Poly (A) binding protein enhances the binding affinity of potyvirus VPg to eukaryotic initiation factor eIF4F and activates in vitro translation

Abstract

Viral protein linked to the genome (VPg) of turnip mosaic virus (TuMV) interacts with eIF4F and plays an important role in cap-independent initiation of protein synthesis. In order to understand the importance of PABP on the interaction of eIF4F with VPg, we report that PABP enhanced the binding affinity ~3- and 4-fold for eIF4F-VPg and eIF4F·eIF4B-VPg, respectively. PABP enhances rates of protein synthesis in uncapped viral mRNA and correlates with binding affinity of eIF4F with VPg. Temperature dependent (278 K to 298 K) showed ~3-fold increase in eIF4F binding to VPg in presence of PABP and eIF4B. A van't Hoff analysis reveals that eIF4F·eIF4B·PABP binding to VPg was enthalpy-driven and entropy-favorable with 30% increase in enthalpic contribution and 81% decrease in entropic contribution. PABP increased the association rate (4-fold) and decreased the dissociation rate (3-fold) for the eIF4F·eIF4B binding to VPg. PABP significantly decreased the activation energy of eIF4F·eIF4B binding to VPg. When both PABP and eIF4B were present, not only was the energy barrier reduced but the binding rate was faster and dissociation rate was slower for the protein-protein complex formation. These results suggest increased hydrogen bonding and an overall conformational change, and more stable platform for effective viral translation.