The central and C-terminal domains of VPg of Clover yellow vein virus are important for VPg–HCPro and VPg–VPg interactions

Ma. Leonora M. Yambao1, Chikara Masuta1, Kenji Nakahara2 and Ichiro Uyeda1

1 Pathogen Plant Interactions Group, Plant Breeding Science, Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan
2 Plant Genetic Engineering Laboratory, Biotechnology Institute, Akita Prefectural University, Ogata, Akita 010-0444, Japan

Correspondence
Ichiro Uyeda
uyeda{at}res.agr.hokudai.ac.jp


   ABSTRACT
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Interactions between the major proteins of Clover yellow vein virus (ClYVV) were investigated using a GAL4 transcription activator-based yeast two-hybrid system (YTHS). Self-interactions manifested by VPg and HCPro and an interaction between NIb and NIaPro were observed in ClYVV. In addition, a strong HCPro–VPg interaction was detected by both YTHS and by in vitro far-Western blot analysis in ClYVV. A potyvirus HCPro–VPg interaction has not been reported previously. Using YTHS, domains in ClYVV for the VPg self-interaction and the HCPro–VPg interaction were mapped. The VPg C-terminal region (38 amino acids) was important for the VPg–VPg interaction and the central 19 amino acids were needed for the HCPro–VPg interaction.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Clover yellow vein virus (ClYVV) is a member of the genus Potyvirus and has a single-stranded, positive-sense RNA genome of about 10 kb (Takahashi et al., 1997). Mature functional proteins (P1, HCPro, P3, 6K1, CI, 6K2, VPg, NIaPro, NIb and CP) are generated by viral polyprotein processing by three virus-encoded proteases (P1, HCPro and NIa) (Riechmann et al., 1992). Although substantial information has been reported on the similarity of the interactions between potyviral proteins, differences are also observed within members of this genus.

Most potyviral proteins are multifunctional. For example, HCPro is involved in aphid transmission (Atreya et al., 1990), genome replication (Klein et al., 1994; Kasschau et al., 1997), long-distance movement (Cronin et al., 1995; Kasschau et al., 1997; Saenz et al., 2002) and autoproteolysis (Carrington et al., 1989). In addition, it has been demonstrated that HCPro has plasmodesmatal gating (Rojas et al., 1997) and nucleic acid binding properties (Maia & Bernardi, 1996). Recently HCPro has been identified as a suppressor of post-transcriptional gene silencing (reviewed by Marathe et al., 2000; Anandalakshmi et al., 1998; Brigneti et al., 1998; Kasschau & Carrington, 1998).

VPg is also multifunctional and needed for virus replication (Shahabuddin et al., 1988; Murphy et al., 1990, 1991, 1996). It is also needed for virus cell-to-cell and long-distance movement (Nicolas et al., 1997; Schaad et al., 1997; Keller et al., 1998; Rajamäki & Valkonen, 2002). It contains a nuclear localization signal that is important for virus replication (Schaad et al., 1996) and a sequence-non-specific RNA-binding domain (Merits et al., 1998). Furthermore, the plant cap-binding proteins eIF4E and eIF(iso)4E have been found to bind the VPg of potyviruses (Wittmann et al., 1997; Leonard et al., 2000; Schaad et al., 2000). This interaction was found to be important for the replication of the Turnip mosaic virus (TuMV), Lettuce mosaic virus (LMV), Potato virus Y (PVY) and Tobacco etch virus (TEV) in Arabidopsis thaliana and pepper (Duprat et al., 2002; Lellis et al., 2002; Ruffel et al., 2002).

