Isolation of Fidelity Variants of RNA Viruses and Characterization of Virus Mutation Frequency

RNA viruses use RNA dependent RNA polymerases to replicate their genomes. The intrinsically high error rate of these enzymes is a large contributor to the generation of extreme population diversity that facilitates virus adaptation and evolution. Increasing evidence shows that the intrinsic error rates, and the resulting mutation frequencies, of RNA viruses can be modulated by subtle amino acid changes to the viral polymerase. Although biochemical assays exist for some viral RNA polymerases that permit quantitative measure of incorporation fidelity, here we describe a simple method of measuring mutation frequencies of RNA viruses that has proven to be as accurate as biochemical approaches in identifying fidelity altering mutations. The approach uses conventional virological and sequencing techniques that can be performed in most biology laboratories. Based on our experience with a number of different viruses, we have identified the key steps that must be optimized to increase the likelihood of isolating fidelity variants and generating data of statistical significance. The isolation and characterization of fidelity altering mutations can provide new insights into polymerase structure and function^1-3. Furthermore, these fidelity variants can be useful tools in characterizing mechanisms of virus adaptation and evolution^4-7.     

Does Mutational Robustness Inhibit Extinction by Lethal Mutagenesis in Viral Populations?

Lethal mutagenesis is a promising new antiviral therapy that kills a virus by raising its mutation rate. One potential shortcoming of lethal mutagenesis is that viruses may resist the treatment by evolving genomes with increased robustness to mutations. Here, we investigate to what extent mutational robustness can inhibit extinction by lethal mutagenesis in viruses, using both simple toy models and more biophysically realistic models based on RNA secondary-structure folding. We show that although the evolution of greater robustness may be promoted by increasing the mutation rate of a viral population, such evolution is unlikely to greatly increase the mutation rate required for certain extinction. Using an analytic multi-type branching process model, we investigate whether the evolution of robustness can be relevant on the time scales on which extinction takes place. We find that the evolution of robustness matters only when initial viral population sizes are small and deleterious mutation rates are only slightly above the level at which extinction can occur. The stochastic calculations are in good agreement with simulations of self-replicating RNA sequences that have to fold into a specific secondary structure to reproduce. We conclude that the evolution of mutational robustness is in most cases unlikely to prevent the extinction of viruses by lethal mutagenesis.     

RNA levers and switches controlling viral gene expression

RNA viruses are diverse and abundant pathogens that are responsible for numerous human diseases. RNA viruses possess relatively compact genomes and have therefore evolved multiple mechanisms to maximize their coding capacities, often by encoding overlapping reading frames. These reading frames are then decoded by mechanisms such as alternative splicing and ribosomal frameshifting to produce multiple distinct proteins. These solutions are enabled by the ability of the RNA genome to fold into 3D structures that can mimic cellular RNAs, hijack host proteins, and expose or occlude regulatory protein-binding motifs to ultimately control key process in the viral life cycle. We highlight recent findings focusing on less conventional mechanisms of gene expression and new discoveries on the role of RNA structures.     

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.     

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.     

Utility of DNA viruses for studying human host history: case study of JC virus

Microbial pathogens, and viruses in particular, can serve as important complements to traditional genetic markers when investigating the population histories of their human host. The range of mutation rates for DNA viruses suggests that DNA viruses can be useful markers of both recent and ancient events in their host histories. Here, we assess the utility of a well known DNA virus, JC virus (JCV), for investigating human history and demography. Using complete coding viral genomes, we confirm the phylogeographic structure of JCV in populations worldwide and provide coalescent estimates of its evolutionary rate under two alternative models of its history. Using these rate estimates, we compare Bayesian skyline plots of population size changes for JCV to those of its human host as estimated with coding mitochondrial genomes of the latter. These comparisons, when combined with other evidence including a log Bayes Factor model test, show that JCV is evolving rapidly and is therefore tracking the recent history of its human host. These results support the hypothesis that post-World War II societal changes are most likely responsible for the recent demographic patterns observed among different regional JCV populations. In sum, fast evolving DNA viruses, such as JCV, can complement RNA viruses to provide novel insights about the recent history and demography of their human host.     

Imprecise recombinant viruses evolve via a fitness-driven,iterative process of polymerase template-switching events

Recombination is a common feature of many positive-strand RNA viruses, playing an important role in virus evolution. However, to date, there is limited understanding of the mechanisms behind the process. Utilising in vitro assays, we have previously shown that the template-switching event of recombination is a random and ubiquitous process that often leads to recombinant viruses with imprecise genomes containing sequence duplications. Subsequently, a process termed resolution, that has yet to be mechanistically studied, removes these duplicated sequences resulting in a virus population of wild type length genomes. Using defined imprecise recombinant viruses together with Oxford Nanopore and Illumina high throughput next generation sequencing technologies we have investigated the process of resolution. We show that genome resolution involves subsequent rounds of template-switching recombination with viral fitness resulting in the survival of a small subset of recombinant genomes. This alters our previously held understanding that recombination and resolution are independent steps of the process, and instead demonstrates that viruses undergo frequent and continuous recombination events over a prolonged period until the fittest viruses, predominantly those with wild type length genomes, dominate the population.     

