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Pararetroviruses are a group of viruses with a single, open circular, double-stranded DNA genome. They replicate by a process of reverse transcription similar to that of retroviruses, via a pregenomic RNA that is also used as a polycistronic mRNA for expression of viral proteins (32). All plant pararetroviruses belong to the Caulimoviridae family (29). The genome organization (number and distribution of the open reading frames [ORFs]) differs among members of this family. However, all retain several motifs conserved in many types of retroelements (polymerase, primer binding site, aspartic protease, RNase H, and Zn finger) as well as some domains specific to plant pararetroviruses (movement, proline-rich, nucleic acid-binding, and transactivator domains) (15). In addition, they have different virion sizes and shapes as well as very narrow and distinct host ranges, and those transmitted by insects can have different vectors (aphids, mealybugs, and leafhoppers) (2).

Sequence comparison revealed a conserved coiled-coil motif present at the N terminus of the ORF III product (pIII) of all members of the Caulimovirus genus and within ORF II (pII) of badnaviruses and rice tungro bacilliform virus (RTBV) (20). We have named each of these proteins VAP (virion-associated protein) because of the association with the capsid protein in the virion shells of cauliflower mosaic virus (CaMV) (7, 21), commelina yellow mottle virus (CoYMV) (5), and RTBV (16). VAP is essential for the virus life cycle, as shown for CaMV (8, 17) and RTBV (14). In CaMV, the N-terminal 32 residues of the coiled-coil domain induce the formation of a parallel tetramer (20) that is the functional form of VAP in planta (33). CaMV VAP has a non-sequence-specific nucleic acid-binding activity via its C-terminal proline-rich domain (17, 27), and the same region is also involved in interaction with the capsid protein (21, 22). The VAPs of RTBV and cacao swollen shoot virus (CSSV) also contain a C-terminal nucleic acid-binding domain (18, 19), and RTBV VAP interacts with the viral coat protein (14). Despite all of this information, no function had been assigned to these proteins until recently, when interaction of VAP with the aphid transmission factor (ATF) was detected in CaMV (23). This interaction is essential for transmission of the virus by aphids since VAP makes the link between the virus particle and the ATF. However, why VAP is also essential for plant infection remains to be determined.

All VAP homologues contain a coiled-coil motif. Computer analysis based on the method of Lupas et al. (25, 24) was performed to identify coiled-coil motifs in all available sequences of plant pararetroviruses. Coiled-coils are bundles of two or more amphipathic  helices, characterized by a heptad repeat of hydrophobic and hydrophilic amino acids, that are supercoiled together (13). The sequence alignment shown in Fig. 1 expands and updates our previous findings (20) and confirms that a VAP homologue that contains at least one coiled-coil domain exists in all of these viruses. The characteristic heptad repeat is present at the N terminus of the ORF III product of all caulimoviruses, the N terminus of ORF C of the soybean chlorotic mottle virus (soyCMV)-like viruses, within ORF II of RTBV and of all badnaviruses, and at the N terminus of cassava vein mosaic virus (CVMV) ORF IV (4). A single coiled-coil domain exists in the petunia vein clearing virus (PVCV) genome, located in the second half of the polyprotein encoded by ORF I (30). To assess the characteristics and the order of oligomerization of plant pararetroviral VAPs, we selected representative members of four distinct genera of the Caulimoviridae family: RTBV, Cestrum yellow leaf curling virus (CmYLCV) (L. Stavolone, A. Ragozzino, and T. Hohn, Abstr. Book XIth Int. Congr. Virology, abstr. VP23.13, 1999), CVMV, and PVCV. pII was selected for RTBV (16) because although several lines of evidence strongly suggest this protein to be the VAP in this virus, oligomerization has not been formally shown. The genome of CmYLCV has recently been sequenced (L. Stavalone, unpublished data), and we chose this new plant pararetrovirus to represent the soyCMV-like genus, to which it belongs (Stavolone et al., Abstr. Book XIth Int. Cong. Virol., 1999). ORF C most likely encodes the VAP in the members of this genus. ORF IV of CVMV codes for the only protein (pIV) of the virus possessing a heptad repeat at its N terminus and within the size range of plant pararetrovirus VAPs (4). The formation of a coiled-coil is predicted at the highest probability for this domain; therefore, pIV was selected as the most promising candidate to be the VAP of CVMV. In the case of PVCV, the size and the number of proteins encoded by ORF I have not yet been determined; therefore, we designed an arbitrary VAP by selecting from ORF I a region of 134 amino acids that contained the typical heptad periodicity at the N terminus [p (1-134) (see Fig. 4B)]. We used either the Gal4 (Clontech) or the DupLexA (Clontech) yeast two-hybrid system (9) to analyze self-interaction of these proteins and chemical cross-linking {sulfo-bis [2-(succinimido-oxycarbonyloxy)ethyl]sulfone (BSOCOES); Pierce} to test their ability to multimerize.

