Translation Initiation Factors eIF4F and eIFiso4F from Wheat Germ Interact Differently with Potyvirus Genome‐linked Protein, VPg.

The binding between wheat germ eukaryotic translation initiation factors eIF4F or eIFiso4F and viral protein linked to the genome (VPg) of potyvirus were measured and compared. The fluorescence quenching data showed eIF4F (K d = 219.41 nM) has about 2.6‐fold less affinity than eIFiso4F (K d = 84.34 nM) for VPg. However, eIF4F with tobacco etch virus (TEV) RNA (K d = 79.0 nM) showed ~2.3‐fold higher binding affinity as compared to eIFiso4F and TEV RNA (K d = 178.0 nM). In order to determine if these reactions were competitive, addition of TEV RNA to the eIF4F‐VPg and eIFiso4F‐VPg complex was examined and found to increase the protein‐protein affinity. The temperature dependent binding studies revealed that VPg binding to eIF4F and eIFiso4F is enthalpy‐driven and entropy favorable with a large negative ΔH° (−27.83 and −31.03 kJmole −1 ), and positive ΔS° (34.12 and 32.32 Jmole −1 K −1 ). A Lineweaver‐Burk plot indicates noncompetitive or uncompetitive binding between VPg and TEV RNA for both eIF4F and eIFiso4F. Enhancement of eIF4F‐VPg and eIFiso4F‐VPg binding with the addition of TEV RNA suggests that translation initiation involving VPg occurs at internal ribosomal entry sites. Overall these results suggest the direct involvement of viral genome protein (VPg) in protein translation. Supported by Research Center in Minority Institution Award RR‐03037 from the National Center for Research Resources of NIH and National Science Foundation Grant MCB 0413982.