The intricate interplay between RNA viruses and NF-κB

RNA viruses have rapidly evolving genomes which often allow cross-species transmission and frequently generate new virus variants with altered pathogenic properties. Therefore infections by RNA viruses are a major threat to human health. The infected host cell detects trace amounts of viral RNA and the last years have revealed common principles in the biochemical mechanisms leading to signal amplification that is required for mounting of a powerful antiviral response. Components of the RNA sensing and signaling machinery such as RIG-I-like proteins, MAVS and the inflammasome inducibly form large oligomers or even fibers that exhibit hallmarks of prions. Following a nucleation event triggered by detection of viral RNA, these energetically favorable and irreversible polymerization events trigger signaling cascades leading to the induction of antiviral and inflammatory responses, mediated by interferon and NF-κB pathways. Viruses have evolved sophisticated strategies to manipulate these host cell signaling pathways in order to ensure their replication. We will discuss at the examples of influenza and HTLV-1 viruses how a fascinating diversity of biochemical mechanisms is employed by viral proteins to control the NF-κB pathway at all levels.     

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.     

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     

Asymmetric Genome Organization in an RNA Virus Revealed via Graph-Theoretical Analysis of Tomographic Data

Cryo-electron microscopy permits 3-D structures of viral pathogens to be determined in remarkable detail. In particular, the protein containers encapsulating viral genomes have been determined to high resolution using symmetry averaging techniques that exploit the icosahedral architecture seen in many viruses. By contrast, structure determination of asymmetric components remains a challenge, and novel analysis methods are required to reveal such features and characterize their functional roles during infection. Motivated by the important, cooperative roles of viral genomes in the assembly of single-stranded RNA viruses, we have developed a new analysis method that reveals the asymmetric structural organization of viral genomes in proximity to the capsid in such viruses. The method uses geometric constraints on genome organization, formulated based on knowledge of icosahedrally-averaged reconstructions and the roles of the RNA-capsid protein contacts, to analyse cryo-electron tomographic data. We apply this method to the low-resolution tomographic data of a model virus and infer the unique asymmetric organization of its genome in contact with the protein shell of the capsid. This opens unprecedented opportunities to analyse viral genomes, revealing conserved structural features and mechanisms that can be targeted in antiviral drug design.     

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.     

Escape from Prisoner's Dilemma in RNA phage phi6

We previously examined competitive interactions among viruses by allowing the RNA phage phi6 to evolve at high and low multiplicities of infection (ratio of infecting viruses to bacterial cells). Derived high-multiplicity phages were competitively advantaged relative to their ancestors during coinfection, but their fixation caused population fitness to decline. These data conform to the evolution of lowered fitness in a population of defectors, as expected from the Prisoner's Dilemma of game theory. However, the generality of this result is unknown; the evolution of viruses at other multiplicities may alter the fitness payoffs associated with conflicting strategies of cooperation and defection. Here we examine the change in matrix variables by propagating the ancestor under strictly clonal conditions, allowing cooperation the chance to evolve. In competitions involving derived cooperators and their selfish counterparts, our data reveal a new outcome where the two strategies are predicted to coexist in a mixed polymorphism. Thus, we demonstrate that the payoff matrix is not a constant in phi6. Rather, clonal selection allows viruses the opportunity to escape the Prisoner's Dilemma. We discuss mechanisms that may afford selfish genotypes an advantage during intrahost competition and the relevance in our system for alternative ecological interactions among viruses.     

Long-range correlations between DNA bending sites: relation to the structure and dynamics of nucleosomes

It has been established that the precise positioning of nucleosomes on genomic DNA can be achieved, at least for a minority of them, through sequence-dependent processes. However, to what extent DNA sequences play a role in the positioning of the major part of nucleosomes is still debated. The aim of the present study is to examine to what extent long-range correlations (LRC) are related to the presence of nucleosomes. Using the wavelet transform technique, we perform a comparative analysis of the DNA text and of the corresponding bending profiles generated with curvature tables based on nucleosome positioning data. The exploration of a number of eukaryotic and bacterial genomes through the optics of the so-called "wavelet transform microscope" reveals a characteristic scale of 100-200 bp that separates two regimes of different LRC. Here, we focus on the existence of LRC in the small-scale regime (10-200 bp) which are actually observed in eukaryotic genomes, in contrast to their absence in eubacterial genomes. Analysis of viral DNA genomes shows that, like their host's genomes, eukaryotic viruses present LRC but eubacterial viruses do not. There is one exception for genomes of poxviruses (Vaccinia and Melamoplus sanguinipes) which do not replicate in the cell nucleus and do not exhibit LRC. No small-scale LRC are detected in the genomes of all examined RNA viruses, with the exception of retroviruses. These results together with the observation of LRC between particular sequence motifs known to participate in the formation of nucleosomes (e.g. AA dinucleotides) strongly suggest that the 10-200 bp LRC are a signature of the sequence-dependence of nucleosome positioning. Finally, we discuss possible interpretations of these LRC in terms of the physical mechanisms that might govern the positioning and the dynamics of the nucleosomes along the DNA chain through cooperative processes.     