View larger version (37K): [in this window] [in a new window]   FIG. 1.   Prediction of plant pararetrovirus VAP coiled-coil domains. Shown is alignment of N-terminal domains of ORF III of caulimoviruses and ORF C of the soyCMV-like viruses, the putative coiled-coil domains within ORF II of RTBV and badnaviruses, the N-terminal part of CVMV ORF IV, and the putative coiled-coil domain located in the second half of PVCV ORF I. The seven positions of the heptad repeat are labeled a through g above the alignment. Positions a and d (bold) are predominantly occupied by hydrophobic amino acids that form the helix interface, while the others are solvent-exposed polar residues. Coiled-coils form when two or more of these  helices interact via the hydrophobic residues, resulting in oligomerization of the protein. The a and d positions in the putative VAP coiled-coil domains are highlighted. (Modified from reference 20).

Two coiled-coil domains contribute to RTBV pII tetramerization. Existing evidence suggests that pII is the VAP of RTBV (16), and although its self-interaction was not supported in the Ga14 yeast two-hybrid system (14), when ORF II was expressed using the DupLexA two-hybrid system -galactosidase (-Gal) activity was induced, proving that self-association of pII indeed occurs (Fig. 2B). The coiled-coil domain of RTBV pII is located in the middle of the protein, with an additional shorter  helix at the N terminus (Fig. 1). To investigate their relative contributions to self-interaction, deletion mutants in which the -helical domains were removed singly or in combination were tested for the ability to interact with full-length pII in yeast. Deletion of the central coiled-coil [m2 (55-73) (Fig. 2A)] had only a weak effect on the observed interaction, whereas deletion of the N-terminal domain [m1 (7-21) (Fig. 2A)] significantly reduced -Gal activity (Fig. 2B). However, a contribution of the central domain is apparent from the double mutant [m3 (7-21, 55-73) (Fig. 2A)], where -Gal activity was reduced still further (Fig. 2B). To directly visualize oligomerization, wild-type (wt) pII and the three deletion mutants were cloned into the vector pET3a (Novagen) and expressed in Escherichia coli. Following chemical cross-linking and separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), four bands corresponding in size to monomers, dimers, trimers, and tetramers were detected with wt RTBV pII (Fig. 2C). Chemical cross-linking also confirmed that both coiled-coil domains contribute to multimerization of the protein, as all three mutants appeared only as the monomeric form in the gel (Fig. 2C). Since the effect of the deletions was very dramatic compared to the two-hybrid results, we suspected that just the reduction in size or improper folding of the mutants could have affected oligomerization. However, cross-linking of an additional mutant [m4 (1-73) (Fig. 2A)], in which both coiled-coil domains are present but the entire C terminus is missing, yielded four bands in the gel, confirming that a truncated protein containing both coiled-coils can tetramerize (Fig. 2C). The orientation of this tetramer was assessed by oxidative disulfide cross-linking (Pierce) of a synthetic peptide corresponding to the longer coiled-coil pep (55-73) with the sequence (GSCECKQ) added to its N terminus (20). In fact, only multimers of  helices in parallel orientation allow cysteine covalent links and thus can be visualized on the gel. As in the case of the VAP of CaMV, the tetramer of RTBV VAP seems to assemble in a parallel orientation (Fig. 2D). This result also provides additional evidence of the contribution of the central coiled-coil domain to self-interaction of pII.

