Nucleic acid-binding properties and subcellular ... - CiteSeerX

Journal of General Virology (1998), 79, 1273–1280. Printed in Great Britain ...................................................................................................................................................................................................................................................................................

Nucleic acid-binding properties and subcellular localization of the 3a protein of brome mosaic bromovirus Miki Fujita, Kazuyuki Mise, Yasuko Kajiura, Koji Dohi and Iwao Furusawa Laboratory of Plant Pathology, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan

Brome mosaic bromovirus (BMV) 3a protein is required for cell-to-cell movement of the virus in host plants. The BMV 3a protein (B3a) was produced in Escherichia coli using an expression vector. Gel retardation analysis and UV cross-linking experiments demonstrated that B3a bound singlestranded RNA cooperatively without sequence specificity. Binding competition analysis showed that B3a bound to single-stranded nucleic acids more strongly than to double-stranded nucleic acids. De-

Introduction The cell-to-cell movement of plant viruses is assumed to occur through plasmodesmata, which are intercellular connections between plant cells that permit movement of macromolecules (reviewed by Lucas, 1995 ; Carrington et al., 1996). Virus-encoded movement protein (MP) has been shown to be involved in this process. Plant viruses can be categorized by their mechanism of cell-to-cell movement. Several groups of viruses, including tobacco mosaic virus (TMV), do not require coat protein (CP) ; its encoded MP induces an increase in the size exclusion limit of the plasmodesmata permeable space (Ding et al., 1995 ; Lucas, 1995 ; Wolf et al., 1989) and possesses nonspecific single-stranded (ss) RNA-binding activity to shape viral nucleic acids into a transferable form (Citovsky et al., 1990, 1992). Another group, including cowpea mosaic virus (CPMV) and cauliflower mosaic caulimovirus (CaMV), does require CP and its encoded MP participates in the formation of tubular structures which extend from plasmodesmata and in which virus-like particles are detected (van Lent et al., 1991). The fact that CaMV MP also has nucleic acid-binding properties suggests that two mechanisms of movement may exist concomitantly in a single virus infection (Citovsky et al., 1991). Besides these viruses, tobacco etch potyvirus (Dolja et al., 1994) and potato virus X (Oparka et al., 1996) require CP but tubule-mediated virion transport has not been reported. Author for correspondence : Miki Fujita. Fax ­81 75 753 6131. e-mail miki!kais.kyoto-u.ac.jp

0001-5333 # 1998 SGM

letion mutagenesis located a nucleic acid-binding domain to amino acids 189–242. Western blot analysis of fractionated proteins of BMV-infected barley using monoclonal antibodies against B3a indicated that B3a may interact with membrane materials and form complexes in the cytoplasm. Immunogold labelling of thin sections of infected barley tissues revealed that B3a was associated with plasmodesmata and cytoplasmic inclusions.

Brome mosaic virus (BMV), a member of the Bromoviridae, is a tripartite RNA plant virus. It has three separately encapsidated positive-strand genomic RNAs. RNA1 and RNA2 encode nonstructural proteins 1a and 2a, respectively, both of which are required for viral RNA replication (Kroner et al., 1989, 1990). RNA3 encodes the nonstructural 3a protein (B3a) and CP, which are dispensable for RNA replication but required for the spread of infection in plants. Fluorescent in situ hybridization analysis reveals that the 3a gene is involved in virus cell-to-cell movement from initially infected cells (Schmitz & Rao, 1996). B3a dictates host specificity (Mise et al., 1993) and modulates symptom expression in susceptible hosts (Fujita et al., 1996 ; Rao & Grantham, 1995). Recently, BMV has been shown to require CP along with B3a for virus cell-to-cell movement (Schmitz & Rao, 1996) and virus-like particles have been observed in tubules extending from protoplasts transfected with BMV (Kasteel et al., 1997). Speculation concerning the function of B3a has been based on sequence similarity with other viral proteins described above, but has not been studied directly in vitro. To gain information on the MP of BMV, we have examined the nucleic acid-binding properties of B3a produced in Escherichia coli and identified the RNA-binding domain using deletion mutants. Furthermore, we have observed the localization of B3a in infected tissues immunologically by fractionation and electron microscopy, and discuss here the mechanism of BMV cell-tocell movement.

