key: cord-0005300-3m0m47w6 authors: McClintock, Katherine; Lamarre, Alain; Parsons, Valérie; Laliberté, Jean-François; Fortin, Marc G. title: Identification of a 37 kDa plant protein that interacts with the turnip mosaic potyvirus capsid protein using anti-idiotypic-antibodies date: 1998 journal: Plant Mol Biol DOI: 10.1023/a:1005916917228 sha: bd53a99331b79db7de9b1c972db38b5a95b888de doc_id: 5300 cord_uid: 3m0m47w6 Experimental data are provided for the presence of a plant protein that interacts with the capsid protein (CP) of turnip mosaic potyvirus (TuMV). The receptor-like protein was identified by exploiting the molecular mimicry potential of anti-idiotypic antibodies. A single-chain Fv molecule derived from the monoclonal antibody 7A (Mab-7A), which recognizes the CP of TuMV, was produced in Escherichia coli and the recombinant protein was used to raise rabbit antibodies. The immune serum reacted with Mab-7A but not with a monoclonal antibody of the same isotype, indicating that anti-idiotypic antibodies were produced. These anti-idiotypic antibodies recognized a 37 kDa protein from Lactuca sativa. Complex formation between the anti-idiotypic antibodies and the plant protein was inhibited by the CP of TuMV which indicates that the plant protein interacts with the viral protein. The 37 kDa protein was localized in chloroplasts and was detected in other plant species. The infection of susceptible plant hosts by RNA viruses depends on the association of host and viral factors for the replication of the viral genome, the expression of viral genes and the movement of virus throughout the plant [44] . Consequently, plant and viral proteinprotein interactions are presumed to play a pivotal role in disease development. One viral protein believed to interact with host components is the capsid protein (CP). The CP has a structural role but is also necessary for virion translocation in the plant and aphid transmissibility [2, 9, 25, 37, 41] . Furthermore, in the case of alfalfa mosaic virus (AlMV), the CP participates in viral RNA replication [30] . In addition, the CP has been reported to be the interface in the defense response in Nicotiana sylvestris carrying the N 0 resistance gene against tobacco mosaic virus (TMV) [6] and in Solanum tuberosum carrying the Rx resisty In memoriam. ance gene against potato virus X [16, 33] . The plant proteins involved in the interaction with the CP are thought to be early components of a signal transduction pathway similar to that of the TMV resistance N gene of Nicotiana tabacum [42] . However, the identity of the plant components interacting with CPs has not been established. Protein-protein interactions, such as receptor-ligand complexes, have been studied using the idiotype-anti-idiotype network approach [23] . This approach involves the preparation of an anti-ligand monoclonal antibody (Mab) which is subsequently used as immunogen to obtain anti-anti-ligand or antiidiotypic antibodies (anti-ids). If the anti-ligand antibody and the receptor protein interact with the same region of the ligand, a sub-population of the anti-ids could potentially mimic the ligand epitope and bind to the receptor protein. Anti-ids have been used to identify cell surface receptors for hormones and animal viruses [16, 31] and in vaccination [27] . Likewise, Akimitsu et al. [1] used anti-ids for the detection of the plant cell surface receptor for victorin. The anti-idiotypic approach was used in this study to identify plant proteins interacting with the CP of turnip mosaic potyvirus (TuMV). The CP of potyviruses is a three-domain protein with the N-and C-terminal ends exposed on the surface of the virion and the central domain forming the core subunit structure [39] . The highly conserved central region is needed for encapsidation of the viral RNA while the highly variable, surface-exposed, Nterminal and C-terminal regions are involved in hostvector-virus interactions and/or transport functions [2, 9, 25] . Monoclonal antibodies (Mab) have previously been raised against the 33 kDa CP of TuMV [21] . Here, an antibody derivative of Mab TuMV-7A was synthesized. This derivative, known as a single-chain Fv (sc-Fv) molecule [24] , consisted of the heavy-and light-chain variable domains of the parent immunoglobulin joined together by a flexible peptide linker and was expressed in Escherichia coli. A polyclonal serum was raised against the recombinant protein which also reacted with a 37 kDa protein found in chloroplasts of Lactuca sativa. The interaction between the anti-scFv-7A serum and the chloroplast protein was inhibited by the presence of TuMV CP. These characteristics suggest that the chloroplast protein may interact with the CP of TuMV. Bacterial strains and plasmids E. coli strains XL1-Blue (Stratagene) and BL21(DE3) (Novagen) were used as bacterial hosts. The expression