Centro Nacional de Biotecnología (CSIC), Campus de la Universidad Autónoma de Madrid, 28049 Madrid, Spain1
Author for correspondence: Juan Antonio García. Fax +34 915854535. e-mail jagarcia{at}cnb.uam.es
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Abstract |
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Introduction |
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The potyvirus CI protein has been shown to have RNA helicase activity (Laín et al., 1990 ; Eagles et al., 1994
), which is required for virus RNA replication (Fernández et al., 1997
). Moreover, electron microscopy experiments (Rodríguez-Cerezo et al., 1997
; Roberts et al., 1998
) and genetic analysis (Carrington et al., 1998
) have demonstrated the involvement of CI protein in cell-to-cell movement. Several potyvirus proteins have been suggested to interact with cylindrical inclusions on the basis of immunoreaction of these structures with specific antibodies (Langenberg, 1993
; Rodríguez-Cerezo et al., 1993
; Arbatova et al., 1998
).
The yeast two-hybrid system (Fields & Song, 1989 ) was developed to provide a genetic approach to identify proteins that interact physically in vivo and to define contacts among the subunits of multiprotein complexes, as well as to map specific domains within proteins that are responsible for interactions (Fields & Sternglanz, 1994
; Frederickson, 1998
). This system relies on the modular nature of many eukaryotic site-specific transcriptional activators to generate a transcriptional signal from the interaction of a protein (X) fused to a DNA-binding domain (DBD) with another protein (Y) fused to a transcription-activation domain (AD). Interaction between proteins X and Y leads to the transcription of a reporter gene containing a binding site for the reconstructed transcriptional activator. The sensitivity and specificity of the two-hybrid system have been enhanced with the introduction of yeast strains such as PJ69/4a (James et al., 1996
) containing three reporter genes, HIS3, ADE2 and lacZ, each under the control of a different promoter (GAL1, GAL2 and GAL7, respectively), all of which respond to the same activator, GAL4. One of the reporter genes carried by this yeast strain, GAL2ADE2, displays excellent sensitivity and extremely low background, making it ideal for two-hybrid selection.
In the present study, we report the use of a yeast two-hybrid assay and deletion analysis to map a domain involved in the self-interaction of the PPV CI protein.
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Methods |
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Sequential or simultaneous yeast transformations were done by using the lithium acetate method of Gietz et al. (1992) . Interaction between murine p5372-390 (pVA3-1) and SV40 large T antigen84-708 (pTD1-1) was used as a positive control (Clontech).
-Galactosidase activity was tested by colony-lift filter assay (Schneider et al., 1996
) or by a quantitative assay in liquid using the Luminiscent
-galactosidase detection kit II (Clontech). Accumulation levels of
-galactosidase were estimated on the basis of enzymatic activity of known amounts of purified protein.
Plasmid construction. All plasmids used in this study are listed in Table 1
.
Plasmids for full-length protein interactions.
PPV CI (nt 36515556), P3/6K1 (nt 24463651) and CP (nt 85789566) coding regions were amplified from plasmids pMalcNCIcx (CI) (Fernández et al., 1997 ) and pGPPV (P3/6K1 and CP) (Riechmann et al., 1990
) by PCR with Taq DNA polymerase (Amersham) by using the following oligodeoxynucleotides: P3/6K1, 5' CATGCCATGGTCTTGAAGTGGATAAGTG 3' and 5' CGGGATCCTCACTGATGCTGAACAGCCTG 3'; CI, 5' AGCTTGGACGATATAG 3' and 5' CTAGTCAGTCATTGATGG 3'; and CP, 5' CATGCCATGGCTGACGAAAGAG 3' and 5' CGGGATCCCTACACTCCCCTCAC 3'. Restriction sites introduced to facilitate cloning and artificial initiation and termination codons are underlined and in bold, respectively.
