John Maynard Smith compared protein evolution to the game where one word is converted into another a single letter at a time, with the constraint that all intermediates are words: WORD→WORE→GORE→GONE→GENE. In this analogy, epistasis constrains evolution, with some mutations tolerated only after the occurrence of others. To test whether epistasis similarly constrains actual protein evolution, we created all intermediates along a 39-mutation evolutionary trajectory of influenza nucleoprotein, and also introduced each mutation individually into the parent. Several mutations were deleterious to the parent despite becoming fixed during evolution without negative impact. These mutations were destabilizing, and were preceded or accompanied by stabilizing mutations that alleviated their adverse effects. The constrained mutations occurred at sites enriched in T-cell epitopes, suggesting they promote viral immune escape. Our results paint a coherent portrait of epistasis during nucleoprotein evolution, with stabilizing mutations permitting otherwise inaccessible destabilizing mutations which are sometimes of adaptive value.
During evolution, the effect of one mutation on a protein can depend on whether another mutation is also present. This phenomenon is similar to the game in which one word is converted to another word, one letter at a time, subject to the rule that all the intermediate steps are also valid words: for example, the word WORD can be converted to the word GENE as follows: WORD→WORE→GORE→GONE→GENE. In this example, the D must be changed to an E before the W is changed to a G, because GORD is not a valid word.
Similarly, during the evolution of a virus, a mutation that helps the virus evade the human immune system might only be tolerated if the virus has acquired another mutation beforehand. This type of mutational interaction would constrain the evolution of the virus, since its capacity to take advantage of the second mutation depends on the first mutation having already occurred.
Gong et al. examined whether such interactions have indeed constrained evolution of the influenza virus. Between 1968 and 2007, the nucleoprotein—which acts as a scaffold for the replication of genetic material—in the human H3N2 influenza virus underwent a series of 39 mutations. To test whether all of these mutations could have been tolerated by the 1968 virus, Gong et al. introduced each one individually into the 1968 nucleoprotein. They found that several mutations greatly reduced the fitness of the 1968 virus when introduced on their own, which strongly suggests that these ‘constrained mutations’ became part of the virus’s genetic makeup as a result of interactions with ‘enabling’ mutations.
The constrained mutations decreased the stability of the nucleoprotein at high temperatures, while the enabling mutations counteracted this effect. It may, therefore, be possible to identify enabling mutations based on their effects on thermal stability. Intriguingly, the constrained mutations helped the virus overcome one form of human immunity to influenza, suggesting that interactions between mutations might limit the rate at which viruses evolve to evade the immune system.
Overall, these results show that interactions among mutations constrain the evolution of the influenza nucleoprotein in a fashion that can be largely understood in terms of protein stability. If the same is true for other proteins and viruses, this work could lead to a deeper understanding of the constraints that govern evolution at the molecular level.