The Open Reading Frame III Product of Cauliflower Mosaic Virus Forms a Tetramer through a N-terminal Coiled-coil*

The open reading frame III product of cauliflower mosaic virus is a protein of 15 kDa (p15) that is essential for the virus life cycle. It was shown that the 34 N-terminal amino acids are sufficient to support protein-protein interaction with the full-length p15 in the yeast two-hybrid system. A corresponding peptide was synthesized and a recombinant p15 was expressed in Escherichia coli and purified. Circular dichroism spectroscopy showed that the peptide and the full-length protein can assume an α-helical conformation. Analytical centrifugation allowed to determine that p15 assembles as a rod-shaped tetramer. Oxidative cross-linking of N-terminal cysteines of the peptide generated specific covalent oligomers, indicating that the N terminus of p15 is a coiled-coil that assembles as a parallel tetramer. Mutation of Lys22 into Asp destabilized the tetramer and put forward the presence of a salt bridge between Lys22 and Asp24 in a model building of the stalk. These results suggest a model in which the stalk segment of p15 is located at its N terminus, followed by a hinge that provides the space for presenting the C terminus for interactions with nucleic acids and/or proteins.

The open reading frame III product of cauliflower mosaic virus is a protein of 15 kDa (p15) that is essential for the virus life cycle. It was shown that the 34 Nterminal amino acids are sufficient to support proteinprotein interaction with the full-length p15 in the yeast two-hybrid system. A corresponding peptide was synthesized and a recombinant p15 was expressed in Escherichia coli and purified. Circular dichroism spectroscopy showed that the peptide and the full-length protein can assume an ␣-helical conformation. Analytical centrifugation allowed to determine that p15 assembles as a rod-shaped tetramer. Oxidative cross-linking of N-terminal cysteines of the peptide generated specific covalent oligomers, indicating that the N terminus of p15 is a coiled-coil that assembles as a parallel tetramer. Mutation of Lys 22 into Asp destabilized the tetramer and put forward the presence of a salt bridge between Lys 22 and Asp 24 in a model building of the stalk. These results suggest a model in which the stalk segment of p15 is located at its N terminus, followed by a hinge that provides the space for presenting the C terminus for interactions with nucleic acids and/or proteins.
Cauliflower mosaic virus (CaMV) 1 is the type member of the caulimovirus group, a family of plant pararetroviruses (for review, see Ref. 1). The virion is made of 420 subunits of processed forms of the coat protein (44, 39, and 37 kDa) that build an icosahedral viral particle of symmetry T ϭ 7 (2). A small viral protein of 15 kDa, encoded by ORF III (p15) is found associated with the purified virus particle (3). This protein contains a C-terminal proline-rich nonspecific DNA-binding domain that is conserved in the caulimovirus group and similar to the C terminus of the histone-like proteins (4). It was suggested that this domain could be involved in compaction of the viral genomic DNA (4). p15 is essential for the virus life cycle (5)(6)(7), although its exact function remains unknown.
The analysis of the amino acid sequence of the N terminus of p15 predicted a coiled-coil structure. A coiled-coil is a protein structure that allows the oligomerization of a protein via association of two or more ␣-helices. Coiled-coils are found in a large class of fibrous proteins like keratin, myosin, and fibrinogen, and more recently, they have been recognized as a dimerization element of transcription factors (8). Sequences that are capable of forming coiled-coils are characterized by a heptad repeat of seven residues denoted "a" to "g" (9 -11) in which the "a" and "d" positions are occupied predominantly by hydrophobic amino acids, whereas polar residues are found elsewhere. A hydrophobic interface is formed between the helices and represents the major driving force for the stabilization of the oligomer (12). Positions "e" and "g" are frequently charged residues which might contribute to stability, the specificity of helix association, and their relative order of oligomerization (13)(14)(15)(16)(17). There can be two, three, four, or five helices in the bundle, in parallel or in opposite orientation (for review, see Ref. 8).
We have characterized the coiled-coil domain of p15, located at its N terminus, and have shown that the protein assembles into a parallel tetramer through this domain to adopt a rodlike structure.
An EcoRI and an NcoI site at the N terminus and a BglII site at the C terminus were generated by PCR for cloning in pGAD424, pGBT9 (CLONTECH), and pET-3D (Novagen). The entire sequence of each construct was verified by DNA sequencing.