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Plasmids for deletion mapping.
pAS-CI409 and pACT-CI409 were constructed by deleting an ApaLISalI fragment that includes PPV nt 48785555 (the last 226 aa of the CI protein) from pAS-CI and pACT-CI, respectively. Deletion of a PmlISalI fragment that includes PPV nt 41825555 (the last 458 aa of the CI protein) from pAS-CI and pACT-CI yielded pAS-CI177 and pACT-CI177, respectively. pAS-CI407-635 and pACT-CI407-635 were obtained by deleting an NcoIApaLI fragment that includes PPV nt 36514871 (the first 406 aa of the CI protein) from pAS-CI and pACT-CI, respectively. Deletions in the same plasmids of NcoIPmlI fragments (including PPV nt 36514181, which encode the first 177 aa of CI) yielded pAS-CI178-635 and pACT-CI178-635.
pAS-CI134 and pACT-CI134 were constructed by cloning an NcoISphI fragment from pACT-CI (PPV nt 36514051, encoding the first 134 aa of the CI protein) between the NcoI and SmaI sites of pAS2-1 and between the NcoI and SacI sites of pACT-CI, respectively. An NcoINsiI fragment from pACT-CI (PPV nt 36513870, encoding the first 73 aa of the CI protein) was cloned between the NcoI and SmaI sites of pAS2-1 and between the NcoI and SacI sites of pACT2, yielding pAS-CI73 and pACT-CI73. pAS-CI76-177 and pACT-CI76-177 were constructed by inserting an NsiIBamHI fragment from pACT-CI177 (PPV nt 38754182, CI aa 76177) between the SmaI and BamHI sites of pAS2-1 and pACT2, respectively. pACT-CI135-177 was constructed by insertion of an SphIBamHI fragment from pACT-CI177 (PPV nt 40524182, CI aa 135177) between the NcoI and BamHI sites of pACT2. The same PPV sequence was used to generate pAS-CI135-177 by inserting an NdeIBamHI fragment from pACT-CI135-177 in the corresponding sites of pAS2-1.
In all cases, blunting by either Klenow or mung bean nuclease treatment preceded ligation of fragments with non-cohesive ends.
Other plasmids.
pMalcNCI was described previously (Fernández et al., 1997 ). pGGCI was obtained by cloning the PCR-amplified PPV CI coding sequence between the NcoI and SalI sites of pGG5S6N (Simón-Buela et al., 1997
). In pGGCI, the PPV CI coding sequence is under the control of a truncated T7 promoter and the PPV leader sequence (Riechmann et al., 1990
). The accuracy of all the constructs was verified by restriction analysis and by sequencing through the junctions of the ligated fragments.
Recovery of plasmids from yeast.
Total DNA from yeast was prepared by following the method of Hoffman & Winston (1987) and used to transform E. coli DH5
by electroporation with an Electro manipulator 600 (BTX) according to the manufacturers specifications. Colonies were selected in a medium containing ampicillin.
Immunoblot analyses.
Yeast cells were grown in appropriate selective synthetic medium SD (Difco) to reach mid-exponential phase (OD600 of 0·40·6). Total proteins from yeasts were prepared as described in the Yeast Protocols Handbook (Clontech). Yeast cells were lysed with glass beads (425600 µm) in cracking buffer (8 M urea, 5% SDS, 40 mM TrisHCl, pH 8·8, 0·1 mM EDTA and 0·4 mg/ml bromophenol blue) supplemented with -mercaptoethanol and protease inhibitors. Protein samples (between 10 and 20 µg) were fractionated by SDSPAGE through a 12·5% gel, transferred to nitrocellulose membranes and subjected to Western blot analyses. The membranes were probed with monoclonal antibodies against GAL4 DBD and AD domains (Clontech) and then with horseradish peroxidase-conjugated goat anti-mouse IgG (1:5000 dilution, Jackson ImmunoResearch Laboratories). The peroxidase reaction was developed by using the ECL kit (Amersham).
In vitro transcription and translation.
