Magnitude and sign epistasis among deleterious mutations in a positive-sense plant RNA virus

How epistatic interactions between mutations determine the genetic architecture of fitness is of central importance in evolution. The study of epistasis is particularly interesting for RNA viruses because of their genomic compactness, lack of genetic redundancy, and apparent low complexity. Moreover, interactions between mutations in viral genomes determine traits such as resistance to antiviral drugs, virulence and host range. In this study we generated 53 Tobacco etch potyvirus genotypes carrying pairs of single-nucleotide substitutions and measured their separated and combined deleterious fitness effects. We found that up to 38% of pairs had significant epistasis for fitness, including both positive and negative deviations from the null hypothesis of multiplicative effects. Interestingly, the sign of epistasis was correlated with viral protein-protein interactions in a model network, being predominantly positive between linked pairs of proteins and negative between unlinked ones. Furthermore, 55% of significant interactions were cases of reciprocal sign epistasis (RSE), indicating that adaptive landscapes for RNA viruses maybe highly rugged. Finally, we found that the magnitude of epistasis correlated negatively with the average effect of mutations. Overall, our results are in good agreement to those previously reported for other viruses and further consolidate the view that positive epistasis is the norm for small and compact genomes that lack genetic robustness.     

Prevalence of epistasis in the evolution of influenza A surface proteins

The surface proteins of human influenza A viruses experience positive selection to escape both human immunity and, more recently, antiviral drug treatments. In bacteria and viruses, immune-escape and drug-resistant phenotypes often appear through a combination of several mutations that have epistatic effects on pathogen fitness. However, the extent and structure of epistasis in influenza viral proteins have not been systematically investigated. Here, we develop a novel statistical method to detect positive epistasis between pairs of sites in a protein, based on the observed temporal patterns of sequence evolution. The method rests on the simple idea that a substitution at one site should rapidly follow a substitution at another site if the sites are positively epistatic. We apply this method to the surface proteins hemagglutinin and neuraminidase of influenza A virus subtypes H3N2 and H1N1. Compared to a non-epistatic null distribution, we detect substantial amounts of epistasis and determine the identities of putatively epistatic pairs of sites. In particular, using sequence data alone, our method identifies epistatic interactions between specific sites in neuraminidase that have recently been demonstrated, in vitro, to confer resistance to the drug oseltamivir; these epistatic interactions are responsible for widespread drug resistance among H1N1 viruses circulating today. This experimental validation demonstrates the predictive power of our method to identify epistatic sites of importance for viral adaptation and public health. We conclude that epistasis plays a large role in shaping the molecular evolution of influenza viruses. In particular, sites with , which would normally not be identified as positively selected, can facilitate viral adaptation through epistatic interactions with their partner sites. The knowledge of specific interactions among sites in influenza proteins may help us to predict the course of antigenic evolution and, consequently, to select more appropriate vaccines and drugs.     

Epistasis between mutations is host-dependent for an RNA virus

How, and to what extent, does the environment influence the way mutations interact? Do environmental changes affect both the sign and the magnitude of epistasis? Are there any correlations between environments in the variability, sign or magnitude of epistasis? Very few studies have tackled these questions. Here, we addressed them in the context of viral emergence. Most emerging viruses are RNA viruses with small genomes, overlapping reading frames and multifunctional proteins for which epistasis is abundant. Understanding the effect of host species in the sign and magnitude of epistasis will provide insights into the evolutionary ecology of infectious diseases and the predictability of viral emergence.     

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.     

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.     

High fidelity of murine hepatitis virus replication is decreased in nsp14 exoribonuclease mutants

Replication fidelity of RNA virus genomes is constrained by the opposing necessities of generating sufficient diversity for adaptation and maintaining genetic stability, but it is unclear how the largest viral RNA genomes have evolved and are maintained under these constraints. A coronavirus (CoV) nonstructural protein, nsp14, contains conserved active-site motifs of cellular exonucleases, including DNA proofreading enzymes, and the severe acute respiratory syndrome CoV (SARS-CoV) nsp14 has 3'-to-5' exoribonuclease (ExoN) activity in vitro. Here, we show that nsp14 ExoN remarkably increases replication fidelity of the CoV murine hepatitis virus (MHV). Replacement of conserved MHV ExoN active-site residues with alanines resulted in viable mutant viruses with growth and RNA synthesis defects that during passage accumulated 15-fold more mutations than wild-type virus without changes in growth fitness. The estimated mutation rate for ExoN mutants was similar to that reported for other RNA viruses, whereas that of wild-type MHV was less than the established rates for RNA viruses in general, suggesting that CoVs with intact ExoN replicate with unusually high fidelity. Our results indicate that nsp14 ExoN plays a critical role in prevention or repair of nucleotide incorporation errors during genome replication. The established mutants are unique tools to test the hypothesis that high replication fidelity is required for the evolution and stability of large RNA genomes.     

