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.     

Evolution of mutational robustness

We review recent advances in the understanding of the mutation-selection balance of asexual replicators. For over 30 years, population geneticists thought that an expression derived by Kimura and Maruyama in 1966 fully solved this problem. However, Kimura and Maruyama's result is only correct in the absence of neutral mutations. The inclusion of neutral mutations leads to a wealth of interesting new effects, and, in particular, to a selective pressure to evolve robustness against mutations. We cover recent literature on the population dynamics of asexual replicators on networks of neutral genotypes, on the outcompetition of fast replicators by slower ones with better mutational support, and on the probability of fixation at high mutation rates. We discuss empirical evidence for the evolution of mutational robustness, and speculate on its relevance for higher organisms.     

Distributed robustness versus redundancy as causes of mutational robustness

A biological system is robust to mutations if it continues to function after genetic changes in its parts. Such robustness is pervasive on different levels of biological organization, from macromolecules to genetic networks and whole organisms. I here ask which of two possible causes of such robustness are more important on a genome-wide scale, for systems whose parts are genes, such as metabolic and genetic networks. The first of the two causes is redundancy of a system's parts: A gene may be dispensable if the genome contains redundant, back-up copies of the gene. The second cause, distributed robustness, is more poorly understood. It emerges from the distributed nature of many biological systems, where many (and different) parts contribute to system functions. I will here discuss evidence suggesting that distributed robustness is equally or more important for mutational robustness than gene redundancy. This evidence comes from the functional divergence of redundant genes, as well as from large-scale gene deletion studies. I also ask whether one can quantify the extent to which redundancy or distributed robustness contribute to mutational robustness.     

Recombination and the evolution of mutational robustness

Mutational robustness is the degree to which a phenotype, such as fitness, is resistant to mutational perturbations. Since most of these perturbations will tend to reduce fitness, robustness provides an immediate benefit for the mutated individual. However, robust systems decay due to the accumulation of deleterious mutations that would otherwise have been cleared by selection. This decay has received very little theoretical attention. At equilibrium, a population or asexual lineage is expected to have a mutation load that is invariant with respect to the selection coefficient of deleterious alleles, so the benefit of robustness (at the level of the population or asexual lineage) is temporary. However, previous work has shown that robustness can be favoured when robustness loci segregate independently of the mutating loci they act upon. We examine a simple two-locus model that allows for intermediate rates of recombination and inbreeding to show that increasing the effective recombination rate allows for the evolution of greater mutational robustness.     

Selection for thermostability can lead to the emergence of mutational robustness in an RNA virus

Mutational robustness has important evolutionary implications, yet the mechanisms leading to its emergence remain poorly understood. One possibility is selection acting on a correlated trait, as for instance thermostability (plastogenetic congruence). Here, we examine the correlation between mutational robustness and thermostability in experimental populations of the RNA bacteriophage Qβ. Thermostable viruses evolved after only six serial passages in the presence of heat shocks, and genome sequencing suggested that thermostability can be conferred by several alternative mutations. To test whether thermostable viruses have increased mutational robustness, we performed additional passages in the presence of nitrous acid. Whereas in control lines this treatment produced the expected reduction in growth rate caused by the accumulation of deleterious mutations, thermostable viruses showed no such reduction, indicating that they are more resistant to mutagenesis. Our results suggest that selection for thermostability can lead to the emergence of mutational robustness driven by plastogenetic congruence. As temperature is a widespread selective pressure in nature, the mechanism described here may be relevant to the evolution of mutational robustness.     

Selection for mutational robustness in finite populations

We investigate the evolutionary dynamics of a finite population of RNA sequences replicating on a neutral network. Despite the lack of differential fitness between viable sequences, we observe typical properties of adaptive evolution, such as increase of mean fitness over time and punctuated-equilibrium transitions, after initial mutation-selection balance has been reached. We find that a product of population size and mutation rate of approximately 30 or larger is sufficient to generate selection pressure for mutational robustness, even if the population size is orders of magnitude smaller than the neutral network on which the population resides. Our results show that quasispecies effects and neutral drift can occur concurrently, and that the relative importance of each is determined by the product of population size and mutation rate.     