The clone p(⌬34 -107) was generated by a BamHI digest of p15wt cloned into pGAD424 that allowed the deletion of an p15 internal BamHI fragment. The linearized plasmid was ligated and used in the two-hybrid assay. The fusion of the N terminus of p15 was then generated without adding or changing any amino acids of the protein. The construct p  was generated by elution of the EcoRI-BamHI fragment of p15wt in pGAD424 and cloning in the pGAD424 vector linearized with the same enzymes. This cloning generated a fusion of 5 amino acids at the C terminus (MNRRY) of the first 34 amino acids of ORF II of CaMV that are not present in the wt sequence.
The PCR mutagenesis was done according to Vogel and Das (19) with primers 11710 and 11711. The PCR products were cloned in pGAD424 as described previously.
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§ § To whom correspondence should be addressed. The RTBV genome served as template for the PCR amplification of the ORF II. PCR was performed using the forward oligonucleotide 5Ј-ACGTAAGTGCCCATATGAGCGCTGA TTATCCAACTTTCAAG-G-3Ј and the reverse oligonucleotide 5Ј-GCCGCAGGATCCTCATGCT-GGATATTTTCTTTTAATTCC-3Ј. The PCR fragment was digested with NdeI and BamHI and cloned into the procaryotic expression vector pET-3A (Novagen). The resulting plasmid allowed the expression of the complete ORF II product.
Purification of the CaMV p15-The bacterial pellet from 3 liters of culture was resuspended in 50 ml of TP buffer (20 mM Tris, pH 7.0, 20 mM NaCl, 1 mM EDTA, 1 mM EGTA, 5% glycerol, and mixture of inhibitors of proteases (Boehringer Mannheim)), and the cell suspension was lysed by sonication or with a French press. The lysate was heated at 65°C for 15 min and centrifuged at 30,000 ϫ g for 60 min at 4°C. The supernatant was kept and centrifuged at 100,000 ϫ g for 60 min at 4°C. The supernatant was loaded on a CM-Sephacellulose column that was previously pre-equilibrated in TP buffer. p15 was eluted with TP buffer ϩ 100 mM sodium chloride. The eluted protein was diluted 10-fold with 0.5ϫ TP buffer and passed through the column once more. The eluted protein was then diluted 5-fold in TP buffer, pH 8.8, and loaded on a DEAE-Sepharose column. The protein was eluted with TP buffer, pH. 8.8, plus 250 mM NaCl and concentrated with Centriprep P50 (Amicon). This procedure yielded around 10 mg of pure protein/liter of culture medium.
Peptides-The peptide sequences were as follows; pep(wt) is GSCECKQNLNQIQKEVSEILSDQKSMKADIKAILELL, pep(K3 N) is GSCECKQNLNQIQKEVSEILSDQKSMNA DIKAILELL, and pep-(K3 D) is GSCECKQNLNQIQKEVSEILSDQKSMDADIKAILELL. The peptides were synthesized, freeze-dried, dissolved in water to a concentration of 1 mg/ml, and stored as stock solution at -80°C.
SDS-PAGE and Electroblotting-The proteins were mixed with 1/3 of the final volume of loading buffer containing 5% SDS, 30% glycerol, and 0.01% bromphenol blue. The SDS-PAGE was performed as described elsewhere (20).
Analytical Ultracentrifugation-Analytical ultracentrifugation was performed according to Ref. 21. The recombinant protein was analyzed at 20°C in 12.5 mM Tris, pH 8.3, and 90 mM glycine. The protein concentration was adjusted to 0.2 mg/ml. Sedimentation velocity (SV) and equilibrium (SE) of the sample was performed using the Beckman model XLA analytical ultracentrifuge equipped with absorption optics. The SV run was carried out at 56,000 rpm, at 20°C in a 12-mm double-sector cell and recorded at 278 nm. The SE run was carried out in the same cell, as previously mentioned, but filled only about 3 mm filling height in addition to FC-43 bottom fluid and run at 18,000 rpm at 20°C. The molecular mass was calculated using a floating base-line computer program that adjusts the base-line absorbance to obtain the best linear fit of lnA versus r 2 (A, absorption; r, radial distance). In all cases, a partial specific volume of V ϭ 0.73 cm 3 /g 2 and a buffer density of ϭ 1.005 g/cm 3 .
