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J. Biol. Chem., Vol. 281, Issue 38, 28002-28010, September 22, 2006

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Interaction of Genome-linked Protein (VPg) of Turnip Mosaic Virus with Wheat Germ Translation Initiation Factors eIFiso4E and eIFiso4F^*

Mateen A. Khan, Hiroshi Miyoshi, Sibnath Ray, Tomohide Natsuaki^¶, Noriko Suehiro^¶, and Dixie J. Goss¹

From the Department of Chemistry, Hunter College and the Graduate Center of the City University of New York, New York, New York 10021, Department of Microbiology, St. Marianna University School of Medicine, Kawasaki 216-8511, Japan, and ^¶Genome Research Institute, Utsunomiya University, Utsunomiya 321-8505, Japan

The interaction between VPg of turnip mosaic virus and wheat germ eukaryotic translation initiation factors eIFiso4E and eIFiso4F (the complex of eIFiso4E and eIFiso4G) were measured and compared. The fluorescence quenching data showed the presence of one binding site on eIFiso4E for VPg. Scatchard analysis revealed the binding affinity (K_a) and average binding sites (n) for VPg were (8.51 ± 0.21) x 10⁶ M^–1 and 1.0, respectively. The addition of eIFiso4G to the eIFiso4E increased the binding affinity 1.5-fold for VPg as compared with eIFiso4E alone. However, eIFiso4G alone did not bind with VPg. The van't Hoff analyses showed that VPg binding is enthalpy-driven and entropy-favorable with a large negative H°(–29.32 ± 0.13 kJmol^–1) and positive S° (36.88 ± 0.25 Jmol^–1K^–1). A Lineweaver-Burk plot indicates mixed-type competitive ligand binding between VPg and anthraniloyl-7-methylguanosine triphosphate for eIFiso4E. Fluorescence stopped-flow studies of eIFiso4E and eIFiso4F with VPg show rapid binding, suggesting kinetic competition between VPg and m⁷G cap. The VPg protein binds much faster than cap analogs. The activation energies for binding of eIFiso4E and eIFiso4F with VPg were 50.70 ± 1.27 and 75.37 ± 2.95 kJmol^–1 respectively. Enhancement of eIFiso4F-VPg binding with the addition of a structured RNA derived from tobacco etch virus suggests that translation initiation involving VPg occurs at internal ribosomal entry sites. Furthermore, the formation of a protein-RNA complex containing VPg suggests the possibility of direct participation of VPg in the translation of the viral genome.

Received for publication, June 7, 2006 , and in revised form, July 14, 2006.

^* This work was supported in part by National Science Foundation Grant MCB 0413982 (to D. J. G.), Research Center in Minority Institution Award RR-03037 from the National Center for Research Resources of the National Institutes of Health, and Grant-in-aid for Scientific Research 18580045 (to H. M.) from the Ministry of Education, Science, Sports, and Culture of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Dept. of Chemistry, Hunter College of the City University of New York, New York, NY 10021. Tel.: 212-772-5383; Fax: 212-772-5332; E-mail: dgoss{at}hunter.cuny.edu.

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