Adaptations of an RNA virus to increasing thermal stress

Environments can change in incremental fashions, where a shift from one state to another occurs over multiple organismal generations. The rate of the environmental change is expected to influence how and how well populations adapt to the final environmental state. We used a model system, the lytic RNA bacteriophage Φ6, to investigate this question empirically. We evolved viruses for thermostability by exposing them to heat shocks that increased to a maximum temperature at different rates. We observed increases in the ability of many heat-shocked populations to survive high temperature heat shocks. On their first exposure to the highest temperature, populations that experienced a gradual increase in temperature had higher average survival than populations that experienced a rapid temperature increase. However, at the end of the experiment, neither the survival of populations at the highest temperature nor the number of mutations per population varied significantly according to the rate of thermal change. We also evaluated mutations from the endpoint populations for their effects on viral thermostability and growth. As expected, some mutations did increase viral thermostability. However, other mutations decreased thermostability but increased growth rate, suggesting that benefits of an increased replication rate may have sometimes outweighed the benefits of enhanced thermostability. Our study highlights the importance of considering the effects of multiple selective pressures, even in environments where a single factor changes

Adaptations of an RNA virus to increasing thermal stress

<div><p>Environments can change in incremental fashions, where a shift from one state to another occurs over multiple organismal generations. The <i>rate</i> of the environmental change is expected to influence how and how well populations adapt to the final environmental state. We used a model system, the lytic RNA bacteriophage Φ6, to investigate this question empirically. We evolved viruses for thermostability by exposing them to heat shocks that increased to a maximum temperature at different rates. We observed increases in the ability of many heat-shocked populations to survive high temperature heat shocks. On their first exposure to the highest temperature, populations that experienced a gradual increase in temperature had higher average survival than populations that experienced a rapid temperature increase. However, at the end of the experiment, neither the survival of populations at the highest temperature nor the number of mutations per population varied significantly according to the rate of thermal change. We also evaluated mutations from the endpoint populations for their effects on viral thermostability and growth. As expected, some mutations did increase viral thermostability. However, other mutations <i>decreased</i> thermostability but increased growth rate, suggesting that benefits of an increased replication rate may have sometimes outweighed the benefits of enhanced thermostability. Our study highlights the importance of considering the effects of multiple selective pressures, even in environments where a single factor changes.</p></div

Temperature regimes for experimental evolution with varying rates of thermal change.

<p>Points are offset vertically at 50°C for purposes of visualization. Temperature increments were chosen such that the ancestral virus would experience constant (i.e., linear) decreases in its probability of survival. A control regime of heat shocks at a constant 25°C (not shown) accounted for evolutionary change under transfer conditions. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0189602#pone.0189602.s002" target="_blank">S2 Table</a>.</p

Relative competitive fitness of two double mutants and their constituent single mutants.

<p>Bar heights represent the mean of three replicates; error bars denote standard deviation. After a Bonferroni correction for the number of comparisons, relative competitive fitnesses of the mutants do not differ from the ancestor’s. We note that addition of the double mutations did not change the overall relationship between relative competitive fitness and <i>T</i>₅₀ shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0189602#pone.0189602.g009" target="_blank">Fig 9</a>.</p

Summary of effects of single mutations from endpoint populations on viral thermostability and competitive fitness.

<p>Summary of effects of single mutations from endpoint populations on viral thermostability and competitive fitness.</p

Average percent survival of populations on first transfer at 50°C.

<p>Sudden populations first experienced 50°C on Transfer 1, Moderate populations on Transfer 17, and Gradual populations on Transfer 32. Error bars represent the standard deviation of percent survival. Mean survivals are significantly different between Sudden and Gradual populations (Tukey’s post-hoc test, p = 0.03).</p

Estimation of the thermal kill curve of the ancestral Φ6 genotype.

<p>Temperatures from 44–52°C are shown to visualize the drop in viral survival. A) On three separate days, a cell-free lysate was exposed to a 5-minute heat shock at each temperature and plated before and afterward to calculate percent survival (points). (Note that, because of stochasticity in determining phage titers, survivals occasionally exceed 100%.) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0189602#pone.0189602.e001" target="_blank">Eq 1</a> was fitted to the data in R, where the parameters <i>a</i>, <i>T</i>₅₀, and <i>n</i> were estimated by nonlinear least squares (lines). B) Data from each day were then divided by their respective asymptotes <i>a</i> (points). The adjusted data were pooled and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0189602#pone.0189602.e001" target="_blank">Eq 1</a> re-fit to calculate the ancestral thermal kill curve (line). This curve was used for calculation of the thermal regimes and to set null expectations of viral survival during the evolution experiment.</p

Average percent survival at 50°C on the final transfer (Transfer 32) of the experiment.

<p>Error bars represent the standard deviation of percent survival. There are not significant differences in survival between treatments.</p

Data repository: Adaptations of an RNA virus to increasing thermal stress

Data repository and analysis scripts for pre-print version of paper "Adaptations of an RNA Virus to Increasing Thermal Stress.

Mutations in genes 5 and 8 in each population at the end of the evolution experiment.

<p>Mutations in genes 5 and 8 in each population at the end of the evolution experiment.</p