Antigenic diversification is correlated with increased thermostability in a mammalian virus

Abstract

The theory of plastogenetic congruence posits that ultimately, the pressure to maintain function in the face of biomolecular destabilization produces robustness. As temperature goes up so does destabilization. Thus, genetic robustness, defined as phenotypic constancy despite mutation, should correlate with survival during thermal challenge. We tested this hypothesis using vesicular stomatitis virus (VSV). We produced two sets of evolved strains after selection for higher thermostability by either preincubation at 37°C or by incubation at 40°C during infection. These VSV populations became more thermostable and also more fit in the absence of thermal selection, demonstrating an absence of tradeoffs. Eleven out of 12 evolved populations had a fixed, nonsynonymous substitution in the nucleocapsid (N) open reading frame. There was a partial correlation between thermostability and mutational robustness that was observed when the former was measured at 42°C, but not at 37°C. These results are consistent with our earlier work and suggest that the relationship between robustness and thermostability is complex. Surprisingly, many of the thermostable strains also showed increased resistance to monoclonal antibody and polyclonal sera, including sera from natural hosts. These data suggest that evolved thermostability may lead to antigenic diversification and an increased ability to escape immune surveillance in febrile hosts, and potentially to an improved robustness. These relationships have important implications not only in terms of viral pathogenesis, but also for the development of vaccine vectors and oncolytic agents.

Fig. 1. Schematic for experimental evolution

Regimens used to generated the 37-adapted and 40- adapted populations as well as the adapted controls.

Fig. 2. Thermostability of evolved VSV strains at selected temperatures

WT and adapted strains were tested for thermostability (a) at 37°C and (b) 42°C as described in the text. As confirmed by a two-sample t-test, the 37-adapted strains showed an increase in thermostability at 42°C (p = 0.0278), while the 40-adapted strains showed increases in thermostability at both 37°C (p = 0.0026) and 42°C (p = 0.0334). At 37°C the 37-adapted viruses were not more thermostable than control adapted populations (p = 0.9900) while the 40-adapted viruses were more thermostable (p = 0.0103). (*): p < 0.05, (**): p < 0.01.

Fig. 3. Fitness of thermostable VSV strains

Fitness was determined for each adapted strain. As controls, we show eight independent WT replicas passaged 25 times under identical conditions, except for the thermal selective pressure (e.g. without preincubation of virions at 37°C before infection and keeping the temperature at 37°C during infection). Population means were compared using a two-sample t-test (37-adapted p = 0.0242, 40-adapted p = 0.0137). However, both strains also exhibited lower fitness gains than seen in the control populations (37-adapted p = 0.0014, 40-adapted p = 0.0047), suggesting a fitness cost to thermal adaption. (*): p < 0.05, (**): p < 0.01

Fig. 4. Mutational robustness of thermostable VSV strains

(a) The robustness of WT and thermostable strains were tested as survival in the presence of 5FU. There was no significant increase in robustness for 37-adapted viruses (p = 0.1668) but there was an increase in robustness for 40-adapted viruses (p = 0.0057). Adapted controls were also more robust than WT (p = 0.0119), and neither 37-adapted (p = 0.7088) or 40-adapted (p = 0.1245) virus populations were significantly more robust than the adapted controls. (b) The relationship between thermostability at 37°C and robustness for 37- and 40-adapted strains. A Pearson test revealed no correlation between robustness and thermostability at 37°C for this complete set of thermostable strains (r = 0.0081, p = 0.4895). (c) The relationship between thermostability at 42°C and robustness for 37- and 40-adapted strains. A Pearson test revealed a significant correlation between robustness and thermostability at 42°C for the complete set of thermostable strains, represented by the dashed line (r = 0.5370, p = 0.0292). (*): p < 0.05, (**): p < 0.01

Fig. 5. Correlation between robustness and mutation frequency

A Pearson test showed no correlation between mutation frequency, shown as number of mutations per 1000 bases and robustness, expressed as sensitivity to mutagen (r = 0.2663, p = 0.1896).

Fig. 6. I1 sensitivity of thermostable VSV strains

WT and adapted strains were titrated in the presence and absence of I1 Mab. Mutant frequency was determined as the titer in the presence of I1 divided by the full titer in the absence of I1. These ratios were then log-transformed due to a very large range of values. Using a two-sample t-test, no significant difference was seen in the average of any adapted group (37-adapted p = 0.4180, 40-adapted p = 0.3291; adapted controls p = 0.7808). The outliers are 37B and 40F.

Fig. 7. Sensitivity of thermostable VSV strains to polyclonal sera from natural hosts

Sera from two separate pigs, Pig 1 (a), and Pig 2 (b), a horse (c), and a rabbit (d) were used to test antibody sensitivity in WT and the adapted virus strains as described previously. (a) The 37-adapted viruses mean sensitivity to Pig 1 serum was not significantly different than WT (two-sample t-test, p = 0.5451), but the 40-adapted virus mean sensitivity was significantly less, indicated by the greater number of antibody resistant mutants present (p = 0.0418). The outlier is 37D; the most resistant strain among the 40-adapted is 40F. The control-adapted population did not show any significant differences in sensitivity (p = 0.7644). The 40-adapted population was also significantly more resistant to the serum than the control adapted viruses (p = 0.0008).(b) The 37-adapted population was not significantly more resistant to the second pig serum (p = 0.0817) but the 40-adapted population exhibited reduced sensitivity to this serum (p = 0.0176). The most resistant among the 40-adapted strains is 40F. The control-adapted population was not more resistant than ancestral WT (p = 0.2062). Both the 37-adapted (p = 0.0007) and the 40-adapted (p < 0.0001) populations are significantly more resistant to this serum than the control adapted populations. (c) 37-adapted viruses exhibited no difference in sensitivity to the antibodies in the horse serum compared to ancestral WT (p = 0.5856), while the 40-adapted viruses again showed greater resistance (p = 0.0215). The most resistant strain among the 37-adapted strains is 37D, and the least resistant among the 40-adapted strains is 40E. Control adapted populations were not able to be tested against this serum. (d) The 37-adapted viruses mean sensitivity to rabbit serum was significantly less than ancestral WT (p = 0.0222), but the 40-adapted virus mean sensitivity was not significantly different than WT (p = 0.2210). The control adapted populations also showed a significant decrease in sensitivity when compared to WT (p = 0.0390). Neither population was more resistant than the control adapted population (37-adapted p = 0.1529, 40-adapted p = 0.3531).(*): p < 0.05; (***): p < 0.001