Synonymous Mutations at the Beginning of the Influenza A Virus Hemagglutinin Gene Impact Experimental Fitness

J Mol Biol. 2018 Apr 13;430(8):1098-1115. doi: 10.1016/j.jmb.2018.02.009. Epub 2018 Feb 18.


The fitness effects of synonymous mutations can provide insights into biological and evolutionary mechanisms. We analyzed the experimental fitness effects of all single-nucleotide mutations, including synonymous substitutions, at the beginning of the influenza A virus hemagglutinin (HA) gene. Many synonymous substitutions were deleterious both in bulk competition and for individually isolated clones. Investigating protein and RNA levels of a subset of individually expressed HA variants revealed that multiple biochemical properties contribute to the observed experimental fitness effects. Our results indicate that a structural element in the HA segment viral RNA may influence fitness. Examination of naturally evolved sequences in human hosts indicates a preference for the unfolded state of this structural element compared to that found in swine hosts. Our overall results reveal that synonymous mutations may have greater fitness consequences than indicated by simple models of sequence conservation, and we discuss the implications of this finding for commonly used evolutionary tests and analyses.

Keywords: deep mutational scanning; experimental evolution; influenza A virus; selection; synonymous mutations.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Amino Acid Substitution
  • Animals
  • Dogs
  • Evolution, Molecular
  • Genetic Fitness*
  • HEK293 Cells
  • Hemagglutinin Glycoproteins, Influenza Virus / chemistry*
  • Hemagglutinin Glycoproteins, Influenza Virus / genetics*
  • Humans
  • Influenza A Virus, H1N1 Subtype / genetics
  • Influenza A Virus, H1N1 Subtype / growth & development*
  • Madin Darby Canine Kidney Cells
  • Models, Molecular
  • Phylogeny
  • RNA Folding
  • Silent Mutation*
  • Swine
  • Virus Replication


  • H1N1 virus hemagglutinin
  • Hemagglutinin Glycoproteins, Influenza Virus