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Journal of General Virology (1994), 75, 3199 3202. 3199 Printed in Great Britain Mapping of the RNA-binding domain of the alfalfa mosaic virus movement protein Fabrice Schoumacher,t Christine Giovane, Michel Maira,:~ Anne Poirson, Thrr~se Godefroy-Colburn* and Anne Berna Institut de Biologie Moldculaire des Plantes, Universitd Louis Pasteur, 12 rue du G#n#ral Zimmer, 67084 Strasbourg Cedex, France In-frame contiguous deletions were created in the movement protein gene of alfalfa mosaic virus by sitedirected mutagenesis. The mutated movement proteins were expressed in Escherichia coli, extracted and then purified by denaturing gel electrophoresis and then renatured. Their binding ability with RNA was assayed by electrophoretic retardation and u.v.-crosslinking. Results indicated that a domain included within amino acids 36 to 81 was necessary for RNA binding. The alfalfa mosaic virus (AMV) movement protein encoded by RNA3 (P3,300 amino acids) has been shown to bind single-stranded nucleic acids in vitro (Schoumacher et al., 1992a and b), as do several other viral movement proteins. These are the 30K protein of tobacco mosaic virus (TMV) (Citovsky et al., 1990), the gene I product of cauliflower mosaic virus (CaMV; Citovsky et al., 1991), the 35K protein of red clover necrotic mosaic virus (RCNMV) (Osman et al., 1992), the ORF2 (open reading frame 2) product of foxtail mosaic potexvirus (Rouleau et al., 1993) and the putative 17K movement protein of potato leafroll luteovirus (Tacke et al., 1993). The nucleic acid binding domain(s) have been localized in the movement proteins of TMV (Citovsky et al., 1990, 1992)and RCNMV (Osman et al., 1992, 1993; Giesman-Cookmeyer & Lommel, 1993). Deletion mapping identified two binding domains in the TMV movement protein; one of them was active independently of the rest of the molecule and is qualified as 'independent' but the other was not (Citovsky et al., 1992). By the same approach, an independent RNA binding domain was detected in the RCNMV movement protein (Osman et al., 1993). In addition, alaninescanning mutations affecting several other regions of the molecule decreased the affinity of the movement protein for RNA and the cooperativity of binding; mutations in one of these regions also affected the biological function of the protein (Giesman-Cookmeyer & Lommel, 1993). The nucleic acid binding activities of the TMV and CaMV movement proteins are presumed to be relevant to the movement function because, upon binding, the nucleic acid molecule becomes elongated and 'shaped into a form suitable for transport through plasmodesmatal channels' (Citovsky et aL, 1992). However, binding of the RCNMV movement protein was shown recently not to affect the overall length of RNA molecules (Fujiwara et al., 1993). There is no evidence that the mechanisms of transport are identical for all plant viruses; indeed some evidence to the contrary is emerging (Atabekov & Taliansky, 1990; Harrison et al., 1990). In particular, the movement mechanisms of AMV and TMV may differ significantly because AMV, unlike TMV, seems to require its coat protein for cell-to-cell movement (van der Kuyl et al., 1991) and also the AMV movement protein does not have the same nucleic acid binding characteristics as the TMV 30K protein possesses (Schoumacher et al., 1992a and b). We undertook to locate the RNA binding domain(s) of the P3 protein by deletion mapping in order to compare its (their) structure with those of the TMV and RCNMV movement proteins. For this purpose, we created a set of eight contiguous in-frame deletions in the P3 gene ORF and expressed the corresponding proteins in Escherichia coli. The proteins were purified and their RNA binding activity was tested. The set of deletions of the P 3 0 R F shown in Fig. 1 was obtained by site-directed mutagenesis (creation of unique HindIII sites) and segment reassortment. Mutagenesis was carried out on the plasmid designated pSel 1 .P3N (Schoumacher et al., 1992b). The latter had been constructed by placing the complete P 3 0 R F between the SalI and B a m H I sites of a pAlter-1 phagemid t Present address: Friedrich Miescher Institute, P.O. Box 2543, CH-4002 Basel, Switzerland. Present address: Laboratoire de Grnrtique Mol~culaire des Eucaryotes, ~1, rue Humann, 67085 Strasbourg Cedex, France. 