The helper component of Cauliflower mosaic virus is encoded by viral gene II. This protein (P2) is dispensable for virus replication but required for aphid transmission. The purification of P2 has never been reported, and hence its biochemical properties are largely unknown. We produced the P2 protein via a recombinant baculovirus with a His tag fused at the N terminus. The fusion protein was purified by affinity chromatography in a soluble and biologically active form. Matrix-assisted laser desorption time-of-flight mass spectrometry demonstrated that P2 is not posttranslationally modified. UV circular dichroism revealed the secondary structure of P2 to be 23% α-helical. Most α-helices are suggested to be located in the C-terminal domain. Using size exclusion chromatography and aphid transmission testing, we established that the active form of P2 assembles as a huge soluble oligomer containing 200 to 300 subunits. We further showed that P2 can also polymerize as long paracrystalline filaments. We mapped P2 domains involved in P2 self-interaction, presumably through coiled-coil structures, one of which is proposed to form a parallel trimer. These regions have previously been reported to also interact with viral P3, another protein involved in aphid transmission. Possible interference between the two types of interaction is discussed with regard to the biological activity of P2.
Most plant viruses are transmitted from one host to another by insect vectors (23), the majority in a noncirculative manner (25). In this mode of transmission, virus particles are acquired while the vector feeds on infected plants and are specifically retained in the food canal of the mouthparts (stylets). The virus is subsequently released from the stylets and inoculated to a new host when the insect vector feeds on another plant. This process implies that specific interactions occur between virus particles and the cuticle lining of the vector's stylets. Two distinct molecular strategies mediating these interactions can be distinguished (24). In the capsid strategy, a motif within the virus coat protein directly recognizes a binding site in the vector stylets, whereas in the helper strategy, binding is not direct but is mediated by a so-called helper component (HC), a viral nonstructural protein acting as a reversible molecular bridge between virus and vector (12). The helper strategy is frequently adopted by plant viruses, and although aphid transmission has been extensively studied for the genera Caulimovirus(4) and Potyvirus (26), the biochemical and structural features of HCs in these two virus groups remain largely unknown.
For Cauliflower mosaic virus (CaMV), the best-studied member of the genus Caulimovirus, the HC is encoded by viral gene II (1, 32), whose expression product is a polypeptide of 18 kDa designated P2. Biologically active P2 from various CaMV isolates has been produced in the baculovirus-insect cell expression system (2, 3, 8). Aphids that first acquired this baculovirus-expressed P2 (P2-loaded aphids) by artificial feeding through Parafilm membranes were able to transmit several P2-deficient, and therefore nontransmissible, CaMV isolates. An initially puzzling result was that P2-loaded aphids could subsequently acquire the nontransmissible virus from infected plants or crude extracts thereof but not from purified virion-containing solutions (3). This was later explained by the finding that P2 does not bind directly to the CaMV coat protein but rather binds to the capsid-associated protein P3 (the product of viral gene III), an additional nonstructural factor that is absolutely mandatory for successful aphid transmission and that is lost upon virus purification (16, 17). The region of P2 involved in P3 binding was mapped to the C-terminal 60 amino acids, which are predicted to form two short α-helices, designated α1 and α2, separated by an 8-amino-acid loop. These two helices were further suggested to be capable of engaging in protein-protein interactions via coiled-coil structures (16; for a review on coiled coils, see reference19).
Apart from interacting with the vector and with P3, there is also evidence that the rather small P2 protein (159 amino acid residues) interacts with itself. When expressed in insect cells, P2 was found to be associated with highly organized cytoplasmic structures, referred to as paracrystals (5). Small amounts of biologically active P2 could be reversibly solubilized from these paracrystals, and since such structures had also been observed in CaMV-infected plant cells (27), it was suggested that they may act as a reservoir of HC. The aggregation of P2 into paracrystalline structures could be considered indirect evidence for the existence of one or several domains involved in P2 self-interaction. Unfortunately, the lack of a satisfactory purification procedure, due to the poor solubility of this protein (8), made it impossible to conclude that P2 is the only constituent of the observed paracrystals and has thus far thwarted all attempts to analyze its biochemistry and structure.
In this paper, we describe for the first time the purification of active CaMV HC, thus allowing its biochemical characterization. We show that biological activity is associated with a high-molecular-weight oligomeric form of P2 and, further, that polymerization into huge paracrystalline filaments is an intrinsic property of this protein. We characterize P2 self-interactions involved in such polymerization and map the motifs responsible to the C-terminal region that is also involved in binding to P3. We confirm experimentally that the secondary structure of this domain is mainly α-helical and that both putative α1 and α2 helices are involved in P2 self-association. Possible interference between P2-P2 and P2-P3 binding is discussed.
MATERIALS AND METHODS
Plasmids.
The baculovirus transfer plasmid p119His, allowing cloning of foreign genes in frame with the coding sequence of a series of six histidines (His tag), is described elsewhere (M. Drucker, R. Froissart, E. Hébrard, M. Uzest, M. Ravallec, P. Espérandieu, J. C. Mani, M. Pugniere, F. Roquet, and S. Blanc, submitted for publication). To construct plasmid pHP2, the coding sequence of gene II of CaMV (strain Cabb B-JI) was PCR amplified using forward and reverse primers including NotI andPstI restriction sites, respectively, and the resulting PCR fragment was cloned into the corresponding sites in p119His. Plasmid pP2H was constructed similarly, except that BglII andNcoI sites were included in the forward and reverse primers, respectively.
