5,6-Dihydro-5-aza-2'-deoxycytidine potentiates the anti-HIV-1 activity of ribonucleotide reductase inhibitors.

The nucleoside analog 5,6-dihydro-5-aza-2'-deoxycytidine (KP-1212) has been investigated as a first-in-class lethal mutagen of human immunodeficiency virus type-1 (HIV-1). Since a prodrug monotherapy did not reduce viral loads in Phase II clinical trials, we tested if ribonucleotide reductase inhibitors (RNRIs) combined with KP-1212 would improve antiviral activity. KP-1212 potentiated the activity of gemcitabine and resveratrol and simultaneously increased the viral mutant frequency. G-to-C mutations predominated with the KP-1212-resveratrol combination. These observations represent the first demonstration of a mild anti-HIV-1 mutagen potentiating the antiretroviral activity of RNRIs and encourage the clinical translation of enhanced viral mutagenesis in treating HIV-1 infection.

A common, non-optimal phenotypic endpoint in experimental adaptations of bacteriophage lysis time.

BACKGROUND: Optimality models of evolution, which ignore genetic details and focus on natural selection, are widely used but sometimes criticized as oversimplifications. Their utility for quantitatively predicting phenotypic evolution can be tested experimentally. One such model predicts optimal bacteriophage lysis interval, how long a virus should produce progeny before lysing its host bacterium to release them. The genetic basis of this life history trait is well studied in many easily propagated phages, making it possible to test the model across a variety of environments and taxa. RESULTS: We adapted two related small single-stranded DNA phages, ΦX174 and ST-1, to various conditions. The model predicted the evolution of the lysis interval in response to host density and other environmental factors. In all cases the initial phages lysed later than predicted. The ΦX174 lysis interval did not evolve detectably when the phage was adapted to normal hosts, indicating complete failure of optimality predictions. ΦX174 grown on slyD-defective hosts which initially entirely prevented lysis readily recovered to a lysis interval similar to that attained on normal hosts. Finally, the lysis interval still evolved to the same endpoint when the environment was altered to delay optimal lysis interval. ST-1 lysis interval evolved to be ~2 min shorter, qualitatively in accord with predictions. However, there were no changes in the single known lysis gene. Part of ST-1's total lysis time evolution consisted of an earlier start to progeny production, an unpredicted phenotypic response outside the boundaries of the optimality model. CONCLUSIONS: The consistent failure of the optimality model suggests that constraint and genetic details affect quantitative and even qualitative success of optimality predictions. Several features of ST-1 adaptation show that lysis time is best understood as an output of multiple traits, rather than in isolation.

Understanding Primers and Polymerase Chain Reaction by Using a Kinesthetic Paper Model.

Performing a PCR often does not teach students what happens in a PCR. This kinesthetic, hands-on exercise allows students to simulate by hand the steps of a PCR. Each step of the first two cycles is modeled with template DNA and primers formed from paper, with additional newly synthesized DNA strands written in chalk. At the end of the exercise, students design primers to amplify the chosen sequence. They also attain a greater understanding of the restrictions that limit DNA polymerase activity, which are of general importance to molecular biology.

Layers of evolvability in a bacteriophage life history trait.

Functional redundancy in genomes arises from genes with overlapping functions, allowing phenotypes to persist after gene knockouts. Evolutionary redundancy or evolvability of a genome is one step removed, in that functional redundancy is absent but the genome has the potential to evolve to restore a lost phenotype. Exploring the extent to which this recovery alters gene networks can illuminate how functional gene interactions change through time. Here, the evolvability of lysis was studied in bacteriophage T7, revealing hidden functional interactions. Lysis is the destruction of host cell wall and membranes that releases progeny and is therefore essential for phage propagation. In most phages, lysis is mediated by a two-component genetic module: a muralytic enzyme that degrades the bacterial cell wall (endolysin) and a holin that permeabilizes the inner membrane to allow the endolysin access to the cell wall. T7 carries one known holin, one endolysin, and a second muralytic enzyme that plays little role in lysis by wild-type phage. If the primary endolysin is deleted, the second muralytic enzyme evolves to restore normal lysis after selection for faster growth. Here, a second level of evolutionary redundancy was revealed. When the second muralytic enzyme was prevented from adapting in a genome lacking the primary endolysin, the phage reevolved lysis de novo in the absence of any known muralytic enzymes by changes in multiple genes outside the original lysis module. This second level of redundancy proved to be evolutionarily inferior to the first, and both result in a lower fitness and slower lysis than wild-type T7. Deletion of the holin gene delayed lysis time modestly; fitness was restored by compensatory substitutions in genes that lack known roles in lysis of the wild type.