0001-2569 © 1994 SGM Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sat, 17 Jun 2017 13:24:01 3200 Short communication P3 wt 0 50 I I I ~ i KM 100 150 200 250 1 I I I I KI I I KL I KL 300 (a) C I 1 2 3 4 5 6 7 8 9 KL KI MGKL P3A[1 20] ~v P3A[21 34] ~KL P3A[36-811 ~ P3,5182-112] I rI._L. KI tL I lq P3A[113-156] I P3,5[157-213] [ [L ] Iq [L I K~ P3A[215-2431 [ P3,51244-300] I Fig. 1. Mutagenesis of the P 3 0 R F . The wild-type P 3 0 R F is designated P3 wt and the deleted ORFs are designated P3A[x-y], where x and y indicate respectively the first and the last amino acid of the deleted portion, as numbered in the wild-type sequence (Ravelonandro et al., 1984). The positions of the HindIII restriction sites that were either originally present ( ~ ) or created by mutagenesis are represented by vertical bars in the upper diagram (P3 wt), with the original dipeptides indicated underneath and amino acid numbering given above. In the deleted ORFs, the remaining portions of the wild-type sequence are represented by rectangles above which the inserted amino acids are indicated. The amino acids that border the deletions are shown within the rectangles, with changes indicated in bold type. (a) I 2 (b) 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 Fig. 2. Analysis of the P3-related proteins purified from recombinant E. coli. Proteins prepared from recombinant E. coli expressing the wild- type or the mutant P3 genes (about 150 ng) were subjected to SDS PAGE in a 10% gel (Laemmli, 1970). The gel was blotted to nitrocellulose by contact, then stained with Coomassie Brilliant Blue (a), and the nitrocellulose membrane was processed for detection of P3related proteins with an anti-P3 serum as described by Schoumacher et al. (1992a) (b). The E. coli-expressed proteins were: full-length P3 (lane 1), P3A[1-20] (lane 2), P3A[21-34] (lane 3), P3A[36-81] (lane 4), P3A[82 112] (lane 5), P3A[113-156] (lane 6), P3A[157-213] (lane 7), P3A[215-243] (lane 8) and P3A[244~300] (lane 9). The arrowhead points to the position of the full-length P3 protein. (formerly pSelect; Promega Biotec) from which the HindIII site had been eliminated, and by creating an NcoI restriction site at the start codon in order to (b) Fig. 3. U.v.-crosslinking assay. The radioactive RNA (10 ng in 20 lal) was incubated with 15 to 30 ng of the different proteins (lanes 1 to 9, same as in Fig. 2) or without protein (lane C). The open microtubes were then subjected to u.v.-light (3.6J) in the 'Stratalinker 1800' apparatus (Stratagene). Unprotected RNA was digested for 30 min at 37 °C by 500 ng of ribonuclease A and the protein carrying residual RNA was analysed as in Fig. 2, except that the gel was dried and autoradiographed without staining. (a) shows the autoradiogram and (b) the immunoblot. facilitate subsequent insertion of the ORF in the expression vector pET-3d (Studier et al., 1990). After eliminating conservatively the natural HindIII restriction site of the P3 gene (encoding a KL dipeptide at positions 112 to 113 of the amino acid sequence), several other hexanucteotides, encoding identical or similar dipeptides (KL, KI or KM at nucleotide positions 35 to 36, 81 to 82, 156 to 157, 213 to 214 and 242 to 243) were substituted to create unique HindIII sites. In addition, a mutant (designated 'A') was created by inserting a HindIII site immediately upstream of the twenty-first codon. The original pSel 1 .P3N plasmid and the mutagenized plasmids derived from it were digested by HindlII (in