The C terminus of the movement protein of Brome mosaic virus controls the requirement for coat protein in cell-to-cell movement and plays a role in long-distance movement

Atsushi Takeda, Masanori Kaido, Tetsuro Okuno and Kazuyuki Mise

Laboratory of Plant Pathology, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan

Correspondence
Kazuyuki Mise
kmise{at}kais.kyoto-u.ac.jp


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The 3a movement protein (MP) plays a central role in the movement of Brome mosaic virus (BMV). To identify the functional regions in BMV MP, 24 alanine-scanning (AS) MP mutants of BMV were constructed. Infectivity of the AS mutants in the host plant Chenopodium quinoa showed that the central region of BMV MP is important for viral movement and both termini of BMV MP have effects on the development of systemic symptoms. A green-fluorescent-protein-expressing RNA3-based BMV vector containing a 2A sequence from Foot-and-mouth disease virus was also constructed. Using this vector, two AS mutants that showed more efficient cell-to-cell movement than wild-type BMV were identified. The MPs of these two AS mutants, which have mutations at their C termini, mediated cell-to-cell movement independently of coat protein (CP), unlike wild-type BMV MP. Furthermore, a BMV mutant with a truncation in the C-terminal 42 amino acids of MP was also able to move from cell to cell without CP, but did not move systemically, even in the presence of CP. These results and an encapsidation analysis suggest that the C terminus of BMV MP is involved in the requirement for CP in cell-to-cell movement and plays a role in long-distance movement. Furthermore, the ability to spread locally and form virions is not sufficient for the long-distance movement of BMV. The roles of MP and CP in BMV movement are discussed.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Plant viral movement proteins (MPs) play central roles in virus cell-to-cell and long-distance movement in plants (reviewed by Lazarowitz & Beachy, 1999) and are classified into three types on the basis of their requirement for coat protein (CP) and the form they take during cell-to-cell movement (Callaway et al., 2001). First, some viral MPs function in viral cell-to-cell movement independently of CP. Viruses with this type of MP, e.g. Tobacco mosaic virus (TMV; Dawson et al., 1988) and Cowpea chlorotic mottle virus (CCMV; Rao, 1997), move from cell to cell as an MP–viral RNA complex. Second, some MPs require CP but not virion formation for cell-to-cell movement. For example, Cucumber mosaic virus (CMV; Blackman et al., 1998; Kaplan et al., 1998; Schmitz & Rao, 1998) has this type of MP and is thought to move from cell to cell as an MP–CP–viral RNA complex. Third, some viral MPs require encapsidation-competent CP in cell-to-cell movement. Viruses with this type of MP, e.g. Cowpea mosaic virus (CPMV; Kasteel et al., 1993; Wellink & Van Kammen, 1989) and Brome mosaic virus (BMV; Kasteel et al., 1997; Okinaka et al., 2001; Rao & Grantham, 1996), move from cell to cell as virions through tubular structures induced by MP. It is interesting that related viruses belonging to the family Bromoviridae have different types of MP, as discussed previously (Callaway et al., 2001); CCMV, CMV and BMV belong to the same family, but the MP of each is classified differently into the three types described above.

