1 Institut National de la Recherche Scientifique, Institut Armand-Frappier, 531 boulevard des Prairies, Laval, Québec, Canada H7V 1B7
2 Department of Plant Science, McGill University, 21 111 Lakeshore, Ste-Anne-de-Bellevue, Québec, Canada H9X 3V9
Correspondence
Jean-François Laliberté
jean-francois.laliberte{at}inrs-iaf.uquebec.ca
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ABSTRACT |
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INTRODUCTION |
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Animal viruses have elaborated strategies that allow the preferential translation of viral mRNAs (Bushell & Sarnow, 2002), with the consequence that host protein synthesis is usually blocked. This phenomenon is known as host translation shutdown. Plant virus infection and its impact on cell protein synthesis has, however, not been studied as extensively as animal virus infections (Aranda & Maule, 1998
). The most-studied example of a block in translation involves the animal picornaviruses. The inhibition is mediated by the cleavage of eIF4G by viral or cellular proteinases. Hydrolysis of eIF4G prevents eIF4E from binding to the 43S ribosome subunit complex and thus precludes the recruitment of capped cellular mRNAs (Lamphear et al., 1995
; Liebig et al., 1993
). However, this presumption has recently been challenged (Ali et al., 2001
). Equally, adeno- and influenza viruses inhibit host-cell protein synthesis by inactivating eIF4E through dephosphorylation (Huang & Schneider, 1991
; Feigenblum & Schneider, 1993
). Finally, the ring Z protein of the lymphocytic choriomeningitis virus was shown to interact with eIF4E and to repress host mRNA translation (Campbell Dwyer et al., 2000
).
RNA viruses may also recruit translation factors to increase their translation efficiency further. A case in point is the rotavirus NSP3 protein. This interacts with the 3' end of rotavirus mRNAs, which are capped but not polyadenylated (Poncet et al., 1993, 1994
). NSP3 also interacts with eIF4G (Piron et al., 1998
). Further studies have shown that enhancement of rotavirus mRNA translation requires the simultaneous interaction of NSP3 with eIF4G and the mRNA 3' end (Vende et al., 2000
). During rotavirus infection, PABP is probably evicted from eIF4G, impairing the translation of polyadenylated host mRNAs (Piron et al., 1998
). Finally, the VPg (viral protein linked to the genome) of the Norwalk virus has been shown to bind eIF3, suggesting a role for the viral protein in translation initiation complex recruitment (Daughenbaugh et al., 2003
).
Turnip mosaic virus (TuMV) is a member of the genus Potyvirus (Riechmann et al., 1992). Potyviruses have a plus-sense, single-stranded RNA genome of about 10 kb in length, a poly(A) tail at the 3' end and a VPg covalently linked to the 5' end. The genome encodes one large polyprotein, which is processed into at least ten mature proteins by three viral proteinases (Pro) (Riechmann et al., 1992
). Several functions have been attributed to the potyvirus VPg. First, the viral protein and its precursor form, VPg-Pro, interact with, and have a stimulating effect on the activity of, the viral RNA-dependent RNA polymerase (RdRp), suggesting participation in virus replication (Daros et al., 1999
; Fellers et al., 1998
; Hong et al., 1995
; Li et al., 1997
). Additionally, interaction between VPg and eIF4E has been reported for Tobacco etch virus (TEV) (Schaad et al., 2000
) and TuMV (Léonard et al., 2000
; Wittmann et al., 1997
). The importance of this factor for TuMV infection has been shown in mutant Arabidopsis thaliana plants that do not express eIF(iso)4E (Lellis et al., 2002
; Duprat et al., 2002
). Although these plants had a normal phenotype, they were immune to TuMV. Finally, VPg has also been shown to have a role in overcoming viral resistance in plants (Borgstrom & Johansen 2001
; Johansen et al., 2001
; Keller et al., 1998
; Masuta et al., 1999
; Nicolas et al., 1996
, 1997
; Rajamaki & Valkonen, 1999
; Schaad et al., 1997b
). In the case of pepper and lettuce, the recessive resistance gene against Potato virus Y and Lettuce mosaic virus has been identified as encoding eIF4E (Ruffel et al., 2002
; Nicaise et al., 2003
).