In silico predicted robustness of viroid RNA secondary structures. II. Interaction between mutation pairs

Viroids are plant subviral pathogens whose genomes are constituted by a single-stranded and covalently closed small RNA molecule that does not encode for any protein. Most of the 29 described viroid species fold into a rodlike or quasi-rodlike structure, whereas a few of them fold as highly branched structures. In a previous study, we used RNA thermodynamic secondary structure prediction algorithms to compare the mutational robustness of all viroid species. Here we used the same approach to explore the sign and strength of epistasis among pairs of random mutations. We found that antagonistic interactions were more abundant than synergistic ones. However, despite their lower frequency, synergistic interactions tended to be more intense. Mutational robustness and the intensity of epistasis were correlated such that viroid species with large average mutational effects showed stronger antagonistic epistasis, whereas viroids with mild average mutational effects showed weaker antagonistic interactions. The strength of antagonistic epistasis decreased with genome complexity as a consequence of the gained robustness of duplicated genomes. In good agreement with our previous finding of an evolutionary trend toward increased robustness, we now found a trend toward reduced antagonistic epistasis.     

Comparative restriction endonuclease maps of proviral DNA of the primate type C simian sarcoma-associated virus and gibbon ape leukemia virus group.

Extrachromosomal DNA was purified from canine thymus cells acutely infected with different strains of infectious primate type C viruses of the woolly monkey (simian) sarcoma helper virus and gibbon ape leukemia virus group. All DNA preparations contained linear proviral molecules of 9.1 to 9.2 kilobases, at least some of which represent complete infectious proviral DNA. Cells infected with a replication-defective fibroblast-transforming sarcoma virus and its helper, a replication-competent nontransforming helper virus, also contained a 6.6- to 6.7-kilobase DNA. These proviral DNA molecules were digested with different restriction endonucleases, and the resultant fragments were oriented to the viral RNA by a combination of partial digestions, codigestion with more than one endonuclease, digestion of integrated proviral DNA, and hybridization with 3'- and 5'-specific viral probes. The 3'- and 5'-specific probes each hybridized to fragments from both ends of proviral DNA, indicating that, in common with those of other retroviruses, these proviruses contain a large terminal redundancy at both ends, each of which consists of sequences derived from both the 3' and 5' regions of the viral RNA. The proviral sequences are organized 3',5'-unique-3',5'. Four restriction enzymes (KpnI, SmaI, PstI, and SstI) recognized sites within the large terminal redundancies, and these sites were conserved within all the isolates tested. This suggests that both the 3' and 5' ends of the genomic RNA of these viruses are extremely closely related. In contrast, the restriction sites within the unique portion of the provirus were not strongly conserved within this group of viruses, even though they were related along most of their genomes. Whereas the 5' 60 to 70% of the RNA of these viruses was more closely related by liquid hybridization experiments than was the 3' 30 to 40%, restriction sites within this region were not preferentially conserved, suggesting that small sequence differences or point mutations or both exist throughout the entire unique portion of the genome among these viruses.     

The quasispecies (extremely heterogeneous) nature of viral RNA genome populations: biological relevance--a review

We review evidence that cloned (or uncloned) populations of most RNA viruses do not consist of a single genome species of defined sequence, but rather of heterogeneous mixtures of related genomes (quasispecies). Due to very high mutation rates, genomes of a quasispecies virus population share a consensus sequence but differ from each other and from the consensus sequence by one, several, or many mutations. Viral genome analyses by sequencing, fingerprinting, cDNA cloning etc. indicate that most viral RNA populations (quasispecies) contain all possible single and double genomic site mutations and varying proportions of triple, quadruple, etc. site mutations. This quasispecies structure of RNA virus populations has many important theoretical and practical implications because mutations at only one or a few sites may alter the phenotype of an RNA virus.     