BMV is a well-studied positive-sense single-stranded RNA plant virus (Ahlquist, 1999). It is an established model for the study of plant RNA viruses and has been used for in-depth studies of viral gene expression, recombination and replication (Kao & Sivakumaran, 2000). However, the cell-to-cell and long-distance movements of BMV are not as well characterized as those of some other plant viruses. The genome of BMV is divided into three RNA components. RNA1 and RNA2 encode the proteins, 1a and 2a, respectively, which are required for viral RNA replication. Dicistronic RNA3 encodes the 3a protein, which is an MP of BMV (Rao, 1997), and CP. CP is translated from subgenomic RNA4, which is transcribed from minus-strand RNA3 (Miller et al., 1985). BMV MP binds to single-stranded nucleic acids (Fujita et al., 1998; Jansen et al., 1998), localizes in the plasmodesmata (Fujita et al., 1998), co-localizes with replication proteins in the BMV infection-inducing inclusion bodies (Dohi et al., 2001) and induces tubular structures on the surfaces of protoplasts (Sanchez-Navarro & Bol, 2001). Moreover, BMV MP is involved in host specificity (De Jong et al., 1995; Fujita et al., 1996, 2000; Mise et al., 1993; Mise & Ahlquist, 1995; Sasaki et al., 2001) and modulates symptom expression in susceptible host plants (Fujita et al., 1996; Rao & Grantham, 1995a). As mentioned, BMV MP is multifunctional and requires CP for cell-to-cell movement (Rao, 1997; Rao & Grantham, 1995b; Sasaki et al., 2003; Schmitz & Rao, 1996).

To gain further insight into the function of BMV MP, we used alanine-scanning (AS) mutagenesis (Cunningham & Wells, 1989) to map the BMV MP sequences that influence virus infectivity and symptom development. We then developed a BMV vector carrying the green fluorescent protein (GFP) gene to visualize BMV movement. We identified two BMV mutants with cell-to-cell movement more efficient than that of wild-type BMV. Furthermore, we showed that three BMV mutants can move from cell to cell independently of CP. An encapsidation analysis suggested that the capacities for virion formation and local spread are not sufficient to allow the long-distance movement of BMV, and that the C terminus of BMV MP plays a role in long-distance movement. We discuss the roles of MP and CP in BMV movement.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
cDNA clones.
The plasmids pB1TP3, pB2TP5 and pB3TP8 contain the full-length cDNA of RNAs 1, 2 and 3, respectively, of the wild-type BMV M1 strain (Janda et al., 1987). A series of plasmids, pB3ASn (‘n’ represents the position of the first amino acid residue in the mutated region within the BMV MP gene, Table 1), were generated by PCR-mediated site-directed mutagenesis of pB3TP8 using the appropriate primers. We selected positions in the MP gene that encode two or three consecutive charged residues or residues with high surface probability (Emini et al., 1985) to be changed to alanine codons (Table 1). The CLUSTAL W program (Thompson et al., 1994) was used to identify conserved residues in the MP of four bromoviruses, BMV (Ahlquist et al., 1981), CCMV (Allison et al., 1989), Broad bean mottle virus (Romero et al., 1992) and Spring beauty latent virus (Fujisaki et al., 2003). The plasmid pB3B3a-FS contains a two-nucleotide frameshift mutation introduced into the ClaI site of the 3a MP gene of pB3TP8.


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Table 1. BMV MP residues affected by AS mutagenesis

Sequences encoding affected amino acid residues were changed to alanine codons in each AS mutant. The underlined amino acid residues are conserved among the four bromoviruses BMV, CCMV, Broad bean mottle virus and Spring beauty latent virus. Positions affected by mutagenesis are defined as conserved if at least one of the residues is invariant in the 3a protein encoded by the four bromoviruses.

 
To construct pB3{Delta}3a/GFP and pB3{Delta}CP/GFP2, a GFP gene, GFPmut1 (Cormack et al., 1996), was precisely substituted for the MP gene in pB3TP8 and for the gene of a truncated but functional CP in pB3RS2 (Sacher & Ahlquist, 1989), respectively. The plasmid pB3GAC2 is a derivative of pB3{Delta}CP/GFP2 and encodes a fusion protein consisting of GFP and CP with an insertional sequence of a 2A oligopeptide from Foot-and-mouth disease virus (FMDV) (QLLNFDLLKLAGDVESNPGP) (Ryan & Drew, 1994). The fusion protein can be translated from RNA4 derived from pB3GAC2, and GFP–2A protein and CP can be produced simultaneously by 2A-mediated intraribosomal cleavage (de Felipe et al., 2003). The plasmid pB3TGF32 contains the GFP gene upstream from a duplicated intercistronic region inserted upstream from the start codon of the MP gene. The plasmid pB3B3a-FS-GAC2 contains a two-nucleotide frameshift mutation, which was introduced into the ClaI site of the MP gene of pB3GAC2. The sequence encoding an FMDV 2A peptide, except for the dipeptide QF, was deleted from pB3GAC2 to create pB3GC2 from which the GFP–CP fusion protein is expressed. The plasmid pB3AC2 encodes a fusion protein consisting of an FMDV 2A oligopeptide and CP.