Plasmid pGEX-GII and mutant derivatives allowing bacterial expression of glutathione S-transferase (GST)–P2 fusions have been described previously (28). Two additional mutants were produced. The mutation mod (plasmid pGEX-GIImod) corresponds to the replacement of four amino acids of P2 at positions 102, 105, 109, and 116 with glutamic acid residues; the mutation +4 (plasmid pGEX-GII + 4) corresponds to the insertion of four amino acids (Ser-His-Gly-Ser) between amino acids 146 and 147 of P2 (Fig.1). In the GST-P2mod protein, the mutated positions correspond to hydrophobic residues located in the putative α1 helix and suspected to be involved in a coiled-coil interaction. In GST-P2 + 4, the insertion of four amino acids in the middle of the predicted α2 helix should change the overall structure of that motif and change the phase of the α helix, thus compromising any possible coiled-coil formation.
Fig. 1.
Fig. 1. Modifications introduced in P2. The P2 protein is schematically represented at the top. The empty boxes correspond to the predicted helices α1 and α2. The amino acid sequence of the C-terminal domain of P2 encompassing the α1 and α2 regions is listed below the diagram of P2. Hydrophobic residues at positions possibly involved in coiled-coil formation are underlined. The four mutations engineered in mod constructs as well as the four amino acids inserted in +4 constructs are also indicated. The bottom line represents the complete amino acid sequence of HP2Cter.
To construct plasmids pP2::PhoA, pP2 + 4::PhoA, and pP2mod::PhoA, where P2 was fused to the N terminus of alkaline phosphatase (PhoA), we used the PhoA*Color SYSTEM kit (Q. BIOgene). The coding sequences of CaMV gene II and derivatives were PCR amplified from the corresponding pGEX-GII, pGEX-GII + 4, or pGEX-GIImod templates and cloned in the pQUANTagen(kx) expression vector as indicated in the PhoA*Color SYSTEM kit user's manual.
To express the C-terminal 60 amino acids of P2, we cloned PCR fragments corresponding to the region of CaMV Cabb B-JI gene II between nucleotides 1648 and 1827 (nucleotide numbering is according to reference 9) into either pET 3a (Novagen) or pQUANTagen(kx). The former (plasmid pHP2Cter) was cloned usingEcoRI and BamHI restriction sites and expresses the peptide as a C-terminal fusion to a His tag (Fig. 1) whose sequence was introduced in the forward primer. The latter (plasmid pP2Cter::PhoA) was cloned using SalI andBglII restriction sites and expresses the same peptide as an N-terminal fusion to PhoA.
All plasmid constructs were verified by sequencing.
Recombinant baculoviruses.
Recombinant baculoviruses were obtained by cotransfecting Spodoptera frugiperda (Sf9) cells with either pHP2 or pP2H and AcSLP10 baculovirus DNA, followed by plaque purification assays, as previously described (6). pHP2 and pP2H give rise to baculovirus recombinants expressing P2 fused to a His tag at the N (HP2) and C (P2H) termini, respectively. The construction and cloning of the recombinant baculovirus expressing native P2 has been described previously (2).
Protein purification from Sf9 cells.
Approximately 2 × 107 Sf9 cells were infected with either P2-, HP2-, or P2H-encoding baculovirus recombinants at a multiplicity of infection of 10 and harvested after 48 h of incubation at 28°C. Cells were subjected to a freeze-thaw cycle at −20°C, crushed using a syringe with a 26-gauge needle (Terumo) in 1 ml of DB5 buffer {50 mM HEPES [pH 7.0], 500 mM LiSO4, 0.5 mM EGTA, 0.2% [wt/vol] 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate [CHAPS]}, and then diluted in the same buffer to a final volume of 25 ml. After being shaken for 2 h at 4°C, the extract was ultracentrifuged at 100,000 × g for 30 min. The supernatant was stirred for 1 h at 4°C after addition of 1 ml of Ni-nitrilotriacetic acid resin (Qiagen) preequilibrated with DB5 buffer. The slurry was then transferred to a 1-ml column and rinsed with 3 volumes of DB5 buffer. The protein was finally eluted with 2 ml of DB5 buffer supplemented with 500 mM imidazole, dialyzed in DB5, and concentrated in the dialysis tubing on polyethylene glycol 20,000 powder.
Protein purification from bacteria.
Escherichia coli (500-ml culture) transformed with pHP2Cter was induced with 1.5 mM IPTG (isopropyl-β-d-thiogalactopyranoside) for 5 h, pelleted, resuspended in 25 ml of PBS-A buffer (phosphate-buffered saline [PBS] buffer [4.3 mM Na2HPO4, 1.4 mM KH2PO4, 137 mM NaCl, 2.7 mM KCl, pH 7.3] supplemented with 20 mM β-mercaptoethanol and 10 mM imidazole), and frozen at −20°C. The bacteria were then thawed and sonicated before centrifugation for 30 min at 6,000 ×g, and the supernatant was subjected to a 20-min period at 70°C prior to an additional identical centrifugation. The heat-stable proteins in the supernatant were mixed with 2 ml of Ni-nitrilotriacetic acid resin (Qiagen) preequilibrated with PBS-A buffer and stirred gently for 3 h at 4°C. The slurry was then transferred to a 1-ml column and rinsed with 2 volumes of PBS-A, followed by 2 additional volumes of PBS supplemented with 1 M NaCl and then by 3 volumes of PBS. The protein was finally eluted with 2 ml of PBS supplemented with 500 mM imidazole and dialyzed in water. Under these conditions, the HP2Cter peptide precipitates and can be resuspended in various buffers, as indicated.
Expression of P2::PhoA and derivatives and preparation of the corresponding bacterial periplasmic extracts were performed according to the PhoA*Color SYSTEM kit user's manual (Q. BIOgene).