Reconstruction of natural RNA sequences from RNA shape, thermodynamic stability, mutational robustness, and linguistic complexity by evolutionary computation

The process of designing novel RNA sequences by inverse RNA folding, available in tools such as RNAinverse and InfoRNA, can be thought of as a reconstruction of RNAs from secondary structure. In this reconstruction problem, no physical measures are considered as additional constraints that are independent of structure, aside of the goal to reach the same secondary structure as the input using energy minimization methods. An extension of the reconstruction problem can be formulated since in many cases of natural RNAs, it is desired to analyze the sequence and structure of RNA molecules using various physical quantifiable measures. In prior works that used secondary structure predictions, it has been shown that natural RNAs differ significantly from random RNAs in some of these measures. Thus, we relax the problem of reconstructing RNAs from secondary structure into reconstructing RNAs from shapes, and in turn incorporate physical quantities as constraints. This allows for the design of novel RNA sequences by inverse folding while considering various physical quantities of interest such as thermodynamic stability, mutational robustness, and linguistic complexity. At the expense of altering the number of nucleotides in stems and loops, for example, physical measures can be taken into account. We use evolutionary computation for the new reconstruction problem and illustrate the procedure on various natural RNAs.     

Evolution favors protein mutational robustness in sufficiently large populations

Background  

An important question is whether evolution favors properties such as mutational robustness or evolvability that do not directly benefit any individual, but can influence the course of future evolution. Functionally similar proteins can differ substantially in their robustness to mutations and capacity to evolve new functions, but it has remained unclear whether any of these differences might be due to evolutionary selection for these properties.     

Viral RNA gymnastics

Retroviruses have a stretch of RNA that dimerizes during viral particle formation. A new study suggests that RNA flexibility in the monomeric form may facilitate dimerization or other RNA-dependent viral functions.     

A genetic background with low mutational robustness is associated with increased adaptability to a novel host in an RNA virus

Although mutational robustness is central to many evolutionary processes, its relationship to evolvability remains poorly understood and has been very rarely tested experimentally. Here, we measure the evolvability of Vesicular stomatitis virus in two genetic backgrounds with different levels of mutational robustness. We passaged the viruses into a novel cell type to model a host‐jump episode, quantified changes in infectivity and fitness in the new host, evaluated the cost of adaptation in the original host and analyzed the genetic basis of this adaptation. Lineages evolved from the less robust genetic background demonstrated increased adaptability, paid similar costs of adaptation to the new host and fixed approximately the same number of mutations as their more robust counterparts. Theory predicts that robustness can promote evolvability only in systems where large sets of genotypes are connected by effectively neutral mutations. We argue that this condition might not be fulfilled generally in RNA viruses.     

Epstein-Barr virus RNA. VI. Viral RNA in restringently and abortively infected Raji cells.

Nuclear and polyadenylated RNA fractions of Raji cells are encoded by larger fractions of Epstein-Barr virus DNA (35 and 18%, respectively) than encode polyribosomal RNA (10%). Polyribosomal RNA is encoded by DNA mapping at 0.05 X 10(8) to 0.29 X 10(8), 0.63 X 10(8) to 0.66 X 10(8), and 1.10 X 10(8) to 0.03 X 10(8) daltons. An abundant, small (160-base), non-polyadenylated RNA encoded by EcoRI fragment J (0.05 X 10(8) to 0.07 X 10(8) daltons) is also present in the cytoplasm of Raji cells. After induction of early antigen in Raji cells, there was a substantial increase in the complexity of viral polyadenylated and polyribosomal RNAs. Thus, nuclear RNA was encoded by 40% of Epstein-Barr virus DNA, and polyadenylated and polyribosomal RNAs were encoded by at least 30% of Epstein-Barr virus DNA. Polyribosomal RNA from induced Raji cells was encoded by Epstein-Barr virus DNAs mapping at 0.05 X 10(8) to 0.29 X 10(8), 0.63 X 10(8) to 0.66 X 10(8), and 1.10 X 10(8) to 0.03 X 10(8) daltons and also by DNAs mapping within the long unique regions of Epstein-Barr virus DNA at 0.39 X 10(8) to 0.49 X 10(8), 0.51 X 10(8) to 0.59 X 10(8), 0.66 X 10(8) to 0.77 X 10(8), and 1.02 X 10(8) to 1.05 X 10(8) daltons.     