The AS mutation was introduced into pB3GAC2 to create pB3ASn-GAC2. The mutations in the MP gene within pB3AS262 and pB3AS267 were introduced into pB3{Delta}CP/GFP2 to create pB3AS262-{Delta}CP/GFP2 and pB3AS267-{Delta}CP/GFP2, respectively. The nucleotides GAA encoding Glu-262 in the BMV MP gene of pB3TP8 were substituted with TAG, creating pB3C{Delta}42. This mutation was introduced into pB3{Delta}CP/GFP2 and pB3GAC2 to create pB3C{Delta}42-{Delta}CP/GFP2 and pB3C{Delta}42-GAC2, respectively.

The cDNAs generated by PCR were sequenced using a DNA sequencer (Applied Biosystems, model 310). All cDNA plasmids were linearized with EcoRI and capped viral RNA transcripts were synthesized as described previously (Kroner & Ahlquist, 1992). The transcript from the plasmid containing the RNA3 variant is referred to by its plasmid name without the prefix ‘p’ and in parentheses after ‘B3’. For example, RNA3 produced from pB3AS12 is designated B3(AS12). In all experiments, RNA3 derivatives were inoculated together with wild-type RNA1 and RNA2.

Analysis of viral RNAs and proteins.
Isolation, inoculation and incubation of barley protoplasts (Hordeum vulgare cv. Hinodehadaka) were performed as described previously (Damayanti et al., 1999), except for a modification in the protoplast medium which was buffered by the addition of 5 mM MES, pH 6·5. Total and virion fraction RNAs were extracted from infected protoplasts and Northern blot analysis was performed as described previously (Damayanti et al., 1999) with a DIG-labelled probe for the detection of positive-strand RNAs (Sasaki et al., 2001). Total proteins were separated by electrophoresis on 12·5 or 15 % polyacrylamide gels containing 0·1 % SDS (Laemmli, 1970). MP and CP were detected using a mouse monoclonal antibody raised against MP (Fujita et al., 1998) and rabbit anti-BMV antiserum (PVAS178, ATCC), respectively.

Inoculation experiments in plants.
BMV RNA3-derivatives were inoculated onto Chenopodium quinoa plants as described previously (Nagano et al., 1999). Inoculated and upper uninoculated leaves were harvested at 14 days post-inoculation (p.i.). Viral RNAs were detected by tissue printing analysis, as described previously (Mise et al., 1993). The leaves were then ground with 7 ml sample buffer (71·4 mM Tris/HCl, pH 6·8, 2·3 % SDS, 11·4 % glycerol, 0·01 % bromophenol blue, 5·7 % 2-mercaptoethanol) per 1 g of harvested leaves. GFP fluorescence in the inoculated leaves of harvested C. quinoa plants was observed under an epifluorescence microscope (Axioscope, Carl Zeiss) equipped with an FITC filter set (Carl Zeiss).


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Infectivity of alanine-scanning MP mutants of BMV
To identify and map the functional regions of BMV MP, AS mutagenesis (Cunningham & Wells, 1989) was carried out. Twenty-four AS mutants were constructed and assayed in vivo for their ability to potentiate the spread of BMV infection. To examine the effects of AS mutations on virus amplification, we inoculated AS mutants into protoplasts and examined the accumulation of CP 24 h p.i. Fig. 1(a) shows that the accumulation of CP of the 24 AS mutants in infected protoplasts was equivalent to that of wild-type BMV.