Transmission assays.
The conditions for aphid (Myzus persicae Sulz.) rearing, plant culture of turnips (Brassica rapa cv. Just Right), virus propagation, and aphid transmission tests were as previously described (3) unless otherwise indicated. We used a P2-deficient CaMV isolate named Del-S (R. Froissart and S. Blanc, unpublished results). This nontransmissible isolate is a derivative of CaMV Cabb-S harboring a deletion of 421 bp within the coding sequence of gene II similar to that found in the naturally occurring CM4-184 strain (11).
Electrophoresis.
Protein electrophoresis was performed as described by Laemmli (14). Transfer onto nitrocellulose membranes was carried out using a semidry electroblotting apparatus (Ancos) according to the manufacturer's instructions.
Size exclusion chromatography.
Purified HP2 was centrifuged at 100,000 × g for 30 min immediately before size exclusion chromatography in DB5 buffer with Superose 6 prep-grade medium resin (Pharmacia) in an XK70 column (Pharmacia). Gel runs were carried out on an ÄKTA Prime system (Pharmacia) at 4°C with a flow rate of 0.1 ml/min. The columns were calibrated with purified CaMV virus particles (approximately 20,000 kDa), thyroglobulin (669 kDa), ferritin (443 kDa), aldolase (160 kDa), bovine serum albumin (66 kDa), and cytochrome c (12.4 kDa).
Peptide analysis.
The synthetic peptide pep(α1) (GSCECKQLKEIKSLLEAQNTRIKSLEKAIQSLENKI) was prepared, stored, cross-linked, and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Sephadex G50 medium (Pharmacia) chromatography precisely as described previously (15). The calculated molecular mass of pep(α1) is 4.075 kDa.
The GST-P2C3 fusion protein (28) was purified using a prepacked Sepharose 4B column (Pharmacia), digested with factor Xa (Biolabs), and centrifuged through a Centricon 30 (Amicon). The filtrate contained the released P2C3 peptide comprising the C-terminal part of α1 and the entire α2 helix (P2 amino acids 119 to 159). A nuclear magnetic resonance (NMR) spectrum was recorded for peptide P2C3 in PBS (with 5% D2O), at a concentration of 2.5 mg/ml. The acquisition was done at 305 K on a Bruker AMX 600 spectrometer, with 512 scans and 1 s of presaturation to suppress the water signal.
MALDI-TOF mass spectrometry.
Mass measurements were carried out on a Brucker BIFLEX matrix-assisted laser desorption time-of-flight (MALDI-TOF) mass spectrometer and done in linear mode (13, 22). A saturated solution of α-cyano-4-hydroxycinnamic acid in acetone was used as a matrix. A first layer of fine matrix crystals was obtained by spreading and fast evaporation of 0.5 μl of matrix solution. Subsequently, on this fine layer of crystals, a droplet of 0.5 μl of water solution was deposited; afterwards, 0.5 μl of sample solution was added and then a second 0.2-μl droplet of matrix-saturated solution in 50% H2O–50% acetonitrile was added (13).
UV-CD.
All UV circular dichroism (UV-CD) measurements were recorded with a CD6 spectropolarimeter (Jobin-Yvon). The far-UV-CD spectra were recorded in a thermostated quartz cell with a 0.1-cm path length, in steps of 0.1 nm, at various protein concentrations. Two successive scans were averaged.
In vitro protein-protein interactions.
Interaction between P2, or derivatives thereof, and P3 was assessed essentially as described earlier (16), except that the P3 we used was expressed via a baculovirus recombinant in Sf9 insect cells (R. Froissart, M. Drucker, and S. Blanc, unpublished data).
To test P2-P2 interaction, we took advantage of the fusion protein P2::PhoA (PhoA*Color SYSTEM; Q. BIOgene). GST-P2 and derivatives were separated by SDS-PAGE and transferred onto nitrocellulose membranes. After the membranes were blocked with TBS-Blotto (Tris-buffered saline [TBS] [50 mM Tris-HCl {pH 7.4} and 200 mM NaCl] supplemented with 5% skim milk powder and 0.1% Tween 20), they were incubated overnight with the periplasmic extract of P2::PhoA-producing bacteria (dilution of 1:10 in TBS-Blotto). After three rinses of 15 min with TBS, protein-protein interactions were directly detected using the nitroblue tetrazolium–5-bromo-4-chloro-3-indolylphosphate-p-toluidine salt color reaction.
Electron microscopy.
Crude extracts from Sf9 cells producing P2 as well as purified soluble HP2 were trapped on carbon-coated microscopy grids, negatively stained with 2% ammonium molybdate, and observed in a Zeiss (Jena) EM 10C/RC electron microscope at 80 kV.
RESULTS
Purification of active CaMV HC.
We expressed P2 in the baculovirus-insect cell system as N- and C-terminal fusions with a His tag to allow its purification by affinity chromatography. A series of combinations of various salts, buffers, detergents, and pHs were assessed to determine the combination that would satisfactorily solubilize P2 while preserving its biological activity (not shown). The optimal buffer, DB5, described in Materials and Methods, resulted in the purification of HP2 and P2H (P2 with N- and C-terminal His tags, respectively) to apparent homogeneity (Fig.2A). The His tag fusion differentially modified the electrophoretic mobility of the protein depending on its position at either the N- or C-terminal extremity of P2. However, the identity of the purified polypeptides was confirmed by immunoreaction with a P2 antiserum (Fig. 2B). Some minor bands with increased molecular mass (between 20 and 25 kDa) were also specifically recognized by the P2 antiserum, in both HP2 and P2H. The possible significance of these minor forms of P2 is discussed below. Minor bands with lower molecular mass are degradation products and were seen to similar extents in P2, HP2, and P2H.
