Potyvirus genome-linked protein, VPg, directly affects wheat germ in vitro translation: interactions with translation initiation factors eIF4F and eIFiso4F.
Potyvirus genome linked protein, VPg, interacts with translation initiation factors eIF4E and eIFiso4E, but its role in protein synthesis has not been elucidated. We show that addition of VPg to wheat germ extract leads to enhancement of uncapped viral mRNA translation and inhibition of capped viral mRNA translation. This provides a significant competitive advantage to the uncapped viral mRNA. To understand the molecular basis of these effects, we have characterized the interaction of VPg with eIF4F, eIFiso4F, and a structured RNA derived from tobacco etch virus (TEV RNA). When VPg formed a complex with eIF4F, the affinity for TEV RNA increased more than 4-fold compared with eIF4F alone (19.4 and 79.0 nm, respectively). The binding affinity of eIF4F to TEV RNA correlates with translation efficiency. VPg enhanced eIFiso4F binding to TEV RNA 1.6-fold (178 nm compared with 108 nm). Kinetic studies of eIF4F and eIFiso4F with VPg show approximately 2.6-fold faster association for eIFiso4F.VPg as compared with eIF4F.VPg. The dissociation rate was approximately 2.9-fold slower for eIFiso4F than eIF4F with VPg. These data demonstrate that eIFiso4F can kinetically compete with eIF4F for VPg binding. The quantitative data presented here suggest a model where eIF4F.VPg interaction enhances cap-independent translation by increasing the affinity of eIF4F for TEV RNA. This is the first evidence of direct participation of VPg in translation initiation.
Khan
Mateen A
MA
Department of Chemistry, Hunter College and the Graduate Center of the City University of New York, New York, New York 10065.
Miyoshi
Hiroshi
H
Department of Microbiology, St. Marianna University School of Medicine, Kawasaki 216-8511, Japan.
Gallie
Daniel R
DR
Department of Biochemistry, University of California, Riverside, California 92521-0129.
Goss
Dixie J
DJ
Department of Chemistry, Hunter College and the Graduate Center of the City University of New York, New York, New York 10065. Electronic address: dgoss@hunter.cuny.edu.
D002457:Cell Extracts; D039562:Eukaryotic Initiation Factor-4F / Q000378:metabolism*; D005453:Fluorescence; D017930:Genes, Reporter; D007700:Kinetics; D008156:Luciferases / Q000378:metabolism; D050505:Mutant Proteins / Q000378:metabolism; D011485:Protein Binding; D014176:Protein Biosynthesis; D012315:RNA Caps / Q000378:metabolism; D012367:RNA, Viral / Q000235:genetics; D012261:Ribonucleoproteins / Q000378:metabolism*; D014908:Triticum / Q000378:metabolism*; D017361:Viral Nonstructural Proteins / Q000378:metabolism*
D002457:Cell Extracts; D039562:Eukaryotic Initiation Factor-4F; D050505:Mutant Proteins; D012315:RNA Caps; D012367:RNA, Viral; D012261:Ribonucleoproteins; D017361:Viral Nonstructural Proteins; C102206:genome-linked viral protein, potato virus Y; D008156:Luciferases