Protein–protein interactions play important roles in the virus infection cycle. Several interactions between potyviral proteins have been studied using the yeast two-hybrid system (YTHS) (Guo et al., 2001). However, the reported interactions between major proteins are not necessarily common to all potyviruses, and there are many inconsistent observations among the potyviruses that have been analysed by YTHS (Hong et al., 1995; Li et al., 1997; Merits et al., 1999; Urcuqui-Inchima et al., 1999; Choi et al., 2000; Guo et al., 2001; Lopez et al., 2001). In this report, we describe a novel protein–protein interaction between HCPro and VPg in ClYVV that has not been reported in any other potyvirus, suggesting that the HCPro–VPg interaction participates in some function unique to ClYVV. Since the reported results for protein–protein interactions of other potyviruses are not always applicable to ClYVV, the present work will serve as a molecular basis for further characterization of ClYVV proteins.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Viruses and plants.
ClYVV no. 30 is maintained as a clone, pClYVV, in our laboratory (Takahashi et al., 1997). It was originally isolated from French bean (Phaseolus vulgaris) (Uyeda et al., 1975) and induces lethal necrosis on broad bean (Vicia faba). The infected tissues used in this paper were all prepared from infected broad beans.

Yeast two-hybrid system and plasmid construction.
Yeast two-hybrid assays were carried out according to the MATCHMAKER Two-Hybrid System 3 protocol (Clontech). The yeast (Saccharomyces cerevisiae) strain AH109 was used to determine protein–protein interactions. The DNA binding domain (BD) vector pAS2-1 and the activation domain (AD) vector pACT2 were used throughout. HCPro cDNA was inserted in-frame into the BD between the SmaI and SalI sites of pAS2-1 and in-frame into the AD between the BamHI and SacI sites of pACT2. Wild-type VPg cDNA and its deletion clones were inserted in-frame into the BD between the EcoRI and BamHI sites of pAS2-1, or into the BamHI and EcoRI sites of pACT2. PCR primers were designed to add the required restriction endonuclease recognition sequences so that the PCR products could be cloned in-frame into the AD or BD gene. Recombinant plasmids were transformed into E. coli strain JM109. Plasmid DNA was isolated by Quantum Prep Plasmid Miniprep Kit (Bio-Rad). Competent cells of S. cerevisiae strain AH109 were transformed simultaneously with pACT2 and pAS2-1 recombinant DNAs by the lithium acetate method (Gietz et al., 1995). Yeast cells were plated on a high-stringency selective medium without tryptophan, leucine, histidine and adenine, and their {beta}-galactosidase activities were tested at 30 °C on the same selective media containing X-{alpha}-Gal. Yeast cells co-transformed with pAS2-1 and pACT2 without inserts were used as a negative control, whereas those co-transformed with pTD1-1 [AD simian virus 40 (SV40) large T antigen] and pVA3-1 (BD mouse p53) were used as a positive control.

Anti-VPg polyclonal antibody production.
Due to the insolubility of the full-length VPg expressed in E. coli, the 5' (aa 1–100) and 3' (aa 153–191) halves of the VPg gene were separately cloned into the pGEX vector (Amersham) to produce glutathione S-transferase (GST)–VPg fusion proteins in E. coli JM109. The former protein was designated VPgN and the latter VPgC. Each purified fusion protein was used to immunize BALB/c mice.

Anti-HCPro monoclonal antibody production.
The entire HCPro gene was cloned into pMAL-c2 (New England Biolabs) to express the maltose-binding protein (MBP)–HCPro fusion protein in E. coli JM109. The fusion protein was purified with an MBP affinity column and used to produce a monoclonal antibody against HCPro, essentially as described by Inoue et al. (1984).

Western blot analysis.
Total proteins were prepared from either the leaves or stem tissues of infected plants. Tissues were homogenized in denaturing buffer containing 2 % SDS and 5 % {beta}-mercaptoethanol (Laemmli, 1970) or in PBS. The extracts were boiled for 5 min and centrifuged to collect the supernatants. Equal amounts of protein (~10 µg) were separated by SDS-PAGE. Proteins were then transferred to nitrocellulose membranes (Amersham Pharmacia Biotech) and the blots were probed with anti-HCPro monoclonal antibody. Proteins were visualized using anti-mouse secondary antibodies conjugated to alkaline phosphatase, followed by treatment with NBT/BCIP.