the P3 gene ORF) and by E c o R V (at a unique site located about 580 nucleotides upstream of the NcoI site). The internal deletions were created by reassembling the small E c o R V HindIII fragment and the large H i n d l I I - E c o R V fragment from two mutants carrying the HindIII site at successive locations. To obtain the N-terminally deleted gene, a linker carrying an NcoI site with the ATG Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sat, 17 Jun 2017 13:24:01 3201 Short communication in phase with the rest of the sequence was inserted upstream of the HindIII site of mutant A. To obtain the C-terminally-deleted gene, a TGA triplet followed by a B a m H I site was inserted after the 243rd codon. The sequence of 150 to 200 nucleotides bordering the mutations was confirmed with the dideoxynucleotide 'T7' chain termination system (Pharmacia), primed by the next mutagenic oligonucleotide downstream of the deletion created, or by a universal primer hybridizing to the phage T7 promoter adjacent to the polylinker of pAlter-1. Cloning techniques used in these and subsequent operations were as described by Sambrook et al. (1989). The coding sequences to be expressed, obtained as N c o I - B a m H I fragments from the pSell .P3N-derived plasmids, were inserted between the NcoI and the B a m H I sites of the expression vector pET3d. The recombinant plasmids were cloned into E. coli strain BL21(DE3) and the proteins were extracted from inclusion bodies of the IPTG-induced bacteria with a urea-containing buffer (Schoumacher et al., 1992b). They were purified by SDS-PAGE in a 10 % gel (Laemmli, 1970), eluted from the gel according to Hager & Burgess (1980), renatured by dialysis against 50 mu-Tris-HC1, pH 7.5, containing 10 mM-2-mercaptoethanol and 1% Tween-20 and stored at - 2 0 °C after addition of glycerol (50% v/v final concentration). The purified mutant proteins were analysed in parallel with the full-length protein (Schoumacher et al., 1992b) by denaturing electrophoresis (Fig. 2a) and immunoblotting with an anti-P3 serum (Fig. 2b). A~ 0 scanning of the gel shown in Fig. 2(a) indicated that all these proteins were at least 90 % pure. All the mutants reacted with the anti-P3 serum in the presence of 1% Tween-20, although the efficiency of detection was variable. The renatured deleted proteins were tested for their ability to bind radioactively labelled RNA, using u.v.crosslinking and electrophoretic retardation assays adapted from Citovsky et al. (1990). The incubations were done for 1 h at 27 °C in 40 mM-HEPES-NaOH, pH 7.0 (containing 12.5 mM-Tris-HC1, 2'5 mM-2-mercaptoethanol, 0.25 % Tween-20 and 25 % glycerol originating from the protein sample), in polypropylene microtubes which had been treated with dimethyldichlorosilane (2% solution in trichlorethane, obtained from LKB) and rinsed with deionized water. The RNA (141 nucleotides, collinear to the 5' end of AMV RNA3) was synthesized by the phage T7 RNA polymerase from an HaeIII fragment of the transcription plasmid 3BS(12.0) (Dore et al., 1990), in the presence of [~-a2P]UTP (0"05 Ci/pmol). The u.v.-crosslinking assay showed that all the deleted proteins (Fig. 3 lanes 2 to 9), except P3A[36-81] (Fig. 3 lane 4), could bind RNA as did full-length P3 (Fig. 3 lane C 1 2 3 4 5 6 7 8 9 Fig. 4. Electrophoretic retardation of R N A by the P3-related proteins. The radioactive R N A (0.5 ng in 20 gl) was incubated without protein (lane C) or with 25 to 30 ng of the different proteins (lanes 1 to 9, as in Fig. 2). The samples were electrophoresed for 1 h at 13 V / c m in a 6 % polyacrylamide gel (acrylamide:bisacrylamide 36:1) made in 20 mMTris acetic acid, 0.5 mN-EDTA, pH 7.5. ~fhe gel was dried and autoradiographed. The positions of free and completely retarded R N A are indicated respectively by the closed triangle and the arrowhead. 