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Fig. 1. Accumulation of CP in protoplasts and plants inoculated with AS MP mutants of BMV. Protoplasts and plants were inoculated with BMV RNA1 and RNA2 together with RNA3 derivatives with AS mutations in the MP gene. To detect BMV CP, total protein from (a) equal numbers of protoplasts (3·0x103) and (b) equal amounts (0·4 mg fresh weight) of the leaves of C. quinoa were loaded onto gels. The names of the AS mutants are indicated above each lane without the prefix ‘AS’. Samples from the leaves inoculated with wild-type BMV diluted 10-fold (WT 1/10) or 100-fold (WT 1/100) were loaded to estimate the relative accumulation of CP in leaves inoculated with the AS mutants. Total proteins from leaves inoculated with buffer or an MP-frameshift mutant were loaded into lanes Mock and FS, respectively. (I) and (U) indicate inoculated and upper uninoculated leaves, respectively.

 
To examine the infectivity of the AS mutants in a host plant, we inoculated AS mutants onto C. quinoa plants and observed systemic symptoms at 14 days p.i. In C. quinoa, wild-type BMV induced chlorotic local lesions on the inoculated leaves, and leaf distortions and chlorotic local lesions on the upper uninoculated leaves (Table 2). Infection of ten AS mutants caused systemic symptoms (Table 2) and five of these ten AS mutants induced systemic symptoms distinct from those induced by wild-type BMV; B3(AS280) and B3(AS281) induced more severe leaf distortions than did wild-type BMV; B3(AS12) and B3(AS27) induced chlorotic local lesions and mild leaf distortions; B3(AS244) induced small faint chlorotic local lesions, but no leaf distortions. These systemic symptoms induced by AS mutants on C. quinoa were reproduced in three independent experiments. Symptomatic leaves were always accompanied by viral RNA signals detected by tissue print analysis (data not shown). These results suggest that BMV MP plays a role in symptom expression in C. quinoa plants.


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Table 2. Systemic infectivity of AS MP mutants of BMV in C. quinoa

C. quinoa leaves were inoculated with BMV RNA1 and RNA2 together with the RNA3 derivative having AS mutations in the MP gene. Symptoms and CP accumulation were examined in both inoculated and upper uninoculated leaves at 14 days p.i. NLL, necrotic local lesions; CLL, chlorotic local lesions; SCLL, small chlorotic local lesions; SFCLL, small faint chlorotic local lesions; VSLD, very severe leaf distortions; SLD, severe leaf distortions; MLD, mild leaf distortions. Viral RNAs were detected by tissue print assay in all leaves displaying symptoms. The accumulation of CP was examined by Western blot analysis. Representative data are in Fig. 1(b). (+) and (–) indicate the presence and absence of CP, respectively. The number of (+) shows relative amount of CP as follows: (++++), the accumulation of CP was equal to that of wild-type BMV; The CP accumulation was 10-fold (+++) and 100-fold (++) lower than that of wild-type BMV; (+), the accumulation of CP was less than (++).

 
To investigate whether the severity of systemic symptoms depends on the degree of cell-to-cell movement in upper uninoculated leaves, we examined the accumulation of CP in infected plants. The accumulation of CP in inoculated and upper uninoculated leaves is considered to reflect the degree of cell-to-cell movement because the accumulation of CP from the 24 AS mutants in the protoplasts was equivalent to that of wild-type BMV (Fig. 1a). The accumulation of CP in upper uninoculated leaves was similar to that of wild-type BMV in the seven AS mutants that induced severe or very severe leaf distortions (Fig. 1b and Table 2). On the other hand, in upper uninoculated leaves infected with the three AS mutants that induced mild or no leaf distortions, the amount of CP was considerably lower than that in the upper uninoculated leaves of plants infected with wild-type BMV (Fig. 1b and Table 2). These results suggest that the induction of leaf distortions requires efficient cell-to-cell movement in the upper uninoculated leaves of C. quinoa. B3(AS244), which induced no leaf distortions, systemically infected C. quinoa plants (Table 2). In B3(AS244)-inoculated leaves, CP accumulated to a level 100-fold less than the CP that accumulated in the wild-type BMV-inoculated leaves (Fig. 1b), suggesting that efficient cell-to-cell movement in inoculated leaves is not necessarily required for the long-distance movement of BMV.