Extraction of yeast proteins.
Yeast cells were grown in appropriate selective synthetic medium SD (Difco) to reach the mid-exponential phase (OD600=0·6–0·8). Total proteins from yeast were prepared as described in the Yeast Protocols Handbook (Clontech). Yeast cells were lysed with glass beads (425–600 µm) in cracking buffer (8 M urea, 5 % SDS, 40 mM Tris/HCl, pH 8·8, 0·1 mM EDTA and 0·4 mg bromophenol blue ml-1) supplemented with {beta}-mercaptoethanol and protease inhibitors. Protein samples (10–20 µg) were fractionated by SDS-PAGE through a 12·5 % gel, transferred to nitrocellulose membranes and subjected to immunoblot analysis. The membranes were probed with antibodies against GAL4 AD or BD (1 : 200 dilution; Santa Cruz Biotechnology) and then with alkaline phosphatase-conjugated goat anti-rabbit IgG (1 : 250 dilution; Bio-Rad).

Far-Western blot analysis.
The VPg–HCPro interaction was further examined in vitro by far-Western blotting. Two different probes, GST–VPg (VPgN+VPgC) and MBP–HCPro, were used. Both probes were produced in E. coli JM109. Bacterially expressed proteins and total protein extracts from ClYVV-infected or healthy stem tissues were separated by SDS-PAGE and the fractionated proteins were then transferred on to nitrocellulose membranes. Alternatively, bacterially expressed proteins were directly dot-blotted on to membranes. The blots were first incubated in binding buffer (0·2 M PBS) containing E. coli-produced probe proteins (GST–VPg or GST only and MBP–HCPro or MBP only). After being probed with these proteins, the membranes were extensively washed in PBS and treated with specific antibodies (anti-GST–VPg polyclonal antibodies or anti-HCPro monoclonal antibody). All the membranes were treated with alkaline phosphatase-conjugated anti-mouse IgG (Bio-Rad) and finally immersed in the substrate solution containing BCIP/NBT as described above.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Interactions between ClYVV proteins
Interactions between ClYVV-encoded proteins (P1, HCPro, P3, CI, VPg, NIaPro, NIb and CP) were examined using YTHS. Yeast strain AH109 has three reporter genes, HIS3, ADE2 and lacZ, under the control of the promoter sequences from the GAL1, GAL2 and MEL1 genes, respectively. The use of three reporters eliminates false positives since artificial interactions are not able to activate all of the promoter sequences. To acquire an initial understanding of the interrelationships among ClYVV-encoded proteins, we cloned each of the eight ORFs into both the pACT2 and pAS2-1 plasmids so that each protein could be expressed as a fusion protein with either the GAL4 AD or the DNA BD. AH109 yeast cells transformed with the recombinant pAS2-1 DNAs carrying viral genes and the empty pACT2 vector were first tested for self-activation on the selective medium lacking tryptophan, leucine, histidine and adenine. Yeast cells did not grow on the synthetic medium in any of the control experiments, which were observed for up to 5 days. The same result was obtained when the reciprocal combination of the empty pAS2-1 vector and the recombinant pACT2 DNAs carrying viral genes was tested. Among the different genes evaluated for protein–protein interactions, strong positive interactions were observed between HCPro and HCPro, VPg and Vpg, NIaPro and NIb, and VPg and HCPro (Table 1). While the first three interactions have been found previously in potyviruses (Thornbury et al., 1985; Li et al., 1997; Wang & Pirone, 1999; Guo et al., 1999, 2001; Oruetxebarria et al., 2001), the VPg–HCPro interaction has not previously been described in a potyvirus. Dimerization of HCPro was further indicated by Western blot analysis (Fig. 1) using a monoclonal antibody against HCPro. In addition to the 50 kDa monomer band, a distinct 100 kDa band was detected, suggesting the existence of strong HCPro dimerization. However, in the present study we cannot exclude the possibility that HCPro could form a heterodimer with either a viral or host protein.


