an empirical framework and from the associated theoretical point of view.
To distinguish continuous from discontinuous evolutionary change, a relation of nearness between phenotypes is needed. Such
a relation is based on the probability of one phenotype being accessible from another through changes in the genotype. This
nearness relation is exemplified by calculating the shape neighborhood of a transfer RNA secondary structure and provides
a characterization of discontinuous shape transformations in RNA. The simulation of replicating and mutating RNA populations
under selection shows that sudden adaptive progress coincides mostly, but not always, with discontinuous shape transformations.
The nature of these transformations illuminates the key role of neutral genetic drift in their realization.
The nucleotide sequence of 32P-RNA from Q beta phage clones was sampled by two-dimensional polyacrylamide gel electrophoresis of the RNAase T1-resistant oligonucleotides (T1 fingerprinting). About 15% of the clones derived from a multiply passaged Q beta population showed fingerprint patterns which deviated from that of the RNA from the total population. All deviations examined could be attributed to one and, less frequently, to two or more nucleotide transitions. Since the fingerprinting technique allows the analysis of only about 10% of the RNA sequence, we estimate that each viable phage genome in a multiply passaged population differs in one to two positions from the "average" sequence of the parental population. Several deviant clones were tested by growth competition against a "wildtype" population, after 10-20 generations, the resulting phage showed the "wild-type" T1 fingerprint pattern. We propose that a Q beta phage population is in a dynamic equilibrium, with viable mutants arising at a high rate (Batschelet, Domingo and Weissmann, 1976; Domingo, Flavell and Weissmann, 1976) on the one hand, and being strongly selected against on the other. The genome of Q beta phage cannot be described as a defined unique structure, but rather as a weighted average of a large number of different individual sequences.
All life that is known to exist on Earth today and all life for which there is evidence in the geological record seems to be of the same form--one based on DNA genomes and protein enzymes. Yet there are strong reasons to conclude that DNA- and protein-based life was preceded by a simpler life form based primarily on RNA. This earlier era is referred to as the 'RNA world', during which the genetic information resided in the sequence of RNA molecules and the phenotype derived from the catalytic properties of RNA.
The folding of RNA sequences into secondary structures is a simple yet biophysically grounded model of a genotype-phenotype map. Its computational and mathematical analysis has uncovered a surprisingly rich statistical structure characterized by shape space covering, neutral networks and plastogenetic congruence. I review these concepts and discuss their evolutionary implications.
The method of calculation is shown wherebt a formula has been derived that approximately the ratio of the rate of accumulation of advantageous mutant genes in a population that undergoes recombination to the rate in an otherwise non-recombining one. A table is given showing the ratios thus found for different frequencies of advantageous mutations and different degrees of their advantage. It is shown that this calculation does not apply for mutant genes that act advantageously only when in some special combinations with one or more other mutant genes, and that as far as these cases of special synergism are concerned recombining lines have no evolutionary advantage over non-recombining ones. Other limitations of the formula are pointed out and assessed.