Poly(A)-Binding Protein Increases the Binding Affinity and Kinetic Rates of Interaction of Viral Protein Linked to Genome with Translation Initiation Factors eIFiso4F and eIFiso4F·4B Complex

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.