Abstract
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.
Highlights
Summary of Kd and anisotropy values All values are the mean of three independent experiments
Summary of Kd,app and unwinding values All values are the mean of three independent experiments
The calculated apparent affinity of the eIF4G682–1599/A/B complex to the Sabin 3 IRES is reduced by 2.5-fold compared with the WT IRES. This is consistent with the reduced affinity we observed for eIF4G binding to the Sabin 3 mutant PV dV using our anisotropy assay
Summary
To fully understand the extent to which the Sabin mutations reduce initiation factor affinity for the PV IRES. It has been suggested that variation in eIF4G and/or PTB protein levels in different cell types could provide a molecular basis for observed tissue tropism effects of Sabin mutant PV strains and reporter genes [4, 21, 22]. This indicates that this mutant functions by reducing the affinity of the components for the IRES rather than the activity of the bound components To directly test this affinity model in growing cells, we overexpressed a functional truncation of eIF4GI (eIF4G557–1599) to determine whether it can overcome the translation defect caused by the Sabin 3 mutation of a PV–luciferase replicon. Our results provide insight into the molecular mechanism of PV Sabin attenuation and support a model in which variations in the intracellular concentration of eIF4G can be responsible for cell type–specific translation defects of a PV Sabin 3 mutant
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