1). This was confirmed by the electrophoretic retardation assay (Fig. 4). Indeed all the mutant proteins except P3A[36-81] (Fig. 4 lane 4) decreased the mobility of RNA to some extent. Those which did bind RNA could be placed in two groups according to the degree of retardation observed. The first group comprising P3A[1-20] (Fig. 4 lane 2), P3A[21-34] (lane 3), P3A[l13-156] (lane 6), P3A[215-243] (lane 8) and P3A[244-300] (lane 9) seemed to bind RNA as cooperatively as did full-length P3 (lane 1) because the only detectable species were the free and the fully retarded RNA. However, complete retardation of the bound RNA may have been due in some cases to aggregation of the protein. Aggregation, or incorrect conformation of the protein, could also cause the low efficiency of binding observed in lane 9 of Fig. 4. Indeed, P3A[244-300] had the characteristics of thermodynamically unstable proteins (Schein, 1990): poor solubility (of the order of 5 to 10 ~tg/ml after centrifugation for 20 min at 15000 g) and tendency to adsorb to polypropylene in spite of the presence of detergent. The lack of RNA binding activity of P3A[36-81] could also have been caused by incorrect renaturation. This seems unlikely however, because the latter protein did not aggregate or adsorb to plastic. An additional argument against improper renaturation is that this mutant had the same antibody reactivity as fulllength P3 (Fig. 2). The second group of mutants, P3A[82-112] (Fig. 4, lane 5) and P3A[157-213] (Fig. 4 lane 7) bound the RNA with sufficient affinity to decrease its mobility but a significant proportion of the complexes (66% in the case of P3A[82-112] and 34% in the case of P3A[157 213], evaluated by A600 measurement) were incompletely retarded, indicating a low degree of cooperativity or a low binding constant. A similar Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sat, 17 Jun 2017 13:24:01 3202 Short communication behaviour has been observed with some alanine-scanning mutants of the RCNMV movement protein (GiesmanCookmeyer & Lommel, 1993). In summary the region spanning amino acids 36 to 81 of P3 appears necessary for RNA binding. For reasons explained above, we think it is unlikely that this region enables binding by an indirect effect on the conformation of the polypeptides as does the 65 to 86 amino acid region of TMV 30K (Citovsky et al., 1992). Thus the amino acids 36 to 81 region of P3 should interact directly with the RNA. Indeed, it includes the major portion of a positively charged domain: (69) K+E K+K+SILNR +MLPK+IGQR+MYVH+H+H + (90). Moreover, amino acids 69 to 72 have a higher surface probability than any other region of P3 according to Emini et al. (1985). The positive charge and high probability of surface exposure are shared by the independent RNA binding domains of the TMV and RCNMV movement proteins (Citovsky et al., 1992; Osman et al., 1993), but this is the only similarity. Other experiments now in progress will determine the relevance of the RNA binding domain of P3 to its biological activity. We thank Drs W. F. Studier (Brookhaven National Laboratory, Upton N.Y., U.S.A.) for the pET vector and E. coli strain BL21(DE3), L. Pinck and J.-M. Dore (IBMP, Strasbourg, France) for plasmid 3BS(12.0) and C. Stussi-Garaud (IBMP) for critical reading of the manuscript. This work was supported by the Commission of the European Communities in the context of a BRIDGE contract no. BIOT-CT90-0156 and F.S. was supported by a fellowship from the Minist~re de la Recherche et de la Technologic. References ATABEKOV, J. G. & TALIANSKY, M. E. (1990). Expression of a plant virus-coded transport function by different viral genomes. Advances in Virus Research 38, 201-248. CITOVSKY, V., KNORR, D., SCHUSTER, G. & ZAMBRYSKI,P. (1990). The P30 movement protein of tobacco mosaic virus is a single-strand nucleic acid binding protein. Cell 60, 637-647. CITOVSKY, V., KNORR, D. & ZAMBRYSKI,P. (1991). Gene I, a potential cell-to-cell movement locus of cauliflower mosaic virus, encodes an RNA-binding protein. Proceedings of the National Academy of Sciences, U.S.A. 88, 2476-2480. CITOVSKY, V., WONG, M. L., SHAW, A., PRASAD,B. V. V. & ZAMBRYSKI, P. (1992). Visualization and characterization of tobacco mosaic virus movement protein binding to single-stranded nucleic acids. Plant Cell 4, 397-411. DORE, J.