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Methods + Expression and purification of B3a and its deletion mutants. Plasmid pBC3KM11 (Mise et al., 1993) was digested with BamHI and BglII, and the resulting 0±9 kb fragment, which contains the entire region of the 3a gene, was cloned into the BamHI site of the T7 polymerase vector pET3b (Novagen) to generate pEB1. An NdeI site was introduced at the translation initiation codon of the 3a gene in pBC3KM11 by PCR using the sense primer BC3NdeI (5« GGATCCATATGTCTAAGA 3«, where the NdeI site is underlined) and the antisense primer KM11-4 (5« GCGGGTTCTGATTCTTC 3«). The PCRamplified fragment was digested with NdeI and ClaI and then replaced with the corresponding fragment of pEB1 to generate pB3a. The NdeI restriction site of pET3b induces the translation initiation signal so that plasmid pB3a expresses the 3a protein without alterations to the coding sequence. To obtain a set of deletions of the 3a ORF (Fig. 1), we used a series

Fig. 1. Schematic illustration of the deletion mutants constructed within B3a and their ability to bind ss RNA. The position of PstI restriction sites introduced by mutagenesis (∆) are represented in the upper diagram (WT) with amino acid numbering given above. The deleted region and the remaining portions of the wild-type sequence are represented by horizontal lines and shadowed rectangles, respectively.

Fig. 3. Electrophoretic gel retardation assay for RNA binding by B3a. The indicated amount of B3a was incubated in 10 µl of buffer B with 1 ng of 32 P-labelled transcripts derived from BMV RNA3 cDNA clone (A) and bacterial expression vector (B), and the mixture was electrophoresed in a 4 % nondenaturing polyacrylamide gel. (C) Competition analysis with B3a. B3a (400 ng in 10 µl) was incubated with 1 ng of labelled ss RNA probe either in the absence (lane 2) or presence (lanes 3–10) of nonlabelled competitors : ss RNA (BMV RNA, lanes 3 and 4), ds RNA (RDV RNA, lanes 5 and 6), ss DNA (heat-denatured plasmid DNA fragment, lanes 7 and 8), ds DNA (plasmid DNA fragment, lanes 9 and 10). Two levels of competitor were used : 250 ng (lanes 3, 5, 7 and 9) or 1 µg (lanes 4, 6, 8 and 10). Lane 1, probe only. Fig. 2. Analysis of the expression and purification of wild-type and deletion mutants of B3a. Proteins were analysed on a 15 % polyacrylamide–SDS gel and visualized by Coomassie blue staining. The positions of the molecular mass markers (kDa) are indicated.

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Properties of BMV 3a protein (A)

(B)

Fig. 4. (A) RNA-binding properties of B3a deletion mutants were analysed by UV cross-linking assay. (B) Amino acid sequence of the RNA-binding domain of B3a, and sequence homology between B3a and CMV MP RNA-binding domains. Homologies are as defined by Melcher (1990). Basic amino acid residues (H, K, R) are shown (­). Numbers in parentheses indicate amino acid positions.