Monitoring Insect Transposable Elements in Large Double-Stranded DNA Viruses Reveals Host-to-Virus and Virus-to-Virus Transposition

The mechanisms by which transposable elements (TEs) can be horizontally transferred between animals are unknown, but viruses are possible candidate vectors. Here, we surveyed the presence of host-derived TEs in viral genomes in 35 deep sequencing data sets produced from 11 host–virus systems, encompassing nine arthropod host species (five lepidopterans, two dipterans, and two crustaceans) and six different double-stranded (ds) DNA viruses (four baculoviruses and two iridoviruses). We found evidence of viral-borne TEs in 14 data sets, with frequencies of viral genomes carrying a TE ranging from 0.01% to 26.33% for baculoviruses and from 0.45% to 7.36% for iridoviruses. The analysis of viral populations separated by a single replication cycle revealed that viral-borne TEs originating from an initial host species can be retrieved after viral replication in another host species, sometimes at higher frequencies. Furthermore, we detected a strong increase in the number of integrations in a viral population for a TE absent from the hosts’ genomes, indicating that this TE has undergone intense transposition within the viral population. Finally, we provide evidence that many TEs found integrated in viral genomes (15/41) have been horizontally transferred in insects. Altogether, our results indicate that multiple large dsDNA viruses have the capacity to shuttle TEs in insects and they underline the potential of viruses to act as vectors of horizontal transfer of TEs. Furthermore, the finding that TEs can transpose between viral genomes of a viral species sets viruses as possible new niches in which TEs can persist and evolve.     

Viral induction and suppression of RNA silencing in plants

RNA silencing in plants and insects can function as a defence mechanism against invading viruses. RNA silencing-based antiviral defence entails the production of virus-derived small interfering RNAs which guide specific antiviral effector complexes to inactivate viral genomes. As a response to this defence system, viruses have evolved viral suppressors of RNA silencing (VSRs) to overcome the host defence. VSRs can act on various steps of the different silencing pathways. Viral infection can have a profound impact on the host endogenous RNA silencing regulatory pathways; alterations of endogenous short RNA expression profile and gene expression are often associated with viral infections and their symptoms. Here we discuss our current understanding of the main steps of RNA-silencing responses to viral invasion in plants and the effects of VSRs on endogenous pathways. This article is part of a Special Issue entitled: MicroRNAs in viral gene regulation.     

Determining the frequency and mechanisms of HIV-1 and HIV-2 RNA copackaging by single-virion analysis

HIV-1 and HIV-2 are derived from two distinct primate viruses and share only limited sequence identity. Despite this, HIV-1 and HIV-2 Gag polyproteins can coassemble into the same particle and their genomes can undergo recombination, albeit at an extremely low frequency, implying that HIV-1 and HIV-2 RNA can be copackaged into the same particle. To determine the frequency of HIV-1 and HIV-2 RNA copackaging and to dissect the mechanisms that allow the heterologous RNA copackaging, we directly visualized the RNA content of each particle by using RNA-binding proteins tagged with fluorescent proteins to label the viral genomes. We found that when HIV-1 and HIV-2 RNA are present in viral particles at similar ratios, ～10% of the viral particles encapsidate both HIV-1 and HIV-2 RNAs. Furthermore, heterologous RNA copackaging can be promoted by mutating the 6-nucleotide (6-nt) dimer initiation signal (DIS) to discourage RNA homodimerization or to encourage RNA heterodimerization, indicating that HIV-1 and HIV-2 RNA can heterodimerize prior to packaging using the DIS sequences. We also observed that the coassembly of HIV-1 and HIV-2 Gag proteins is not required for the heterologous RNA copackaging; HIV-1 Gag proteins are capable of mediating HIV-1 and HIV-2 RNA copackaging. These results define the cis- and trans-acting elements required for and affecting the heterologous RNA copackaging, a prerequisite for the generation of chimeric viruses by recombination, and also shed light on the mechanisms of RNA-Gag recognition essential for RNA encapsidation.