Fig. 2.
Fig. 2. Purification of HP2 and P2H. P2 was fused to a His tag at either its N (HP2) or C (P2H) terminus and produced in Sf9 cells via a baculovirus recombinant. (A) Purified HP2 (lane 2) and P2H (lane 3) were analyzed by SDS–12% PAGE and stained with Coomassie blue. A crude extract from insect cells infected with a P2-encoding baculovirus recombinant was loaded in lane 1. (B and C) The same proteins as in panel A were transferred onto nitrocellulose membranes and immunodetected with a P2 antiserum (B) or subjected to a P3 binding assay as described in Materials and Methods (C). The positions of molecular weight markers (in thousands) are shown on the left.
While fusing the His tag to the C terminus of P2 totally abolished biological activity, a similar fusion to the N terminus did not. Indeed, Table 1 shows that P2- and HP2-loaded aphids, but not P2H-loaded ones, transmitted the nontransmissible CaMV isolate Del-S from plant to plant. In addition, the ability to bind P3 is not affected in HP2 but is totally abolished in P2H (Fig. 2C). In agreement with previous results reported by Leh et al. (16), the loss of P3 binding alone can explain the lack of P2H biological activity.
Table 1.
Table 1. Biological activity testing of purified HP2 and P2H
Aphids were allowed to feed for 15 min on the feeding solution to be tested. P2 was from a crude extract of baculovirus-infected Sf9 cells as described previously (10). HP2 and P2H, purified in DBS buffer, were offered at a concentration of 0.5 mg/ml.
b
Aphids were transferred onto plants infected by a nontransmissible CaMV isolate (Del-S) for 15 min prior to a final transfer onto healthy test plants. Ten aphids were used for each test plant.
These results represent the first successful purification of a biologically active form of the HC of CaMV (HP2), thus allowing further biochemical characterization. Because of its lack of HC biological activity, further characterization of P2H is not reported here.
Experimental evidence for α-helical structure in CaMV HC.
Although the previously published models of the secondary structure of P2 (16, 21) predict the presence of α-helices, no consistent experimental data have been available thus far. To investigate the secondary structure of the CaMV HC experimentally, purified HP2 was analyzed by UV-CD spectroscopy as described in Materials and Methods (Fig. 3A). The UV spectrum showed minima at around 208 and 222 nm, confirming the presence of an α-helical conformation. Further calculations carried out with the K2D program (20) indicated a total α-helix content of 23% (±5%). For concentrations of HP2 ranging from 2 to 10 μM, the molar CD intensity remained constant (data not shown).
Fig. 3.
Fig. 3. UV-CD spectroscopy and MALDI-TOF mass spectrometry of HP2. (A) Purified HP2 was dialyzed in DB5 buffer and analyzed at a concentration of 5.1 μM by far-UV-CD spectroscopy in which two successive scans were averaged. (B) Purified HP2 was subjected to MALDI-TOF mass spectrometry. For each peak, the mass in daltons is indicated. The spectrum shows the presence of the trimer (3M), the dimer (2M), the monomer (M), and multicharged ions (3M/2 and M/2).
CaMV HC is not posttranslationally modified.
In MALDI-TOF mass spectrometry (Fig. 3B), the monomeric mass of HP2 was measured as 19.035 kDa, a value slightly lower than the 19.152 kDa calculated from the amino acid sequence. Hence, we conclude that the active form of CaMV HC does not exhibit any posttranslational modification other than removal of the methionine at amino acid position 1. Although MALDI-TOF mass spectrometry does not usually preserve noncovalent oligomers of a protein, additional peaks of higher molecular mass were detected in the HP2 spectrum shown in Fig. 3B. These correspond to di- and trimeric forms of HP2 and suggest that strongly associated oligomers composed of at least three subunits may be present in solution.
Biologically active HC of CaMV forms a huge oligomer.
The apparent molecular mass of purified HP2 was determined by gel filtration (Fig. 4). Surprisingly, when gels with exclusion limits of around 90 kDa (AcA 54; Ultrogel) and 750 kDa (AcA 34; Ultrogel) were used, all HP2 eluted in the void volume of the column (not shown). In a gel with an exclusion limit of around 20,000 kDa, the vast majority of HP2 eluted as one large peak with a small shoulder. The apparent molecular mass for this peak was calculated to be on the order of 5,000 kDa (Fig. 4), corresponding to an association of 200 to 300 HP2 subunits.
Fig. 4.
Fig. 4. Size exclusion chromatography of HP2. The elution profile of soluble HP2 at a concentration of 1.12 mg/ml was recorded in Superose 6 (prep grade) (Pharmacia) as described in Materials and Methods. The column was calibrated using, as standard molecular mass markers, purified CaMV virions (20,000 kDa), thyroglobulin (669 kDa), ferritin (443 kDa), aldolase (160 kDa), bovine serum albumin (66 kDa), and cytochrome c (12.4 kDa). The position of the elution peak of each marker protein is indicated above the graph together with the corresponding molecular mass (in kilodaltons).
The soluble HP2 applied to the gel filtration column contained HC biological activity (the results are those shown in Table 1). The fact that it elutes in totality with an apparent mass of 5,000 kDa suggests that the active form of HP2 is present, in solution, as a huge oligomer. Moreover, aphid transmission testing of all eluted fractions showed that the HC activity was found only in the fraction collected between 48 and 50 ml, precisely matching the HP2 peak mentioned above (Fig. 4). Unfortunately, the transmission rate was as low as 2% (data not shown), and this, we believe, can be explained by an inevitable dilution of the sample during gel filtration. Concentrating the fractions prior to HC activity testing was not possible for reasons described below.
