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.     

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.     

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.     

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.     

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.     

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.     

Viral suppressors of RNA silencing

The infection and replication of viruses in the host induce diverse mechanisms for combating viral infection. One of the best-studied antiviral defence mechanisms is based on RNA silencing. Consistently, several viral suppressors of RNA silencing (VSRs) have been identified from almost all plant virus genera, which are surprisingly diverse within and across kingdoms, exhibiting no obvious sequence similarities. VSRs efficiently inhibit host antiviral responses by interacting with the key components of cellular silencing machinery, often mimicking their normal cellular functions. Recent findings have revealed that the impact of VSRs on endogenous pathways is more complex and profound than had been estimated thus far. This review highlights the current understanding of and new insights into the mechanisms and functions of plant VSRs.     

Viral suppression of RNA silencing

Gene silencing (RNA silencing) plays a fundamental role in antiviral defense in plants, fungi and invertebrates. Viruses encode proteins that suppress gene silencing to counter host defense. Viral suppressors of RNA silencing (VSRs) have been identified from almost all plant virus genera and some viruses of insects and mammals. Recent studies have revealed that VSRs counter host defense and interfere with host gene regulation by interacting with RNA or important components of the RNA silencing pathway. Here, we review the current understanding of the complex mechanisms of VSRs that have been revealed by recent studies.     

Viral escape from antisense RNA

RNA coliphage SP was propagated for several generations on a host expressing an inhibitory antisense RNA complementary to bases 31–270 of the positive-stranded genome. Phages evolved that escaped inhibition. Typically, these escape mutants contained 3–4 base substitutions, but different sequences were observed among different isolates. The mutations were located within three different types of structural features within the predicted secondary structure of SP genomic RNA: (i) hairpin loops; (ii) hairpin stems; and (iii) the 5' region of the phage genome complementary to the antisense molecule. Computer modelling of the mutant genomic RNAs showed that all of the substitutions within hairpin stems improved the Watson–Crick pairing of the stem. No major structural rearrangements were predicted for any of the mutant genomes, and most substitutions in coding regions did not alter the amino acid sequence. Although the evolved phage populations were polymorphic for substitutions, many substitutions appeared independently in two selected lines. The creation of a new, perfect, antisense RNA against an escape mutant resulted in the inhibition of that mutant but not of other escape mutants nor of the ancestral, unevolved phage. Thus, at least in this system, a population of viruses that evolved to escape from a single antisense RNA would require a cocktail of several antisense RNAs for inhibition.     

Cooperative object transport with a swarm of e-puck robots: robustness and scalability of evolved collective strategies

Cooperative object transport in distributed multi-robot systems requires the coordination and synchronisation of pushing/pulling forces by a group of autonomous robots in order to transport items that cannot be transported by a single agent. The results of this study show that fairly robust and scalable collective transport strategies can be generated by robots equipped with a relatively simple sensory apparatus (i.e. no force sensors and no devices for direct communication). In the experiments described in this paper, homogeneous groups of physical e-puck robots are required to coordinate and synchronise their actions in order to transport a heavy rectangular cuboid object as far as possible from its starting position to an arbitrary direction. The robots are controlled by dynamic neural networks synthesised using evolutionary computation techniques. The best evolved controller demonstrates an effective group transport strategy that is robust to variability in the physical characteristics of the object (i.e. object mass and size of the longest object’s side) and scalable to different group sizes. To run these experiments, we designed, built, and mounted on the robots a new sensor that returns the agents’ displacement on a 2D plane. The study shows that the feedback generated by the robots’ sensors relative to the object’s movement is sufficient to allow the robots to coordinate their efforts and to sustain the transports for an extended period of time. By extensively analysing successful behavioural strategies, we illustrate the nature of the operational mechanisms underpinning the coordination and synchronisation of actions during group transport.     

Viral IRES RNA structures and ribosome interactions

In eukaryotes, protein synthesis initiates primarily by a mechanism that requires a modified nucleotide 'cap' on the mRNA and also proteins that recruit and position the ribosome. Many pathogenic viruses use an alternative, cap-independent mechanism that substitutes RNA structure for the cap and many proteins. The RNAs driving this process are called internal ribosome-entry sites (IRESs) and some are able to bind the ribosome directly using a specific 3D RNA structure. Recent structures of IRES RNAs and IRES-ribosome complexes are revealing the structural basis of viral IRES' 'hijacking' of the protein-making machinery. It now seems that there are fundamental differences in the 3D structures used by different IRESs, although there are some common features in how they interact with ribosomes.     

