Elsevier

Biochimie

Volume 90, Issue 10, October 2008, Pages 1427-1434
Biochimie

Research paper
Turnip mosaic virus VPg interacts with Arabidopsis thaliana eIF(iso)4E and inhibits in vitro translation

https://doi.org/10.1016/j.biochi.2008.03.013Get rights and content

Abstract

The interaction between turnip mosaic virus (TuMV) viral protein linked to the genome (VPg) and Arabidopsis thaliana eukaryotic initiation factor (iso)4E (eIF(iso)4E) was investigated to address the influence of potyviral VPg on host cellular translational initiation. Affinity chromatographic analysis showed that the region comprising amino acids 62–70 of VPg is important for the interaction with eIF(iso)4E. In vitro translation analysis showed that the addition of VPg significantly inhibited translation of capped RNA in eIF(iso)4E-reconstituted wheat germ extract. This result indicates that VPg inhibits cap-dependent translational initiation via binding to eIF(iso)4E. The inhibition by VPg of in vitro translation of RNA with wheat germ extract did not depend on RNase activity. Our present results may indicate that excess VPg produced at the encapsidation stage shuts off cap-dependent translational initiation in host cells by inhibiting complex formation between eIF(iso)4E and cellular mRNAs.

Introduction

Most nuclear-encoded mRNAs in eukaryotic cells possess a cap structure consisting of m7GpppN (where N is any nucleotide) at the 5′ terminus. Initiation of protein synthesis proceeds by progressive assembly of initiation complexes, each stage being catalyzed by a different set of initiation factors. Eukaryotic initiation factor 4E (eIF4E) directly contacts the mRNA by binding to the cap and associating with eIF4 G. EIF4 G in turn binds to eIF4A, PABP (poly(A)-binding protein) and eIF3 to form the 48S initiation complex [1], [2].

Recently it has been reported that the viral protein linked to the genome (VPg) of potyvirus may play a role in viral translation and replication, by serving as an analog of the cap structure of mRNAs. It may initiate viral translation and inhibit host cell translation through its interaction with the cap-binding proteins eIF4E and eIF(iso)4E [3], [4], [5], [6], [7].

Turnip mosaic virus (TuMV) is a member of the genus Potyvirus, in the family Potyviridae [8], within the picorna-like plant viruses [9], and is well known to infect cruciferous plants including Arabidopsis thaliana [10]. Potyviruses have a messenger polarity ssRNA genome, a poly(A) tail, and VPg covalently attached to the 5′ terminus. Studies support a biological role for VPg linked to the viral RNA in virions. The covalent tyrosine residue-mediated linkage between VPg and viral RNA is necessary for infectivity [11], and the level of infectivity of viral RNA released from virions during disassembly can be increased or decreased by in vitro degradation of VPg [12]. VPg has been implicated indirectly in cell-to-cell movement of the virus through plasmodesmata, and the results of substitutional or mutational analysis among different strains also indicate that it is required for cell-to-cell and long-distance movement [13], [14], [15], [16], [17], [18]. In several potyviruses, VPg has been identified as an avirulence factor for recessive resistance genes in various plants [19], [20], [21], [22]. The recessive resistance genes pvr2 of Capsicum annuum, mo1 of Lactuca sativa and sbm1 of Pisum sativum L. correspond to the eIF4E gene [23], [24], [25].

Multiple isoforms of eIF4E exist in plants, mammals, Drosophila, and Caenorhabditis elegans [26], [27], [28], [29]. Wheat germ and other plants express two related, but distinct, forms of the cap binding complex, designated as eIF4F and eIFiso4F. Each consists of two subunits, eIF4E and eIF4 G, and eIF(iso)4E and eIFiso4 G. eIF4F and eIFiso4F function in supporting cap-dependent in vitro translation [30]. The amino acid sequences of eIF4E and eIF(iso)4E are approximately 50% conserved and include two tryptophan residues concerned with recognition of the cap-structure on mRNA [31], [32]. The two plant proteins have a number of functional similarities, in that both eIF4F and eIFiso4F support translation in vitro, facilitate ATP-dependent helicase activity, and exhibit RNA-dependent ATPase activity [33], [34], [35]. EIF4F was found to support translation of RNA containing secondary structure in the noncoding region better than did eIFiso4F [36]. Moreover, binding studies with oligonucleotides suggest that eIF4F binding is sensitive to the presence of secondary structure, and that eIFiso4F exhibits a binding preference for linear structures [37]. In vitro translation using the tobacco etch virus 5′ leader to direct cap-independent translation specifically depended on eIF4 G and not eIFiso4 G [38]. Direct binding studies showed that binding affinity for eIF4 G and eIFiso4 G correlated with translational efficiency [39].

