1 Laboratory of Virology, Department of Plant Sciences, Wageningen University, Binnenhaven 11, 6709 PD Wageningen, The Netherlands
2 Laboratory of Molecular Biology, Department of Plant Sciences, Wageningen University, Dreijenlaan 3, 6703 HA Wageningen, The Netherlands
Correspondence
Jan van Lent
jan.vanlent{at}wur.nl
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ABSTRACT |
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INTRODUCTION |
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A specific affinity of the viral MP for the CPs or virions is probably essential not only for entrapment of virions during tubule assembly, but also for targeted transport of virions from the cytoplasmic site of assembly to the plasmodesma. Indirect evidence for a specific interaction between the C terminus of the CPMV MP and virions was presented by Lekkerkerker et al. (1996), who showed that a mutant virus encoding an MP lacking the C-terminal 18 amino acids (residues 313331) was able to form tubules in protoplasts which did not contain virus particles, and the mutant was not able to spread systemically in plant tissue. Furthermore, for Grapevine fanleaf virus (GFLV), another member of the family Comoviridae, Belin et al. (1999)
found that the nine C-terminal amino acids of the MP must be of the same virus origin as the CP for successful systemic spread, and they also proposed a requirement for specific interactions between the movement protein (2BMP) and the coat protein (2CCP).
We describe here further investigations of the affinity of the CPMV MP for virions and for the individual viral CPs by in vitro binding assays, i.e. ELISA and blot overlay assays, using purified MP as a probe.
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METHODS |
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To isolate the complete MP-coding sequence plasmid pM19GFP2A (Gopinath et al., 2000), containing the full-length cDNA of CPMV RNA-2, was used as starting material. The MP fragment was synthesized by PCR using primers 5'-GGGGTACCATGGAAAGCATTA TGAGC-3' and 5'-GGGCTAAGCTTTAGGCCTATTGTGGAAAAGC-3'. A KpnI site was introduced at the 5' end directly before the start codon of the MP sequence and a HindIII site including a stop codon at the 3' end (indicated in bold type). To generate
C48MP, the same forward primer was used and a stop codon and HindIII site were placed at amino acid position 298 using primer 5'-GGGCTAAGCTTCTAAGAAATAGAGTATTTCAA-3'. The fragment encoding
N289MP was amplified using primers 5'-GGGGTACCGGAGAAAGTTTGAAATACTCT-3' and the reverse primer that was used for the full-length construct.
The fragments were first cloned into the pGEM-T Easy vector (Promega), excised with KpnI and HindIII and inserted downstream of the polyhedrin promoter and an N-terminal 6xHis-tag of plasmid pFASTBAC-HT (Gibco BRL). The constructs were sequenced to confirm the integrity of the insert. The resulting plasmids were named pFASTBAC-MP, pFASTBAC-N289MP and pFASTBAC-
C48MP. Recombinant viruses, expressing the MP or the two truncated variants, were constructed using the Bac-to-Bac system protocol (Gibco BRL) and the viruses were designated AcNPV-MP, AcNPV-
N289MP and AcNPV-
C48MP.
Sf21 cells were infected with each recombinant virus and at 36 h or 48 h p.i. the cells were harvested and checked for expression of the proteins by Western blot analysis (Gopinath et al., 2000) and immunofluorescence (essentially as described by Kasteel et al., 1996
) using goat anti-rabbit conjugated to Alexa 488 as fluorochrome (Molecular Probes). Fluorescence was recorded in a Zeiss LSM 510 laser scanning microscope through excitation with blue laser light at 488 nm and emission through a 505530 nm bandpass filter.
Purification of MP.
