Adaptation to fluctuating temperatures in an RNA virus is driven by the most stringent selective pressure

Abstract

The frequency of change in the selective pressures is one of the main factors driving evolution. It is generally accepted that constant environments select specialist organisms whereas changing environments favour generalists. The particular outcome achieved in either case also depends on the relative strength of the selective pressures and on the fitness costs of mutations across environments. RNA viruses are characterized by their high genetic diversity, which provides fast adaptation to environmental changes and helps them evade most antiviral treatments. Therefore, the study of the adaptive possibilities of RNA viruses is highly relevant for both basic and applied research. In this study we have evolved an RNA virus, the bacteriophage Qβ, under three different temperatures that either were kept constant or alternated periodically. The populations obtained were analyzed at the phenotypic and the genotypic level to characterize the evolutionary process followed by the virus in each case and the amount of convergent genetic changes attained. Finally, we also investigated the influence of the pre-existent genetic diversity on adaptation to high temperature. The main conclusions that arise from our results are: i) under periodically changing temperature conditions, evolution of bacteriophage Qβ is driven by the most stringent selective pressure, ii) there is a high degree of evolutionary convergence between replicated populations and also among populations evolved at different temperatures, iii) there are mutations specific of a particular condition, and iv) adaptation to high temperatures in populations differing in their pre-existent genetic diversity takes place through the selection of a common set of mutations.

Figure 1. Description of the evolution experiment designed to study the adaptation of bacteriophage Qβ to constant or deterministically fluctuating temperatures.

The same virus population (Qβ₀) was used to found 5 replicated evolutionary lineages that were evolved in parallel during 15 transfers either at constant (populations Qβ_37, Qβ_30, and Qβ₄₃)_, or fluctuating temperatures (populations Qβ_F1, and Qβ_F5) under the conditions described in Materials and Methods. Replicate populations were labelled with the subscripts (1) and (2). Each evolved population was analyzed at the phenotypic level (determination of fitness at 37°C, 30°C, and 43°C), and at the genotypic level (determination of the consensus sequence from nucleotide 250 to 4180).

Figure 2. Description of the evolution experiment designed to study the influence of the pre-existent genetic diversity on the adaptation of bacteriophage Qβ to high temperature.

The clonal population of the virus, cQβ, obtained upon expression of the plasmid pBRT7Qβ (see Materials and Methods), was propagated at 37°C during 10 serial transfers to yield populations cQβ₃₇₍₁₎ and cQβ₃₇₍₂₎. Population cQβ₃₇₍₁₎ was subjected to 10 additional transfers at 43°C, to yield populations cQβ_37-43(1) and cQβ_37-43(2). The virus cQβ was also adapted to 43°C without previous optimization at 37°C. Adaptation took place through 15 serial transfers to render populations cQβ₄₃₍₁₎ and cQβ₄₃₍₂₎. All evolved populations were characterized at the genotypic level through their consensus sequences (from nucleotide 250 to 4180).

Figure 3. Fitness values of bacteriophage Qβ populations evolved at constant temperatures.

Fitness values at 37°C (upper panel), 30°C (medium panel), and 43°C (lower panel) of the two replicas of populations Qβ_37, Qβ_30, and Qβ_43. Each bar represents the average of three parallel determinations carried out as described in Materials and Methods. The error bars represent the standard deviation. The fitness value obtained for the ancestor population, Qβ_0, at each temperature is indicated by a discontinuous line. Thus, bars above this line represent fitness increases relative to the ancestor and bars below the line correspond to fitness decreases. Asterisks mean that the average fitness for a given population is significantly higher (black ones) or lower (red ones) than the fitness value of population Qβ₀ (P<0.05, Student' t test).

Figure 4. Fitness values of bacteriophage Qβ populations evolved at fluctuating temperatures.

Fitness values at 37°C (upper panel), 30°C (medium panel), and 43°C (lower panel) of the two replicas of populations Qβ_F1, and Qβ_F5. Each bar represents the average of three parallel determinations carried out as described in Materials and Methods. The error bars represent the standard deviation. The fitness value obtained for the ancestor population Qβ₀ at each temperature is indicated by a discontinuous line. Thus, bars above this line represent fitness increases relative to the ancestor and bars below the line correspond to fitness decreases. Asterisks mean that the average fitness for a given population is significantly higher (black ones) or lower (red ones) than the fitness value of population Qβ₀ (P<0.05, Student' t test).

Figure 5. Genetic map of bacteriophage Qβ.

The map shows the genes for the 4 proteins encoded by the virus [A2 (from nt 61 to nt 1320), coat (from nt 1344 to nt 1742), A1 (from nt 1344 to nt 2330, and replicase (from nt 2352 to nt 4118)], and the non-translated regions. The protein A1 synthesizes when the stop codon of the coat protein is read through by a trp-tRNA, giving rise to an elongated protein that incorporates at low amount in the capsid.