Paul E. Turner

Prisoner's dilemma in an RNA virus

The evolution of competitive interactions among viruses was studied in the RNA phage φ6 at high and low multiplicities of infection (that is, at high and low ratios of infecting phage to host cells). At high multiplicities, many phage infect and reproduce in the same host cell, whereas at low multiplicities the viruses reproduce mainly as clones. An unexpected result of this study was that phage grown at high rates of co-infection increased in fitness initially, but then evolved lowered fitness. Here we show that the fitness of the high-multiplicity phage relative to their ancestors generates a pay-off matrix conforming to the prisoners dilemma strategy of game theory. In this strategy, defection (selfishness) evolves, despite the greater fitness pay-off that would result if all players were to cooperate. Viral cooperation and defection can be defined as, respectively, the manufacturing and sequestering of diffusible (shared) intracellular products. Because the low-multiplicity phage did not evolve lowered fitness, we attribute the evolution of selfishness to the lack of clonal structure and the mixing of unrelated genotypes at high multiplicity.

Pleiotropic Costs of Niche Expansion in the RNA Bacteriophage Φ6

Natural and experimental systems have failed to universally demonstrate a trade-off between generalism and specialism. When a trade-off does occur it is difficult to attribute its cause to antagonistic pleiotropy without dissecting the genetic basis of adaptation, and few previous experiments provide these genetic data. Here we investigate the evolution of expanded host range (generalism) in the RNA virus Φ6, an experimental model system allowing adaptive mutations to be readily identified. We isolated 10 spontaneous host range mutants on each of three novel Pseudomonas hosts and determined whether these mutations imposed fitness costs on the standard laboratory host. Sequencing revealed that each mutant had one of nine nonsynonymous mutations in the Φ6 gene P3, important in host attachment. Seven of these nine mutations were costly on the original host, confirming the existence of antagonistic pleiotropy. In addition to this genetically imposed cost, we identified an epigenetic cost of generalism that occurs when phage transition between host types. Our results confirm the existence in Φ6 of two costs of generalism, genetic and environmental, but they also indicate that the cost is not always large. The possibility for cost-free niche expansion implies that varied ecological conditions may favor host shifts in RNA viruses.

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.

Escape from Prisoner’s Dilemma in RNA Phage Φ6

We previously examined competitive interactions among viruses by allowing the RNA phage Φ6 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 Φ6. 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.

Robustness promotes evolvability of thermotolerance in an RNA virus

BackgroundThe ability for an evolving population to adapt to a novel environment is achieved through a balance of robustness and evolvability. Robustness is the invariance of phenotype in the face of perturbation and evolvability is the capacity to adapt in response to selection. Genetic robustness has been posited, depending on the underlying mechanism, to either decrease the efficacy of selection, or increase the possibility of future adaptation. However, the true effect of genetic robustness on evolvability in biological systems remains uncertain.ResultsHere we demonstrate that genetic robustness increases evolvability of thermotolerance in laboratory populations of the RNA virus φ6. We observed that populations founded by robust clones evolved greater resistance to heat shock, relative to populations founded by brittle (less-robust) clones. Thus, we provide empirical evidence for the idea that robustness can promote evolvability in this environment, and further suggest that evolvability can arise indirectly via selection for robustness, rather than through direct selective action.ConclusionOur data imply that greater tolerance of mutational change is associated with virus adaptability in a new niche, a finding generally relevant to evolutionary biology, and informative for elucidating how viruses might evolve to emerge in new habitats and/or overcome novel therapies.

Phage selection restores antibiotic sensitivity in MDR Pseudomonas aeruginosa

Increasing prevalence and severity of multi-drug-resistant (MDR) bacterial infections has necessitated novel antibacterial strategies. Ideally, new approaches would target bacterial pathogens while exerting selection for reduced pathogenesis when these bacteria inevitably evolve resistance to therapeutic intervention. As an example of such a management strategy, we isolated a lytic bacteriophage, OMKO1, (family Myoviridae) of Pseudomonas aeruginosa that utilizes the outer membrane porin M (OprM) of the multidrug efflux systems MexAB and MexXY as a receptor-binding site. Results show that phage selection produces an evolutionary trade-off in MDR P. aeruginosa, whereby the evolution of bacterial resistance to phage attack changes the efflux pump mechanism, causing increased sensitivity to drugs from several antibiotic classes. Although modern phage therapy is still in its infancy, we conclude that phages, such as OMKO1, represent a new approach to phage therapy where bacteriophages exert selection for MDR bacteria to become increasingly sensitive to traditional antibiotics. This approach, using phages as targeted antibacterials, could extend the lifetime of our current antibiotics and potentially reduce the incidence of antibiotic resistant infections.