Translation products were synthesized in a rabbit reticulocyte lysate system (Promega) in the presence of [35S]methionine, as recommended by the manufacturer. The templates were synthetic RNAs (0·2 µg) obtained by in vitro transcription (Ampliscribe T7 RNA transcription kit, Epicentre) of plasmid pGGCI linearized with PvuII to obtain full-length CI and with PvuII and ApaLI to obtain the CI409 protein fragment. Translation products were analysed by SDSPAGE as described above, followed by treatment with Amplify (Amersham) and autoradiography of the dried gels at room temperature.
In vitro pull-down assay.
E. coli JM109 cells harbouring pMal-c (encoding maltose-binding protein, MBP) and pMalcNCI (encoding an MBPCI fusion protein) were grown, induced and lysed essentially as described previously (Fernández et al., 1995 ). Appropriate amounts of the crude extract (containing approximately 8 µg of the recombinant proteins) were loaded onto amylose resin columns (New England Biolabs) equilibrated in 10 mM TrisHCl (pH 7·4), 1 mM EDTA, 1 M NaCl. Non-retained proteins were removed by successive washes with the same buffer containing 1, 0·5 and 0·2 M NaCl and no NaCl. Aliquots of 75 µl of the amylose resin with immobilized MBP or MBPCI proteins were packed in cone tips. Two µl 35S-labelled in vitro translation products, diluted in 50 µl 0·2 M NaCl washing buffer, were loaded in each minicolumn and left for 20 min at 4 °C. Minicolumns were washed twice with 200 µl 10 mM TrisHCl (pH 7·4) and resin-bound proteins were eluted with the same buffer containing 10 mM maltose. The samples were precipitated with trichloroacetic acid (TCA), resuspended in 20 µl loading buffer (125 mM TrisHCl, pH 6·8, 2% SDS, 0·1% bromophenol blue, 6 M urea and 5%
-mercaptoethanol), boiled for 5 min and analysed by SDS12·5% PAGE, Coomassie blue staining and autoradiography.
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Results |
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Immunoelectron microscopy experiments have suggested that the CI protein could interact with the proteins P3/6K1 (Rodríguez-Cerezo et al., 1993 ) and CP (Langenberg, 1993
). However, we were not able to detect CIP3/6K1 interactions by two-hybrid assays using pACT-CI, pAS-CI, pACT-P3/6K1 and pAS-P3/6K1 in both reciprocal crosses. We also analysed CINIb, CICP and CINIa interactions, transforming yeast with pACT-CI and pAS-NIb or pAS-CP, as well as with pAS-CI and pGAD-NIa, with negative results for all the combinations. In this set of experiments, the only interaction detected was that between NIb and NIa, which has already been reported for two other potyviruses, tobacco vein mottling virus (Hong et al., 1995
) and tobacco etch virus (Li et al., 1997
).
The fact that CI aggregates in the cytoplasm of infected cells, forming pinwheel inclusions, suggests strongly that it is able to self-interact. Thus, it was surprising that we could not detect the CICI interaction in the two-hybrid system with pACT-CI and pAS-CI, regardless of whether the plasmids were introduced in the yeast simultaneously or sequentially. Since enhanced expression of one or both partners might affect the efficiency of interaction in the two-hybrid system either positively or negatively, we also assayed other plasmids that provide different levels of expression of the ADCI or DBDCI fusion products, always with negative results (data not shown). The same negative results were obtained when CICI and CIP3/6K1 interactions were analysed by using fusion proteins with the DBD of LexA or when different yeast strains were used in the experiments (data not shown).
In order to rule out the possibility that the above lack of interaction was the result of instability of either of the plasmids expressing the AD or DBD fusion proteins, single E. coli colonies were analysed after transformation with DNA from yeast doubly transformed with pACT-CI+pAS-CI, pACT-CI+pAS-P3/6K1 or pACT-P3/6K1+pAS-CI. The presence of both partners was detected for the three pairs of plasmids, although the pACT-type plasmid predominated in all cases (results not shown). In agreement with this result, Western blot analysis of yeast transformed with pACT-CI+pAS-CI demonstrated expression of both CIAD and CIDBD fusion proteins but appeared to indicate that CIAD accumulated at a higher level than CIDBD (Fig. 1). Nevertheless, a rigorous quantification of the amounts of fusion proteins in yeast cells would require exact measurement of the sensitivity of each monoclonal antibody, a question that we have not addressed.
