Molecular mechanism of poliovirus Sabin vaccine strain attenuation

J Biol Chem. 2018 Oct 5;293(40):15471-15482. doi: 10.1074/jbc.RA118.004913. Epub 2018 Aug 20.


Recruitment of poliovirus (PV) RNA to the human ribosome requires the coordinated interaction of the viral internal ribosome entry site (IRES) and several host cellular initiation factors and IRES trans-acting factors (ITAFs). Attenuated PV Sabin strains contain point mutations in the PV IRES domain V (dV) that inhibit viral translation. Remarkably, attenuation is most apparent in cells of the central nervous system, but the molecular basis to explain this is poorly understood. The dV contains binding sites for eukaryotic initiation factor 4G (eIF4G) and polypyrimidine tract-binding protein (PTB). Impaired binding of these proteins to the mutant IRESs has been observed, but these effects have not been quantitated. We used a fluorescence anisotropy assay to reveal that the Sabin mutants reduce the equilibrium dissociation constants of eIF4G and PTB to the PV IRES by up to 6-fold. Using the most inhibitory Sabin 3 mutant, we used a real-time fluorescence helicase assay to show that the apparent affinity of an active eIF4G/4A/4B helicase complex for the IRES is reduced by 2.5-fold. The Sabin 3 mutant did not alter the maximum rate of eIF4A-dependent helicase activity, suggesting that this mutant primarily reduces the affinity, rather than activity, of the unwinding complex. To confirm this affinity model of attenuation, we show that eIF4G overexpression in HeLa cells overcomes the attenuation of a Sabin 3 mutant PV-luciferase replicon. Our study provides a quantitative framework for understanding the mechanism of PV Sabin attenuation and provides an explanation for the previously observed cell type-specific translational attenuation.

Keywords: RNA-binding protein; eukaryotic initiation factor 4A (eIF4A); eukaryotic translation initiation; eukaryotic translation initiation factor 4G (eIF4G); internal ribosome entry site (IRES); plus-stranded RNA virus; poliovirus; vaccine; virulence.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Baculoviridae / genetics
  • Baculoviridae / immunology
  • Base Sequence
  • Cloning, Molecular
  • Escherichia coli / genetics
  • Escherichia coli / immunology
  • Eukaryotic Initiation Factor-4A / genetics
  • Eukaryotic Initiation Factor-4A / immunology
  • Eukaryotic Initiation Factor-4G / genetics*
  • Eukaryotic Initiation Factor-4G / immunology
  • Gene Expression
  • Genes, Reporter
  • Genetic Vectors / chemistry
  • Genetic Vectors / metabolism
  • HeLa Cells
  • Humans
  • Internal Ribosome Entry Sites
  • Luciferases / genetics
  • Luciferases / metabolism
  • Mutation*
  • Nucleic Acid Conformation
  • Poliovirus / genetics*
  • Poliovirus / immunology
  • Poliovirus Vaccine, Oral / biosynthesis
  • Poliovirus Vaccine, Oral / genetics*
  • Poliovirus Vaccine, Oral / immunology
  • Polypyrimidine Tract-Binding Protein / genetics*
  • Polypyrimidine Tract-Binding Protein / immunology
  • Protein Biosynthesis*
  • Recombinant Proteins / genetics
  • Recombinant Proteins / immunology
  • Sequence Alignment
  • Sf9 Cells
  • Spodoptera
  • Vaccines, Attenuated


  • Eukaryotic Initiation Factor-4G
  • Internal Ribosome Entry Sites
  • Poliovirus Vaccine, Oral
  • Recombinant Proteins
  • Vaccines, Attenuated
  • Polypyrimidine Tract-Binding Protein
  • Luciferases
  • Eukaryotic Initiation Factor-4A