CaMV HC polymerizes as paracrystalline filaments.
During attempts to concentrate purified HP2 in DB5 buffer beyond 1 mg/ml, it invariably precipitated, indicating that the concentration limit of our experimental conditions had been reached. Microscopic examination of the precipitates revealed the presence of huge paracrystal bundles resembling those found in crude extracts of P2-producing Sf9 cells and in viroplasm-enriched fractions prepared from CaMV-infected plants (Fig. 5A and B) (5). The soluble fraction remaining in the supernatant after centrifugation (100,000 × g for 30 min) still contained HP2 at a concentration of approximately 1 mg/ml. Surprisingly, negative staining of this supernatant for electron microscopy revealed the presence of numerous long paracrystalline filaments (Fig. 5C and D). Whether polymerization of HP2 occurred spontaneously in the soluble fraction or was nucleated on the carbon film covering the microscopy grids could not be determined.
Fig. 5.
Fig. 5. Electron microscopy of HP2 polymers. (A and B) Paracrystal bundles were sedimented as described in the text, trapped on a microscopy grid, and negatively stained with 2% ammonium molybdate. (C and D) Purified soluble HP2 was prepared as described in the text, and the protein present in the resulting solution was trapped on a microscopy grid and stained similarly. Bars, 30 nm (A), 100 nm (C), and 12 nm (B and D).
These findings demonstrate that formation of highly organized paracrystals is an intrinsic property of the active form of the CaMV HC, requiring no additional viral or cellular factors. This polymerization activity has so far thwarted all attempts to determine P2 atomic structure by crystallography or NMR.
CaMV HC interacts with itself via the C-terminal domain.
The results of mass spectrometry, size exclusion chromatography, and electron microscopy provide ample evidence for the existence of a P2-P2 interaction that would explain oligomerization and paracrystal formation. A novel in vitro protein blotting-protein overlay assay was developed, as described in Materials and Methods, to identify P2-P2 interaction domains (Fig. 6).
Fig. 6.
Fig. 6. P2-P2 interactions. (A) Schematic outline of native, truncated, or mutated versions of P2 fused to GST as described in Materials and Methods. The GST protein is represented as an oval (not to scale), whereas the lines correspond to the amino acid sequence of P2; helices α1 and α2 are symbolized by boxes. The four point mutations in α1 are represented by vertical lines in GST-P2mod. The 4-amino-acid insertion in α2 is represented by a inverted V in GST-P2 + 4. (B to F) Fusion proteins produced in E. coli were separated by SDS-PAGE and stained with Coomassie blue (B) or transferred onto nitrocellulose membranes (C to F). Numbers in panel A correspond to lanes in panels B to F; 10 μg of total protein was loaded in each lane. The membranes were probed with periplasmic extracts from bacteria producing P2::PhoA (C), P2 + 4::PhoA (D), P2mod::PhoA (E), and P2Cter::PhoA (F). In panel F, lanes 1 and 2 are from the same gel as lanes 3 to 9, for which the contrast was augmented in order to visualize the weaker signals in lanes 6 and 9. The positions of molecular weight markers (in thousands) are indicated on the right.
For this purpose, the C-terminal extremity of P2 was fused to alkaline phosphatase, and the resulting P2::PhoA protein was used to identify putative self-interacting regions in P2. A domain corresponding to the C-terminal 60 amino acids was recognized by the P2::PhoA fusion (Fig. 6C, lane 2). This region is predicted to form two successive short α-helices, designated α1 (amino acids 101 to 128) and α2 (amino acids 137 to 159), separated by a loop of 8 amino acids including two helix-breaking prolines (16). Deletions (Fig. 6C, lanes 3, 4, and 6) or modifications (lanes 8 and 9) in putative α1 or α2 severely reduced but did not totally abolish the interaction with P2::PhoA, suggesting that both presumed helices are involved and that both can also independently interact. Consistent with this conclusion, the interaction between P2 and the P2::PhoA probe was totally abolished only if, in the former, both the α1 and α2 regions were mutated (Fig. 6C, lanes 5 and 7).
As mentioned in Materials and Methods, either only α1 or only α2 remains intact in the P2 + 4::PhoA and P2mod::PhoA fusion proteins, and these two constructs revealed another interesting facet of the P2-P2 interaction. The α1-preserving P2 + 4::PhoA probe could interact only with P2 derivatives retaining wild-type α1 (Fig. 6D, lanes 1, 2, 6, and 9). Similarly, with the α2-preserving P2mod:PhoA probe, interaction was detected only with P2 derivatives harboring an unmodified α2 (Fig. 6E, lanes 1, 2, 3, 4, and 8). Taken together, these results suggest the existence of a self-interaction for both α1 and α2.
When P2Cter::PhoA was used as a probe, the signal was still strong on GST-P2 derivatives containing both α1 and α2 (Fig. 6F, lanes 1 and 2). Although signals were very faint, the other lanes in Fig. 6F show that the P2Cter::PhoA probe binds to GST-P2 fusion derivatives carrying wild-type α1 sequences (lanes 6 and 9) but not to those in which α2 is present alone (lanes 3, 4, and 8). This result suggests that sequences in the N-terminal portion of P2::PhoA, which are absent in P2Cter::PhoA, are also required for a proper α2-driven interaction. In Fig. 6F, the band at a position slightly above that of the GST-P2C3 fusion in lane 3 is a nonspecific staining also seen in lanes 6 and 8 and to a lesser extent in all other lanes. In lane 8, we have no explanation for the band reproducibly appearing at around 30 kDa, a molecular mass that does not correspond to GST-P2mod.