The His-tagged MP and C48MP were purified from AcNPV-MP- and AcNPV-
C48MP-infected cells using a Talon Cell Thru column (Clontech). Infected and uninfected insect cells were pelleted and resuspended in 5 ml lysis buffer (50 mM sodium phosphate, 6 M guanidine.HCl, 300 mM NaCl, pH 8·0) containing 30 µg E64 protease inhibitor ml-1 (Roche). Cells were ruptured by pulse sonication for 6x30 s with 30 s intervals and then centrifuged at 1200 r.p.m. in an SS34 Sorvall rotor for 20 min to pellet cell debris. The supernatant was collected and loaded onto a 2 ml bed volume Talon Cell Thru column, previously equilibrated with lysis buffer. The column was first washed with 30 ml lysis buffer followed by 5 ml washing buffer (45 mM sodium phosphate, 5·4 M guanidine.HCl, 270 mM NaCl, 10 mM imidazole, pH 8·0). MP or
C48MP was then eluted under denaturing conditions, with 3 ml of washing buffer containing 200 mM imidazole. The eluted proteins were immediately renatured by a sequence of dialysis steps against renaturing buffer (20 mM Tris/HCl, pH 7·6, 1 mM EDTA, 10 % glycerol, 100 mM NaCl, 7 mM 2-mercaptoethanol) containing decreasing concentrations of guanidine.HCl. Starting with 6 M guanidine.HCl for 6 h, the concentration was halved every 6 h by adding an equal volume of cold renaturing buffer until 750 mM. Except for the first dialysis, which was done at room temperature, all dialysis steps were done at 4 °C. Samples were then dialysed against pure renaturing buffer to remove guanidine.HCl. Protein concentration was determined by Bradford protein assay (Bio-Rad) using BSA as a standard. Purified MP was stored in 50 µl aliquots of 0·10·2 mg ml-1 at -80 °C until further use.
Antibodies.
Three antibodies were available to detect the His-tagged CPMV MP. A monoclonal antibody against 6x His (anti-His) obtained from Clontech could be used to detect denatured forms but not renatured forms of the MP and C48MP. A rabbit polyclonal antibody against a peptide consisting of the 30 C-terminal amino acids of the CPMV MP (denoted anti-48K; Wellink et al., 1987
) was most specific to the viral MP, but was not suitable to detect the C-terminally truncated
C48MP. A rabbit polyclonal antibody generated against E. coli-expressed 58 kDa protein from CPMV RNA-2, which contains the entire 48 kDa MP sequence (denoted anti-58K; Kasteel, 1999
), was able to detect both the MP and
C48MP but generally showed some additional bands in Western blots. Insect cell lysates and purified MP and
C48MP protein fractions were resolved on 12 % SDS-PAGE gels (Laemmli, 1970
), which were stained with Coomassie blue or electroblotted (0·04 mA for 1 h at room temperature) to PVDF Immobilon P membrane (Amersham) in transfer buffer (50 mM Tris base, 192 mM glycine, 20 % methanol, 0·01 % SDS, pH 9·5). On the latter, the His-tagged MP and
C48MP were detected with an anti-6x His monoclonal antibody (Clontech) or anti-MP polyclonal antibody.
ELISA-based binding assay.
Purified MP and C48MP were tested for binding to virions in an ELISA assay. Wells of Nunc Axisorp F96 immunoplates were coated with 150 µl of 6·6 µg purified CPMV ml-1 coating buffer (0·05 M sodium carbonate, pH 9·6) overnight at 4 °C. Controls consisted of purified MP and similar amounts of purified virions of the related comoviruses Cowpea severe mosaic virus (CPSMV) and Red clover mottle virus (RCMV) and the unrelated Tobacco mosaic virus (TMV). After rinsing the plates three times with PBS, pH 7·2, containing 0·05 % (v/v) Tween 20 (PBS/Tween), the wells were blocked with PBS/Tween containing 5 % (w/v) dry non-fat milk for 1 h. The plates were then incubated with 150 µl of 13 µg ml-1 purified MP or
C48MP in PBS/Tween containing 1 % dry non-fat milk or PBS/Tween as a control for 90 min at room temperature. The plates were rinsed three times with PBS/Tween and incubated with anti-48K or anti-58K antibodies at 37 °C for 1 h. The plates were again rinsed and incubated with secondary antibody conjugated to alkaline phosphatase at 37 °C for 1 h. After rinsing, 150 µl substrate (1 mg p-nitrophenyl phosphate, disodium salt, ml-1 0·01 M diethanolamine buffer, pH 9·6) was added per well. Reactions were quantified by reading the absorbance at 405 nm using a Bio-Tek Instruments EL 312 ELISA-reader. Samples giving an ELISA reading higher than the average of six control readings plus three times the standard deviation were considered positive, those with lower readings negative.
Blot overlay assay.