of plasmids designated pBC3OMn (n ¯ 1–7) (kindly provided by N. Okitsu and Y. Fujita, Kyoto University, Kyoto, Japan) which each have a unique PstI site at various locations in the 3a ORF of pBC3KM11. The PstI (CTGCAG) sites were introduced into pBC3KM11 by inserting four nucleotides (TGCA) between two adjacent codons. The third nucleotide of the upstream codon was substituted with a cytosine if required. In all cases, this substitution did not cause a change in the amino acid encoded by the upstream codon. The first nucleotide of the downstream codon selected was a guanosine. The internal deletions were created by reassembling the small NdeI–PstI fragment and the large NdeI–PstI fragment from two mutants carrying the PstI site at successive locations. Four extra nucleotides in the PstI site were eliminated by PstI digestion, treatment with T4 DNA polymerase and subsequent religation to generate pBC3OMn.n+ (n ¯ 1–6). To create the deletion mutant pB∆1, " the small BglII–PstI fragment of pBC3OM1 was inserted into the BamHI– NcoI sites of pET15b (Novagen) after first repairing the noncompatible PstI and NcoI ends with T4 DNA polymerase. The plasmid was digested with XbaI and HindIII, and cloned into the XbaI–HindIII sites of pET3b. To create mutants pB∆2, pB∆3 and pB∆4, an NdeI site was introduced into pBC3OMn.n+ (n ¯ 1–3), respectively, by PCR as above. The PCR " fragment containing deletions in the 3a ORF was digested with NdeI and BglII and cloned into the NdeI–BamHI sites of pET3b. Mutant pB∆5 was created by ligation of the large ScaI fragment of pBC3OM . with the %& small ScaI fragment of pB3a while mutants pB∆6 and pB∆7 were generated by ligation of large ClaI fragments of pBC3OM . and &' pBC3OM . , respectively, with the small ClaI fragment of pB3a. To '( create mutant pB∆8, pBC3OM8 linearized with PstI digestion and bluntended with T4 DNA polymerase was first ligated to a phosphorylated linker ‘ BglII–amber ’ (5« TAGCAGATCTGCTA 3«) containing a TAG stop codon and a BglII site. The resulting plasmid was digested with BamHI and BglII and the resulting small fragment was cloned into the BamHI site of pET3b. The small ClaI fragment of the resulting plasmid was used to replace the corresponding ClaI fragment of pB3a. To express 3a proteins, the pB3a and pB∆n (n ¯ 1–8) plasmids were transfected into E. coli strain BL21(DE3) (Studier & Moffatt, 1986) and overexpressed as indicated in the manufacturer’s instructions (Novagen). The cells were harvested and lysed by sonication for 15 min at 4 °C (sonic source Kubota 201M, 150 W) in buffer B (50 mM Tris–HCl pH 8±0,

Fig. 5. Subcellular localization of B3a in infected barley tissues. Secondary leaves of BMV-infected barley (4 days post-inoculation) were fractionated into CW (lane 1), 1000 g pellet (P1, lane 2), 30 000 g pellet (P30, lane 3), 30 000 g supernatant (S30, lane 4), 100 000 g pellet (P100, lane 5) and 100 000 g supernatant (S100, lane 6). Total protein of mockinoculated barley is shown in lane 7. For all fractions, protein equivalent to 2±5 mg of leaves was used.

2 mM EDTA, 100 mM NaCl). The insoluble fraction was pelleted by centrifugation at 3000 g for 5 min at 4 °C. The pellets were washed twice with buffer B containing 0±5 % Triton X-100 and 10 mM EDTA, and then washed twice with buffer B. Proteins were solubilized with buffer B containing 8 M urea and renatured by dialysis at 10 °C against buffer B. The insoluble debris resulting from the dialysis was pelleted and the concentration of the soluble fractions was determined by the Bradford assay (Bradford, 1976) using BSA (fraction V, Sigma) as a standard. + Gel retardation analysis and UV cross-linking assay. pB3TP8 (Janda et al., 1987), containing a full-length cDNA copy of RNA3 of BMV, was digested with ClaI, and the first 0±6 kb of the BMV RNA3 were uniformly labelled with [α-$#P]UTP in an in vitro transcription reaction using T7 RNA polymerase. Similarly, the bacterial vector plasmid pBluescript II KS+ (Stratagene) was digested with DdeI and 0±6 kb labelled transcript was synthesized using T7 RNA polymerase. The resulting RNA transcripts were purified over a Sephadex G-50 (Phar-

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Fig. 6. For legend see facing page.