vector pET22b (Novagen) was used for recombinant protein expression in E. coli. The scFv derivative of Mab-7A was prepared essentially as described [5] with modifications: first-strand cDNA was prepared from 10 ऌg of total RNA using murine leukemia virus reverse transcriptase (BRL) and the constant heavy-chain-1 primer MOCG12FOR (5 0 -CTCAATTTTCTTGTCCACCTTGGTGC-3 0 ) and constant ऊ chain primer CKFOR (5 0 -CTCATTCCTGT-TGAAGCTCTTGAC-3 0 ). Thermus aquaticus DNA polymerase (Bio/Can Scientific) was used for amplification and reaction conditions were as recommended by the manufacturer. Polymerase chain reaction (PCR) amplifications of the V L V H coding regions were carried out independently. The cDNAs were amplified for 30 cycles and each cycle consisted of a 1 min denaturation (94 ae C), 1 min annealing (60 ae C) and a 2 min extension (72 ae C). Primers VH1FOR-2 (5 0 -TG-AGGAGCAGGTGACCGTGGTCCCTTGGCCCC-3 0 ) and VH1BACK (5 0 -AGGTSMARCTGCAGSAGTC-WGG-3 0 : where S = C or G; M = A or G; W = A or T) were used to amplify the V H region. Primers VK2BACK (5 0 -GAC-ATTGAGCTCACCCAGTCTCCA-3 0 ) and VK4FOR (consisting of an equimolar mix of MJK1FONX, 5 0 -CCGTTTGATTTCCAGCTTGGTGCC-3 0 ; MJK2-FONX, 5 0 -CCGTTTTATTTCCAGCTTGGTCCC-3 0 ; MJK4FONX, 5 0 -CCGTTTTATTTCCAACTTTGTC-CC-3 0 ; MJK5FONX, 5 0 -CCGTTTCAGCTCCAGCT-TGGTCCC-3 0 ) were used to amplify the V K region. The linker DNA was similarly amplified from pscFvD1.3 using primers MO-LINK-BACK and MO-LINK-FOR (complementary to VH1FOR-2 and VK2BACK, respectively). Gel-purified V H and V K amplified fragments (100 ng each) were mixed with 20 ng of the linker DNA fragment encoding the peptide (Gly4Ser)3 in a 50 ऌl reaction mixture without primers and amplified for seven cycles (94 ae C for 1 min, 72 ae C for 2.5 min) with Vent DNA polymerase (New England Biolabs) to randomly join the fragments, then amplified for 23 cycles (94 ae C for 1 min, 60 ae C for 2 min, and 72 ae C for 2 min) using 25 pmol each of VH1BACK and VK4FOR, which contained NcoI and NotI restriction sites respectively. The NcoI/NotI scFv gene fragment was subcloned into identically restricted pET22b to create pETscFv-7A. E. coli BL21(DE3), harboring pETscFv-7A, was grown in Luria Broth with ampicillin (100 ऌg/ml) at 37 ae C. At A 600 = 0:5, 0.4 mM isopropyl-ae-D-thiogalactopyranoside (IPTG) was added and the cells were incubated for an additional 2 h at 22 ae C after which they were harvested by centrifugation at 4000 ࣾ g. The cells were resuspended in 50 mM Tris-HCl pH 8.0, 1.0 mM EDTA, 100 mM NaCl and lysed with 2 mg/ml lysozyme and several freeze-thawing cycles. Inclusion bodies were purified according to Sambrook et al. [38] . Recombinant scFv-7A was purified by SDS-PAGE in a 12% acrylamide gel. After staining the gel with Coomassie brilliant blue R-250 in water, the band was excised and passed through a 21G hypodermic needle. Rabbits were immunized as described in Robbins et al. [35] . Western blot analyses were performed as described [38] using polyvinylidene difluoride (PVDF) or nitrocellulose membranes. ELISAs were performed at room temperature except where noted. Plates were washed six times with phosphate buffered saline (4.3 mM Na 2 HPO 4 , 1.4 mM KH 2 PO 4 , 137 mM NaCl, 2.7 mM KCl, pH 7.0) containing 0.1% Tween-20 (PBS-T) between each incubation. Proteins suspended in coating/extraction buffer (15 mM Na 2 CO 3 , 35 mM NaHCO 3 pH 9.6, 2% polyvinylpyrrolidone (PVP)) were bound to microtiter plates (Falcon) for 4 h and wells were then blocked in 3% BSA-PBS-T for 4 h. Plates were subsequently incubated 1 h with the rabbit serum and developed using anti-rabbit IgG conjugated with alkaline phosphatase or horse-radish peroxidase. For competitive ELISA, two-fold serial dilutions of E. coli lysate in conjugate buffer (PBS-T, 2% PVP, 0.2% BSA) were added and incubated overnight at 4 ae C. Pellet (P-100) and soluble (S-100) fractions were obtained by grinding 7 g of fresh lettuce leaves in 35 ml of ice-cold 50 mM Tris-HCl pH 8.0, 5.0 mM EDTA, 0.1% 2-mercaptoethanol for one minute in a blender. The extract was centrifuged at 20 000 ࣾ g for 15 min at 4 ae C, and the resulting supernatant was further centrifuged at 100 000 ࣾ g for 45 min at 4 ae C. The pellet (P-100) was resuspended in 1.0 ml 50 mM Tris-HCl pH 8.0, 5 mM EDTA. Chloroplasts were isolated from 50 g of young lettuce leaves according to the method of Palmer et al. [32] . The chloroplast suspension was adjusted to 60 mM Na 2 CO 3 pH 10.0, 60 mM DTT and 2% SDS and insoluble material was removed by centrifugation at 14 400ࣾg for 30 min at 4 ae C. Mitochondria were purified using the extraction protocol outlined by Hanson et al. [19] : two mitochondrial fractions were collected, the 1.6 M/2.0 M sucrose interface (M1) and the 1.2 M/1.6 M interface (M2). In this study, a scFv molecule derived from the Mab-7A cell line [21] was