RNA substrate specificity and structure-guided mutational analysis of bacteriophage T4 RNA ligase 2

Here we report that bacteriophage T4 RNA ligase 2 (Rnl2) is an efficient catalyst of RNA ligation at a 3'-OH/5'-PO(4) nick in a double-stranded RNA or an RNA.DNA hybrid. The critical role of the template strand in approximating the reactive 3'-OH and 5'-PO(4) termini is underscored by the drastic reductions in the RNA-sealing activity of Rnl2 when the duplex substrates contain gaps or flaps instead of nicks. RNA nick joining requires ATP and a divalent cation cofactor (either Mg or Mn). Neither dATP, GTP, CTP, nor UTP can substitute for ATP. We identify by alanine scanning seven functionally important amino acids (Tyr-5, Arg-33, Lys-54, Gln-106, Asp-135, Arg-155, and Ser-170) within the N-terminal nucleotidyl-transferase domain of Rnl2 and impute specific roles for these residues based on the crystal structure of the AMP-bound enzyme. Mutational analysis of 14 conserved residues in the C-terminal domain of Rnl2 identifies 3 amino acids (Arg-266, Asp-292, and Glu-296) as essential for ligase activity. Our findings consolidate the evolutionary connections between bacteriophage Rnl2 and the RNA-editing ligases of kinetoplastid protozoa.     

Accurate Strand-Specific Quantification of Viral RNA

The presence of full-length complements of viral genomic RNA is a hallmark of RNA virus replication within an infected cell. As such, methods for detecting and measuring specific strands of viral RNA in infected cells and tissues are important in the study of RNA viruses. Strand-specific quantitative real-time PCR (ssqPCR) assays are increasingly being used for this purpose, but the accuracy of these assays depends on the assumption that the amount of cDNA measured during the quantitative PCR (qPCR) step accurately reflects amounts of a specific viral RNA strand present in the RT reaction. To specifically test this assumption, we developed multiple ssqPCR assays for the positive-strand RNA virus o''nyong-nyong (ONNV) that were based upon the most prevalent ssqPCR assay design types in the literature. We then compared various parameters of the ONNV-specific assays. We found that an assay employing standard unmodified virus-specific primers failed to discern the difference between cDNAs generated from virus specific primers and those generated through false priming. Further, we were unable to accurately measure levels of ONNV (−) strand RNA with this assay when higher levels of cDNA generated from the (+) strand were present. Taken together, these results suggest that assays of this type do not accurately quantify levels of the anti-genomic strand present during RNA virus infectious cycles. However, an assay permitting the use of a tag-specific primer was able to distinguish cDNAs transcribed from ONNV (−) strand RNA from other cDNAs present, thus allowing accurate quantification of the anti-genomic strand. We also report the sensitivities of two different detection strategies and chemistries, SYBR® Green and DNA hydrolysis probes, used with our tagged ONNV-specific ssqPCR assays. Finally, we describe development, design and validation of ssqPCR assays for chikungunya virus (CHIKV), the recent cause of large outbreaks of disease in the Indian Ocean region.     

Gel Electrophoresis of Avian Leukosis and Sarcoma Viral RNA in Formamide: Comparison with Other Viral and Cellular RNA Species

Class a and class b 30 to 40S RNA subunits obtained by heat dissociation from the 60 to 70S RNA complex of avian tumor viruses were compared with several RNA standards by electrophoresis in formamide-polyacrylamide gels. Class a RNA was found to have a lower electrophoretic mobility and hence probably a higher molecular weight than class b RNA. The absolute molecular weight of class a and b RNA could not be determined with accuracy, because the relationship between logarithm of molecular weight and mobility of the RNA standards was not linear. The size of class a RNA fell into the range of 2.4 x 10(6) to 3.4 x 10(6) daltons and that of class b into the range of 2.2 x 10(6) to 2.9 x 10(6) daltons, depending on the standards used. The possible biological significance of this difference is discussed.