3D gene of foot-and-mouth disease virus. Conservation by convergence of average sequences

The nucleotide sequence of the 3D (polymerase) gene of eight epidemiologically related isolates of foot-and-mouth disease virus of serotype C1 is reported. The genetic heterogeneity of 3D RNA is compared with that of the VP1-coding RNA of the same viruses. Regression lines of substitutions per nucleotide that distinguish any pair of viruses as a function of the time interval between the corresponding isolations show: (1) the slope (substitutions/nucleotide per month) is 2.1 times larger for the VP1 RNA than for the 3D RNA region; (2) the intercept with the ordinate (substitutions/nucleotide) for VP1 RNA is indistinguishable from that for 3D RNA. Thus, the average heterogeneity of the VP1-coding region is very similar to that of the 3D-coding region only among co-circulating viruses. Nine mutations and points of heterogeneity occurred within nucleotide residues 883 to 1026, which encode an amino acid segment, extremely conserved among many different RNA viruses. The results suggest that, rather than due to inherently lower mutability, the conservation of 3D genes is caused by a limitation in the fixation of substitutions in viable genomes.     

Dynamics of mutation and recombination in a replicating population of complementing, defective viral genomes

In a previous study, we documented that serial passage of a biological clone of foot-and-mouth disease virus (FMDV) at high multiplicity of infection (moi) in cell culture resulted in viral populations dominated by defective genomes that included internal in-frame deletions, affecting the L and capsid-coding regions, and were infectious by complementation. In the present study, analyses of the defective genomes present in individual viral plaques, and of consensus nucleotide sequences determined for the entire genomes of sequential samples, have revealed a continuous dynamics of mutation and recombination. At some points of high genetic instability, multiple minority genomes with different internal deletions co-existed in the population. At later passages, a new defective RNA arose and displaced a related, previously dominant RNA. Nucleotide sequences of the different genomic forms found in sequential isolates have revealed an accumulation of mutations at an average rate of 0.12 substitutions per genome per passage. At the regions around the deletion sites, substantial, minor or no nucleotide sequence identity is found, suggesting relaxed sequence requirements for the occurrence of internal deletions. Competition experiments indicate a selective advantage of late phase defective genomes over their precursor forms. The defective genome-based FMDV retained an expansion of host cell tropism, undergone by the standard virus at a previous stage of the same evolutionary lineage. Thus, despite a complex dynamics of mutation and recombination, and phases of high genetic instability, a biologically relevant phenotypic trait was stably maintained after the evolutionary transition towards a primitive genome segmentation. The results extend the concept of a complex spectrum of mutant genomes to a complex spectrum of defective genomes in some evolutionary transitions of RNA viruses.     

Mechanisms for RNA Capture by ssDNA Viruses: Grand Theft RNA

Viruses contain three common types of packaged genomes; double-stranded DNA (dsDNA), RNA (mostly single and occasionally double stranded) and single-stranded DNA (ssDNA). There are relatively straightforward explanations for the prevalence of viruses with dsDNA and RNA genomes, but the evolutionary basis for the apparent success of ssDNA viruses is less clear. The recent discovery of four ssDNA virus genomes that appear to have been formed by recombination between co-infecting RNA and ssDNA viruses, together with the high mutation rate of ssDNA viruses provide possible explanations. RNA–DNA recombination allows ssDNA viruses to access much broader sequence space than through nucleotide substitution and DNA–DNA recombination alone. Multiple non-exclusive mechanisms, all due to the unique replication of ssDNA viruses, are proposed for this unusual RNA capture. RNA capture provides an explanation for the evolutionary success of the ssDNA viruses and may help elucidate the mystery of integrated RNA viruses in viral and cellular DNA genomes.     

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.     

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.     