Circular Dichroism-CD spectra were recorded on a Cary 61 spectropolarimeter at 20°C. For far UV CD (250 -195 nm), thermostated quartz cells of 0.1 or 0.2 cm path length were used. Mean residue ellipticity values ([Q]MRW) were calculated (22) by using the expression: where Q is the measured ellipticity in degrees, c is the protein concentration in mg/ml, d is the path length in cm, M r is the relative molecular mass, and Naa is the number of amino acids per protein.
Oxidative Disulfide Cross-linking-Synthesized peptides were lyophilized and resuspended at a concentration of 1 mg/ml in water. To analyze the formation of hetero-oligomers, the peptides were mixed in the presence of 1 mM reduced glutathione in 200 mM Tris, pH 7.5 or 8.8, 200 mM NaCl, and 1 mM EDTA, heated to 37°C for 10 min., and then cooled to 4°C on ice. The samples were prewarmed at the appropriate temperature for 5 min prior to oxidation by the addition of 10 mM oxidized glutathione. The free cysteines were blocked by the addition of 100 mM iodoacetamide for 15 min at the same temperature. Products were analyzed by Tricine-SDS-polyacrylamide gel electrophoresis (20) and visualized by Coomassie Blue staining.
Chemical Cross-linking-The cross-linking was performed in 20 mM sodium phosphate buffer, 150 mM NaCl, pH 7 (phosphate-buffered saline) at a concentration of 0.2 mg/ml with 5 mM or 10 mM of sulfo-GMBS for 2 h at 4°C according to the manufacturer's protocol (Pierce). Products were analyzed by Tricine-SDS-polyacrylamide gel electrophoresis as described before. Cross-linking of the ORF II product of RTBV was done with 5 mM bis-2-sulfosuccinimidooxycarbonyloxyethyl sulfone using the same procedure (Pierce).
Computer Modeling-The ORF III coiled-coil tetramer was modeled using the computer graphics program TURBO-FRODO (23). The crystal structure of GCN4-tetramer (24) was taken as a template for constructing the model. The structure was further refined by using X-PLOR program (25, 26) using a procedure already described (27).
Mass Spectrometry-Liquid chromatography interfaced with electrospray ionization-mass spectrometry was performed using a Rheos 4000 chromatograph with a Jasco 975 UV detector. The system was equipped with a Vydac C8 column (1 ϫ 250 mm) interfaced with Sciex API 300. The mass spectrometer was equipped with an electrospray ion source. The Vydac C8 column was equilibrated in 5% solvent A (2% CH 3 CN, 0.05% trifluoroacetic acid) and solvent B (80% CH 3 CN, 0.045% trifluoroacetic acid) using a linear gradient from 5 to 60% B in 30 min at a flow rate of 50 l/min. The ion spray voltage was 5500 V, and the gas pressure was 40 p.s.i. The mass spectrometer was operated in single quadrupole operating mode. The mass range from 300 to 2400 Da was scanned with a step size of 0.5 kDa and a dwell time of 0.75 ms.

Mapping the Interaction of CaMV p15 with Itself in the Yeast
Two-hybrid System-The yeast two-hybrid system (28) was used to study the interaction of p15 with itself. The ORF III sequence of CaMV was fused in frame with the C terminus of the GAL4 DNA binding domain in the vector pGBT9 to give pGBT III and to the C terminus of the GAL4 activation domain in the vector pGAD424 to give pGAD III. The yeast reporter strain was co-transformed with the plasmids, and ␤-galactosidase activity could be detected less than 1 h after colony lift with To map the domain of interaction, deletions were made in pGAD III (Fig. 1A). The proteins p(1-118), p(1-82), and p(1-34), named after the corresponding amino acids of p15, harbor a C-terminal deletion and induce a ␤-galactosidase activity in the yeast two-hybrid system. The protein p(⌬34 -107) harbors an internal deletion of 73 amino acids and does not affect the interaction with full-length p15, as seen by induction of ␤-galactosidase activity. However, no activity was observed if the first 33 amino acids were deleted in the protein p(34 -129), suggesting that the interaction domain of p15 is located at the N terminus of the protein.