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Properties of BMV 3a protein macia) column. Typically, 1 ng of transcripts and 0±05–2 µg of proteins in a total volume of 10 µl were used in a binding reaction. Reaction mixtures were incubated in buffer B for 30 min on ice, and the mixture was either subjected to PAGE in nondenaturing 4 % polyacrylamide gels in TBE buffer (Sambrook et al., 1989) or irradiated with UV light for 10 min using a UV light source (CSL-4LC ; Cosmo Bio, 4 W at 254 nm), treated with RNase A and analysed by SDS–PAGE (Laemmli, 1970). The gels were dried and autoradiographed or analysed by a digital radioactive imaging analyser (Fujix BAS2000 ; Fuji). For competition analysis, BMV RNA (ss RNA), rice dwarf virus (RDV) RNA [double-stranded (ds) RNA], SmaIdigested and heat-denatured vector plasmid pUC118 (Takara Shuzo) (ss DNA) and SmaI-digested pUC118 (ds DNA) were used as competitors. + Production of monoclonal antibodies. Mouse monoclonal antibody specific to B3a was produced using E. coli-expressed B3a as immunogen. B3a was purified as above and injected into BALB}c mice intraperitoneally. Spleen cells of the immunized mice and sp2}O-Ag14 myeloma cells were fused by polyethylene glycol as previously described (Galfre et al., 1977). Two clones of anti-B3a monoclonal antibodies, designated B3-1 and B3-16, were used for subsequent experiments. + Subcellular fractionation of barley and immunodetection. The systemic host barley (Hordeum vulgare cv. Hinode-hadaka) was grown in a plant growth room as previously described (Fujita et al., 1996), infected with BMV-M1 (Ahlquist et al., 1984) and plant tissue extract was fractionated as previously described (Rouleau et al., 1994). Briefly, 1 g of leaf tissue from BMV-infected barley secondary leaves (4 days postinoculation) was homogenized with a pestle and mortar in 4 ml homogenization buffer. The homogenate was filtered through Miracloth (Calbiochem) to obtain the cell wall (CW) fraction. The filtrate was centrifuged at 1000 g for 10 min to recover the pellet fraction (P1). The supernatant solution was recentrifuged at 30 000 g for 30 min to generate a supernatant fraction (S30) and pellet fraction (P30). S30 was further centrifuged at 100 000 g for 60 min to generate a supernatant fraction (S100) and pellet fraction (P100). The CW fraction was washed with homogenization buffer containing 2 % Triton X-100. Proteins were extracted from each fraction as previously described (Laemmli, 1970), and separated by SDS–PAGE. Immunoblot analysis was carried out as previously described (Towbin, 1979) using an Immobilon-P transfer membrane (Millipore). B3a was detected using mouse monoclonal antibody B3-1 and an alkaline phosphatase-conjugated goat anti-mouse secondary antibody. + Immunocytochemical methods. Barley secondary leaves infected with BMV-KU2 (Nagano et al., 1997) or BMV-M1 were cut and fixed with 4 % (w}v) paraformaldehyde and 0±1 % (v}v) glutaraldehyde. After dehydration, the specimens were soaked in propyleneoxide and embedded in an LR White resin (London Resin). Ultrathin sections of the specimens were collected on Formvar-filmed stainless meshes and treated with TTBS (20 mM Tris–HCl pH 7±5, 0±5 M NaCl, 0±05 %, v}v, Tween 20) containing 3 % (w}v) skim milk, followed by treatment with a mixture of supernatants of hybridoma cell culture for B3-1 or B3-16 at 1 : 10 dilution with TTBS. After washing, the sections were treated with goldconjugated anti-mouse IgG goat immunoglobulin (Zymet) diluted 1 : 50

with 50 mM Tris–HCl pH 7±4, containing 150 mM NaCl, 1 % (w}v) BSA and 0±05 % (w}v) sodium azide. Finally the sections were stained with a solution of uranyl acetate and lead citrate, and examined under the electron microscope (Hitachi 7100F).

Results Expression and purification of the 3a proteins

B3a was expressed in E. coli cells carrying plasmid pB3a, which contains the coding region of B3a linked to a strong bacteriophage T7 promoter. Induction of expression of the T7 RNA polymerase resulted in the synthesis of a protein with the expected size (molecular mass 32 kDa). B3a formed insoluble aggregates which could be separated from the soluble bacterial proteins by centrifugation. However, B3a could be solubilized in 8 M urea and remained soluble after removal of urea. The purified B3a gave a single band on SDS–PAGE followed by Coomassie blue staining (Fig. 2, lane1). The deletion mutants of B3a using plasmids pB∆n (n ¯ 1–8) were expressed and purified as above (Fig. 2, lanes 2–9, respectively).

