Interaction of VPg-Pro of Turnip mosaic virus with the translation initiation factor 4E and the poly(A)-binding protein in planta

Simon Léonard1, Catherine Viel1, Chantal Beauchemin1, Nicole Daigneault1, Marc G. Fortin2 and Jean-François Laliberté1

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


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The viral protein linked to the genome (VPg) of Turnip mosaic virus (TuMV) interacts in vitro with the translation eukaryotic initiation factor (eIF) 4E. In the present study, we investigated the consequence of TuMV infection on eIF4E expression. Two isomers are present in plants, namely eIF4E and eIF(iso)4E. Expression of the latter was detected in both TuMV-infected and mock-inoculated Brassica perviridis plants, but expression of eIF4E was found only in infected plants. Membranes from TuMV-infected or mock-inoculated tissues were separated by sucrose gradient centrifugation and fractions were collected. Immunoblot analyses showed that 6K2-VPg-Pro/VPg-Pro polyproteins were associated with endoplasmic reticulum membranes and were the viral forms likely to interact with eIF(iso)4E and eIF4E. In planta interaction between 6K2-VPg-Pro/VPg-Pro and eIF(iso)4E/eIF4E was confirmed by co-purification by metal chelation chromatography. The poly(A)-binding protein (PABP) was also found to co-purify with VPg-Pro. Direct interaction between VPg-Pro and PABP was shown by an ELISA-based binding assay. These experiments suggest that a multi-protein complex may form around VPg-Pro of TuMV.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Translation of viral RNAs by the host machinery is a crucial event in the virus cell cycle and proceeds essentially as for cellular mRNAs (Gale et al., 2000; Thompson & Sarnow, 2000). One of the first steps in translation is the recruitment of mRNAs by the translation eukaryotic initiation factor (eIF) 4F complex. eIF4E is a member of this complex and recognizes the cap structure (m7GpppN, where N is any nucleotide) at the 5' end of mRNAs. The other members of the eIF4F complex are eIF4G and eIF4A. In conjunction with additional proteins, the eIF4F complex links mRNAs to ribosomes and promotes the search for the translation start site. Among the proteins that interact with components of eIF4F is the poly(A)-binding protein (PABP) (Le et al., 1997; Tarun et al., 1997). A functional consequence of this interaction is an increase in the affinity of PABP for the poly(A) tail and of eIF4F for the 5' cap structure of mRNAs (Gallie, 1998). In addition, association of PABP with eIF4F results in the circularization of mRNAs, which facilitates multiple rounds of translation (Sachs, 2000).

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.


   METHODS
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METHODS
RESULTS
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Microorganisms and media.
Manipulations of bacteria as well as nucleic acids and proteins were done by standard methods (Sambrook et al., 1989). E. coli XL-1 Blue was used for subcloning and E. coli BL21 (DE3) (Novagen) was used for protein expression.

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 20–45 % 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).


   RESULTS
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INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Expression of eIF4E isomers following TuMV infection
There are at least two isomers of initiation factor 4E in plants, namely eIF4E and eIF(iso)4E (Browning, 1996). They share significant sequence homology, but their respective role in translation initiation is unknown. Furthermore, the functional state of eIF4E is altered following infection by certain animal viruses (Campbell Dwyer et al., 2000; Feigenblum & Schneider, 1993; Huang & Schneider, 1991). The consequence of TuMV infection on the expression state of the eIF4E isomers was thus investigated. B. perviridis plants at the three-leaf stage were mock inoculated or TuMV infected. Ten days after inoculation, the leaf that developed next to the inoculated leaf was harvested. Infection was confirmed by immunoblot analysis using a rabbit serum raised against the capsid protein of TuMV (data not shown). The harvested leaf was minced in homogenization buffer and the extract centrifuged at 3 700 g. Proteins from the supernatant were separated by SDS-PAGE and subjected to immunoblot analysis using a rabbit serum raised against a recombinant form of eIF(iso)4E from A. thaliana. eIF4E and eIF(iso)4E of A. thaliana have a calculated size of 26·5 and 22·5 kDa, respectively (Rodriguez et al., 1998). Although the size for the B. perviridis isomers was not known, it was expected to be very similar, since both plants belong to the same family. Fig. 1 shows that eIF(iso)4E was detected in mock-inoculated plants, migrating at the same position as recombinant eIF(iso)4E of A. thaliana. This protein was retained on m7GTP Sepharose, which confirmed its cap-binding property (data not shown). eIF(iso)4E was also detected in TuMV-infected plants along with an additional protein that had the same molecular mass as recombinant eIF4E of A. thaliana. Cross-reaction of the anti-eIF(iso)4E serum with eIF4E can be expected since the two proteins are highly homologous (Rodriguez et al., 1998) and a rabbit serum raised against wheat eIF(iso)4E recognized both eIF4E and eIF(iso)4E of A. thaliana (Ruud et al., 1998). TuMV infection thus induced the production of eIF4E, while the expression level of eIF(iso)4E did not appear to change significantly compared with its level in mock-inoculated plants. Interestingly, a protein migrating ahead of eIF(iso)4E was detected in infected leaves, but the nature of this species was not investigated further.



