Phosphorylation of translation initiation factor eIFiso4E promotes translation through enhanced binding to potyvirus VPg.

Abstract

Interactions of phosphorylated eIFiso4E binding to VPg as a function of temperature and ionic strength were assessed employing fluorescence spectroscopic. Phosphorylation increased the binding affinity ∼3.5-fold between VPg and eIFiso4E under equilibrium conditions. Binding affinity of VPg for eIFiso4Ep correlates with the ability to enhance in vitro protein synthesis. Addition of VPg and eIFiso4Ep together to Dep WGE enhances the translation for both uncapped and capped mRNA. However, capped mRNA translation was inhibited with addition of eIFiso4Ep alone in dep WGE, suggesting that phosphorylation prevents the cap binding and favours the VPg binding to promotes translation. Temperature dependence showed that the phosphorylated form of the eIFiso4E is preferred for complex formation. A van't Hoff analysis reveals that eIFiso4Ep binding to VPg was enthalpy driven (ΔH = -43.9 ± 0.3 kJ.mol-1) and entropy-opposed (ΔS = -4.3 ± 0.1 J.mol-1K-1). Phosphorylation increased the enthalpic contributions ∼33% for eIFiso4Ep-VPg complex. The thermodynamic values and ionic strength dependence of binding data suggesting that phosphorylation increased hydrogen-bonding and decreased hydrophobic interactions, which leads to more stable complex formation and favour efficient viral translation. Overall these data correlate well with the observed translational data and provide more detailed information on the translational strategy of potyviruses.