RNA-binding properties

Binding of B3a to ss RNA was assayed by electrophoretic gel retardation in polyacrylamide gels. Fig. 3 (A) and (B) illustrate the binding of B3a to two kinds of $#P-labelled 0±6 kb RNA, transcribed from a BMV RNA3 cDNA clone or bacterial vector plasmid (pBluescript), respectively. At low protein concentrations, the RNA3 fragment probe migrated to the same distance as the protein-free RNA, but when 0±4 µg or more of B3a was added, the probe barely entered the gel matrix (Fig. 3 A), indicating that the RNA formed a complex with B3a. Moreover, B3a also bound to pBluescript-derived transcript in the same manner (Fig. 3 B), suggesting that B3a has no sequence specificity in binding to RNAs. When B3a was heat-treated prior to assay, RNA-binding activity of B3a was abolished (data not shown). The relative stability of B3a–RNA complexes at different salt concentrations was analysed by a UV cross-linking assay. B3a bound RNA maximally at 50 mM NaCl ; at 200 mM NaCl, the B3a–RNA complexes were fully dissociated (data not shown). We also investigated whether B3a binds preferentially to particular types of nucleic acid. Competition binding assays were performed by incubating $#P-labelled RNA probe and unlabelled competitor nucleic acids together with the B3a, followed by gel retardation analysis. The ss nucleic acids were able to compete efficiently with the labelled transcript for

Fig. 6. Intracellular localization of B3a in BMV-infected barley leaf tissues using anti-B3a and gold-conjugated antibodies. Each section was prepared from secondary leaves of mock-inoculated (A) or BMV-KU2 infected (B–F) barley tissues (4 days postinoculation). No specific labelling was observed in mock-inoculated barley tissue (A). Gold labels are present within the electron-dense inclusions in cytoplasm (B) and longitudinal (C, D) or cross (E, F) sections of plasmodesmata. Bars, 200 nm. W, Cell wall ; C, chloroplast ; In, electron-dense inclusions.

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binding to B3a at low mass ratio (Fig. 3 C, lanes 3, 4, 7 and 8), while ds nucleic acids were able to compete only at higher mass ratio (Fig. 3 C, lanes 5, 6, 9 and 10), suggesting that B3a binds to ss nucleic acids more strongly than to ds nucleic acids. RNA binding by B3a deletion mutants

To map the B3a domains involved in RNA binding, we expressed and purified a series of deletion mutants of B3a (Fig. 2, lanes 2–9). The RNA-binding activity was assessed by a UV cross-linking assay as described in Methods. A comparison of the RNA-binding activity of B3a deletion mutants 1–8 showed that mutant 6 (∆189–242) failed to bind RNA, while the other mutants all bound RNA at readily detectable levels (Fig. 4 A). Subcellular localization of B3a in barley tissues

The monoclonal antibodies raised against the bacterially produced B3a allowed investigation of its subcellular localization in BMV-infected barley. On Western blots, both of the two monoclonal antibodies, B3-1 and B3-16, reacted with B3a synthesized in E. coli and in barley plants infected with BMVM1 or KU2 strain, while no proteins were immunoreactive in total protein from mock-inoculated plant homogenates (data not shown). We conclude that the monoclonal antibodies specifically react with B3a. To determine the intracellular localization of B3a, BMV-infected barley leaves were fractionated by differential centrifugation to produce CW, 1000 g pellet (P1), 30 000 g pellet (P30) and 30 000 g supernatant (S30) fractions and were then analysed by Western blotting for their B3a content. Immunodetection revealed that B3a was found predominantly in the P30 and S30 fractions (Fig. 5, lanes 3 and 4), although trace amounts of B3a were also detected in the CW fraction (Fig. 5, lane 1). When the S30 fraction was further fractionated by 100 000 g centrifugation, part of the B3a was pelleted into the P100 fraction (Fig. 5, lane 5). Intracellular localization

To investigate further the location of B3a in planta, ultrathin sections of the non-inoculated secondary leaves of mockinoculated and BMV-infected barley were immunogoldlabelled using the anti-B3a antibodies. In mock-infected barley tissues, no specific labelling with anti-B3a antibodies was observed (Fig. 6 A). B3a was found to be associated with electron-dense inclusions in the cytoplasm (Fig. 6 B). These inclusions were BMV infection-specific and contained membranous materials and some vesicle-like regions. B3a was also observed in the longitudinal (Fig. 6 C, D) and cross sections (Fig. 6 E, F) of plasmodesmata. No specific labelling with antiB3a antibodies was present inside cellular organelles such as the chloroplasts, mitochondria and microbodies, and the central vacuoles were devoid of labelling. There was no difference between results from inoculations with BMV-KU2 and BMVM1 strains (data not shown).