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Table 1. Protein–protein interactions between ClYVV proteins in the yeast two-hybrid system

 


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Fig. 1. Detection of HCPro isolated from infected tissues using an anti-HCPro monoclonal antibody. Proteins were extracted in PBS and separated by SDS-PAGE. Two bands of 50 and 100 kDa were detected in ClYVV-infected leaves of broad beans after extraction in PBS. H, healthy leaves; ClYVV, upper systemic leaves infected with ClYVV. The positions of molecular mass markers are indicated on the left.

 
Identification of the domain of VPg involved in the VPg–HCPro interaction
To define the protein domains involved in this interaction, a series of deletions in VPg were made. All truncated forms of VPg, in either BD or AD, were again evaluated for autoactivation of the marker genes in yeast. When transformed with a deletion mutant and an empty vector in reciprocal combinations, no growth was observed in the selective medium. The truncated VPgs, as well as the full-length HCPro expressed from either the BD or AD, were all tested by Western blot analysis to confirm stable expression of those proteins in yeast (Fig. 2). The nature of the deletion mutant constructs is shown in Fig. 3. Fifty and eighty amino acids from the N terminus of VPg were deleted, creating BD 51–191 and BD 81–191, respectively. The constructs containing the N-terminal domain (BD 1–99) and the central domain (BD 77–153) were also prepared. AH109 yeast cells transformed with these plasmids and a plasmid containing the full-length HCPro (either in BD or AD) turned blue when transferred on to a medium containing X-{alpha}-Gal, whereas cells transformed with BD 1–80 and AD HCPro did not grow. These results indicate that a short peptide between positions 80 and 99 (19 amino acids) is important for VPg–HCPro interaction (Fig. 3).



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Fig. 2. Western blot analysis of ClYVV protein expression in yeast cells. Total protein was prepared from yeast transformed with the plasmids indicated above the lanes and from non-transformed yeast (-). (A) Proteins extracted from VPg–HCPro interactions as shown in Fig. 3. (B) Representative proteins extracted from VPg–VPg interactions as shown in Fig. 4. After SDS-PAGE and transfer to a nitrocellulose membrane, the fusion proteins were detected by immunoreaction with polyclonal antibodies against the BD (upper panels) and AD (lower panels). The positions of the proteins expected from the respective plasmids are indicated by arrows. The sizes are shown on the left. A white arrow indicates a possible processed protein from the AD VPg.

 


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Fig. 3. Estimation of the domain sequence responsible for the VPg–HCPro interaction using the yeast two-hybrid system. (A) Genome organization of ClYVV. Some functional domains of VPg are indicated based on TEV and PVA research results (Schaad et al., 1997; Rajamäki & Valkonen, 2002). The nuclear localization signal and genome amplification are shown by a hatched box; the tyrosine residue bound to the viral 5' end is shown by a vertical line; and the region involved in long-distance movement is shown by a black box. (B) Protein–protein interactions between VPg deletion constructs and HCPro. ++, Colonies turned blue within 2 h; +, colonies turned blue between 2 and 6 h; -, no colonies appeared on the selective medium. Numbers inside the boxes are the amino acid positions relative to the intact VPg.

 
In vitro interaction of VPg and HCPro
As described above, the ClYVV VPg was shown to interact with HCPro using the YTHS, and the VPg domain important for the VPg–HCPro interaction was also mapped by the same method. The interaction was further examined in vitro by far-Western blots. As shown in Fig. 4(A), the MBP–HCPro fusion protein corresponding to 92 kDa (left membrane, uppermost arrow) was clearly detected, but MBP alone was not detectable (left membrane), indicating specific positive interactions between MBP–HCPro and GST–VPg (VPgN+VPgC). The lower faint bands may be degradation products of MBP–HCPro. In the same membrane, total protein extracts from ClYVV-infected tissues containing VPg and HCPro were also analysed. Two bands corresponding to the molecular masses of HCPro (50 kDa, upper arrow) and VPg (22 kDa) were visualized. No bands were found from total proteins from the healthy tissues.