Six AS mutants (AS46, AS69, AS201, AS208, AS240 and AS301) induced symptoms only in the inoculated leaves of C. quinoa (Table 2), suggesting that these AS mutants move from cell to cell, but not systemically. To confirm our visual observations, we examined the accumulation of CP. As expected, CP was detected only in the leaves inoculated with the six AS mutants (Fig. 1b). We have never detected CP in leaves inoculated with an MP-frameshift mutant (see FS in Fig. 1b), which suggests that the six AS mutants do move from cell to cell, but do not move systemically. In contrast, eight AS mutants (AS57, AS82, AS83, AS107, AS135, AS169, AS221 and AS227) did not induce symptoms (Table 2) and no viral RNAs (data not shown) or CP (Fig. 1b) were detected, even in the inoculated leaves, suggesting that these eight AS mutants do not move from cell to cell.

Visualization of the cell-to-cell movement of BMV using GFP
To further analyse the movement behaviour of the AS mutants, we attempted to visualize BMV movement using the GFP gene. In the family Bromoviridae, several RNA3 derivatives which express GFP have been used to visualize cell-to-cell movement in CMV (Canto et al., 1997) and Alfalfa mosaic virus (AMV) (Sanchez-Navarro & Bol, 2001; Sanchez-Navarro et al., 2001). Similarly, three RNA3-based BMV vectors, B3({Delta}3a/GFP), B3({Delta}CP/GFP2) and B3(TGF32), were inoculated onto C. quinoa together with wild-type RNA1 and RNA2. However, unlike the CMV system, co-inoculation with B3({Delta}3a/GFP) plus B3({Delta}CP/GFP2) (trans-complementation of MP and CP from two different RNA3 constructs) did not show multicellular GFP fluorescence on the inoculated leaves of C. quinoa at 5 days p.i. (data not shown). Furthermore, unlike the AMV system, inoculation with B3(TGF32), which contains the GFP gene inserted adjacent to the 5' leader sequence and a duplicated subgenomic promoter sequence, also showed no multicellular GFP fluorescence (data not shown). We then tested the other derivative, B3(GAC2), which encodes a GFP–FMDV 2A–CP fusion protein (Fig. 2a) and detected foci with multiple fluorescent cells on the leaves inoculated with B3(GAC2) at 5 days p.i. (Table 3). However, we failed to observe systemic spread of GFP fluorescence until 14 days p.i. (data not shown). The GFP fluorescence induced by B3(GAC2-FS), which expresses a dysfunctional BMV MP, was confined to single cells (see FS in Table 3), suggesting that the cell-to-cell movement of B3(GAC2) depends on the function of MP. Passive diffusion of the GFP fusion proteins (GFP–2A–CP and GFP–2A) was not observed under our experimental conditions using fully expanded source leaves of C. quinoa. Therefore, we used B3(GAC2) to examine the capacity of the MP mutants for cell-to-cell movement.