Lytic bacteriophage have diverse indirect effects in a synthetic cross-feeding community.

Bacteriophage shape the composition and function of microbial communities. Yet it remains difficult to predict the effect of phage on microbial interactions. Specifically, little is known about how phage influence mutualisms in networks of cross-feeding bacteria. We mathematically modeled the impacts of phage in a synthetic microbial community in which Escherichia coli and Salmonella enterica exchange essential metabolites. In this model, independent phage attack of either species was sufficient to temporarily inhibit both members of the mutualism; however, the evolution of phage resistance facilitated yields similar to those observed in the absence of phage. In laboratory experiments, attack of S. enterica with P22vir phage followed these modeling expectations of delayed community growth with little change in the final yield of bacteria. In contrast, when E. coli was attacked with T7 phage, S. enterica, the nonhost species, reached higher yields compared with no-phage controls. T7 infection increased nonhost yield by releasing consumable cell debris, and by driving evolution of partially resistant E. coli that secreted more carbon. Our results demonstrate that phage can have extensive indirect effects in microbial communities, that the nature of these indirect effects depends on metabolic and evolutionary mechanisms, and that knowing the degree of evolved resistance leads to qualitatively different predictions of bacterial community dynamics in response to phage attack.

Optimal foraging by bacteriophages through host avoidance.

Optimal foraging theory explains diet restriction as an adaptation to best utilize an array of foods differing in quality, the poorest items not worth the lost opportunity of finding better ones. Although optimal foraging has traditionally been applied to animal behavior, the model is easily applied to viral host range, which is genetically determined. The usual perspective for bacteriophages (bacterial viruses) is that expanding host range is always advantageous if fitness on former hosts is not compromised. However, foraging theory identifies conditions favoring avoidance of poor hosts even if larger host ranges have no intrinsic costs. Bacteriophage T7 rapidly evolved to discriminate among different Escherichia coli strains when one host strain was engineered to kill infecting phages but the other remained productive. After modifying bacteria to yield more subtle fitness effects on T7, we tested qualitative predictions of optimal foraging theory by competing broad and narrow host range phages against each other. Consistent with the foraging model, diet restriction was favored when good hosts were common or there was a large difference in host quality. Contrary to the model, the direction of selection was affected by the density of poor hosts because being able to discriminate was costly.

Songs about cancer, gene expression, and the biochemistry of photosynthesis.

These three biology songs can be used for educational purposes to teach about biochemical concepts. They touch on three different topics: (1) cancer progression and germ cells, (2) gene expression, promoters, and repressors, and (3) electronegativity and the biochemical basis of photosynthesis. © 2017 by The International Union of Biochemistry and Molecular Biology, 46(1):98-99, 2018.

Testing optimality with experimental evolution: lysis time in a bacteriophage.

Optimality models collapse the vagaries of genetics into simple trade-offs to calculate phenotypes expected to evolve by natural selection. Optimality approaches are commonly criticized for this neglect of genetic details, but resolution of this disagreement has been difficult. The importance of genetic details may be tested by experimental evolution of a trait for which an optimality model exists and in which genetic details can be studied. Here we evolved lysis time in bacteriophage T7, a virus of Escherichia coli. Lysis time is equivalent to the age of reproduction in an organism that reproduces once and then dies. Delaying lysis increases the number of offspring but slows generation time, and this trade-off renders the optimum sensitive to environmental conditions: earlier lysis is favored when bacterial hosts are dense, later lysis is favored when hosts are sparse. In experimental adaptations, T7 evolved close to the optimum in conditions favoring early lysis but not in conditions favoring late lysis. One of the late lysis adaptations exhibited no detectable phenotypic evolution despite genetic evolution; the other evolved only partly toward the expected optimum. Overall, the lysis time of the adapted phages remained closer to their starting values than predicted by the model. From the perspective of the optimality model, the experimental conditions were expected to select changes only along the postulated trade-off, but a trait outside the trade-off evolved as well. Evidence suggests that the model's failure ultimately stems from a violation of the trade-off, rather than a paucity of mutations.

Interrelationship between HIV-1 fitness and mutation rate.