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Fig. 4. Far-Western blot analysis of the VPg–HCPro interaction. (A) Detection of HCPro using GST–VPg (GST–VPgN+GST–VPgC) as probes. Samples were first separated by SDS-PAGE and transferred to nitrocellulose membranes. Three duplicate membranes were prepared. The left membrane was first overlaid with GST–VPgN+GST–VPgC, then incubated with anti-GST–VPg antibodies. The middle membrane was first overlaid with MBP and then incubated with anti-MBP antibodies. The right membrane was directly incubated with an anti-HCPro monoclonal antibody. The positions of molecular mass markers are indicated on the left. The molecular masses are: MBP–HCPro, 92 kDa; HCPro, 50 kDa; VPg, 22 kDa. (B) Detection of GST–VPg using MBP–HCPro as a probe. E. coli-produced proteins (indicated on the right) were first dot-blotted on to nitrocellulose membranes. The left membrane was probed with MBP–HCPro, the middle with MBP and right was directly incubated with anti-GST–VPg antibodies. The arrow indicates an interaction between VPg and HCPro.

 
The reverse direction for the far-Western analysis was also determined, using the E. coli-expressed MBP–HCPro as a probe (Fig. 4B). When GST–VPg (VPgN+VPgC) was separated by SDS-PAGE, anti-GST–VPg antibodies reacted very weakly to GST–VPg, although they had originally been used to raise the antibodies. Instead, using a dot-blot far-Western assay, it was shown that GST–VPg could interact with anti-GST–VPg antibodies (Fig. 4B, right membrane). The blotted GST–VPgN reacted to the overlaid MBP–HCPro (left membrane, arrow), whereas there was no signal on the blotted GST–VPgC or GST only. No positive signal was found with MBP (middle membrane) suggesting that MBP did not bind to either GST or VPg. These observations are consistent with the domain identified by the YTHS, indicating that the domain of aa 80–99 of VPg (also within GST–VPgN) is important for the VPg–HCPro interaction. However, our in vitro far-Western blot experiments do not exclude the possibility that the C-terminal half may also contribute to the protein–protein interaction in planta.

Identification of the domain of VPg involved in its self-interaction
We also analysed the VPg domain(s) involved in its self-interaction. A series of VPg deletion mutants from both the BD and AD vectors were made (Fig. 5). When the C terminus of VPg was deleted, no growth was observed on the selective medium. To eliminate the possibility that the lack of interaction was due to instability of the proteins expressed in the yeast cells, Western blot analysis was performed (Fig. 2). Comparison of the results obtained from BD 51–191 and BD 81–191 with BD 77–153 and BD 50–153 suggested that the 38 C-terminal amino acids (residues 153–191) are important for VPg self-interaction (Fig. 5). Furthermore, the combination of BD 81–191 and AD 81–191 showed a positive interaction (Fig. 5). The interaction was stronger in both directions when the combination of the region from residues 51 to 191 and the full-length VPg was used. The structural requirement for VPg self-interaction probably favours the entire VPg, although the C-terminal region is indispensable.



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Fig. 5. Estimation of the domain sequence responsible for VPg self-interaction using the yeast two-hybrid system by protein–protein interaction between VPg deletion constructs. ++, Colonies turned blue within 2 h; +, colonies turned blue between 2 and 6 h; -, no colonies appeared on the selective medium. The C-terminal region 154–191 was found to be important for self-interaction of VPg. Numbers inside the boxes indicate the amino acid positions relative to the intact VPg.