Taken together, the results presented in Fig. 6 provide the first direct evidence for a P2-P2 interaction. We demonstrate that a 60-amino-acid-long C-terminal domain plays a pivotal role in this interaction, presumably by the concerted action of helices α1 and α2.
Characterization of the C-terminal region of CaMV HC.
To investigate the oligomerization state of the self-interacting C-terminal domain of P2, we expressed a peptide encompassing the predicted α1 and α2 helices (amino acids 100 to 159) as a C-terminal fusion to a His tag in E. coli. The corresponding HP2Cter peptide was purified, and, to verify that it was correctly folded, a UV-CD spectrum was recorded in DB5 buffer (Fig.7A). A strong signal with typical minima at around 208 and 222 nm revealed the presence of α-helical conformations, which were further calculated to concern 80% (±5%) of the HP2Cter sequence. The presence of HP2Cter oligomers was first suggested by MALDI-TOF mass spectrometry measurements which showed, besides a major peak corresponding to the monomer, the presence of additional peaks precisely at the molecular masses of di- and trimers (Fig. 7B). These peaks are significantly more intense than the artifactual multimeric peaks usually observed with this technique. The fact that they were also visualized in SDS-PAGE analysis (Fig. 7C, lane 1) both confirmed the specificity of dimers and trimers detected by mass spectrometry and indicated that the interaction responsible for the formation of these oligomers is very stable. Moreover, chemical cross-linking with glutaraldehyde greatly augmented the proportions of both dimers and trimers (Fig. 7C, lanes 2 and 3).
Fig. 7.
Fig. 7. Characterization of the C-terminal domain of P2. (A) Purified HP2Cter was dialyzed in DB5 buffer and analyzed at a concentration of 9.9 μM by far-UV-CD spectroscopy in which two successive scans were averaged. (B) A similarly purified sample was subjected to MALDI-TOF mass spectrometry. For each peak, the mass (in daltons) is indicated. The spectrum shows the presence of the trimer (3M), the dimer (2M), the monomer (M), and multicharged ions (3M/2). (C) Purified HP2Cter (10 μg) was visualized on a Coomassie blue-stained SDS–15% polyacrylamide gel, either before cross-linking or after 5 and 10 min of cross-linking with 0.5% glutaraldehyde (lanes 1, 2, and 3, respectively). (D) The pep(α1) peptide (approximately 5 μg), was loaded on an SDS-Tricine gel and stained with Coomassie blue either untreated (lane 1) or cross-linked with sulfo-GMBS at two different concentrations (5 and 10 mM in lanes 2 and 3, respectively). Pep(α1) peptide was also subjected to disulfide oxidative cross-linking for 1 min (lane 4) or 5 min (lane 5). The positions of molecular weight markers (in thousands) in panels C and D are indicated on the left. Numbers on the right indicate the positions of the monomeric (bands 1), dimeric (bands 2), and trimeric (bands 3) forms of HP2Cter (C) and pep(α1) (D).
Since both putative helices α1 and α2 are implicated in self-interaction (Fig. 6), we produced each of the corresponding regions independently for further characterization. Gel filtration in Sephadex G-50 medium and under native conditions showed that pep(α1) eluted in totality at a molecular mass corresponding to a trimer, together with cytochrome c (12.4 kDa) (not shown). Cross-linking of pep(α1) withN-(γ-maleimidobutyryloxy)sulfosuccinimide ester (sulfo-GMBS) (Pierce) confirmed the presence of a major band corresponding to a trimer in SDS-Tricine gel electrophoresis (Fig. 7D, lanes 2 and 3). In pep(α1), the α1 sequence (P2 amino acids 100 to 130) is preceded by the sequence GSCECK. The two cysteines in this extension allow the orientation of the interacting molecules to be assessed by oxidative disulfide cross-linking as described previously (15). Figure 7D (lanes 4 and 5) shows that 1 min of oxidation was sufficient to cross-link the majority of pep(α1) into a trimer, thus indicating that the three α1 helices involved are associated in parallel orientation. We also observed the formation of tetramers and pentamers, but the corresponding bands were not as prominent as that of the trimer.
The α2-containing P2C3 peptide (see Materials and Methods) was produced and purified in order to assess its putative oligomerization through α2 self-interaction. An NMR spectrum, established as indicated in Materials and Methods, did not show a significant amount of slowly exchanging NH signals among the ca. 40 expected for a peptide of that size, eliminating the possibility of the presence of a persistent and stable α2 self-association.
DISCUSSION
The high insolubility of the CaMV HC has been a longstanding problem (3, 5, 7, 8) preventing its purification and hence biochemical and structural characterization. We determined conditions for solubilizing P2 and showed that an N-terminal His tag fusion allows the purification of the resulting HP2 from baculovirus-infected Sf9 cells without compromising HC activity. An apparent quantitative difference (Table 1) in the HC efficiency of P2 versus that of HP2 was not investigated statistically. However, repeated aphid transmission testing seems to confirm that HP2 is not as efficient as native P2 (not shown). It is possible that the His tag fusion slightly affects some properties of the N terminus of P2. Unfortunately, both the structure of that region and its associated functions are thus far totally obscure.