View larger version (30K): [in this window] [in a new window]   FIG. 2.   Mapping of domains involved in self-interaction and tetramerization of RTBV VAP. (A) RTBV ORF II wt product and mutants are depicted by boxes with amino acids numbers indicated. The hatched boxes represent the two predicted coiled-coil domains (replaced with a diagonal line if deleted). (B) Interaction of the wt product with itself and with deletion mutants in the DupLexA yeast two-hybrid system (Clontech). The test was performed according to the manufacturer's instructions, using the cloning vectors pEG202-NLS and pJG4-5 (12) to transform the yeast strain EGY48 (p8op-lacZ). -Gal activity was estimated by filter assay by monitoring the appearance of a blue color after 2 h (++, dark blue; +/, very light blue). Activities in liquid assay given in -Gal units are also shown. nt, not tested. (C) Chemical cross-linking of wt and mutant RTBV VAP. Coding regions were cloned in the vector pET3a (Novagen). The bacterial pellet from induced cells (E. coli BL21) was resuspended in 150 mM NaCl (pH 7), lysed by brief sonication, and centrifuged. The supernatant was collected for the assay. Disulfide cross-linking of this preparation was performed in 20 mM sodium phosphate buffer-150 mM NaCl (pH 7) at a concentration of 0.2 mg/ml with 0.5 mM or 5 mM sulfo-BSOCOES for 2 h at 4°C according to the manufacturer's protocol (Pierce). Products were analyzed by SDS-PAGE, and proteins were detected by Western blotting using an anti-pII antibody (kindly provided by A. Druka and R. Hull, John Innes Centre, Norwich, United Kingdom). ni, not induced. (D) Oxidative cross-linking of the internal RTBV coiled-coil [pep (55-73)]. Positions of the monomeric (1), dimeric (2), trimeric (3), and tetrameric (4) cross-linked products are marked at the right (20).

Complex interactions are involved in CmYLCV pC oligomerization. Self-association of CmYLCV pC was clearly observed in the yeast Gal4 two-hybrid system (Fig. 3B). Two deletion mutants of CmYLCV pC [m1 (24-179) and m2 (9-19) (Fig. 3A)] could still interact with the wt pC with complete deletion of the region of heptad periodicity (m1 mutant), giving rise to a level of -Gal activity of around 50% of that produced by a partial deletion (m2) (Fig. 3B). This result suggested that region 1-23 is involved in the interaction but that sequences other than this coiled-coil domain could also participate. Looking for other possible domains, we found that two coiled-coil segments are predicted, albeit with a lower probability, between positions 24 and 50 and that another short  helix is located at the C terminus of pC (amino acids 158 to 170). N-terminal truncation of pC to position 50 [m5 (50-179) (Fig. 3A)] abolished the interaction with wt C (Fig. 3B), suggesting a role for region 24-50 in self-association (see also results for m1 and m6). In addition, the reduced -Gal activity induced by m3 (1-115) compared to m2 shows that the  helix at the C terminus might also play a role in the self-association of the protein (Fig. 3B). This was confirmed by the ability of this region alone [m4 (116-179) (Fig. 3A)] to interact strongly with itself (data not shown) as well as retain a weak association with wt pC (Fig. 3B). Thus, the entire region 1-49 seems to be involved in self-interaction of CmYLCV pC, and the C-terminal region could also contribute to this association. Chemical cross-linking of the CmYLCV pC (as described for RTBV) proved that the protein could exist in the form of a tetramer (Fig. 3C). Both m1 (24-179) and m2 (9-19) yielded only one band in the gel, although a faint band with a size consistent with that of a dimer was detectable for m2 (Fig. 3C). Together the results of both assays suggest that the domain responsible for tetramerization of the CmYLCV pC is located in the first coiled-coil (positions 1 to 23) but that at least other two regions of the protein contribute to stability of the oligomer.

View larger version (25K): [in this window] [in a new window]   FIG. 3.   Mapping of domains involved in self-interaction and tetramerization of CmYLCV VAP. (A) CmYLCV ORF C wt product and mutants are depicted by boxes with amino acids numbers indicated. The hatched boxes represent the predicted coiled-coil domain (replaced with a diagonal line if deleted), and the grey boxes represent the second heptad repeat. The black boxes correspond to the short - helix located between amino acid positions 158 and 170 (IGEMSELLQNLVKI). (B) Interaction of wt VAP with itself and with the deletion mutants in the Gal4 yeast two-hybrid system (Clontech). Sequences were cloned into plasmids pACTIIst and pAS (kindly provided by P. Legrain, Institut Pasteur, Paris, France) (10), and the assay was carried out in the yeast strain HF7c according to the manufacturer's instructions. -Gal activity was estimated by filter assay by monitoring the appearance of a blue color after 6 h (++, dark blue; +/, very light blue; , no color). Activities in liquid assay are given in -Gal units. Yeast transfected with wt VAP constructs produced very small colonies compared to the others and could not be tested in liquid assay. nt, not tested. (C) Chemical cross-linking of CmYLCV wt and mutant VAP. CmYLCV coding regions (wt and mutants), fused at the carboxyl terminus with an influenza virus hemagglutinin epitope tag (11), were cloned into the vector pET3d (Novagen). Sample preparation and disulfide cross-linking were performed as described in the legend to Fig. 2. Products were analyzed by SDS-PAGE, and proteins were detected by Western blotting using monoclonal antibody HA.11 (BAbCo). ni, not induced. The asterisk indicates the possible m2 mutant dimer.