Random mutagenesis was performed on the p15 gene using a modified PCR reaction with Taq polymerase. The fidelity of DNA synthesis of Taq polymerase can be altered by modification of the buffer composition. The presence of metal ions as well as altered nucleotide ratios have been shown to decrease the fidelity of DNA synthesis (19,29). Following PCR mutagenesis, a bank of mutated DNA fragments encoding the p15 gene were cloned into the pGAD vector and cotransformed in yeast with the wt p15 sequence in pGBT III. Five colonies that did not show ␤-galactosidase activity were selected. Their plasmids were isolated and sequenced. One of the five colonies was an empty pGBT vector. The other four plasmids revealed mutations that introduced early stop codons (pM1, pM2, and pM5) or a frameshift ending at a new stop codon (pM3) (Fig. 1B). All the mutant proteins terminated within the first 34 nucleotides, which was the minimal domain of interaction with the fulllength p15 mapped in the experiment (Fig. 1A). These mutants could probably not interact with p15 because they are too short. This result reinforces the importance of the N terminus of p15 in the protein-protein interaction. Since only mutations found close to the N terminus of p15 could influence the interaction, it is tempting to suggest that only this region is involved in the oligomerization of the protein.
Determination of the Oligomeric Structure of p15-When expressed in Escherichia coli BL21(DE3) cells, p15 represented more than 20% of the total protein. The purified protein was analyzed by analytical ultracentrifugation to determine the quaternary structure. The calculation yielded a molecular mass of 57 kDa in 12.5 mM Tris pH 8.3 and 90 mM glycine. The sedimentation coefficient of p15 was of 2.8 s 0 20,w . The combined analytical ultracentifugation data show that the protein is a tetramer and has a rodlike shape.
The p15 N-terminal Peptide Forms an ␣-Helical Coiled-coil-The analysis of the amino acid sequence of the N terminus of p15 revealed a heptad repeat that is characteristic of a coiledcoil (Fig. 1C). To test the ability of this region to form a coiledcoil structure, a peptide corresponding to residues 3-32 of p15 with a molecular mass of 4149 Da was synthesized. The presumptive coiled-coil sequence was preceded by the sequence GSCECKQ and called pep(wt) (Fig. 2A). The two cysteines were used to allow disulfide cross-linking of associated peptides. The position of the cysteines at the N terminus of pep(wt) is important to assess the orientation of the ␣-helices to each other in the coiled-coil. Based on the results obtained in the two-hybrid system, it was concluded that the N-terminal 34 amino acids of p15 are involved in the oligomerization of the protein. Furthermore, the analytical centrifugation showed us that p15 is a tetramer.
To investigate the formation of an oligomeric structure, pep(wt) was reduced at 37°C, slowly cooled to 4°C and oxidized with glutathione for different times. After alkylation, the cross-linked peptide complexes were separated by electrophoresis on non-reducing SDS-polyacrylamide gels. One minute of oxidation was sufficient to cross-link the peptide into a parallel tetramer, which is the major cross-linked form observed on the gel (Fig. 2B). Oxidation for longer periods gave similar results (Fig. 2B). We also observed the formation of a tetramer as the highest multimer when pep(wt) was chemically cross-link with sulfo-GMBS (Fig. 2C). The mass of the tetramer is overestimated on the SDS gel when compared with the protein markers. This could be due to the dense packing of the coiled-coil making it resistant to complete denaturation by SDS, thus causing retardation of the protein in the gel because less SDS is bound to it.
To assess the formation of a coiled-coil, the secondary structure of the peptide was examined by CD spectroscopy. The spectrum, recorded at 20°C in 200 mM Tris, pH 7.5, 200 mM NaCl, and 1 mM EDTA at a concentration of 0.5 mg/ml, displayed minima around 208 and 222 nm, characteristic for an  (4) cross-linked products are marked to the left and the position of molecular mass marker proteins on the right. The C lane was not incubated with oxidized glutathione prior to alkylation and denaturation with the SDS-PAGE loading dye. C, chemical cross-linking of pep(wt). Cross-linking was performed with 5 and 10 mM sulfo-GMBS for 2 h at 4°C according to the manufacturer's protocol (Pierce). As a control, peptide cross-linked through oxidation of the 2 N-terminal cysteines for 10 min was done as described in B.
The purified tetramer of p15 was also analyzed by CD spectroscopy. The protein showed a typical ␣-helical spectrum (Fig.  3B) with two minima, at 208 and 222 nm. The calculation predicts an ␣-helix content of 65%, 27% random coil, and 8% ␤-turn.