In the present study, we investigated whether eIF4E expression is affected following TuMV infection and whether VPg and its precursor form, VPg-Pro, could act as a recruitment focal point for other proteins of cellular origin. Our data show that the eIF4E isomers expression profile was indeed modified and that, in addition to eIF(iso)4E and eIF4E, PABP can interact with VPg-Pro in planta.
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METHODS |
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PABP expression in E. coli.
Plasmid pETtagPABPhis encodes PABP2 of A. thaliana (accession no. NM_119572). RT-PCR was performed on total RNA of A. thaliana using a forward (5'-TATATACATATGGCTAGCCCGAATTCGATGGCGCAGGTTCAACTT-3'; NheI and EcoRI sites are underlined) and reverse primer (5'-TATATACTCGAGAGAGAGGTTCAAGGAAGC-3'; XhoI site is underlined). The amplified fragment was digested with EcoRI and XhoI and cloned in the similarly restricted pET21b (Novagen). The resulting PABP was fused at its N-terminal end to the 11 amino acid N-terminal peptide of the T7 gene 10 protein (T7 tag), which is recognized by the anti-T7-tag monoclonal antibody (Novagen). It also had a His tail at its C terminus. pETtagPABPhis was digested with XhoI and the ends were blunted with Klenow fragment giving PABP2 without the His tail. An overnight culture of E. coli BL21 (DE3) was diluted 1 : 100 in fresh medium and incubated at 37 °C until the OD600 reached 0·6. Protein production was then induced at room temperature with 0·4 mM IPTG for 3 h. Purification by metal chelation was performed as in Ménard et al. (1995).
ELISA-based binding assay.
The specified proteins were absorbed to wells of an ELISA plate (100 µl of protein at 10 ng µl-1) by overnight incubation at 4 °C and wells were blocked with 5 % Blotto in PBS. Appropriate proteins were diluted in PBS with 1 % Blotto and 0·2 % Tween 20 and incubated for 1·5 h at 4 °C with the previously coated wells. Detection of retained protein was achieved as in ELISA with the anti-T7-tag antibody and peroxidase-labelled goat anti-mouse immunoglobulin G (KPL). Wells were washed three times with 0·05 % Tween 20 between incubations.
Membrane fractionation.
Membrane recovery from plants was carried out according to Schaad et al. (1997a). Brassica perviridis plants (three-leaf stage) were infected with TuMV or mock-inoculated with PBS. At 10 days post-inoculation, the leaf that developed next to the inoculated leaf was harvested. Leaf tissue (2 g) was minced in 6 ml homogenization buffer [50 mM Tris/HCl, pH 8·0, 10 mM KCl, 3 mM MgCl2, 1 mM EDTA, 1 mM DTT, 0·1 % BSA, 0·3 % dextran, 13 % sucrose, plus one tablet of Complete Mini EDTA-free protease inhibitor cocktail (Roche) per 10 ml of buffer]. The homogenate was filtered through Miracloth and subjected to centrifugation at 3 700 g for 10 min at 4 °C. The supernatant (2·5 ml) was layered on to a 9 ml 2045 % sucrose gradient containing the respective homogenization buffer and subjected to centrifugation at 143 000 g in a Beckman SW41 Ti rotor for 4 h at 4 °C. Fractions (0·9 ml) were collected, diluted 1 : 1 in protein dissociation buffer and subjected to immunoblot analysis following 12·5 % SDS-PAGE. Immunoreactions were detected using the ECL-based secondary-antibody system (Amersham).