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No interactions were detected between any of the CI fragments and full-length CI protein (Fig. 2) or any other PPV replication proteins (P3/6K1, NIa, NIb) or CP (data not shown). However, the N-terminal fragments CI177 and CI409 were able to interact with themselves and with each other (Fig. 2
). We were not able to detect interactions of the C-terminal fragments CI407-635 and CI178-635 either with themselves or with N-terminal fragments of the CI protein.
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Some extra amino acids derived from vector sequences were linked to the C terminus of the corresponding AD and DBD fusion proteins, as a result of the procedure used to obtain plasmids encoding fragments of the N terminus of the CI protein. We have ruled out the possibility that these extra amino acids are involved in the interactions that we have detected, since efficient activation of the reporter genes was also observed when plasmids expressing fusion products that ended exactly at PPV CI residue 177 were used in the assay (data not shown).
Further mapping of the CI interaction domain
The results described above suggest that sequences involved in self-interaction of the CI protein are confined to its first 177 amino acids. To map the CICI interaction domain further, additional interaction experiments were carried out with new fragments from the CI177 N-terminal region cloned in the two-hybrid vectors (Table 1 and Fig. 3
). The correct expression of all new fusion proteins was confirmed by Western blot analysis (data not shown). Reporter gene expression was observed in all the combinations that included pAS-CI73 and pAS-CI134. These plasmids were able to activate the transcription of the reporter genes even in combination with empty pACT2 (Fig. 3
) or in the absence of a second plasmid encoding the GAL4 AD (data not shown); moreover, yeast cells transformed with these plasmids showed high
-galactosidase activity. This indicates that CI73 and CI134 have intrinsic transcription activation activity and, thus, pAS-CI73 and pAS-CI134 were useless for CICI interaction analysis. pACT-CI76-177 by itself did not activate the reporter genes, but together with the empty DBD vector pAS2-1 it was able to confer on yeast the ability to grow in the absence of histidine and adenine, indicating fortuitous interactions between the CI76-177 fragment and pAS2-1 sequences. However, it was possible to differentiate this artifactual reporter gene activation from genuine CICI interactions because yeast cells transformed with pACT-CI76-177 and pAS2-1 did not show
-galactosidase activity, in contrast with yeast cells containing plasmids that expressed interacting CI-fusion proteins. No accidental reporter gene activation was observed for the rest of the plasmids, so they provide unambiguous information on the localization of CICI binding regions.
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In vitro self-interaction of the CI protein
Although the two-hybrid assay revealed protein interactions at the N terminus of the CI protein, it failed to detect self-interaction for the full-length CI protein. To obtain independent evidence of self-interaction of the CI protein at its N-terminal region, we performed pull-down experiments, using CI fused to an MBP tag (MBPCI) and 35S-labelled CI synthesized in an in vitro translation system (Fig. 4).