Because of the discrepancy in apparent molecular weight on the gel, we verified the oligomerization state by mass spectrom-etry. The cross-linked pep(wt) was applied to a reverse phase high performance liquid chromatography interfaced with an electrospray ionization mass spectrometer. The spectrum of peak A (Fig. 4A) showed a mass of 16,585 Da corresponding to the tetramer form with 8 oxidized Cys. Peak B, with a mass of 4759 Da, corresponded to the monomer, taking into account the two attached glutathione on the Cys, and peak C with a mass of 8293 Da corresponds to the dimer with 4 oxidized cysteines.  4. A, mass spectra of pep(wt). Reverse phase chromatography and mass spectra of the cross-linked pep(wt). The various cross-linked forms of pep(wt) were separated on FPLC by reverse phase chromatography. The mass spectra of peak A shows a mass of 16,585 Da corresponding to the tetramer, peak B a mass of 4759 Da corresponding to the monomer, and peak C a mass of 8293 Da corresponding to the dimer. B, schematic "helical wheel" representation of the tetrameric ␣-helical coiled-coil of p15 as seen from the N terminus. The upper part shows the hydrophobic interactions within the tetramer. The dashed line in the bottom part shows the interchain ionic interaction between Lys 22 and Asp 24 found in the "e" and "g" positions, respectively. The solid line shows a putative stabilizing interchain hydrophobic interaction. Dashed arrows also represent the e-g interaction.
The trimer form was previously found as the least abundant form of oligomers on the SDS-polyacrylamide gel (Fig. 4A). The trimer was not detected with the liquid chromatography-electrospray ionization mass spectrometer.
A helical wheel representation of the coiled-coil of p15 (Fig.  4B) highlights the importance of the hydrophobic residues in the formation of the stalk and the contribution of the electrostatic interaction between Lys 22 and Asp 24 in position "e" and "g," respectively, for the stability of the tetrameric coiled-coil. The hydrophobic residues Leu 29 and Leu 31 could also contribute to the stability of the stalk.
Gel Filtration of Pep(wt)-To discard the possibility of an artifact caused by the oxydative cross-linking of pep(wt), the homogeneity of the oligomer of the reduced pep(wt) was verified in native condition by gel filtration on a Sephadex G-50 medium (Pharmacia). Two globular proteins with molecular masses of 25 kDa (chymotrypsinogen A) and 12.4 kDa (cytochrome C) were used as controls. The stalked peptide, assembled as a tetramer (16.585 kDa), was exclusively eluting together with the chymotrypsinogen A (25 kDa) fractions (Fig. 5).
To verify the level of oligomerization of the eluted peptide, fraction 9 was oxidized for 10 min with glutathione at 4°C prior to denaturing and loading on the SDS-PAGE. As expected, a major band corresponding to the parallel tetramer was detected (Fig. 5). We think that the overestimation of the mass of the tetramer by gel filtration is due to the rod-shaped structure of the coiled-coil that increased its migration through the beads as compared with a globular protein.
Lys 22 Is Involved in Electrostatic Interactions That Stabilize the Tetramer-Asp 24 in position "e" and Lys 22 in position "g" are facing each other and could be involved in an electrostatic interaction that stabilizes the tetramer or even determines its oligomerization state (17). To verify this hypothesis, we synthesized a mutant peptide, pep(K22N). At 4°C, this mutant assembled like pep(wt) (Fig. 6). However, when the oxidation and the alkylation of pep(K22N) was performed at 42 or 65°C, the formation of the tetramer was clearly reduced and the formation of dimers increased (Fig. 6). Mutation of Lys 22 to Asp impaired considerably the formation of the tetramer at 4°C (Fig. 6). In this case, the tetramer almost completely disappeared and more dimers and monomers could be detected.

DISCUSSION
The formation of ␣-helical coiled-coil structures is a common mechanism of protein subunit assembly. Well characterized examples are myosin, intermediate filaments, laminin, and transcription factors (30 -32). The ORF III product of CaMV (p15) contains a heptad repeat at its N terminus, typical of a coiled-coil domain. Using the yeast two-hybrid system, we showed that the first 33 amino acids of p15 are sufficient to allow oligomerization of the full-length p15. Oxidative and chemical cross-linking of the stalk peptides (pep(wt)), compris-ing amino acids 3-32 of p15, could form a parallel tetramer as shown by SDS-PAGE and mass spectroscopy. Finally, the CD spectrum of pep(wt) confirmed the ␣-helical structure of the tetramer. The homogeneity of the oligomer of the reduced pep(wt) was verified by gel filtration, suggesting that the peptide is found uniquely as a tetramer in solution.