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RESULTS |
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When the anti-VPg-Pro serum was used, no reactivity was noticed in the 3 700 g supernatant (S3) recovered from mock-inoculated plants (Fig. 2e, lane N). On the other hand, several VPg-Pro-related polypeptides were detected in the S3 extract from TuMV-infected plants (Fig. 2e
, lane S). These species had the expected molecular masses for 6K2-VPg-Pro, VPg-Pro and Pro (see Fig. 2f
for a schematic presentation of TuMV polyprotein). Recombinant forms of these proteins have been previously expressed in E. coli (Ménard et al., 1995
) and migrated to the same positions as the proteins detected in TuMV-infected plants (data not shown). However, no polypeptide corresponding to the mature VPg was detected. The different viral proteins localized to different fractions in the 3 mM MgCl2 sucrose gradient. A subpopulation of VPg-Pro, C-terminally processed VPg-Pro (Ménard et al., 1995
) and Pro were found in the lighter fractions of the sucrose gradient. On the other hand, 6K2-VPg-Pro and the remaining portion of VPg-Pro were found near the bottom of the gradient (fractions 15). In the sucrose gradient fractionation carried out in the absence of MgCl2, the 6K2-VPg-Pro/VPg-Pro peak moved up the gradient by approximately two to three fractions, which was similar to what had been observed for the bottom BiP-containing fractions. This shift thus suggested that 6K2-VPg-Pro/VPg-Pro are present with ribosome-associated ER membranes and are likely to be the protein type to interact with the translation initiation factor(s) in planta.
Co-purification of 6K2-VPg-Pro/VPg-Pro with eIF(iso)4E/eIF4E
Detection of 6K2-VPg-Pro and VPg-Pro in the same sucrose gradient fractions as the initiation factors raises the question of whether the two viral proteins interact with eIF(iso)4E, eIF4E, or both, in planta. VPg-Pro has an intrinsic capacity to bind to nickelagarose resin, the presence of a histidine tail being unnecessary (Ménard et al., 1995). The binding to the resin is mediated by the VPg domain (data not shown). Purification of 6K2-VPg-Pro and VPg-Pro by metal chelation chromatography was thus attempted and the co-purification of both eIF(iso)4E and eIF4E evaluated. For TuMV-infected and mock-inoculated tissues, fractions 15 from a 3 mM MgCl2 membrane fractionation experiment were pooled and loaded on to a nickelagarose column. After washing the resin, the bound proteins were eluted with 100 mM imidazole and analysed by immunoblot assay following SDS-PAGE. Fig. 3
(a) shows that both 6K2-VPg-Pro and VPg-Pro were effectively purified from infected tissues. Similarly, eIF4E and eIF(iso)4E were detected in the eluted protein fraction when the column was loaded with the membrane fractions from TuMV-infected leaves (Fig. 3b
). They were not detected with mock-inoculated membrane fractions, even after prolonged film exposure. This experiment thus indicated that 6K2-VPg-Pro and/or VPg-Pro interact with both eIF(iso)4E and eIF4E in infected cells.
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DISCUSSION |
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The binding domain to eIF(iso)4E is located within VPg (Léonard et al., 2000; Wittmann et al., 1997
). We have shown by membrane co-localization and co-purification experiments that it was 6K2-VPg-Pro and/or VPg-Pro, precursor forms of VPg, that do the actual binding in plants. VPg can exist as a fully processed protein and also as precursor forms, which each may have different functions during virus replication (Riechmann et al., 1992
). In the case of Potato virus A (PVA), several precursor forms of VPg were detected in insect and plant cells, notably P3-6K1-helicase-6K2-VPg-Pro-Pol, helicase-6K2-VPg-Pro-Pol, helicase-6K2-VPg-Pro, VPg-Pro-Pol and VPg-Pro-Pol-CP (Merits et al., 2002
). These high-molecular-mass forms were not detected in the case of TuMV, most likely because the PAGE conditions were not optimal for this size range. Interestingly, 6K2-VPg-Pro, which was clearly detected in TuMV-infected leaves, was not detected in the case of PVA. This may reflect that this form does not exist, or is processed at a much faster rate, in PVA-infected cells. Curiously, we did not detect fully processed VPg in infected leaves. Fully processed VPg would at the very least be found within virions. Detection of this form would, however, require significant loading of material on SDS gels (Murphy et al., 1990