engineered in order to obtain antibodies directed against the paratope of the monoclonal antibody. This strategy was chosen to minimize production of anti-isotypic and anti-allotypic antibod-ies. The TuMV 7A monoclonal cell line was produced using purified TuMV nucleoprotein; for TuMV, the virion is composed of ca. 2000 copies of the CP, surrounding the RNA to which is attached one copy of the virus-encoded VPg protein [21] . The cDNA encoding the scFv molecule was obtained via RT-PCR using RNA extracted from the mouse hybridoma cell line; the amplified fragments coded for the variable domain of both the light and heavy chains. scFv molecules built using the RT-PCR approach used here have been shown to bind their respective antigen similar to that of their parent immunoglobulin molecules [22] . The assembled gene was sequenced and characterized as an IgG 2a-subclass immunoglobulin (data not shown). The Mab-7A had previously been identified as an IgG 2a by immunoanalysis using isotypic-specific antibodies (data not shown). The chimeric gene was cloned into expression vector pET22b for high-level expression in E. coli. A fusion protein of the predicted size (31 kDa) was found to accumulate in inclusion bodies as indicated by SDS-PAGE ( Figure 1A ). An anti-mouse Fab serum (ICN) reacted with the recombinant protein, confirming that an immunoglobulinderived molecule had been synthesized ( Figure 1B) . The recombinant protein was purified and injected into rabbits to raise antibodies. As expected, the resulting serum recognized the scFv-7A protein ( Figure 1C ). Because the scFv-7A molecule is made up exclusively of the variable domains of the parent immunoglobulin, it was expected that anti-ids were predominantly produced. An ELISA was performed to test for their presence in the serum. The wells were coated either with Mab-7A or the control Mab-7-10A of unrelated specificity but of the same isotype (IgG 2a) [7] and incubated with various dilutions of the anti-scFv-7A serum and a non-immune serum. The immune serum reacted preferentially with Mab-7A ( Figure 2 , filled squares), which indicates that scFv-7A was indeed a derivative of Mab-7A and that anti-ids were mainly produced since the rabbit serum did not react with the isotypic control antibody. Since anti-ids were produced against Mab-7A, it was hypothesized that a portion of them would mimic the CP epitope that was recognized by Mab-7A. If this CP epitope interacts with a plant protein, the anti-ids representing the internal image of the CP should also interact with the plant protein. This was verified by Since the plant protein was recognized by an anti-anti-CP serum, the 37 kDa plant protein was thus speculated to interact with the TuMV CP. If this is the case, the viral protein (CP) must be able to compete with the anti-scFv-7A antibodies for the binding of the 37 kDa plant protein. The competition was first per- formed by immunoblotting: total plant proteins were separated by SDS-PAGE, transferred onto PVDF membrane and incubated with the anti-scFv-7A serum in the absence or presence of CP. As control, recombinant scFv-7A was also transferred. Although the signal for the scFv-7A-anti-scFv-7A complex decreased only slightly when CP was present during the incubation, the signal for the 37 kDa protein was only 40% of the original signal (Figure 4) . To confirm the displacement seen by immunoblotting, competition experiments in ELISA plates were performed in which wells were coated with plant proteins and incubated with the anti-scFv-7A serum and with increasing quantities of E. coli lysate containing CP [35] . The CP lysate competed with anti-scFv-7A antibodies for binding the 37 kDa protein, showing an average 35% decrease in signal strength with the addition of lysate containing 1.5 ng of CP, compared to that generated with the negative control lysate containing an equal amount of protein . The displacement was also observed using CP purified from dissociated virus particles (results not shown). The lack of complete displacement could be explained by the higher affinity an immunoglobulin has for its ligand (anti-7a/37 kDa protein) than that found in a protein-protein (CP/37 kDa protein) complex, the complementary determining regions of the immunoglobulin playing an important role in the increased affinity [14] . These experiments suggest that the CP of TuMV is able to interact with the plant protein recognized by anti-ids directed against Mab-7a. The cellular localization of the plant protein was determined. Leaves from L. sativa were homogenized and the cellular content was fractionated by centrifugation into soluble (S-100), membrane (P-100), mitochondria and chloroplast components. The proteins from each fraction were separated by SDS-PAGE and analyzed by immunoblotting. The anti-scFv-7A serum recognized the 37 kDa protein