p15 is a thermostable protein as evidenced by the purification protocol (see "Experimental Procedures"). Electrostatic interactions have been shown to improve thermal stability in coiled-coils (17). The mutation K22N decreased thermal stability. Furthermore, mutation K22D introduced a negative charge that destabilized the tetramer and favored the formation of a dimer. In a tetrameric and pentameric coiled-coil, positions "a" and "d" are substantially buried in the stalk and are close to each other; residues at the "e" and "g" positions are partially buried; residues "b," "c," and "f" remain completely exposed (27). Only dimers were observed when a negative charge was introduced at position e 22 probably because the tetramer stalk cannot accommodate two negative charges in position e 22 and g 24 .
Sequence alignment of related proteins found in other caulimoviruses suggests also the presence of N-terminal coiled-coils. Thus, oligomerization of this class of proteins would seem to be important for its function (Fig. 7A). This is supported by the observation that deletion of 20 amino acids in the coiled-coil of CaMV p15 leads to a non-infectious virus (33). As shown in studies of GCN4 leucine zipper mutants, the "a" and "d" positions exhibit preferences for leucine and isoleucine, respectively, in a tetrameric coiled-coil (12). The coiled-coil domain of p15 shows no strong bias for leucine at the "a" position, while the "d" position, as in the case of the GCN4-tetramer, is occupied by apolar C ␤ -branched residues (mostly Ile). This provides additional support for the idea that the isoleucine at "d" position can favor coiled-coil tetramerization. To visualize the spatial arrangement of the ORF III product tetramer, a model of the three-dimensional structure was developed (Fig. 8A). The data and the modeling suggest that Asp 24 can interact simultaneously with both Lys 22 and Lys 26 . The modeling also suggests that the p15 tetramer has two interhelical salt bridges between charged side chains at the "g" and "b" positions (Asp 17 -Lys 19 , Asp 24 -Lys 26 ) (Fig. 8A). This type of ion pair was also found in the crystal structure of the four-stranded coiled-coil of GCN4 (12) and can provide for an additional interaction favoring the tetramerization of p15. A hydrophobic interaction be- 5. Gel filtration of pep(wt). Separation of the reduced pep(wt) on Sephadex G-50 (Pharmacia). SDS-gel electrophoresis was performed with fractions 6 -18. The two globular protein markers were chymotrypsinogen A (25 kDa) and cytochrome C (12.4 kDa), as shown on the right side of the gel. Fractions 8, 9, and 10 contain the reduced pep(wt). Fraction 9 was oxidized 10 min to cross-link, denatured with SDS loading dye, and loaded on the gel as described before. Lane 9C represents the oxidized cross-linked form of pep(wt) as shown in Fig. 3. The gel was stained with Coomassie Blue.
FIG. 6. Disulfide cross-linking of the stalk peptide mutants. A, reduced pep(wt) and pep(KN) were incubated for 10 min at 4, 42, and 65°C at a protein concentration of 0.5 mg/ml with 10 mM oxidized glutathione and 1 mM reduced glutathione. Samples were alkylated at the temperature of incubation during 10 min and brought slowly to room temperature prior to denaturation and analysis by SDS-PAGE. Positions of the monomeric (1), dimeric (2), trimeric (3), and tetrameric (4) cross-linked products are marked to the left, and molecular mass marker proteins are on the right of the gel.
tween Leu 29 and Leu 31 of CaMV p15 in position "e" and "g" could also contribute to the stability of this complex.
Charged residues are found in the e 22 position of the ORF III product of all the aligned caulimovirus (Fig. 7A). This charge is compensated by an opposite charge in position g 24 and b 26 in carnation etched ring virus, and in g 24 for strawberry vein banding virus. For soybean chlorotic mottle virus, the electrostatic bridge is found at positions e 17 and g 19 between a Glu and an Arg residue. Furthermore, because of the rather close proximity of the "b" and "c" positions in a tetramer stalk (11), it is possible that the soybean chlorotic mottle virus couples Asp 7 -Arg 8 and Lys 21 -Glu 22 and the figwort mosaic virus couple Glu 27 and Arg 28 also contribute to the stability of the pIII coiled-coils by forming salt bridges.