). Alternatively, lack of VPg detection may reflect a high turnover rate or may be artefactual. We thus cannot exclude the possibility of free VPg binding the initiation factors. Our membrane fractionation experiment also indicated that the interaction is likely to take place in ribosome-associated ER membranes. In the case of TEV, Schaad et al. (1997a)
showed an association of 6K2-VPg-Pro with these subcellular membranes and demonstrated that the 6K2 domain is an integral protein of ER membranes. Furthermore, the potyvirus RNA replication complex is associated with ER membranes (Martin & Garcia, 1991
; Schaad et al., 1997a
) and it has been proposed that 6 kDa-containing proteins, notably 6K2-VPg-Pro, would participate in replication (Riechmann et al., 1992
). It appears thus that potyvirus replication and translation are closely linked phenomena involving a common set of proteins that are found in the same subcellular compartment. This close link between replication and translation is in agreement with the finding that another translation initiation factor, eIF3, was present in highly purified replication complexes of both Brome mosaic virus (Quadt et al., 1993
) and Tobacco mosaic virus (Osman & Buck, 1997
). In the case of the latter virus, there is in vitro evidence that the factor interacts with the methyltransferase-like domain of the 126 and 183 kDa replicase proteins (Taylor & Carr, 2000
).
Furthermore, we showed that VPg-Pro can also interact with PABP in planta. PAPB is ubiquitous in eukaryotes and participates in at least three major post-transcriptional processes: initiation of protein synthesis, mRNA turnover and mRNA biogenesis. A. thaliana PABPs are encoded by a very diverse gene family (Belostotsky & Meagher, 1993). The isomer PABP2 is highly expressed in all organs of A. thaliana (Palanivelu et al., 2000b
) and was shown to function in yeast translational processes (Palanivelu et al., 2000a
). This isomer was thus the first choice to test for interaction with the viral protein. It remains to be seen whether the other isomers of PABP are also capable of interacting with VPg-Pro. VPg-Pro and PABP could interact with each other in two separate ways, potentially increasing the overall stability of the complex. First, the interaction can be direct as shown in this study. The other way would be through the connection of the eIF(iso)4E/eIF(iso)4G dimer. Indeed, eIF(iso)4G has been shown to bind PABP (Le et al., 1997
) and can interact with VPg-Pro through the intermediary of eIF(iso)4E (M. G. Fortin, unpublished data). However, this last possibility needs to be experimentally demonstrated. Moreover, the interaction between VPg-Pro and eIF(iso)4E/eIF4E as well as PABP could possibly promote RNA circularization during translation. Circularization has been shown to be necessary for efficient translation of cellular mRNAs (Gallie, 1998
) and also to take place for animal viral RNAs (Michel et al., 2001
). Linkage of VPg-Pro to the viral RNA (Murphy et al., 1990
) and formation of the VPg-ProPABP complex and a likely VPg-ProeIF4EeIF4GPABP complex could bring both ends of the viral RNA in close proximity. It now remains to be demonstrated, through an approach similar to that used for eIF(iso)4E (Lellis et al., 2002
; Duprat et al., 2002
), whether A. thaliana PABP2 knockouts are immune to TuMV infection.
Finally, interaction with eIF(iso)4E, eIF4E and PABP suggests that VPg-Pro may serve as a focal point for translation initiation complex assembly. Preliminary experiments also showed that other proteins interact with the viral protein (C. Viel, unpublished data). This concept of VPg as a protein recruitment factor has recently been suggested for the Norwalk virus VPg (Daughenbaugh et al., 2003). This VPg was shown to interact with eIF3, and pull-down experiments showed that other translation initiation factors were co-purified, notably eIF4GI, eIF4E and eIF2
. To what extent these interactions are directly with VPg or mediated through eIF3 is not yet known. Future experiments will investigate which other proteins are found associated with VPg-Pro of TuMV and if these proteins are the same whether the viral protein is localized in the ER, the cytoplasm or the nucleus.
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ACKNOWLEDGEMENTS |
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Received 7 October 2003;
accepted 28 November 2003.