Effects of population size and mutation rate on the evolution of mutational robustness

It is often assumed that the efficiency of selection for mutational robustness would be proportional to mutation rate and population size, thus being inefficient in small populations. However, Krakauer and Plotkin (2002) hypothesized that selection in small populations would favor robustness mechanisms, such as redundancy, that mask the effect of deleterious mutations. In large populations, by contrast, selection is more effective at removing deleterious mutants and fitness would be improved by eliminating mechanisms that mask the effect of deleterious mutations and thus impede their removal. Here, we test whether these predictions are supported in experiments with evolving populations of digital organisms. Digital organisms are self-replicating programs that inhabit a virtual world inside a computer. Like their organic counterparts, digital organisms mutate, compete, evolve, and adapt by natural selection to their environment. In this study, 160 populations evolved at different combinations of mutation rate and population size. After 10(4) generations, we measured the mutational robustness of the most abundant genotype in each population. Mutational robustness tended to increase with mutation rate and to decline with population size, although the dependence with population size was in part mediated by a negative relationship between fitness and robustness. These results are independent of whether genomes were constrained to their original length or allowed to change in size.     

Selection for robustness in mutagenized RNA viruses

Mutational robustness is defined as the constancy of a phenotype in the face of deleterious mutations. Whether robustness can be directly favored by natural selection remains controversial. Theory and in silico experiments predict that, at high mutation rates, slow-replicating genotypes can potentially outcompete faster counterparts if they benefit from a higher robustness. Here, we experimentally validate this hypothesis, dubbed the “survival of the flattest,” using two populations of the vesicular stomatitis RNA virus. Characterization of fitness distributions and genetic variability indicated that one population showed a higher replication rate, whereas the other was more robust to mutation. The faster replicator outgrew its robust counterpart in standard competition assays, but the outcome was reversed in the presence of chemical mutagens. These results show that selection can directly favor mutational robustness and reveal a novel viral resistance mechanism against treatment by lethal mutagenesis.     

Mechanisms of genetic robustness in RNA viruses

Two key features of RNA viruses are their compacted genomes and their high mutation rate. Accordingly, deleterious mutations are common and have an enormous impact on viral fitness. In their multicellular hosts, robustness can be achieved by genomic redundancy, including gene duplication, diploidy, alternative metabolic pathways and biochemical buffering mechanisms. However, here we review evidence suggesting that during RNA virus evolution, alternative robustness mechanisms may have been selected. After briefly describing how genetic robustness can be quantified, we discuss mechanisms of intrinsic robustness arising as consequences of RNA-genome architecture, replication peculiarities and quasi-species population dynamics. These intrinsic robustness mechanisms operate efficiently at the population level, despite the mutational sensitivity shown by individual genomes. Finally, we discuss the possibility that viruses might exploit cellular buffering mechanisms for their own benefit, producing a sort of extrinsic robustness.     

The effect of mutational robustness on the evolvability of multicellular organisms and eukaryotic cells

Canalization involves mutational robustness, the lack of phenotypic change as a result of genetic mutations. Given the large divergence in phenotype across species, understanding the relationship between high robustness and evolvability has been of interest to both theorists and experimentalists. Although canalization was originally proposed in the context of multicellular organisms, the effect of multicellularity and other classes of hierarchical organization on evolvability has not been considered by theoreticians. We address this issue using a Boolean population model with explicit representation of an environment in which individuals with explicit genotype and a hierarchical phenotype representing multicellularity evolve. Robustness is described by a single real number between zero and one which emerges from the genotype–phenotype map. We find that high robustness is favoured in constant environments, and lower robustness is favoured after environmental change. Multicellularity and hierarchical organization severely constrain robustness: peak evolvability occurs at an absolute level of robustness of about 0.99 compared with values of about 0.5 in a classical neutral network model. These constraints result in a sharp peak of evolvability in which the maximum is set by the fact that the fixation of adaptive mutations becomes more improbable as robustness decreases. When robustness is put under genetic control, robustness levels leading to maximum evolvability are selected for, but maximal relative fitness appears to require recombination.     

RNA 沉默的病毒抑制子

RNA 沉默是一种在真核生物体内普遍保守的、通过核酸序列特异性的相互作用来抑制基因表达的调控机制 . RNA 沉默的一种重要生物学效应是防御病毒的侵染,而针对寄主的这种防御机制,许多植物病毒已演化通过编码 RNA 沉默的抑制子来克服这种防御反应 . 目前,已从植物、动物和人类病毒中鉴定了 20 多种 RNA 沉默的抑制子,围绕抑制子的鉴定和作用机理研究已成为病毒学研究的一个热点 . 对 RNA 沉默抑制子的发现、鉴定方法、作用机理及与病毒病症状形成的关系、动物病毒的沉默抑制子等方面的最新进展做了综述,并对沉默抑制子的应用和存在的问题进行了讨论 .