 

   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Protein–protein interactions between different genes from the genera Potyvirus and Tritimovirus, members of the Potyviridae family, is well documented (Hong et al., 1995; Li et al., 1997; Guo et al., 1999; Merits et al., 1999; Urcuqui-Inchima et al., 1999; Choi et al., 2000; Lopez et al., 2001; Oruetxebarria et al., 2001). However, there has been little information on the virus-encoded proteins of the potyvirus ClYVV. We are currently characterizing the functions of each protein of ClYVV. Although similarities in the sequences within the Potyvirus genus are not necessarily high, some conserved sequences between members of this group have been reported. The conserved proteins should have similar functions in common within this genus. In this study, we found interactions between HCPro and HCPro, VPg and VPg, and NIaPro and NIb. The HCPro–HCPro interaction was initially reported in TEV by Thornbury et al. (1985) using biochemical methods. Using YTHS, HCPro self-interactions have also been reported in Potato virus A (PVA), Pea seed-borne mosaic virus (PSbMV) and LMV (Guo et al., 1999, 2001; Urcuqui-Inchima et al., 1999). Two different domains were found to be important for self-interaction. For PVY and LMV, the N terminus is important, while both the N and C termini are important for PVA (Guo et al., 1999; Urcuqui-Inchima et al., 1999). VPg self-interaction was first shown in PVA (Oruetxebarria et al., 2001) and then in PSbMV (Guo et al., 2001). The NIb protein has been reported to interact with VPg in Tobacco vein mottling virus (TVMV) (Hong et al., 1995). In contrast, it interacts with NIaPro, but not with VPg, in TEV and PSbMV (Li et al., 1997; Guo et al., 2001). Taken together, these results indicate that such interactions are not necessarily common among potyviruses but may be differential, i.e. each potyvirus may have unique protein–protein interaction patterns.

The interaction found between VPg and HCPro in ClYVV is a novel feature, so far not shown for any other Potyvirus. Whether this interaction is required for ClYVV replication, protein synthesis or movement is unclear. Potyviruses encode several proteins with common functions in their infection cycle. At least seven proteins (P1, HCPro, P3, CI, 6K2, VPg and NIb) have been found to be involved in replication (reviewed by Revers et al., 1999), and five (HCPro, CI, 6K2, VPg and CP) are involved in virus movement (Dolja et al., 1994, 1995; Klein et al., 1994; Cronin et al., 1995; Kasschau et al., 1997; Schaad et al., 1997; Andersen & Johansen, 1998; Carrington et al., 1998; Lopez-Moya & Pirone, 1998; Rajamäki & Valkonen, 1999). Although HCPro and VPg have common functions, especially in replication and virus movement, it is conceivable that VPg and HCPro act only cooperatively and not independently in replication and movement.

The involvement of VPg in potyviral movement has been reported in TEV, TVMV and PVA (Schaad et al., 1997; Nicolas et al., 1997; Rajamäki & Valkonen, 1999, 2002). In these systems, these authors indicated that VPg formed a ‘movement complex’ with another viral protein and/or a host factor to function in virus movement. Furthermore, in a separate study VPg was found in an early stage of infection of PVA in the companion cells of infected Solanum commersonii, emphasizing its role in the movement of potyviruses (Rajamäki & Valkonen, 2003). Since our observations indicate that the central domain of VPg is important for its interaction with HCPro and the same domain was reported to be important for viral movement in other potyviruses (Rajamäki & Valkonen, 2002), we speculate that ClYVV VPg and HCPro may play an important role in the virus movement process. In view of the fact that VPg is attached to the 5' end of viral RNA and that HCPro has been identified as a movement protein that increases the size exclusion limit of plasmodesmata (Rojas et al., 1997), the association of VPg and HCPro might facilitate the movement process in a host plant.


   ACKNOWLEDGEMENTS
 
We thank Dr Shigenori Ueda for technical assistance during the initial stage of the YTHS. This work was supported by a Grant-in-Aid for scientific research on priority area (A) Ministry of Education, Culture, Sports, Science and Technology under the title Molecular Mechanisms of Plant–Pathogenic Microbe Interaction toward Production of Disease Resistant Plants. M. L. M. Y. was supported by a graduate student scholarship from the Ministry of Education, Science, Sports and Culture of Japan.


   REFERENCES
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Received 23 April 2003; accepted 9 June 2003.