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Fig. 1. Expression profile of eIF4E isomers following TuMV infection. Recombinant A. thaliana eIF(iso)4E and eIF4E, as well as proteins from mock-inoculated and TuMV-infected leaf extracts were separated by SDS-PAGE and electroblotted on to nitrocellulose. The membrane was probed with a rabbit serum against eIF(iso)4E of A. thaliana.

 
Membrane localization of eIF4E, eIF(iso)4E and VPg-Pro
eIF4E is associated with 48S ribosomal complexes (Hiremath et al., 1989). To determine which VPg-containing protein type is found in ribosome-containing structures, cellular membranes from mock-inoculated and TuMV-infected leaves were fractionated on 20–45 % sucrose gradients. The fractionation was carried out in either the presence or absence of 3 mM MgCl2. The presence of MgCl2 preserves the integrity of ribosomes associated with the endoplasmic reticulum (ER). On the other hand, the absence of MgCl2 promotes the dissociation of ribosomes from the ER, which results in a shift of the ER membranes towards the top of the gradient (Lord et al., 1973). Fractions were collected and first analysed by immunoblot assay using antibodies raised against BiP (a marker of the ER; Cascardo et al., 2000), proteins containing xylose-{beta}1 -> 2-mannose modifications (a marker of the medial- and trans-Golgi) and the tonoplast H+-ATPase. In the presence of 3 mM MgCl2, two BiP-containing peaks were resolved, one near the bottom and one near the top of the gradient (Fig. 2a). This result has previously been described by others (Han & Sanfaçon, 2003; Schaad et al., 1997a), but no explanation was provided concerning the nature of the light BiP-containing peak. This may reflect the tendency of BiP to be released from the ER membranes and to float in the gradient. The heavy BiP-containing fractions contained rRNA (data not shown). The peak moved two to three fractions up the gradient in the absence of MgCl2, which suggested that fractions 1–5 contained the ER, with a proportion of the membranes associated with ribosomes. The proteins containing {beta}-xylosyl were detected near the top of the gradient (Fig. 2b; fractions 11 and 12). Likewise, the tonoplast H+-ATPase was located in fractions 10–12 (Fig. 2c). This indicated that the Golgi apparatus and vacuolar membranes were well resolved from the ER in the sucrose gradient.



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Fig. 2. Detection of proteins in membrane fractions following centrifugation in sucrose gradient. Tissue extracts were prepared and centrifuged on 20–45 % sucrose density gradients in the presence or absence of MgCl2, as indicated. The direction of sedimentation was from right to left, with fraction 12 representing the top of the gradient. Fractions were collected and proteins separated by SDS-PAGE and electroblotted on to nitrocellulose. (a–c) Sucrose gradients in the presence or absence of 3 mM MgCl2 (as indicated) of infected extract and immunoblot analysis using anti-BiP serum (a), anti-{beta}-xylosyl serum (b) and anti-tonoplast H+-ATPase serum (c). (d) Sucrose gradient in the presence of 3 mM MgCl2 of mock-inoculated and TuMV-infected extract and immunoblot analysis using anti-eIF(iso)4E serum. Recombinant eIF(iso)4E from A. thaliana (C) and non-fractionated extracts (S) were analysed. Gel migration position of eIF4E and eIF(iso)4E is indicated on the left. (e) Sucrose gradient in the presence or absence of 3 mM MgCl2 of infected extract and immunoblot analysis using anti-VPg-Pro serum. Recombinant VPg-Pro from TuMV (C), mock-inoculated (N) and TuMV-infected non-fractionated extracts (S) were also analysed. (f) Schematic representation of the TuMV polyprotein. Fully processed proteins are indicated by boxes.