It has been reported that TuMV VPg interacts with the translation initiation factors eIF(iso)4E and eIFiso4F of wheat germ [5], [40], [41]. Earlier studies [4], [5] have shown that interactions between VPg and plant eIF(iso)4E and eIFiso4F are sufficiently strong that VPg competes with cap binding. Moreover, we showed that VPg could displace eIF(iso)4E from m7GTP-Sepharose [4]. Khan et al. proposed that VPg linked covalently to viral genome RNA promotes translation of the viral genome RNA by stabilizing the eIFiso4F–VPg–IRES complex [5]. These results indicate that plant translational initiation factor complexes containing eIF(iso)4E may be disrupted by VPg.

The potyvirus gene products are considered to be generated in equimolar amounts in infected cells [42]. Formation of a potyvirus particle requires up to 2000 units of a single structural coat protein [43]. However, a single VPg protein is attached to the 5′-end of each viral RNA, presumably resulting in an excess of VPg at the encapsidation stage. The excess VPg may interfere with complex formation between eIF(iso)4E and cellular mRNAs. To address the influence of potyviral VPg on translation of mRNA, we investigated the interaction between VPg of the Potyvirus TuMV, and A. thaliana eIF(iso)4E by using affinity chromatography and an in vitro translation system.

Section snippets

Materials

An A. thaliana EST clone (clone no. SQ062g05) encoding eIF(iso)4E was obtained from the Kazusa DNA Research Institute (http://est.kazusa.or.jp/en/plant/arabi/EST/index.html). The TuMV full-length cDNA clone was described previously [44]. Restriction enzymes, KOD Plus DNA Polymerase, Ribonuclease inhibitor ScriptMAX Thermo T7 transcription kit and PROTEIOS Wheat germ cell-free protein synthesis core kit including pEU-DHFR were purchased from Toyobo Co. Ltd. The DNA ligation kit ver.2 and DNase I

Determination of binding site of TuMV VPg to A. thaliana eIF(iso)4E

In the pull-down assay, the expressed eIF(iso)4E binds to m7GTP-Sepharose in the first step. Purified VPg-His6 variant is added to the eIF(iso)4E-bound m7GTP-Sepharose, and the bound eIF(iso)4E is eluted from m7GTP-Sepharose if the VPg-His variant interacts with eIF(iso)4E [4]. We expressed five N-terminally truncated VPg variants and purified them to delineate further the binding site of VPg to eIF(iso)4E (Fig. 1A). EIF(iso)4E was eluted from m7GTP-Sepharose with all of the VPg variants,

Discussion

Our results indicate that the region comprising amino acids 51–70 of VPg is important for the interaction with eIF(iso)4E. Given that the region comprising amino acids 62–93 of VPg was previously reported to be involved in the interaction, we can conclude that the region comprising amino acids 62–70 of VPg is important for the interaction with eIF(iso)4E, as well as Asp-77 of VPg [41]. We then prepared single point mutants of each of amino acids 62–70 of VPg to Ala, and examined their

Acknowledgment

This work was supported in part by Grants-in-Aid for Scientific Research (18580045 and 17208004) from the Ministry of Education, Science, Sports & Culture of Japan.

References (50)

  • M.L. Allen et al.

    Isolation and sequence of the cDNAs encoding the subunits of the isozyme form of wheat protein synthesis initiation factor 4F

    J. Biol. Chem.

    (1992)
  • R.D. Abramson et al.

    Initiation factors that bind mRNA. A comparison of mammalian factors with wheat germ factors

    J. Biol. Chem.

    (1988)
  • S.R. Lax et al.

