Centre de Microbiologie et Biotechnologie, INRS-Institut Armand-Frappier, 531 Boulevard des Prairies, Ville de Laval, Québec, CanadaH7V 1B71
Pacific Agri-Food Research Centre, 4200 Highway 97, Summerland, BC, CanadaV0H 1Z02
Author for correspondence: Hélène Sanfaçon. Fax +1 250 494 0755. e-mail SanfaconH{at}em.agr.ca
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Abstract |
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Main text |
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Viral proteins are likely to participate in the regulation of viral genome translation (Thompson & Sarnow, 2000 ; Gale et al., 2000
). A case in point is the VPg of Turnip mosaic virus (TuMV; genus Potyvirus), which interacts with the eukaryotic initiation factor eIF(iso)4E of Arabidopsis thaliana (Wittmann et al., 1997
; Léonard et al., 2000
). eIF(iso)4E is a plant isomer of eIF4E (Rodriguez et al., 1998
) which binds the cap structure of cellular mRNAs and plays an important role in the regulation of translation initiation (Sonenberg & Gingras, 1998
). The cap analogue m7GTP, but not GTP, inhibits VPgeIF(iso)4E complex formation, suggesting that VPg and cellular mRNAs compete for eIF(iso)4E binding. Plants inoculated with TuMV infectious cDNA containing a mutation in the eIF(iso)4E binding domain of VPg remain symptomless and do not show accumulation of virus coat protein, indicating that there is a correlation between VPgeIF4E binding in vitro and virus viability in planta (Léonard et al., 2000
). The NIa protein (also called VPg-Pro) of Tobacco etch virus also interacts with eIF4E from tomato and tobacco, and the interaction was shown to enhance genome amplification (Schaad et al., 2000
). Although the precise biological function of the VPg (or VPg-Pro)eIF4E interaction remains to be elucidated, it may either play a role in recruiting host factors for the translation and/or replication of the viral RNA or be involved in host translational shut-down, possibly through disruption of the interaction between cellular mRNAs and cap-binding translation initiation factors.
Nepoviruses are closely related to potyviruses in term of their genomic structure and genome expression strategy but differ from potyviruses in at least two significant aspects. First, the nepovirus genome is bipartite, with RNA1 encoding most of the proteins involved in virus replication (including VPg and Pro). Second, nepovirus VPgs are much smaller than potyvirus VPgs, which range from 22 to 24 kDa (Riechmann et al., 1992 ). For example, the VPg of Tomato ringspot virus (ToRSV) is composed of 27 residues (Wang et al., 1999
). There is no amino acid sequence homology between potyvirus and nepovirus VPgs (Mayo & Fritsch, 1994
). It was therefore of interest to determine if the nepovirus VPg, or larger precursor forms, interacts with eIF(iso)4E. One possible precursor of VPg is VPg-Pro. VPg-Pro (the functional equivalent of the potyvirus NIa) was found to accumulate during in vitro translation of larger precursors as a result of inefficient processing of the VPg-Pro cleavage site (Wang et al., 1999
; Wang & Sanfaçon, 2000
), although accumulation of VPg-Pro in infected plants has not yet been demonstrated.