in the chloroplast fraction ( Figure 5, lane 3) . The presence of this 37 kDa protein in chloroplasts of Brassica perviridis, Nicotiana tabacum and Lycopersicon esculentum was tested. Chloroplast proteins were separated by SDS-PAGE and analyzed by western blot using the anti-scFv-7A serum ( Figure 6 ). In all plants tested, the serum reacted with a protein of 37 kDa, with an additional 40 kDa species for L. esculentum. This indicates that the protein bearing the epitope recognized by the anti-ids is highly conserved. This study provides experimental data for the presence of a chloroplast protein that interacts with the CP of TuMV. This plant protein was identified by exploiting the molecular mimicry potential of anti-ids. The concept of anti-ids containing the 'internal image' of the ligand afforded a convenient alternative to its purification. Evidence for such a protein was first the observation that the anti-ids against Mab-7A recognized a 37 kDa plant protein. However, the antibodies involved could have detected a framework feature of Mab-7A found also on the plant protein. The operational criterion supporting the identification of an interacting protein was thus the inhibition of the anti-ids-plant protein complex formation by CP, the antigen used for the production of Mab-7A. This criterion of competitive inhibition between anti-ids and antigen for binding the cellular protein has been used in other studies as evidence that the identified cellular protein is a receptor for the antigen [18, 40, 43] . The 37 kDa protein was associated with chloroplasts of L. sativa and polypeptides of similar molecular mass were detected in B. perviridis, N. tabacum and L. esculentum, with an additional form of higher molecular weight also observed in the latter. The antiids containing the CP internal image should bind only a small region of the 37 kDa protein. It is noteworthy that this epitope was detected on proteins of similar molecular weight in different plant species. The 37 kDa protein may thus play a role in the viral multiplication cycle. Indeed, plant viruses can replicate in most cell types and the presence of the epitope in several plant species suggests a link between the 37 kDa protein and viral infection. Viral infections have long been associated with chloroplasts: symptoms such as mosaics, green islands, yellows or chlorosis are all largely attributable to viral interference with normal chloroplast functions [28] . Some viruses induce structural changes in that organelle; turnip yellow mosaic virus (TYMV) as well as poplar mosaic virus cause their aggregation [4, 11] and tombusviruses cause the development of multivesicular bodies within the chloroplasts [36] . TuMV caused the aggregation of chloroplasts in Chenopodium quinoa and electron microscopic examination revealed a layer of viral particles between the clustered chloroplasts [26] . Replication complexes in chloroplasts have also been reported for AlMV [8] and TYMV [13] . Tobacco etch potyviral RNA as well as potato virus Y CP, helper component protein and RNA have been found within infected leaf chloroplasts [12, 17] . However, there is still little evidence to support the idea that the chloroplast is the principal site of viral replication. Furthermore, there are reports associating CP with chloroplasts. The CP of TMV was shown to accumulate in chloroplasts of infected tobacco plants [34] . Hodgson et al. [20] showed that the selective inhibition of photosystem II (PS II) in spinach by TMV resulted from the association of the CP with the PS II complex. Recently, the YSI/1 mutant of the common strain (U1) of TMV was found to induce a severe yellow mosaic in N. tabacum instead of the light green/dark green mosaic of the parental strain. The CP accumulated several-fold in chloroplasts and the chlorosis was linked to a single amino acid substitution near the Nterminal end of the CP [3] , suggesting that this region of the viral protein was responsible for more severe symptoms. However, the relationship between CP and chloroplasts is not clear. Dramatic effects on chloroplast photosynthetic capacity in vitro after viral infection have been reported, but these can be alleviated with adequate nitrogen supplies to the plant; circumstances that also promote viral replication [45] . Although the purpose of the interaction between the CP of TuMV and the chloroplast protein is not known, it is possible that it is a step towards the sequestration of chloroplast components necessary to fuel viral replication [10, 29, 46] . 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R. McDermott for providing us with cell line TuMV 7A. We thank P. Talbot for helpful discussions and critical reading of the manuscript. This research was supported by grants from the Natural Sciences and Engineering Research Council of Canada to J.F.L. and M.G.F.