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Discussion In this study, we have investigated the functional properties of B3a which are required for BMV cell-to-cell movement. We expressed B3a in E. coli and showed that B3a possesses RNAbinding activity in vitro. Immunological studies using monoclonal antibodies against B3a revealed that B3a localized in plasmodesmata and cytoplasmic inclusions in BMV-infected barley tissues. RNA-binding properties

B3a cooperatively bound RNA in a sequence-nonspecific manner similar to the other viral MPs studied previously. The RNA–protein complexes formed by B3a were more sensitive to NaCl than are those formed by the TMV 30 kDa MP (Citovsky et al., 1990) and were almost as sensitive as those formed by the MPs of CaMV (Citovsky et al., 1991), alfalfa mosaic virus (AlMV) (Schoumacher et al., 1992) and cucumber mosaic virus (CMV) (Li & Palukaitis, 1996). B3a also bound ss DNA, ds RNA and ds DNA and, like AlMV MP (Schoumacher et al., 1992), showed higher affinity to ss than to ds nucleic acids. Furthermore, we showed that there is an RNA-binding domain located between amino acids 189 and 242. This is a predominantly hydrophilic region with relatively high surface probability similar to the RNA-binding domain of TMV (Citovsky et al., 1992), AlMV (Schoumacher et al., 1994), red clover necrotic mosaic virus (Osman et al., 1993), CaMV (Thomas & Maule, 1995) and CMV (Vaquero et al., 1997). This region includes a large number of positivelycharged residues which could form ionic bonds with negatively-charged phosphate groups. BMV and CMV are both members of the family Bromoviridae and an amino acid homology search showed that the RNA-binding domain of B3a is related to that of CMV MP (Melcher, 1990 ; Vaquero et al., 1997) (Fig. 4 B). Additionally, the RNA-binding activity of B3a was affected by heat treatment, suggesting that the activity is structure-dependent, similar to CMV (Li & Palukaitis, 1996). Localization

Subcellular fractionation analysis showed that B3a was present in the CW fraction. Consistent with this, immunogold labelling of ultrathin sections of BMV-infected barley revealed that B3a was localized in plasmodesmata within the cell wall. These data suggest the involvement of B3a in virus movement through plasmodesmata, including modification of plasmodesmata. However, we have not observed the distinct tubules with virus-like particles in plasmodesmata which are typical of CPMV-like plant viruses (van Lent et al., 1991), and we could not affirm how BMV moves through plasmodesmata. We have shown that B3a was also associated with electrondense inclusions containing membranous materials. This was consistent with the results of subcellular fractionation analysis

Properties of BMV 3a protein

showing that considerable amounts of B3a were present in the P30 fraction which should be rich in cytoplasmic membrane material (Deom et al., 1990). It has previously been shown by immunocytochemical analysis using polyclonal antibody against B3a (Hosokawa et al., 1992) that B3a localizes in cytoplasmic inclusions characteristic of BMV infections. We have confirmed this using monoclonal antibodies against B3a. In subcellular fractionation studies, at least part of the B3a in the 30 000 g supernatant fraction was pelleted by 100 000 g centrifugation, suggesting that B3a may form complexes, rather than exist as free protein. These complexes could be associations between B3a and fragmented subcellular structures such as the endoplasmic reticulum. Alternatively, these structures might be nucleoproteins involved in BMV cell-tocell movement as speculated for TMV (Citovsky et al., 1990). Cell-to-cell movement of BMV

The results presented here suggest that B3a may form complexes with BMV RNA which function as intermediates in virus cell-to-cell movement. On the other hand, recent studies have provided evidence that BMV requires CP for successful cell-to-cell movement (Schmitz & Rao, 1996) and possesses tubule-forming properties (Kasteel et al., 1997), suggesting that BMV moves through tubules assembled in plasmodesmata as virus particles. Similarly, CaMV and AlMV MP also induce tubular structures (Linstead et al., 1988 ; Kasteel et al., 1996, 1997) and possess RNA-binding activity (Citovsky et al., 1991 ; Schoumacher et al., 1992). This suggests that at least two mechanisms of movement may co-exist in a single virus infection (Citovsky et al., 1991), and that BMV infection may be an example. We thank Paul Ahlquist for the cDNA clones of BMV M1 strain, Nahoko Okitsu and Yasunari Fujita for plasmid clones pBC3OM 1–7 and for helpful discussions. This work was supported by the Research for the Future program of the Japan Society for the Promotion of Science under grant JSPS-RFTF96L00603. M. F. was supported by Research Fellowships of the Japan Society for the Promotion of Science for Young Scientists.