 
In mock-inoculated tissue, eIF(iso)4E sedimented at a position near the bottom of the gradient in the presence of 3 mM MgCl2 (Fig. 2d; fractions 1–5). The sucrose gradient sedimentation of eIF(iso)4E in TuMV-infected plants was similar. On the other hand, two eIF4E-containing peaks were resolved, one near the bottom of the gradient (fractions 1 and 2) and one in the middle (fractions 5–8). These last fractions also contained rRNA (data not shown). This dual distribution may reflect the position of eIF4E in polysomes and monosomes in the sucrose gradient (Davies & Abe, 1995). No shift of the initiation factors to lighter fractions was observed in the absence of MgCl2 (data not shown), which was expected since they are associated with ribosomes and not with ER membranes. Localization of eIF(iso)4E in fractions 1–5 supports the notion that these fractions contained ribosomes. Interestingly, the anti-eIF(iso)4E-reacting protein of low molecular mass was found in the top fractions where no rRNA was detected.

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 1–5). 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 nickel–agarose 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 1–5 from a 3 mM MgCl2 membrane fractionation experiment were pooled and loaded on to a nickel–agarose 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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Fig. 3. Co-purification of 6K2-VPg-Pro/VPg-Pro with eIF(iso)4E and eIF4E by metal chelation chromatography. Fractions 1–5 from a sucrose gradient centrifugation experiment were pooled, and the membranes solubilized by the addition of 0·5 % Tween-20 and loaded on to a column containing 0·4 ml nickel–agarose resin. Proteins were eluted with 100 mM imidazole, separated by SDS-PAGE and electroblotted on to nitrocellulose. The membrane was probed using (a) anti-VPg-Pro serum and (b) anti-eIF(iso)4E serum. Wells were loaded with proteins derived from membrane fractions of mock-inoculated tissue, proteins derived from membrane fractions of TuMV-infected tissue, eluted proteins from mock-inoculated tissue and eluted proteins from TuMV-infected tissue.

 
VPg-Pro interaction with PABP
Interaction of 6K2-VPg-Pro/VPg-Pro with eIF(iso)4E and eIF4E in planta raises the possibility that other factors involved in the initiation of translation might be, directly or indirectly, associated with the viral protein. One candidate protein is PABP. A rabbit serum raised against PABP2 of A. thaliana (Palanivelu et al., 2000b) was used to detect PABP co-purification with 6K2-VPg-Pro/VPg-Pro. Fig. 4 shows that a B. perviridis 69 kDa PABP2-like isomer can be co-purified from infected tissues. This protein was not purified by metal chelation chromatography from mock-inoculated membrane fractions. This experiment then indicates that 6K2-VPg-Pro and/or VPg-Pro interact with PABP in planta. The higher concentration of PABP in infected plants was found not to be significant in subsequent experiments.



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Fig. 4. Co-purification of PABP2 by metal chelation chromatography. The experimental approach was as described in Fig. 3. The membrane was probed using anti-PABP2 serum. Wells were loaded with proteins derived from the membrane fraction of mock-inoculated tissue, proteins derived from the membrane fraction of TuMV-infected tissue, eluted proteins from mock-inoculated tissue and eluted proteins from TuMV-infected tissue.

 
PABP co-purification with 6K2-VPg-Pro/VPg-Pro may be the result of direct interaction with the viral protein or through the intermediary of another protein that directly interacts with VPg-Pro. ELISA-based binding assays with recombinant proteins were carried out to investigate direct interaction. PABP2 of A. thaliana was produced in E. coli as a T7-tagged, His-tailed fusion protein and purified by metal chelation chromatography. ELISA plate wells were coated either with VPg-Pro or metal chelation chromatography-purified proteins from a control E. coli lysate containing pET21b. The coated wells were then incubated with increasing concentrations of T7-tagged His-tailed PABP2. Complex retention was detected using an anti-T7-tag antibody. Fig. 5(a) shows that proteins from the E. coli lysate did not interact with PABP2. However, a saturation binding curve was observed for VPg-Pro. This experiment then indicates that VPg-Pro interacts directly with PABP2. Binding conferred by the presence of the His tail was excluded, as complex formation between VPg-Pro and PABP2 was equally observed, whether PABP was His-tailed or not (Fig. 5b). The T7 tag has been shown previously not to interact with VPg-Pro (Wittmann et al., 1997).