Badnaviruses have a genomic organization related to the one of caulimoviruses. ORF II of badnaviruses has been suggested to be the homologue of the ORF III of caulimoviruses based on sequence homology and function of their C-terminal nucleic acid binding domain (7). We could also find a putative coiledcoil domain in the ORF II of the badnavirus RTBV in the middle of the protein (Fig. 7B). The purification and chemical cross-linking of the ORF II product showed that also this protein oligomerizes as a tetramer (data not shown). It is tempting to extrapolate this feature to the other badnavirus (Fig. 7B). The putative coiled-coil of CoYMV shows some residues of the same charge in positions b 57 and c 58 , and b 64 and c 65 , respectively. This arrangement would destabilize the tetrameric coiled-coil; however, the virus might have compensated this deficiency by an increase in length of the coiled coil structure. We can also find putative salt bridges between the couples e 58 -g 60 and e 65 -g 67 in the sugarcane bacilliform virus, b 55 -c 56 in cacao swollen shoot virus, e 60 -g 62 in the RTBV and e 67 -g 69 in CoYMV coiled-coil. Potential hydrophobic stabilizing interactions are found between positions e 46 and g 48 in the CoYMV, and between positions e 51 and g 53 in the sugarcane bacilliform virus coiled-coils.
All the coiled-coils presented in Fig. 7 (A and B), with the exception of RTBV, which has the smallest coiled-coil, are interrupted once with a Gln or Thr in position "a" or "d" in the stalk of the multimer. This seems to be a common feature of other coiled-coils, and the nature of this phenomenon is not understood. The assembly domain of cartilage oligomeric matrix protein forms a homo-pentamer coiled-coil with a conserved Gln in the interior "d" position. The substitution of this residue for Leu improved the thermal stability of the coiled-coil and did not lead to any difference in the assembly of the multimer (34). It is possible that nature favors the presence of a Gln or Thr in this position to allow protein turnover in the cell (34). The tetramer structure has a continuous central channel with a radius that varies from 1.0 to 1.3 Å. The radius of the pore excludes a 1.4-Å radius of a water molecule probe or an anion, for example chloride, which was found in a pentameric coiled-coil (35). However, the oxygen atoms of Gln 18 , in the CaMV p15 coiled-coil, could form hydrogen bounds in the stalk and possibly provide a higher specificity for a parallel coiledcoil structure (Fig. 8B) (34).
The coefficient of sedimentation and the evidences brought FIG. 7. Alignment of coiled-coils from different plant pararetroviruses. A, alignment of the ORF III N-terminal domain of different caulimoviruses. GCN4.mut sequence corresponds to the yeast GCN4 leucine zipper mutant that forms a tetramer. B, alignment of the putative coiled-coils of the ORF II of the known plant badnaviruses. The residues found in position "a" and "d" that are part of the core of the tetramer coiled-coil are shown in black cartridges and bold. The residues in bold and italics outside the black cartridge are charged residues that could be involved in a interchain ionic interaction. The hydrophobic residues outside the black cartridge with a black point below the letter could be involved in stabilizing a hydrophobic bond.
FIG. 8. A, stereo view of a modeled structure of the ORF III coiled-coil domain. Negatively and positively charged side chains are in red and blue, polar and apolar side chains are in green and black, and the disulfide-bonded cysteines are in yellow. Ionic bonds between the side chains are denoted by dotted lines. This figure as well as Fig. 2 were generated with the program MOLSCRIPT (37). B, axial projection of the tetramer model looking from the C terminus. The projection shows the specific network of hydrogen bonds between glutamines 18 (shown by a ball-and-stick representation) in a tetramer arrangement. by the oxidative and chemical cross-linking as well as the gel filtration of pep(wt) suggest that the protein adopts a rodlike structure presenting the C-terminal functional domain. The C terminus is likely to be important for the function since a deletion of the last 5 amino acids of the protein completely abolishes the viability of the virus (33). The rod-shaped structure of the coiled-coil probably extends to the two central Cys residues (Cys 64 and Cys 66 ) because of an extension of the amphipathic ␣-helix (Fig. 1C). The amino acid sequence after those two Cys is rich in the ␣-helix breakers Gly and Pro. This region of the protein probably serves as a hinge, providing the space necessary for the C-terminal domain to bind nucleic acids (4). The tetramerization of p15 through a coiled-coil domain can improve its binding affinity for nucleic acid (36), because it links together identical subunits which enhance the affinity for the substrate. Furthermore, this feature might be important to modify the structure of the viral genomic DNA prior to assembly (4). It is not excluded that the tetramerization of p15 could also play a role in the specificity of recognition of nucleic acids.