PABP and eIF4B Effects Binding Affinity and Kinetic Rates of Genome Linked Viral Protein (VPg) with Eukaryotic Initiation Factor 4F

Abstract

Translation initiation is a critical early step in the viral replication cycle. Interaction of eukaryotic translational initiation factors with VPg of Turnip mosaic virus (TuMV) plays an important role in the cap‐independent initiation of protein synthesis. To understand this process in potyviruses, we investigated the equilibrium binding, thermodynamic parameters and rate constants for the interaction of VPg with eIF4F in the presence of PABP and eIF4B using fluorescence and stopped‐flow spectroscopy. Equilibrium studies showed that PABP increased VPg binding to eIF4F about 3‐fold; however, both PABP and eIF4B together enhanced VPg binding to eIF4F about 4‐fold, showing an additive effect rather than the large increase in affinity shown for cap binding. Temperature dependent studies showed the increase in VPg binding to eIF4F in the presence of PABP and eIF4B was enthalpy‐driven and entropy‐favorable. PABP and eIF4B decreased the entropic contribution for VPg binding to eIF4F. The decrease in entropy for the formation of eIF4F·4Bmiddot;PABP‐VPg complex suggested weakened hydrophobic interactions for complex formation and an overall conformational change. Rate constant for eIF4F interaction with VPg in the presence of PABP and eIF4B was 4‐fold faster; and dissociation rate was 3‐fold slower as compared to eIF4F alone. The temperature dependence of the VPg binding to eIF4F was significantly reduced in activation energy in the presence of PABP and eIF4B. 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 formation of eIF4F‐4B‐PABP‐VPg complex suggests more stable platform for further initiation complex formation. These results further support the importance of PABP and eIF4B interaction to eIF4F with VPg for efficient viral translation.Support or Funding InformationNational Science Foundation MCB‐1513737 (to D.J.G.) and Alfaisal University Research Support IRG‐16414 (to M.A.K.)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.