References Ahlquist, P., French, R., Janda, M. & Loesch-Fries, L. S. (1984).

Multicomponent RNA plant virus infection derived from cloned viral cDNA. Proceedings of the National Academy of Sciences, USA 81, 7066–7070. Bradford, M. M. (1976). A rapid and sensitive method for the quantities of protein utilizing the principle of protein dye binding. Analytical Biochemistry 72, 248–254. Carrington, J. C., Kasschau, K. D., Mahajan, A. K. & Schaad, M. C. (1996). Cell-to-cell and long-distance transport of viruses in plants. Plant

Cell 8, 1669–1681.

Citovsky, V., Knorr, D. & Zambryski, P. (1991). Gene I, a potential cell-

to-cell movement locus of cauliflower mosaic virus, encodes an RNAbinding protein. Proceedings of the National Academy of Sciences, USA 88, 2476–2480. Citovsky, V., Wong, M. L., Shaw, A. L., 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. Deom, C. M., Schubert, K. R., Wolf, S., Holt, C. A., Lucas, W. J. & Beachy, R. N. (1990). Molecular characterization and biological function

of the movement protein of tobacco mosaic virus in transgenic plants. Proceedings of the National Academy of Sciences, USA 87, 3284–3288. Ding, B., Li, Q., Nguyen, L., Palukaitis, P. & Lucas, W. J. (1995).

Cucumber mosaic virus 3a protein potentiates cell-to-cell trafficking of CMV RNA in tobacco plants. Virology 207, 345–353. Dolja, V., Haldeman, R., Robertson, N., Dougherty, W. & Carrington, J. (1994). Distinct functions of capsid protein in the assembly and

movement of tobacco etch potyvirus in plants. EMBO Journal 13, 1482–1491. Fujita, Y., Mise, K., Okuno, T., Ahlquist, P. & Furusawa, I. (1996). A single codon change in a conserved motif of a bromovirus movement protein gene confers compatibility with a new host. Virology 223, 283–291. Galfre, G., Howe, S. C., Milstein, C., Butcher, G. W. & Howard, J. C. (1977). Antibodies to major histocompatibility antigens produced by

hybrid cell lines. Nature 266, 550–552. Hosokawa, D., Kobayashi, K., Horikoshi, M. & Furusawa, I. (1992).

Immunogold localization of coat protein and 3a protein of brome mosaic virus in infected barley cells. Annals of the Phytopathological Society of Japan 58, 773–779. Janda, M., French, R. & Ahlquist, P. (1987). High efficiency T7 polymerase synthesis of infectious RNA from cloned brome mosaic virus cDNA and effects of 5« extensions on transcript infectivity. Virology 158, 259–262. Kasteel, D. T. J., Perbal, M.-C., Boyer, J.-C., Wellink, J., Goldbach, R. W., Maule, A. J. & van Lent, J. W. M. (1996). The movement proteins

of cowpea mosaic virus and cauliflower mosaic virus induce tubular structures in plant and insect cells. Journal of General Virology 77, 2857–2864. Kasteel, D. T. J., van der Wel, N. N., Jansen, K. A. J., Goldbach, R. W. & van Lent, J. W. M. (1997). Tubule-forming capacity of the movement

proteins of alfalfa mosaic virus and brome mosaic virus. Journal of General Virology 78, 2089–2093. Kroner, P., Richards, D., Traynor, P. & Ahlquist, P. (1989). Defined mutations in a small region of the brome mosaic virus 2a gene cause diverse temperature-sensitive RNA replication phenotypes. Journal of Virology 63, 5302–5309. Kroner, P., Young, B. M. & Ahlquist, P. (1990). Analysis of the role of brome mosaic virus 1a protein domains in RNA replication, using linker insertion mutagenesis. Journal of Virology 64, 6110–6120. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685. Li, Q. & Palukaitis, P. (1996). Comparison of the nucleic acid- and NTPbinding of the movement protein of cucumber mosaic cucumovirus and tobacco mosaic tobamovirus. Virology 216, 71–79.