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Fig. 5. VPg-Pro interaction with PABP of A. thaliana as demonstrated by ELISA-based binding assay. (a) Wells were coated with 1·0 µg purified VPg-Pro ({bullet}), metal chelation-purified E. coli lysate containing pET21b vector ({blacksquare}) or with Blotto only ({blacktriangleup}) and then incubated with increasing concentrations of purified T7-tagged His-tailed PAPB. Retention of the complex was detected using anti-T7-tag antibodies. (b) Wells were coated with 1·0 µg metal chelation-purified E. coli lysate containing pET21b only (lanes 1 and 3) or purified VPg-Pro (lanes 2 and 4) and incubated with 50 µg of an E. coli lysate expressing T7-tagged His-tailed PABP (lanes 1 and 2) or expressing T7-tagged PABP (lanes 3 and 4). Retention of the complex was detected using anti-T7-tag antibodies. Values are means of four replicates from a typical experiment. Error bars represent the standard deviation.

 

   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Infection of B. perviridis by TuMV modified the expression profile of the eIF4E isomers. Only eIF(iso)4E was detected in mock-inoculated, healthy leaves, while both isomers were present in infected tissues. A previous study demonstrated that the eIF4E and eIF(iso)4E mRNAs accumulated differentially in A. thaliana tissues (Rodriguez et al., 1998). The eIF4E mRNA was expressed in flower, cauline leaf, leaf and stem tissues, but the relative abundance in root tissues was very low. On the other hand, the eIF(iso)4E mRNA, although detected in all tissues analysed, was particularly abundant in floral organs and in young developing tissues. This is in contrast to our result on protein expression. Dinkova et al. (2000) noticed that eIF4E expression was under post-transcriptional control in maize, and preliminary data in our laboratory indicate that this is also the case in B. perviridis. Post-transcriptional control is adopted when a cell has to respond quickly to a particular stressful situation, without bringing into play nuclear pathways for mRNA synthesis. An additional eIF(iso)4E-related protein was also detected in infected leaves and may correspond to another isomer of the initiation factor (Ruud et al., 1998) or may be a cleavage product of the initiation factor. This protein does not appear to be associated with ribosomes and it is not known whether it has any cap-binding activity. Differential expression supports the notion that eIF(iso)4E and eIF4E carry out distinct cellular functions (Browning, 1996). For instance, Gallie & Browning (2001) proposed that eIF4F (a higher-order protein complex containing eIF4E) may promote translation under cellular conditions in which cap-dependent translation is inhibited. This statement is appealing in light of the fact that viral infection often leads to cap-dependent inhibition of host mRNA translation (Bushell & Sarnow, 2002; Gale et al., 2000). Other proteins have been shown to increase during plant viral infections (Aranda et al., 1996; Havelda & Maule, 2000). Although the mechanism and purpose of induction remain unknown, it was proposed that this increase in expression could prepare the cell for the biosynthetic demands of virus replication (Maule et al., 2002). It is thus possible that eIF4E production in B. perviridis is linked to the cellular response needed to adjust to the pressure on protein synthesis caused by TuMV infection. This is supported by the work of Lellis et al. (2002) and Duprat et al. (2002) who have isolated A. thaliana lines bearing mutations in the gene encoding eIF(iso)4E. These lines had a normal phenotype, even though they did not produce eIF(iso)4E. It was noted that the amount of eIF4E had increased significantly in the transposon-mutated line (Duprat et al., 2002). The mutant A. thaliana may thus have compensated for the lack of eIF(iso)4E by an increased synthesis of eIF4E to keep up with the demand of protein synthesis associated with normal plant development. Additionally, absence of eIF(iso)4E rendered these lines resistant to TuMV. We thus suggest that TuMV infection of B. perviridis leads to the inactivation of eIF(iso)4E or to its monopolization for viral protein synthesis. The pressure by the virus on the host protein synthesis machinery would then have to be relieved by de novo synthesis of eIF4E to fulfil the needs of the plant.

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-Pro–PABP complex and a likely VPg-Pro–eIF4E–eIF4G–PABP 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{alpha}. 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.


   ACKNOWLEDGEMENTS
 
S. L. and C. V. contributed equally to this work. We wish to thank A. Vitale for the anti-BiP, M. Boutry for the anti-tonoplast H+-ATPase, A. Sturm for the anti-{beta}-xylosyl and D. A. Belostotsky for the anti-PAPB2 sera. We are also grateful to Dr H. Sanfaçon for critical reading of the manuscript. This work was supported by NSERC of Canada and the Fonds FCAR awarded to J.-F. L. and M. G. F.


   REFERENCES
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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Received 7 October 2003; accepted 28 November 2003.