Previous experiments, in which P2 was expressed in Sf9 cells by a baculovirus recombinant in the presence of tunicamycin or radiolabeled orthophosphate, indicated that the active form of the CaMV HC is neither phosphorylated nor N glycosylated (S. Blanc, unpublished data). The MALDI-TOF spectrometry data presented here show a molecular mass for the HP2 monomer slightly less than that calculated from its amino acid sequence, thus confirming that such posttranslational modifications are not required for HC activity. The minor bands of higher molecular mass detected by SDS-PAGE in both HP2 and P2H preparations (Fig. 2B) were not seen with MALDI-TOF spectrometry, probably due to their low abundance. Such forms of P2 are not observed when native P2 is baculovirus expressed in Sf9 cells (Fig. 2) and have never been reported for CaMV-infected plants. Since their presence does not correlate with HC biological activity, they may be artifacts from the baculovirus-insect cell expression system.
Our UV-CD study of HP2 secondary structure reveals that the 60-amino-acid C-terminal domain is nearly completely α-helical (80% ± 5%). This result is in perfect agreement with the model previously published by Leh et al. (16). Furthermore, this 80% α-helical content in the C-terminal domain could fully account for the 23% (±5%) that we found in the entire HP2 molecule, thus also confirming the prediction by Modjtahedi et al. (21) that most α-helical motifs of P2 are located in the C terminus.
Alpha helices and their possible association through the formation of coiled-coil structures have been extensively documented (18). The α1 region of P2 contains motifs typical of coiled-coil structures and is demonstrated here to self-assemble, most likely into a parallel trimer. In cross-linking experiments, however, minor bands corresponding to tetra- and pentamers were also visible. Since the formation of tetrameric (15) or pentameric (29) coiled coils has been reported previously, the possible existence of α1 oligomers containing more than three molecules could not be totally ruled out.
The trimeric form of HP2Cter, as revealed by chemical cross-linking experiments and MALDI-TOF spectrometry, can easily be explained by the α1 self-association described above. That the suggested α2-α2 interaction (Fig. 6E) also participates in the oligomerization of HP2Cter is considered unlikely. Indeed, binding of P2::PhoA to α2 requires sequences located in the N-terminal half of P2 (Fig.6F), thus indicating that the α2-driven P2-P2 association is a more integrated property that requires several motifs in the entire molecule. The involvement of the corresponding domain in the polymerization of full-length P2, however, is confirmed by previously published results reporting that mutations in α2 prevented the formation of P2 paracrystals (28).
While the HC involved in aphid transmission of potyviruses (HC-Pro) has been demonstrated to be functional as a soluble dimer, the situation appears to be much more complex in the case of caulimoviruses. By using an experimental approach similar to that described for potyviruses (30, 31), i.e., purification and gel filtration followed by an aphid transmission assay, we show that the HC activity of CaMV is most likely associated with a huge soluble P2 oligomer containing 200 to 300 subunits. The HC biological activity of the corresponding fraction from gel filtration was very low. It was impossible to load more soluble HP2 on the column because a higher concentration of protein would have triggered the formation of a precipitate. Moreover, we did not concentrate the eluted fractions, as this could have modified the oligomeric form of HP2 present in the solution.
Because it seems inherently obvious that P2-P2 interactions are also required to assemble this soluble active form of the CaMV HC, we have investigated a possible correlation between the P2-P2 interaction and HC activity. Unfortunately, we could not directly answer this question. Indeed, mutations affecting α1-α1 interaction resulted in a marked decrease of P2 expression to undetectable levels both in the baculovirus-insect cell system and in plants infected with a mutant CaMV, although the corresponding RNA was present (data not shown). Hence, beyond biological activity, P2-P2 association appears to be required for stability and correct folding of the protein. The effect of the α2-driven P2-P2 interaction on HC activity was not tested because we have previously shown that point mutations that alter the α2 helix totally abolish not only the formation of P2 paracrystals (28) but also the P2-P3 interaction (16). Thus, it would be impossible to distinguish whether disruption of P2-P2 or of P2-P3 interactions was responsible for the loss of HC activity.
This last point highlights an interesting aspect of the work presented here; that is, P2 interacts with itself via amino acid motifs that are also involved in binding to P3. The P2-P3 interaction described by Leh et al. (16) and the P2-P2 interaction reported here are surprisingly similar. Both are very strong when both predicted helices α1 and α2 are present and weaker when only α2 remains. Nevertheless, under our experimental conditions, a striking difference can be observed. In contrast to P2::PhoA (Fig. 6), P3 was never reported to bind to a P2 derivative in which the α2 region is either mutated or deleted (13). For this reason, it seems most probable that α2 is the primary motif of P2 recognized by P3. How the P2-P3 interaction interferes with α2-driven P2-P2 interaction remains to be investigated. Whether binding of P3 initiated on α2 can extend to α1 as speculated by Leh et al. (16) is also unknown, but in that case, P3 would presumably have to displace the rather stable α1-α1 interaction characterized here. Finally, the N-terminal region of P3 that interacts with P2 is also responsible for P3 tetramer formation (15). Whether the form of P3 that binds to P2 is mono- or tetrameric is also unknown. In any case, it is predictable that interaction with P3 will modify the structure of P2 oligo- or polymers and vice versa. Consequently, elucidation of the precise molecular mechanisms of CaMV aphid transmission will require further studies to gain a better understanding of how P3 influences and regulates the functions of P2.
ACKNOWLEDGMENTS
Eugénie Hébrard and Martin Drucker contributed equally to this work.
We are extremely grateful to Q. BIOgene for kindly providing the Color*PhoA SYSTEM kit. The seeds of turnip cv. Just Right were kindly provided by Takii Europe B.V. We thank P. Espérandieu for aphid transmission testing.
REFERENCES
1.