The predicted coiled-coil domains of CVMV pIV and PVCV pI are responsible for tetramerization. Yeast two-hybrid assays were performed to test CVMV pIV self-interaction, but neither in the Gal4 nor in the DuplexA system was any -Gal activity observed. However, we know that the yeast double-hybrid system can fail to detect protein-protein interactions, as in the case of RTBV VAP in the Gal4 system (14) and CaMV ATF-VAP (D. Leclerc, unpublished observation). Therefore, we tested oligomerization of CVMV pIV expressed in E. coli. Electrophoretic fractionation after chemical cross-linking yielded four bands corresponding in size to monomers, dimers, trimers, and tetramers of the protein (Fig. 4C). The involvement of the predicted coiled-coil domain in the oligomerization of CVMV pIV was shown by the inability of a mutant lacking this domain [m1 (40-201) (Fig. 4A)] to oligomerize (Fig. 4C). In the case of CVMV pIV, it seems that the property of assembling as a tetramer is conferred by a single long heptad periodicity. This could explain why a mutant containing only a partial deletion of this domain [m2 (6-24) (Fig. 4A)] could still cross-link to form a dimer (Fig. 4C).

View larger version (35K): [in this window] [in a new window]   FIG. 4.   Mapping of the domain involved in the self-interaction and tetramerization of CVMV VAP and PVCV ORF (1-134) product. (A) CVMV ORF IV wt product and mutants. (B) PVCV ORF I, ORF (1-134), and mutants. Boxes with amino acids numbers indicated depict wt and mutants. The hatched boxes represent the predicted coiled-coil domain (replaced with a diagonal line if deleted). The grey box illustrates the virus-encoded ORF I product. (C and D) Chemical cross-linking of CVMV ORF IV wt and mutants (C) and of PVCV ORF (1-134) wt and mutants (D). ni, not induced. For methods, see the legend to Fig. 3.

Implications for VAP function. Protein assembly in nature commonly occurs via the formation of coiled-coil structures (for a review, see reference 3). The coiled-coil is a highly versatile protein folding and oligomerization motif and therefore highly adaptable (3). Coiled-coil assembly can be modulated by pH, phosphorylation, or interaction with ions, mostly depending on the position of its charged residues (3). In this respect it is not surprising that mutants still partially interacting in the two-hybrid system can no longer tetramerize. Protein mutations and deletions can lead to conformational changes that alter the exposure of crucial amino acid residues to the surrounding environment. The versatility of the coiled-coil motif supports a variety of biological functions, and this mechanism of oligomerization and interaction with other proteins is used by several viral proteins, both structural and functional (1, 6, 26, 28, 31). One of these is the VAP of plant pararetroviruses. Our results suggest that based on their ability to form tetramers, the products of CmYLCV ORF C and CVMV ORF IV are the homologues of CaMV VAP and that a homologue of this protein is probably also encoded within ORF I of PVCV. We also provide additional evidence of common properties between RTBV and CaMV VAPs. Self-interaction of plant pararetrovirus VAPs can involve more than one domain. For example, CaMV VAP contains two coiled-coil domains at the N terminus (Fig. 1), and although one is dispensable for self-interaction in the two-hybrid system (20), a mutant harboring a deletion of this coiled-coil is not infectious in plants (17). As indicated by our results, regions of VAPs other than the major coiled-coil domain possibly contribute to tetramer stability. Alternatively, they could be involved in vivo in heteromeric interactions with other viral or cellular proteins and thus have some regulatory functions. A protein with the features of a VAP seems to be present in all members of the Caulimoviridae family. This supports the idea that aiding aphid transmission cannot be the only function of VAP; only some plant pararetroviruses are in fact transmissible by aphids, while others (e.g., PVCV and CVMV) do not need any insect vector at all. In addition, transmission by aphids is a dispensable function for the virus life cycle, while VAP has been shown to be essential for infection of the host plant (8, 14, 17). We propose that the tetramerization that is a conserved feature of the VAP in plant pararetroviruses has an important biological significance. VAP could act as the "arm" of the virus particle by keeping its C terminus anchored into the capsid shell (21) and exposing the tetramer for interaction with other proteins. Efforts are under way to detect additional proteins interacting with the VAP coiled-coil structure that could uncover further, and perhaps major, functions of these proteins.