Abstract
VPg of turnip mosaic virus (TuMV) was previously shown to interact with translation initiation factor eIFiso4F and play an important role in mRNA translation [Khan, M. A., et al. (2008) J. Biol. Chem. 283, 1340-1349]. VPg competed with cap analogue for eIFiso4F binding and competitively inhibited cap-dependent translation and enhanced cap-independent translation to give viral RNA a significant competitive advantage. To gain further insight into the cap-independent process of initiation of protein synthesis, we examined the effect of PABP and/or eIF4B on the equilibrium and kinetics of binding of VPg to eIFiso4F. Equilibrium data showed the addition of PABP and/or eIF4B to eIFiso4F increased the binding affinity for VPg (K-d = 24.3 +/- 1.6 nM) as compared to that with eIFiso4F alone (K-d = 81.3 +/- 0.2.4 nM). Thermodynamic parameters showed that binding of VPg to eIFiso4F was enthalpy-driven and entropy-favorable with the addition of PABP and/or eIF4B. PABP and eIF4B decreased the entropic contribution by 67% for binding of VPg to eIFiso4F. The decrease in entropy involved in the formation of the eIFiso4F.4B.PABP-VPg complex suggested weakened hydrophobic interactions for complex formation and an overall conformational change. The kinetic studies of eIFiso4F with VPg in the presence of PABP and eIF4B show 3-fold faster association (k(2) = 182 +/- 9.0 s(-1)) compared to that with eIFiso4F alone (k(2) = 69.0 +/- 1.5 s(-1)). The dissociation rate was 3-fold slower (k(-2) = 6.5 +/- 0.43 s(-1)) for eIFiso4F with VPg in the presence of PABP and eIF4B (k(-2) = 19.0 +/- 0.9 s(-1)). The addition of PABP and eIF4B decreased the activation energy of eIFiso4F with VPg from 81.0 +/- 3.0 to 44.0 +/- 2.4 kJ/mol. This suggests that the presence of both proteins leads to a rapid, stable complex, which serves to sequester initiation factors.