Differences in replication fidelity, as well as mutator and antimutator strains, suggest that virus mutation rates are heritable and prone to natural selection. Human immunodeficiency virus type 1 (HIV-1) has many distinct advantages for the study of mutation rate optimization given the wealth of structural and biochemical data on HIV-1 reverse transcriptase (RT) and mutants. In this study, we conducted parallel analyses of mutation rate and viral fitness. In particular, a panel of 10 RT mutants-most having drug resistance phenotypes-was analyzed for their effects on viral fidelity and fitness. Fidelity differences were measured using single-cycle vector assays, while fitness differences were identified using ex vivo head-to-head competition assays. As anticipated, virus mutants possessing either higher or lower fidelity had a corresponding loss in fitness. While the virus panel was not chosen randomly, it is interesting that it included more viruses possessing a mutator phenotype rather than viruses possessing an antimutator phenotype. These observations provide the first description of an interrelationship between HIV-1 fitness and mutation rate and support the conclusion that mutator and antimutator phenotypes correlate with reduced viral fitness. In addition, the findings here help support a model in which fidelity comes at a cost of replication kinetics and may help explain why retroviruses like HIV-1 and RNA viruses maintain replication fidelity near the extinction threshold.

Experimental evolution of a bacteriophage virus reveals the trajectory of adaptation across a fecundity/longevity trade-off.

Life history theory attempts to account for how organisms lead their lives, balancing the conflicting demands of reproduction and survival. Here, we track the genomic and phenotypic evolution of the bacteriophage virus T7 across a postulated fecundity/longevity constraint. We adapted T7 to a challenging survival environment (6M urea). Our evolved strain displayed a significant improvement in propagule survival, coupled with a significant loss of fecundity (reduced growth rate on host cells). However, the increased resistance to urea did not generalise to increased resistance against temperature stress, highlighting that propagule durability is environment dependent. Previous comparative studies predicted that changes in propagule resistance would be mediated by changes in capsid proteins or gene deletions. In contrast, we found that point mutations in internal core protein genes (6.7 and 16) were responsible for the increased urea resistance of our evolved strain. Prior to the emergence of the 6.7 and 16 mutations, a distinct set of 5-point mutations peaked at over 20% prevalence before attenuating, suggestive of negative epistatic interactions during adaptation. Our results illustrate that parasites can adapt to specific transmission environments, and that this adaptation can impose costs on the subsequent ability to exploit host cells, potentially constraining durable parasites to lower virulence.

The phenotype-fitness map in experimental evolution of phages.

Evolutionary biologists commonly interpret adaptations of organisms by reference to a phenotype-fitness map, a model of how different states of a phenotype affect fitness. Notwithstanding the popularity of this approach, it remains difficult to directly test these mappings, both because the map often describes only a small subset of phenotypes contributing to total fitness and because direct measures of fitness are difficult to obtain and compare to the map. Both limitations can be overcome for bacterial viruses (phages) grown in the experimental condition of unlimited hosts. A complete accounting of fitness requires 3 easily measured phenotypes, and total fitness is also directly measurable for arbitrary genotypes. Yet despite the presumed transparency of this system, directly estimated fitnesses often differ from fitnesses calculated from the phenotype-fitness map. This study attempts to resolve these discrepancies, both by developing a more exact analytical phenotype-fitness map and by exploring the empirical foundations of direct fitness estimates. We derive an equation (the phenotype-fitness map) for exponential phage growth that allows an arbitrary distribution of lysis times and burst sizes. We also show that direct estimates of fitness are, in many cases, plausibly in error because the population has not attained stable age distribution and thus violates the model underlying the phenotype-fitness map. In conjunction with data provided here, the new understanding appears to resolve a discrepancy between the reported fitness of phage T7 and the substantially lower value calculated from its phenotype-fitness map.

Evolutionary robustness of an optimal phenotype: re-evolution of lysis in a bacteriophage deleted for its lysin gene.

Optimality models are frequently used to create expectations about phenotypic evolution based on the fittest possible phenotype. However, they often ignore genetic details, which could confound these expectations. We experimentally analyzed the ability of organisms to evolve towards an optimum in an experimentally tractable system, lysis time in bacteriophage T7. T7 lysozyme helps lyse the host cell by degrading its cell wall at the end of infection, allowing viral escape to infect new hosts. Artificial deletion of lysozyme greatly reduced fitness and delayed lysis, but after evolution both phenotypes approached wild-type values. Phage with a lysis-deficient lysozyme evolved similarly. Several mutations were involved in adaptation, but most of the change in lysis timing and fitness increase was mediated by changes in gene 16, an internal virion protein not formerly considered to play a role in lysis. Its muralytic domain, which normally aids genome entry through the cell wall, evolved to cause phage release. Theoretical models suggest there is an optimal lysis time, and lysis more rapid or delayed than this optimum decreases fitness. Artificially constructed lines with very rapid lysis had lower fitness than wild-type T7, in accordance with the model. However, while a slow-lysing line also had lower fitness than wild-type, this low fitness resulted at least partly from genetic details that violated model assumptions.