The binding properties of MP and C48MP were further explored using blot overlay assays essentially as described by Chen et al. (2000)
. Ten µg of purified MP or
C48MP and different virus suspensions (CPMV, RCMV, CPSMV and TMV) were resolved on a 12 % SDS-PAGE gel and electroblotted onto a PVDF Immobilon P membrane. The membrane was washed twice in denaturating buffer (6 M guanidine.HCl, 2 mM EDTA, 50 mM DTT, 50 mM Tris/HCl, pH 8·3) for 10 min at room temperature. The proteins were slowly renatured in serially diluted renaturating buffer (10 mM Tris/HCl, pH 7·4, with 150 mM NaCl, 2 mM EDTA, 2 mM DTT and 0·1 %, v/v, Nonidet P-40) containing, respectively, 4, 3, 2, 1 and 0 M guanidine.HCl each for 10 min at 4 °C. The renatured blot was blocked overnight in renaturating buffer containing 5 % (w/v) non-fat dry milk. To test MP and
C48MP binding, the blot was incubated for 90 min at room temperature with 10 µg ml-1 of either protein in renaturating buffer containing 1 % non-fat dry milk and 5 % (v/v) glycerol. The membrane was washed four times for 5 min with washing buffer (TBS with 0·2 %, v/v, Triton X-100), incubated for 1 h at room temperature with anti-48K or anti-58K antibody and washed again four times for 5 min in washing buffer. The membrane was then incubated with the secondary antibody for 1 h at room temperature and washed four times for 5 min with washing buffer. Antibody binding was detected using the ECL Western blotting kit according to the Amersham protocol. Alternatively, blots containing the MP were probed with purified CPMV and virion binding was detected using anti-CPMV antiserum in a similar procedure.
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RESULTS |
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Binding of MP to virions
Binding of the MPs to virions of several different viruses was tested in an ELISA assay. The wells were coated with purified virus suspensions or buffer and incubated with MP or C48MP, and bound MP was detected using anti-58K antibodies. Average results of three repetitions are illustrated in Fig. 3
. The MP showed binding only to homologous CPMV virions, but not to virions of the related comoviruses RCMV and CPSMV or to virions of the unrelated TMV. The truncated
C48MP did not show binding even to the homologous CPMV virions.
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DISCUSSION |
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Heterologous expression of viral MPs in insect cells and subsequent column purification appears to be a suitable method to obtain functional probes for in vitro binding studies. Earlier studies have shown that upon expression of CPMV MP, but also MPs of other viruses employing a similar mechanism of tubule-guided virion movement, the MP retains its tubule-forming capacity in insect cells (Kasteel et al., 1996; Storms et al., 1995
). Here it was also shown that a truncated form of the MP, containing the tubule-forming domain but devoid of the virion-binding domain, retained its biological function. The MPs are located at the plasma membrane and assemble into tubules protruding from the cell surface into the culture medium, similar to MP in virus-infected plant cells (Van Lent et al., 1991
; Bertens et al., 2003
). Attempts to express a peptide of 52 C-terminal amino acids, constituting the putative virion-binding domain, were not successful. The peptide was either not expressed or synthesized in amounts below the detection limit.
Purification of the expressed MPs was only feasible after unfolding the proteins in the insect cell lysate by a denaturing procedure prior to column filtration. Denaturing apparently exposed the His-tagged N terminus sufficiently for efficient binding to the Talon column. Prior to their use as probes in binding assays, the MPs were renatured again. The process of denaturing and renaturing apparently did not affect the intermolecular binding of the MPs (essential for tubule assembly), as in blot overlay assays the MP probe showed binding to the blotted truncated C48MP (Fig. 2D, E
).
A remarkable outcome of the binding assays is the specificity of the MP for its homologous virion. No binding occurred even to virions of the related comoviruses RCMV and CPSMV (for similarities between comoviruses see Haudenshield & Palukaitis, 1998). These results suggest that in vivo only homologous virions can be moved from cell to cell by the viral transport tubule and that the MP is not able to assist the movement of virions even of related viruses. Little, but supportive, evidence is available for this hypothesis from a study performed by Belin et al. (1999)
on the nepovirus GFLV, showing that nine C-terminal residues of the 2B movement protein must be of the same virus origin as the 2C coat protein for successful systemic spread.
In Western blot overlay assays, binding of the MP occurs only to the CPMV L protein but not to the S protein (Fig. 5). The significance of this binding for the mechanism of virion movement remains to be elucidated, but the capacity of the MP to bind in vitro to L protein molecules may also indicate a possible early interaction between the MP and this coat protein, i.e. this may occur in the membranous cytopathic structure where CPMV replication, viral protein synthesis and virion assembly take place (De Zoeten et al., 1974
; Carette et al., 2000
, 2002
). MP may thus also be involved in the targeting of virions to the plasmodesma. It is still uncertain how the virion-containing transport tubules are assembled, i.e. how MP and virions are targeted to the plasma membrane/plasmodesma. Recently, Pouwels et al. (2002a)
showed that neither the cytoskeleton nor the secretory pathway plays an essential role in this process.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 15 January 2003;
accepted 14 April 2003.