-M., ERNY, C. & PINCK, L. (1990). Biologically active transcripts of alfalfa mosaic virus RNA3. FEBS Letters 264, 183-186. EMINI, E.A., HUGHES, J.V., PERMOW, D.C. & BO~ER, J. (1985). Induction of hepatitis A virus-neutralizing antibody by a virusspecific synthetic peptide. Journal of Virology 55, 836-839. FUJIWARA,T., GIESMAN-COOKMEYER,D., DING, B., LOMMEL, S. A. & LUCAS, W.J. (1993). Cell-to-cell trafficking of macromolecules through plasmodesmata potentiated by the red clover necrotic mosaic virus movement protein. Plant Cell 5, 1783-1794. GIESMAN-COOKMEYER, D. & LOMMEL, S. A. (1993). Alanine-scanning mutagenesis of a plant virus movement proteins identifies three functional domains. Plant Cell 5, 973-982. I-IAGER, D.A. & BURGESS, R.R. (1980). Elution of proteins from sodium dodecyl sulfate-polyacrylamide gels, removal of sodium dodecyl sulfate, and renaturation of enzymatic activity: results with the sigma subunit of Escherichia coli RNA polymerase, wheat germ DNA topoisomerase, and other enzymes. Analytical Biochemistry 109, 76-86. HARRISON, B. D., BARKER, H. & DERRICK, P. M. (1990). Intercellular spread of potato leafroll luteovirus: effects of co-infection and plant resistance. In Recognition and response in plant-virus interactions NATO ASI Series Vol. H41, pp. 405-414. Edited by R. S. S. Fraser. Berlin: Springer-Verlag. LAEMMLI, U.K. (I970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, London 227, 680-685. OSMAN, T. A. M., HAYES, R.J. & BUCK, K. W. (1992). Cooperative binding of the red clover necrotic mosaic virus movement protein to single-stranded nucleic acids. Journal of General Virology 73, 223-227. OSMAN, T. A. M., TH6MMES,P. & BUCK, K. W. (1993). Localization of a single-stranded RNA-binding domain in the movement protein of red clover necrotic mosaic dianthovirus. Journal of General Virology 74, 2453-2457. RAVELONANDRO,M., PINCK, M. & PINCK, L. (1984). Complete sequence of RNA3 from alfalfa mosaic virus, strain S. Biochimie 66, 395-402. ROULEAU, M., BANCROFT, J. B. & MACKIE, G. A. (1993). Purification and characterization of the potexviral movement protein ORF2. Abstracts of the IXth International Congress of Virology (Glasgow, U K . , 8 13 August) p. 99. SAMBROOK, J., FRITSCH, E.F. & MANIATIS, T. (1989). Molecular Cloning. A Laboratory Manual, 2nd edn. New York: Cold Spring Harbor Laboratory. SCHEIN, C. H. (1990). Solubility as a function of protein structure and solvent components. Bio/Technology 8, 308-316. SCHOUMACHER, F., ERNY, C., BERNA, A., GODEFROY-COLBURN, T. & STUSm-GARAUO, C. (1992a). Nucleic-acid-binding properties of the alfalfa mosaic virus movement protein produced in yeast. Virology 188, 896-899. SCHOUMACHER, F., GAGEY, M.-J., MAIRA, M., STUSSI-GARAUD,C. & GODEFROY-COLBURN, T. (1992b). Binding of RNA by the alfalfa mosaic virus movement protein is biphasic. FEBS Letters 308, 231-234. STUDIER, F. W., ROSENBERG,m. H., DUNN, J. J. & DUBENDORFF,J. W. (1990). Use of T7 RNA polymerase to direct expression of cloned genes. Methods in Enzymology 185, 60-89. TACKE, E., PROFER,D., SCHMITZ,J. & ROHDE, W. (1993). The potato leafroll luteovirus (PLRV) 17K protein functions as a putative transport protein and mediates resistance in transgenic tobacco. Abstracts of the IXth International Congress of Virology (Glasgow, U.K., 8-13 August) p. 100. VAN DER KUYL, A. C., NEELEMAN, L. & BOL, J. F. (1991). Complementation and recombination between alfalfa mosaic virus RNA3 mutants in tobacco plants. Virology 183, 731-738. (Received 19 October 1993; Accepted 28 June 1994) Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sat, 17 Jun 2017 13:24:01