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Studies on interaction between potyvirus VPg and plant eIF(iso)4E

Research Project

Project/Area Number 16580031
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field Plant pathology
Research InstitutionSt.Marianna-University School of Medicine

Principal Investigator

MIYOSHI Hiroshi  St.Marianna Univ.Sch.of Med., Dept.Microbiol., Assistant Professor, 医学部, 助手 (80322519)

Co-Investigator(Kenkyū-buntansha) NATSUAKI Tomohide  Utsunomiya Univ., Dept.Plant Pathol., Professor, 農学部, 教授 (10134264)
TOMOO Koji  Osaka Univ.of Pharm.Sc., Dept.Phys.Chem., Lecturer, 薬学部, 講師 (70257898)
Project Period (FY) 2004 – 2005
Project Status Completed (Fiscal Year 2005)
Budget Amount *help
¥3,700,000 (Direct Cost: ¥3,700,000)
Fiscal Year 2005: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 2004: ¥2,800,000 (Direct Cost: ¥2,800,000)
KeywordsArabidopsis thaliana / Potyvirus / translation initiation factor / genome-linked protein / 翻訳開始因子
Research Abstract

I.Expression, purification and crystallization of Arabidopsis thaliana translation initiation factor eIF(iso)4E and turnip mosaic virus (TuMV) genome-linked protein (VPg).
Expression and purification of A.thaliana eIF(iso)4E and TuMV VPg were established by using E.coli - pET vector system. Crystallization of A.thaliana eIF(iso)4E and TuMV VPg have been still investigating and three-dimensional of A.thaliana eIF(iso)4E was generated by using the X-ray structure of human eIF4E (Tomoo, K.et al., Biochim Biophys Acta, 2005) as the template.
II.Binding analyses for the interaction between plant virus VPg and plant translational initiation factors.
Affinity chromatography on m^7GTP-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 chan … More ged to Leu, eIF(iso)4E lost the ability to form a complex with both VPg and m^7GTP-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, H.et al., Biochimie, 2006).
Furthermore, kinetic analysis for the interaction between TuMV VPg and wheat eIF(iso)4E was investigated by fluorescence titration collaborating with Prof.D.J Goss (The City University of New York). Scatchard analysis revealed the binding affinity (K_a) and average binding sites (n) for VPg were 25.14x10^6 M^<-1> and 0.85,respectively. Addition of eIFiso4G to the eIFiso4E increases 1.5 times the binding affinity with VPg as compared to the eIFiso4E alone. However, eIFiso4G alone did not bind with VPg. Addition of m^7G cap to the eIFiso4E-VPg or eIFiso4E-iso4G-VPg complex further increased the binding affinity. The van't Hoff analyses showed that VPg binding is enthalpy-driven and entropy favorable with a large negative ΔH°(-28.34 kJmole^<-1>), and positive ΔS°(38.40 Jmole^<-1>K^<-1>). These results suggest that the VPg binding site is located on or near the cap recognition pocket on eIFiso4E. Further, the formation of a cap binding complex containing VPg suggests the possibility of direct participation of VPg in the translation of the viral genome (ASBMB Annual Meeting 2006). Less

Report

(3 results)
  • 2005 Annual Research Report   Final Research Report Summary
  • 2004 Annual Research Report
  • Research Products

    (12 results)

All 2006 2005 2004 Other

All Journal Article (12 results)

  • [Journal Article] Binding analyses for the interaction between plant virus genome-linked protein (VPg) and plant translational initiation factors2006

    • Author(s)
      三好 洋
    • Journal Title

      Biochimie 88・3-4

      Pages: 329-340

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2005 Final Research Report Summary
  • [Journal Article] Binding analyses for the interaction between plant virus genome-linked protein (VPg) and plant translational initiation factors.2006

    • Author(s)
      Miyoshi H, Suehiro N, Tomoo K, Muto S, Takahashi T, Tsukamoto T, Ohmori T, Natsuaki T.
    • Journal Title

      Biochimie 88(3-4)

      Pages: 329-340

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2005 Final Research Report Summary
  • [Journal Article] Structural basis for mRNA Cap-Binding regulation of eukaryotic initiation factor 4Eby 4E-binding protein, studied by spectroscopic, X-ray crystal structural, and molecular dynamics simulation methods2005

    • Author(s)
      友尾幸司
    • Journal Title

      Biochim Biophys Acta 1753・2

      Pages: 191-208

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2005 Final Research Report Summary
  • [Journal Article] A simplified method for obtaining plant viral RNA for RT-PCR.2005

    • Author(s)
      末廣典子ら
    • Journal Title

      J Virol Methods. 125・1

      Pages: 67-73

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2005 Final Research Report Summary
  • [Journal Article] Structural basis for mRNA Cap-Binding regulation of eukaryotic initiation factor 4E by 4E-binding protein, studied by spectroscopic, X-ray crystal structural, and molecular dynamics simulation methods.2005

    • Author(s)
      Tomoo K, Matsushita Y, Fujisaki H, Abiko F, Shen X, Taniguchi T, Miyagawa H, Kitamura K, Miura K, Ishida T.
    • Journal Title

      Biochim Biophys Acta 1753(2)

      Pages: 191-208

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2005 Final Research Report Summary
  • [Journal Article] A simplified method for obtaining plant viral RNA for RT-PCR.2005

    • Author(s)
      Suehiro N, Matsuda K, Okuda S, Natsuaki T.
    • Journal Title

      J Virol Methods 125(1)

      Pages: 67-73

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2005 Final Research Report Summary
  • [Journal Article] Structural basis for mRNA Cap-Binding regulation of eukaryotic initiation factor 4E by 4E-binding protein, studied by spectroscopic, X-ray crystal structural, and molecular dynamics simulation methods2005

    • Author(s)
      友尾幸司
    • Journal Title

      Biochim Biophys Acta 1753・2

      Pages: 191-208

    • Related Report
      2005 Annual Research Report
  • [Journal Article] A simplified method for obtaining plant viral RNA for RT-PCR.2005

    • Author(s)
      末廣典子
    • Journal Title

      J Virol Methods. 125・1

      Pages: 67-73

    • Related Report
      2005 Annual Research Report
  • [Journal Article] An important determinant of the ability of Turnip mosaic virus to infect Brassica spp. and/or Raphanus sativus is in its P3 protein.2004

    • Author(s)
      末廣典子ら
    • Journal Title

      J Gen Virol. 85・7

      Pages: 2087-2098

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2005 Final Research Report Summary
  • [Journal Article] An important determinant of the ability of Turnip mosaic virus to infect Brassica spp. and/or Raphanus sativus is in its P3 protein.2004

    • Author(s)
      Suehiro N, Natsuaki T, Watanabe T, Okuda S.
    • Journal Title

      J Gen Virol. 85(Pt 7)

      Pages: 2087-2098

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2005 Final Research Report Summary
  • [Journal Article] An important determinant of the ability of Turnip mosaic virus to infect Brassica spp. and/or Raphanus sativus is in its P3 protein.2004

    • Author(s)
      末廣典子
    • Journal Title

      J Gen Virol. 85・7

      Pages: 2087-2098

    • Related Report
      2005 Annual Research Report
  • [Journal Article] Binding analyses for the interaction between plant virus genome-linked protein (VPg) and plant translational initiation factors

    • Author(s)
      三好 洋
    • Journal Title

      Biochimie (印刷中)

    • Related Report
      2005 Annual Research Report

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Published: 2004-04-01   Modified: 2016-04-21  

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