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Fig. 2. Multiplication of GFP–2A-tagged BMV and its variants in protoplasts. (a) Schematic representation of cDNA constructs of BMV RNA3 variants. In pB3GAC2, the cDNA construct used for the synthesis of wild-type RNA3 (pB3TP8) was modified to express the GFP–FMDV 2A peptide (QLLNFDLLKLAGDVESNPGP)–CP fusion protein. The sequence encoding an FMDV 2A peptide, except for the dipeptide QF, was deleted from pB3GAC2 to create pB3GC2 from which the GFP–CP fusion protein was expressed. The GFP coding sequence except for its initiation codon was deleted from pB3GAC2 to create pB3AC2 from which the 2A–CP fusion protein was expressed. Closed boxes represent the BMV MP and CP coding sequences. Open and hatched boxes represent the GFP and the 2A peptide coding sequences, respectively. Single lines indicate the surrounding noncoding regions of RNA3. The bent arrow indicates the transcription start site of subgenomic RNA. (b–d) Protoplasts were inoculated with BMV RNA1 and RNA2 transcripts together with wild-type RNA3 transcript (lane 5) or the transcripts of RNA3 variants: pB3GAC2 (lane 1), pB3GC2 (lane 2) and pB3AC2 (lane 3). Protoplasts were inoculated with buffer only (lane 4). At 24 h p.i., total RNA fraction (b), virion RNA fraction (c) and total protein (d) were prepared from the protoplasts. Positive-strand BMV RNAs were then detected in (b) total RNA and (c) virion RNA fractions. The relative amounts of samples loaded are indicated below the photograph. The expected positions of the BMV RNAs are indicated on the right side of the photograph. (d) Total proteins from equal numbers of protoplasts (3·0x103 for CP and 6·0x103 for MP) were loaded. An asterisk shows an extra band that reacted with anti-CP antiserum.

 

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Table 3. Analysis of cell-to-cell movement of AS MP mutants of BMV expressing GFP

B3(GAC2), which is a BMV RNA3 derivative expressing GFP, was used for the analysis of cell-to-cell movement of the AS mutants. C. quinoa leaves were inoculated with B3(GAC2), B3(B3a-FS-GAC2) or B3(ASn-GAC2) together with BMV RNA1 and RNA2. B3(GAC2) and B3(B3a-FS-GAC2) encode wild-type MP (WT) and an MP with a frameshift mutation (FS), respectively. The data represent the number of infection foci containing the indicated number of green fluorescent cells at 5 days p.i.

 
Analysis of cell-to-cell movement of AS mutants using the B3(GAC2) vector
To analyse the movement behaviour of the AS mutants, we introduced 24 AS mutations into the MP gene on B3(GAC2) to construct B3(ASn-GAC2) and tested their infectivity. Epifluorescence microscopic analyses showed that B3(ASn-GAC2) which contained functional MP for systemic spread in the context of B3(ASn) moved from cell to cell (Tables 2 and 3); B3(ASn-GAC2) which contained dysfunctional MP in the context of B3(ASn) infected subliminally (Tables 2 and 3). Interestingly, two B3(GAC2) derivatives, B3(AS262-GAC2) and B3(AS267-GAC2), displayed more efficient cell-to-cell movement than B3(GAC2) (Table 3). Similarly, recent studies of TMV MP using a DNA shuffling strategy showed that a few mutations in the MP gene increased the cell-to-cell movement of a TMV variant which carries the GFP gene (Toth et al., 2002). Moreover, one of those TMV MP mutants accumulated to higher levels in infected cells by blocking MP degradation (Gillespie et al., 2002). From these studies, we hypothesized that the MPs of B3(AS262-GAC2) and B3(AS267-GAC2) possess a feature similar to that of the TMV MP mutant (Gillespie et al., 2002). To test this, protoplasts were inoculated with B3(AS262-GAC2) and B3(AS267-GAC2) and the accumulation of MP was examined at 24 h p.i. However, the accumulation of MP was equivalent in B3(AS262-GAC2), B3(AS267-GAC2) and B3(GAC2) inoculations (data not shown). These results suggest that the mechanism that increases the efficiency of cell-to-cell movement in B3(AS262-GAC2) and B3(AS267-GAC2) is distinct from that reported previously for the MP mutant of TMV (Toth et al., 2002; Gillespie et al., 2002).

