Binding analyses for the interaction between plant virus genome-linked protein (VPg) and plant translational initiation factors.
The turnip mosaic virus (TuMV) genome-linked protein (VPg) and Arabidopsis thaliana translation initiation factors were expressed and purified in order to investigate their binding properties and kinetics. Affinity chromatography on m(7)GTP-sepharose showed that bound A. thaliana eIF(iso)4E was eluted with crude TuMV VPg. Further column studies with purified VPg and other A. thaliana eIF4E isoforms showed that VPg preferentially bound eIF(iso)4E. Structural data implicate Trp-46 and Trp-92 in eIF(iso)4E in cap recognition. When Trp-46 or Trp-92 were changed to Leu, eIF(iso)4E lost the ability to form a complex with both VPg and m(7)GTP-sepharose. This suggests that the VPg-binding site is located in or near the cap-recognition pocket on eIF(iso)4E. Affinity constants for the interactions with eIF(iso)4E of VPg and capped RNA oligomer were determined using surface plasmon resonance (SPR). The K(D) values showed that the binging affinity of VPg for eIF(iso)4E is stronger than that of capped RNA. This suggests that viral VPg can interfere with formation of a translational initiation complex on host plant cellular mRNA by sequestering eIF(iso)4E. Further experiments with affinity chromatography showed that VPg forms a ternary complex with eIF(iso)4E and eIF(iso)4G. Thus, VPg may participate in viral translational initiation by functioning as an alternative cap-like structure.
Miyoshi
Hiroshi
H
Department of Microbiology, St. Marianna University School of Medicine, 2-16-1 Sugao, Miyamae, Kawasaki 216-8511, Japan. hmiyoshi@marianna-u.ac.jp
Suehiro
Noriko
N
Tomoo
Koji
K
Muto
Shinji
S
Takahashi
Tsubasa
T
Tsukamoto
Toshiro
T
Ohmori
Taku
T
Natsuaki
Tomohide
T
D000595:Amino Acid Sequence; D000818:Animals; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D002846:Chromatography, Affinity; D018360:Crystallography, X-Ray; D039561:Eukaryotic Initiation Factor-4E / Q000235:genetics / Q000378:metabolism*; D039642:Eukaryotic Initiation Factors / Q000235:genetics / Q000378:metabolism*; D018506:Gene Expression Regulation, Plant; D018745:Genome, Plant; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D011485:Protein Binding; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D012313:RNA / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012685:Sepharose / Q000031:analogs & derivatives / Q000378:metabolism; D016415:Sequence Alignment; D020349:Surface Plasmon Resonance; D013997:Time Factors; D017806:Tymovirus; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
D029681:Arabidopsis Proteins; D039561:Eukaryotic Initiation Factor-4E; D039642:Eukaryotic Initiation Factors; D020033:Protein Isoforms; D011994:Recombinant Proteins; D014764:Viral Proteins; C511734:eIF(iso)4E protein, Arabidopsis; C068914:guanosine triphosphate-agarose; D012313:RNA; D006160:Guanosine Triphosphate; D012685:Sepharose