Armour S. L., Melcher U., Pirone T. P., Lyttle D. J., and Essenberg R. C. Helper component for aphid transmission encoded by region II of cauliflower mosaic virus DNA.Virology 129 1983 25–30
Blanc S., Cerutti M., Chaabihi H., Louis C., Devauchelle G., and Hull R. Gene II product of an aphid-nontransmissible isolate of cauliflower mosaic virus expressed in a baculovirus system possesses aphid transmission factor activity.Virology 192 1993 651–654
Blanc S., Cerutti M., Usmany M., Vlak J. M., and Hull R. Biological activity of cauliflower mosaic virus aphid transmission factor expressed in a heterologous system.Virology 192 1993 643–650
Blanc, S., E. Hébrard, M. Drucker, and R. Froissart. Molecular basis of vector transmission: caulimoviruses.In K. Harris, O. P. Smith, and J. E. Duffus (ed.), Virus-insect-plant interactions, in press. Academic Press, San Diego, Calif.
Blanc S., Schmidt I., Kuhl G., Esperandieu P., Lebeurier G., Hull R., Cerutti M., and Louis C. Paracrystalline structure of cauliflower mosaic virus aphid transmission factor produced both in plants and in a heterologous system and relationship with a solubilized active form.Virology 197 1993 283–292
Chaabihi H., Ogliastro M. H., Martin M., Giraud C., Devauchelle G., and Cerutti M. Competition between baculovirus polyhedrin and p10 gene expression during infection of insect cells.J. Virol. 67 1993 2664–2671
Espinoza A. M., Markham P. G., Maule A. J., and Hull R. In vitro biological activity associated with the aphid transmission factor of cauliflower mosaic virus.J. Gen. Virol. 68 1988 1819–1830
Espinoza A. M., Usmany M., Pirone T. P., Harvey M., Woolston C. J., Medina V., Vlak J. M., and Hull R. Expression of cauliflower mosaic virus ORF II in a baculovirus system.Intervirology 34 1992 1–12
Howarth A. J., Gardner R. C., Messing J., and Shepherd R. J. Nucleotide sequence of naturally occurring deletion mutants of cauliflower mosaic virus.Virology 112 1981 678–685
Kiselar J. G. and Downard K. M. Preservation and detection of specific antibody-peptide complexes by matrix-assisted laser desorption ionization mass spectrometry.J. Am. Soc. Spectrom. 11 2000 746–750
Leclerc D., Burri L., Kajava A. V., Mougeot J. L., Hess D., Lustig A., Kleemann G., and Hohn T. The open reading frame III product of cauliflower mosaic virus forms a tetramer through a N-terminal coiled-coil.J. Biol. Chem. 273 1998 29015–29021
Leh V., Jacquot E., Geldreich A., Haas M., Blanc S., Keller M., and Yot P. Interaction between cauliflower mosaic virus ORFIII product and the coat protein is required for transmission of the virus by aphids.J. Virol. 75 2001 100–106
Leh V., Jacquot E., Geldreich A., Hermann T., Leclerc D., Cerrutti M., Yot P., Keller M., and Blanc S. Aphid transmission of cauliflower mosaic virus requires the viral PIII protein.EMBO J. 18 1999 7077–7085
Modjtahedi N., Volovitch M., Mazzolini L., and Yot P. Comparison of the predicted secondary structure of aphid transmission factor for transmissible and non-transmissible cauliflower mosaic virus strains.FEBS Letter. 181 1985 223–228
Moniatte M., Van der Goot F. G., Buckley J. T., Pattus F., and Van Dorsselaer A. Characterisation of the heptameric pore-forming complex of the Aeromonas toxin aerolysin using MALDI-TOF mass spectrometry.FEBS Lett. 384 1996 269–372
Pirone, T. P., and K. L. Perry.Aphids—non-persistent transmission. In R. T. Plumb (ed.), Advances in botanical research, vol. 30, in press. Academic Press, New York, N.Y.
Raccah, B., H. Huet, and S. Blanc. Potyviruses.In K. Harris, J. E. Duffus, and O. P. Smith (ed.), Virus-insect-plant interactions, in press. Academic Press, San Diego, Calif.
Rodriguez D., Lopez-Abella D., and Diaz-Ruiz J. R. An electron microscopic study of Cauliflower mosaic virus-induced viroplasms: unusual structures within the viroplasms matrix with possible functional significance in the viral replication cycle.J. Ultrastruct. Mol. Struct. Res. 100 1988 118–125
Schmidt I., Blanc S., Esperandieu P., Kuhl G., Devauchelle G., Louis C., and Cerutti M. Interaction between the aphid transmission factor and virus particles is a part of the molecular mechanism of cauliflower mosaic virus aphid transmission.Proc. Natl. Acad. Sci. USA 91 1994 8885–8889
Simmerman H. K. B., Kobayashi Y. M., Autry J. M., and Jones L. R. A leucine zipper stabilizes the pentameric membrane domain of phospolamban and forms a coiled-coil pore structure.J. Biol. Chem. 271 1996 5941–5946
Thornbury D. W., Hellman G. M., Rhoads R. E., and Pirone T. P. Purification and characterization of potyvirus helper component.Virology 144 1985 260–267
Woolston C. J., Covey S. N., Penswick J. R., and Davies J. W. Aphid transmission and a polypeptide are specified by a defined region of the cauliflower mosaic virus genome.Gene 23 1983 15–23
Hebrard E, Drucker M, Leclerc DHohn T, Uzest M, Froissart R, Strub J, Sanglier S, van Dorsselaer A, Padilla A, Labesse G, Blanc S 2001. Biochemical Characterization of the Helper Component of Cauliflower Mosaic Virus. J Virol75:.
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