Citovsky, V., Knorr, D., Schuster, G. & Zambryski, P. (1990). The P30

Linstead, P. J., Hills, G. J., Plaskitt, K. A., Wilson, I. G., Harker, C. L. & Maule, A. J. (1988). The subcellular localization of the gene 1 product of

movement protein of tobacco mosaic virus is a single-stranded nucleic acid binding protein. Cell 60, 637–647.

cauliflower mosaic virus is consistent with a function associated with virus spread. Journal of General Virology 69, 1809–1818.

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M. Fujita and others Lucas, W. J. (1995). Plasmodesmata : intracellular channels for macro-

molecular transport in plants. Current Opinion in Cell Biology 7, 673–680. Melcher, U. (1990). Similarities between putative transport proteins of plant viruses. Journal of General Virology 71, 1009–1018. Mise, K., Allison, R. F., Janda, M. & Ahlquist, P. (1993). Bromovirus movement protein genes play a crucial role in host specificity. Journal of Virology 67, 2815–2823. Nagano, H., Okuno, T., Mise, K. & Furusawa, I. (1997). Deletion of the C-terminal 33 amino acids of cucumber mosaic virus movement protein enables a chimeric brome mosaic virus to move from cell to cell. Journal of Virology 71, 2270–2276. Oparka, K. J., Roberts, A. G., Roberts, I. M., Prior, D. A. M. & Santa Cruz, S. (1996). Viral coat protein is targeted to, but does not gate,

plasmodesmata during cell-to-cell movement of potato virus X. Plant Journal 10, 805–813. Osman, T. A. M., Tho$ mmes, 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. Rao, A. L. N. & Grantham, G. L. (1995). Biological significance of the seven amino-terminal basic residues of brome mosaic virus coat protein. Virology 211, 42–52. Rouleau, M., Smith, R. J., Bancroft, J. B. & Mackie, G. A. (1994).

Purification, properties, and subcellular localization of foxtail mosaic potexvirus 26-kDa protein. Virology 204, 254–265. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning : A Laboratory Manual, 2nd edn. Cold Spring Harbor, NY : Cold Spring Harbor Laboratory. Schmitz, I. & Rao, A. L. N. (1996). Molecular studies on bromovirus

BCIA

capsid protein. I. Characterization of cell-to-cell movement-defective RNA3 variants of brome mosaic virus. Virology 226, 281–293. Schoumacher, F., Erny, C., Berna, A., Godefroy-Colburn, T. & StussiGaraud, C. (1992). Nucleic acid binding properties of the alfalfa mosaic

virus movement protein produced in yeast. Virology 188, 896–899. Schoumacher, F., Giovane, C., Maira, M., Poirson, A., GodefroyColburn, T. & Berna, A. (1994). Mapping of the RNA-binding domain

of the alfalfa mosaic virus movement protein. Journal of General Virology 75, 3199–3202. Studier, F. W. & Moffatt, B. A. (1986). Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. Journal of Molecular Biology 189, 113–130. Thomas, C. L. & Maule, A. J. (1995). Identification of the cauliflower mosaic virus movement protein RNA-binding domain. Virology 206, 1145–1149. Towbin, H. (1979). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets : procedure and some applications. Proceedings of the National Academy of Sciences, USA 76, 4350–4354. van Lent, J., Storms, M., van der Meer, F., Wellink, J. & Goldbach, R. (1991). Tubular structures involved in movement of cowpea mosaic

virus are also formed in infected cowpea protoplasts. Journal of General Virology 72, 2615–2623. Vaquero, C., Liao, Y.-C., Na$ hring, J. & Fischer, R. (1997). Mapping of the RNA-binding domain of the cucumber mosaic virus movement protein. Journal of General Virology 78, 2095–2099. Wolf, S., Deom, C. M., Beachy, R. N. & Lucas, W. J. (1989). Movement protein of tobacco mosaic virus modifies plasmodesmatal size exclusion limit. Science 246, 337–339. Received 18 November 1997 ; Accepted 19 January 1998