    ATPase activities of wheat germ initiation factors 4A, 4B, and 4F

    J. Biol. Chem.

    (1986)
  • D.R. Gallie et al.

    eIF4G functionally differs from eIFiso4G in promoting internal initiation, cap-independent translation, and translation of structured mRNAs

    J. Biol. Chem.

    (2001)
  • S. Ray et al.

    Tobacco etch virus mRNA preferentially binds wheat germ eukaryotic initiation factor (eIF) 4G rather than eIFiso4G

    J. Biol. Chem.

    (2006)
  • S. Wittmann et al.

    Interaction of the viral protein genome linked of turnip mosaic potyvirus with the translational eukaryotic initiation factor (iso) 4E of Arabidopsis thaliana using the yeast two-hybrid system

    Virology

    (1997)
  • M.M. Bradford

    A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding

    Anal. Biochem.

    (1976)
  • S. Cotton et al.

    The VPgPro protein of Turnip mosaic virus: In vitro inhibition of translation from a ribonuclease activity

    Virology

    (2006)
  • R. Anindya et al.

    Potyviral NIa proteinase, a proteinase with novel deoxyribonuclease activity

    J. Biol. Chem.

    (2004)
  • M. Aranda et al.

    Virus-induced host gene shutoff in animals and plants

    Virology

    (1998)
  • J.W.B. Hershey et al.

    Translational Control of Gene Expression

    (2000)
  • A.C. Gingras et al.

    eIF4 initiation factors: effectors of mRNA recruitment to ribosomes and regulators of translation

    Annu. Rev. Biochem.

    (1999)
  • I. Goodfellow et al.

    Calicivirus translation initiation requires an interaction between VPg and eIF4E

    EMBO Rep.

    (2005)
  • K.F. Daughenbaugh et al.

    The genome-linked protein VPg of the Norwalk virus binds eIF3, suggesting its role in translation initiation complex recruitment

    EMBO J.

    (2003)
  • P.H. Berger et al.

    Family Potyviridae

  • Cited by (32)

    • The role of the 5' untranslated regions of Potyviridae in translation

      2015, Virus Research
      Citation Excerpt :

      The contribution of PABP in translation was further shown in a PABP-reduced wheat germ lysate, where the addition of recombinant PABP resulted in a 21-fold to about 50-fold increase in TEV mediated translation (Yumak et al., 2010). Recent findings revealed that the viral protein linked to the 5′ end of the genome (VPg) that replaces the 5′ cap and determines infectivity for many potyviruses (Ayme et al., 2006; Qian et al., 2013; Truniger and Aranda, 2009) can affect translation (Eskelin et al., 2011; Khan et al., 2008; Miyoshi et al., 2008). The addition of the TEV VPg with eIF4F to a cap-binding complex-depleted wheat germ extract enhanced TEV-mediated translation by almost 3-fold when compared to eIF4F alone.

    • Next-generation sequencing and micro RNAs analysis reveal SA/MeJA1/ABA pathway genes mediated systemic acquired resistance (SAR) and its master regulation via production of phased, trans-acting siRNAs against stem rot pathogen Macrophomina phaseolina in a RIL population of jute (Corchorus capsularis)

      2014, Physiological and Molecular Plant Pathology
      Citation Excerpt :

      Alternatively, some of the genes involved in the HR may activate a systemic response of the plant or the pathogen may trigger HR to facilitate its colonization in the plant as reported for other pathogens [49]. Several other genes involved in defense such as eIF(iso)4E [50] were also implicated. The candidate genes identified in this study represent a valuable resource for studying the genetic basis underlying resistance to Macrophomina and the identification of the fungal pathogen resistance genes.

    • The genome-linked protein VPg of plant viruses - A protein with many partners

      2011, Current Opinion in Virology
      Citation Excerpt :

      Despite its demonstrated importance for virus replication, it is not yet known which specific step of the replication cycle is affected by the eIF4E–VPg interaction. Although a participation in viral RNA translation is likely [43,44], other functions such as downregulation of host gene expression [45••] may be involved. eIF4E interacts not only with the VPg of potyviruses but also with the VPg of caliciviruses (see accompanying article) and with the VPg–Pro of ToRSV [4], which are distantly related viruses.

    View all citing articles on Scopus
    View full text