Evolutionary Genomics of Host Adaptation in Vesicular Stomatitis Virus

Populations experiencing similar selection pressures can sometimes diverge in the genetic architectures underlying evolved complex traits. We used RNA virus populations of large size and high mutation rate to study the impact of historical environment on genome evolution, thus increasing our ability to detect repeatable patterns in the evolution of genetic architecture. Experimental vesicular stomatitis virus populations were evolved on HeLa cells, on MDCK cells, or on alternating hosts. Turner and Elena (2000. Cost of host radiation in an RNA virus. Genetics. 156:1465-1470.) previously showed that virus populations evolved in single-host environments achieved high fitness on their selected hosts but failed to increase in fitness relative to their ancestor on the unselected host and that alternating-host-evolved populations had high fitness on both hosts. Here we determined the complete consensus sequence for each evolved population after 95 generations to gauge whether the parallel phenotypic changes were associated with parallel genomic changes. We also analyzed the patterns of allele substitutions to discern whether differences in fitness across hosts arose through true pleiotropy or the presence of not only a mutation that is beneficial in both hosts but also 1 or more mutations at other loci that are costly in the unselected environment (mutation accumulation [MA]). We found that ecological history may influence to what extent pleiotropy and MA contribute to fitness asymmetries across environments. We discuss the degree to which current genetic architecture is expected to constrain future evolution of complex traits, such as host use by RNA viruses.

EVOLUTION OF HOST SPECIFICITY DRIVES REPRODUCTIVE ISOLATION AMONG RNA VIRUSES

Abstract Ecological speciation hypotheses claim that assortative mating evolves as a consequence of divergent natural selection for ecologically important traits. Reproductive isolation is expected to be particularly likely to evolve by this mechanism in species such as phytophagous insects that mate in the habitats in which they eat. We tested this expectation by monitoring the evolution of reproductive isolation in laboratory populations of an RNA virus that undergoes genetic exchange only when multiple virus genotypes coinfect the same host. We subjected four populations of the RNA bacteriophage Φ6 to 150 generations of natural selection on a novel host. Although there was no direct selection acting on host range in our experiment, three of the four populations lost the ability to infect one or more alternative hosts. In the most extreme case, one of the populations evolved a host range that does not contain any of the hosts infectible by the wild-type Φ6. Whole genome sequencing confirmed that the resulting reproductive isolation was due to a single nucleotide change, highlighting the ease with which an emerging RNA virus can decouple its evolutionary fate from that of its ancestor. Our results uniquely demonstrate the evolution of reproductive isolation in allopatric experimental populations. Furthermore, our data confirm the biological credibility of simple “no-gene” mechanisms of assortative mating, in which this trait arises as a pleiotropic effect of genes responsible for ecological adaptation.

Viral ecology and the maintenance of novel host use.

Viruses can occasionally emerge by infecting new host species. However, the early phases of emergence can hinge upon ecological sustainability of the virus population, which is a product of both within‐host population growth and between‐host transmission. Insufficient growth or transmission can force virus extinction before the latter phases of emergence, where genetic adaptations that improve host use may occur. We examined the early phase of emergence by studying the population dynamics of RNA phages in replicated laboratory environments containing native and novel host bacteria. To predict the breadth of transmission rates allowing viral persistence on each species, we developed a simple model based on in vitro data for phage growth rate over a range of initial population densities on both hosts. Validation of these predictions using serial passage experiments revealed a range of transmission rates for which the native host was a source and the novel host was a sink. In this critical range of transmission rates, periodic exposure to the native host was sufficient for the maintenance of the viral population on the novel host. We argue that this effect should facilitate adaptation by the virus to utilize the novel host—often crucial in subsequent phases of emergence.

Reassortment in segmented RNA viruses: mechanisms and outcomes

Segmented RNA viruses are widespread in nature and include important human, animal and plant pathogens, such as influenza viruses and rotaviruses. Although the origin of RNA virus genome segmentation remains elusive, a major consequence of this genome structure is the capacity for reassortment to occur during co-infection, whereby segments are exchanged among different viral strains. Therefore, reassortment can create viral progeny that contain genes that are derived from more than one parent, potentially conferring important fitness advantages or disadvantages to the progeny virus. However, for segmented RNA viruses that package their multiple genome segments into a single virion particle, reassortment also requires genetic compatibility between parental strains, which occurs in the form of conserved packaging signals, and the maintenance of RNA and protein interactions. In this Review, we discuss recent studies that examined the mechanisms and outcomes of reassortment for three well-studied viral families — Cystoviridae, Orthomyxoviridae and Reoviridae — and discuss how these findings provide new perspectives on the replication and evolution of segmented RNA viruses.