The interaction between VPg-Pro as well as Pro of ToRSV and eIF(iso)4E of A. thaliana was tested using an ELISA-based binding assay (Wittmann et al., 1997 ; Léonard et al., 2000
). ToRSV has the ability to replicate in A. thaliana (R. I. Hamilton, personal communication). eIF(iso)4E was produced in E. coli and purified by m7GTPSepharose chromatography (Wittmann et al., 1997
). The factor was fused to the N-terminal peptide of the T7 gene-10 protein (T7 tag), which allows its recognition by an anti-T7 tag monoclonal antibody (Novagen). ToRSV proteins were produced in E. coli, purified (Chisholm et al., 2001
) and adsorbed to wells of ELISA plates (1·0 µg per well) by overnight incubation at 4 °C. Purified eIF(iso)4E (2 µg) diluted in 1% Blotto in PBS containing 0·2% Tween was incubated for 1 h at 4 °C in the coated wells. Detection of bound initiation factor was achieved in an ELISA with the anti-T7 tag antibody and peroxidase-labelled goat anti-mouse immunoglobulin G (KPL). VPg-Pro from TuMV was purified as described previously (Wittmann et al., 1997
) and used as positive control. As shown previously (Wittmann et al., 1997
), the VPg-Pro of TuMV interacted with eIF(iso)4E (Fig. 1
). VPg-Pro of ToRSV also interacted with eIF(iso)4E. The interaction was specific for the viral protein since the factor was not retained when wells were coated with an E. coli lysate not containing any VPg-Pro (Wittmann et al., 1997
). An interaction was also detected between the mature Pro and eIF(iso)4E, although retention of the factor was less than for VPg-Pro (0·55 OD units for Pro vs 1·2 OD units for VPg-Pro). This suggests that the interacting domain resides within Pro, but that the presence of VPg increases the affinity of the viral protein for eIF(iso)4E. In contrast, the interacting domain of the TuMV VPg-Pro resides within VPg, and the TuMV VPg-Pro and VPg have the same binding affinity for eIF(iso)4E (Wittmann et al., 1997
).
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To test whether ToRSV proteins and the cap structure of cellular mRNAs compete for eIF(iso)4E binding, the influence of the cap analogue m7GTP on the formation of the VPg-ProeIF(iso)4E and ProeIF(iso)4E complexes was tested. ELISA plate wells were coated with 1·0 µg of viral proteins and incubated with 2·0 µg of eIF(iso)4E and either 10 or 20 mM m7GTP. The cap analogue inhibited the formation of the complexes by approximately 30% at a concentration of 20 µM (Fig. 3). No inhibition was observed with 20 µM of GTP, indicating that the inhibition was cap-related. As previously shown, 20 µM of m7GTP inhibited the interaction of eIF(iso)4E with the VPg-Pro of TuMV by 60% (Léonard et al., 2000
).
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The formation of protein bridges leading to genome circularization may also be an important feature of the translation of plant virus genomes. Recently, the coat protein of Alfalfa mosaic virus was shown to stimulate translation of viral RNAs, presumably by acting as a functional analogue of PABP (Neeleman et al., 2001 ). The nepovirus Pro could similarly participate in genome circularization by acting as a bridging element between host initiation factors and the viral RNAs. Pro is a member of the 3C-like proteinase family and is likely to have RNA-binding properties (Blair et al., 1998
). It could thus interact with the 5' end of the viral genome, as shown for the picornavirus 3C (Gamarnik & Andino, 2000
; Kusov et al., 1997
; Kusov & Gauss-Muller, 1997
; Harris et al., 1994
; Walker et al., 1995
) and the potyvirus NIa proteinases (Daros & Carrington, 1997
). Alternatively, Pro as a precursor protein with VPg may be covalently linked to viral RNAs. In addition to possibly improving translation of the viral RNAs, genome circularization could provide several advantages for virus replication, including the coordination of translation and RNA synthesis, the localization of the viral polymerase at the appropriate start site and a control mechanism for the integrity of the viral genome (Herold & Andino, 2001
). Finally, the interaction of the ToRSV Pro with eIF(iso)4E may also have other biological functions, one of which may be a direct or indirect role in a possible shut-down of host translation.
The presence of the VPg domain on a precursor of the ToRSV Pro regulates the different activity of the proteinase as it enhances its ability to interact with eIF(iso)4E (this study) and decreases its ability to release the movement protein and coat protein from RNA2-derived substrates (Chisholm et al., 2001 ). The results presented here provide additional support for our previous suggestion that the inefficient cleavage at the VPg-Pro site may play an important role in the biology of ToRSV (Chisholm et al., 2001
).
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References |
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Received 11 February 2002;
accepted 5 April 2002.