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Discussion |
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In spite of the fact that CI aggregates to form inclusion bodies in infected cells, we could not detect CICI interactions in any of the versions of the two-hybrid system that we have assayed. We cannot discard the possibility that CI requires additional virus or plant factors to interact and form cylindrical inclusions. In this regard, formation of cylindrical inclusions by CI independently expressed in a heterologous system has not been reported. However, we have been able to demonstrate the CICI interaction directly by a pull-down experiment in vitro. It is well known that some functional interactions cannot be detected in the two-hybrid system, especially for large proteins, due to protein instability in yeast or to masking in the fusion products of domains involved in proteinprotein interaction, nuclear targetting, DNA binding or transcription activation activity. Although we have detected CIAD and CIDBD accumulation in doubly transformed yeast, we have no evidence that they reach the nucleus. It is not unusual that interactions that cannot be reproduced in the two-hybrid system using full-length proteins become apparent when protein fragments are assayed. We have observed that a PPV CI N-terminal fragment of 409 aa (CI409) is able to interact with itself but not with the complete CI. However, CICI409 interaction took place in the pull-down assay, which seems to be a more permissive system for CI self-interaction. A smaller PPV CI N-terminal fragment of 177 aa (CI177) was also able to self-interact in two-hybrid assays. Self-interaction of CI177 appeared to be stronger than CI177CI409 and CI409CI409 interactions, whereas no CI177full-length CI interaction was detected, further supporting the view that sequences outside the binding domain obstruct PPV CICI interaction in the two-hybrid system. No positive interactions were detected with PPV CI C-terminal fragments. Similar but not identical results have been described for the CI protein of WSMV (Choi et al., 2000 ). Thus, full-length WSMV CI protein was not able to self-interact in a two-hybrid system, but it did in an in vitro biochemical assay. Moreover, positive interactions were found in two-hybrid assays using fragments of the WSMV CI protein. In contrast with our results, not only a fragment including the N-terminal 209 aa of the protein, but also a 182 aa-long C-terminal fragment showed binding capacity and both were able to interact with full-length CI protein, although no quantitative data on the binding strength were reported. Differences in the PPV and WSMV CI proteins (they show only 29% sequence identity), fragment sizes and experimental conditions could account for the discrepancies between the two results. Overall, we can conclude that a major domain involved in potyvirus CICI interaction is positioned at the N-terminal region of the protein, although we cannot rule out the possibility that sequences at the C-terminal region of the protein could also contribute to the binding.
Further deletion analysis of the PPV CI177 fragment did not allow us to delimit a precise self-interaction domain. The results suggest that sequences contributing to the binding are spread across several regions of the CI177 fragment. Moreover, the fact that CI135-177 is able to interact with CI177 but not with itself or with CI76-177 when CI135-177 is fused to AD suggests that interactions could be asymmetric, connecting different sequences of each monomer.
Different types of evidence indicate that the potyvirus CI protein is involved in genome amplification and virus movement (Klein et al., 1994 ; Fernández et al., 1997
; Carrington et al., 1998
), but little is known about the involvement of cylindrical inclusions in these functions. Electron microscopy experiments suggest that these structures might be the engines that facilitate virus movement through plasmodesmata (Rodríguez-Cerezo et al., 1997
; Roberts et al., 1998
), but nothing is known of the role of cylindrical inclusions in the replication function of CI. On the other hand, the NTPase and RNA helicase activities of the potyvirus CI protein have been shown to be essential for virus replication (Fernández et al., 1997
). However, extensive mutagenesis analysis has demonstrated that CI mutations that abolish virus replication in isolated cells are not confined to the N-terminal half of the protein, which contains all the motifs conserved in RNA helicases (Carrington et al., 1998
). The deleterious effects on virus replication of mutations that affect RNA helicase activity have precluded the assessment of the involvement of this enzymatic activity in the movement function of the CI protein. CI mutations that affect virus movement without appreciable effects on virus replication have also been described. Two mutations that affect cell-to-cell spread and three mutations that allow the formation of multicellular infection loci in inoculated leaves but prevent systemic virus spread map to the N-terminal 125 aa of the tobacco etch potyvirus CI protein, whereas only one or two mutations that affect long-distance virus movement correspond specifically to amino acids from the C-terminal region (Carrington et al., 1998
). It is tempting to speculate that the fact that most CI mutations with specific effects on virus movement map to the N-terminal region of the protein, the region that we have shown to be involved in CICI self-interaction, may be related to a requirement of CI for cylindrical inclusion formation in order to perform its role in virus spread. It is clear that further research is required to test this hypothesis and to elucidate the relevance of CI aggregation to the enzymatic activities of the protein and its function in virus replication. The data on CICI binding sequences that we describe in this work should help with this task.
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Acknowledgments |
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Footnotes |
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References |
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Received 23 August 2000;
accepted 15 November 2000.