Patterns of evolution and host gene mimicry in influenza and other RNA viruses

It is well known that the dinucleotide CpG is under-represented in the genomic DNA of many vertebrates. This is commonly thought to be due to the methylation of cytosine residues in this dinucleotide and the corresponding high rate of deamination of 5-methycytosine, which lowers the frequency of this dinucleotide in DNA. Surprisingly, many single-stranded RNA viruses that replicate in these vertebrate hosts also have a very low presence of CpG dinucleotides in their genomes. Viruses are obligate intracellular parasites and the evolution of a virus is inexorably linked to the nature and fate of its host. One therefore expects that virus and host genomes should have common features. In this work, we compare evolutionary patterns in the genomes of ssRNA viruses and their hosts. In particular, we have analyzed dinucleotide patterns and found that the same patterns are pervasively over- or under-represented in many RNA viruses and their hosts suggesting that many RNA viruses evolve by mimicking some of the features of their host's genes (DNA) and likely also their corresponding mRNAs. When a virus crosses a species barrier into a different host, the pressure to replicate, survive and adapt, leaves a footprint in dinucleotide frequencies. For instance, since human genes seem to be under higher pressure to eliminate CpG dinucleotide motifs than avian genes, this pressure might be reflected in the genomes of human viruses (DNA and RNA viruses) when compared to those of the same viruses replicating in avian hosts. To test this idea we have analyzed the evolution of the influenza virus since 1918. We find that the influenza A virus, which originated from an avian reservoir and has been replicating in humans over many generations, evolves in a direction strongly selected to reduce the frequency of CpG dinucleotides in its genome. Consistent with this observation, we find that the influenza B virus, which has spent much more time in the human population, has adapted to its human host and exhibits an extremely low CpG dinucleotide content. We believe that these observations directly show that the evolution of RNA viral genomes can be shaped by pressures observed in the host genome. As a possible explanation, we suggest that the strong selection pressures acting on these RNA viruses are most likely related to the innate immune response and to nucleotide motifs in the host DNA and RNAs.     

Two-stepping through time: mammals and viruses

Recent studies have identified ancient virus genomes preserved as fossils within diverse animal genomes. These fossils have led to the revelation that a broad range of mammalian virus families are older and more ubiquitous than previously appreciated. Long-term interactions between viruses and their hosts often develop into genetic arms races where both parties continually jockey for evolutionary dominance. It is difficult to imagine how mammalian hosts have kept pace in the evolutionary race against rapidly evolving viruses over large expanses of time, given their much slower evolutionary rates. However, recent data has begun to reveal the evolutionary strategy of slowly-evolving hosts. We review these data and suggest a modified arms race model where the evolutionary possibilities of viruses are relatively constrained. Such a model could allow more accurate forecasting of virus evolution.     

Are RNA Viruses Adapting or Merely Changing?

RNA viruses and retroviruses fix substitutions approximately 1 million-fold faster than their hosts. This diversification could represent an inevitable drift under purifying selection, the majority of substitutions being phenotypically neutral. The alternative is to suppose that most fixed mutations are beneficial to the virus, allowing it to keep ahead of the host and/or host population. Here, relative sequence diversification of different proteins encoded by viral genomes is found to be linear. The examples encompass a wide variety of retroviruses and RNA viruses. The smoothness of relative divergence spans quasispeciation following clonal infection, to variation among different isolates of the same virus, to viruses from different species or those associated with different diseases, indicating that the majority of fixed mutations likely reflects drift. This held for both mammalian and plant viruses, indicating that adaptive immunity doesn't necessarily shape the relative accumulation of amino acid substitutions. When compared to their hosts RNA viruses evolution appears conservative. Received: 16 November 1999 / Accepted: 10 March 2000     

Low temperature inhibits RNA silencing-mediated defence by the control of siRNA generation

Temperature dramatically affects plant-virus interactions. Outbreaks of virus diseases are frequently associated with low temperature, while at high temperature viral symptoms are often attenuated (heat masking) and plants rapidly recover from virus diseases. However, the underlying mechanisms of these well-known observations are not yet understood. RNA silencing is a conserved defence system of eukaryotic cells, which operates against molecular parasites including viruses and transgenes. Here we show that at low temperature both virus and transgene triggered RNA silencing are inhibited. Therefore, in cold, plants become more susceptible to viruses, and RNA silencing-based phenotypes of transgenic plants are lost. Consistently, the levels of virus- and transgene-derived small (21-26 nucleotide) interfering (si) RNAs-the central molecules of RNA silencing-mediated defence pathways-are dramatically reduced at low temperature. In contrast, RNA silencing was activated and the amount of siRNAs gradually increased with rising temperature. However, temperature does not influence the accumulation of micro (mi) RNAs, which play a role in developmental regulation, suggesting that the two classes of small (si and mi) RNAs are generated by different nuclease complexes.