Virion RNAs were not detected in protoplasts inoculated with B3(GAC2)
To elucidate the mechanism that increases the efficiency of cell-to-cell movement in B3(AS262-GAC2) and B3(AS267-GAC2), we characterized B3(GAC2). First, we examined the accumulation of viral proteins and RNAs in protoplasts inoculated with B3(GAC2). Western blot analysis for B3(GAC2)-inoculated protoplasts detected the uncleaved GFP–2A–CP fusion protein as well as 2A-cleaved CP with a slightly low electrophoretic mobility, probably due to the attachment of a proline residue at the N terminus of CP (Fig. 2d, lane 1). Thus, cleavage by FMDV 2A was incomplete. In the total RNA fraction, the accumulation of viral RNAs (RNA1–RNA4) in B3(GAC2)-inoculated protoplasts was considerably lower than their accumulation in wild-type BMV-inoculated protoplasts (Fig. 2b; compare lane 1 with lane 5). Surprisingly, in the virion RNA fraction, the accumulation of all four BMV RNAs was below the limit of detection in the B3(GAC2)-inoculated protoplasts (Fig. 2c, lane 1), although wild-type RNA1 and RNA2 were easily detected in the total RNA fraction (Fig. 2b, lane 1). These results suggest that a reduction in packaging in B3(GAC2)-infected protoplasts is caused by CP, namely a proline-attached CP cleaved by FMDV 2A or uncleaved GFP–2A–CP fusion protein.

To examine these possibilities, B3(GC2), which encodes a GFP–CP fusion protein (Fig. 2a), and B3(AC2), which encodes an FMDV 2A–CP fusion protein (Fig. 2a), were inoculated into protoplasts and onto C. quinoa plants. In B3(GC2)-inoculated protoplasts, virion RNAs were not detected (Fig. 2c, lane 2). In leaves inoculated with B3(GC2), GFP fluorescence was confined to single cells in most of the infection foci at 5 days p.i. (Table 4). These results suggest that the GFP–CP fusion protein does not encapsidate BMV RNAs nor function in cell-to-cell movement, suggesting that the uncleaved GFP–2A–CP fusion protein in B3(GAC2)-infected cells also does not perform these functions. Furthermore, these results suggest that the CP cleaved by FMDV 2A functions in the cell-to-cell movement of B3(GAC2). On the other hand, virion RNAs were detected in protoplasts inoculated with B3(AC2), which encodes an FMDV 2A–CP fusion protein (Fig. 2c, lane 3). B3(AC2) systemically infected C. quinoa plants with severe leaf distortions, like wild-type BMV (data not shown). These results indicate that CP cleaved by FMDV 2A has the ability to encapsidate viral RNAs and to mediate the cell-to-cell movement and even the long-distance movement of BMV. From these results, it appears that the uncleaved GFP–2A–CP fusion protein inhibits efficient virion formation in B3(GAC2) inoculation.


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Table 4. Analysis of cell-to-cell movement of CP-deficient, MP C terminus mutants of BMV expressing GFP

C. quinoa leaves were inoculated with each RNA3 variant together with BMV RNA1 and RNA2. The data represent the number of infection foci containing green fluorescent cells at 5 days p.i.

 
Two AS mutants have the ability to move from cell to cell independently of CP
The above results showed that a small number of progeny virions could be formed in B3(GAC2)-infected cells and these could move from cell to cell. Despite having poor encapsidation ability, B3(AS262-GAC2) and B3(AS267-GAC2) moved from cell to cell efficiently, suggesting that these mutants have the ability to move from cell to cell independently of virion formation. To test this possibility, the CP-defective constructs B3(AS262-{Delta}CP/GFP2) and B3(AS267-{Delta}CP/GFP2) were inoculated onto C. quinoa leaves. At 5 days p.i., multiple GFP-fluorescent cells were observed in the leaves inoculated with B3(AS262-{Delta}CP/GFP2) or B3(AS267-{Delta}CP/GFP2) (Table 4), although infection with B3({Delta}CP/GFP2) induced infection foci containing single GFP-expressing cells (Table 4). These results indicate that mutant MPs of AS262 and AS267 have the ability to mediate cell-to-cell movement without CP and that a few point mutations in the C terminus of BMV MP change the requirement for CP in the cell-to-cell movement of BMV. We did not detect GFP fluorescence in the upper uninoculated leaves of plants infected with B3(AS262-{Delta}CP/GFP2) or B3(AS267-{Delta}CP/GFP2) until 14 days p.i. (data not shown).

The C terminus of BMV MP plays a role in long-distance movement
As shown above, we identified four amino acid residues at positions 262, 263, 267 and 268 within the C terminus of BMV MP, which are involved in the requirement for CP in cell-to-cell movement. On the other hand, our recent study with CMV MP showed that deletion of the C-terminal 33 amino acids alters the requirement for CP in virus cell-to-cell movement (Nagano et al., 2001). Taken together, these data suggest that deletion of the C terminus of BMV MP also alters the requirement for CP in cell-to-cell movement. To test this, we changed the codon for the amino acid at position 262 of BMV MP into a stop codon and investigated its effect on virus movement. Protoplast experiments showed that the accumulation of viral proteins and RNAs was similar in B3(C{Delta}42) and wild-type BMV inoculations (Fig. 3a–c). We then tested the effect of a C terminus truncation on cell-to-cell movement using the constructs B3(C{Delta}42-GAC2) and B3(C{Delta}42-{Delta}CP/GFP2). In C. quinoa leaves inoculated with B3(C{Delta}42-GAC2) or B3(C{Delta}42-{Delta}CP/GFP2), multiple GFP-fluorescent cells were observed at 5 days p.i. (Table 4). These results demonstrate that a C-terminally-truncated BMV MP has the ability to mediate cell-to-cell movement independently of CP.



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Fig. 3. Multiplication of BMV expressing MP with a C terminus truncation in protoplasts. Protoplasts were inoculated with BMV RNA1 and RNA2 transcripts together with wild-type RNA3 transcript (lane 4) or the transcripts of RNA3 variants: pB3C{Delta}42 (lane 1) and pB3B3a-FS (lane 2). Protoplasts were inoculated with buffer only (lane 3). At 24 h p.i., total RNA fraction (a), virion RNA fraction (b) and total protein (c) were prepared from the protoplasts. Positive-strand BMV RNAs were then detected in (a) total RNA and (b) virion RNA fractions. The positions of the BMV RNAs are indicated on the right side of the photograph. (c) Total proteins from equal numbers of protoplasts (6·0x103 for MP and 3·0x103 for CP) were loaded.

 
B3(C{Delta}42) was then inoculated onto C. quinoa plants to investigate the role of the C terminus of BMV MP in long-distance movement in the presence of CP. At 14 days p.i., the plants inoculated with B3(C{Delta}42) showed no symptoms (data not shown). The inoculated and upper uninoculated leaves of C. quinoa were then harvested and BMV CP was detected by Western blot analysis. In the inoculated leaves, CP was detected after B3(C{Delta}42) inoculation (Fig. 4), suggesting that B3(C{Delta}42) moves from cell to cell without visible symptoms. On the other hand, CP was not detected in the upper uninoculated leaves after B3(C{Delta}42) inoculation (Fig. 4). These results suggest that the C-terminal 42 amino acids of MP are not essential for cell-to-cell movement of BMV, but are indispensable for long-distance movement.



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Fig. 4. Infectivity of BMV expressing MP with a C terminus truncation in plants. Leaves of C. quinoa were inoculated with BMV RNA1 and RNA2 transcripts together with wild-type RNA3 transcript (WT, lanes 7–10) or transcripts of pB3C{Delta}42 (C{Delta}42, lanes 1 and 2) or pB3B3a-FS (FS, lanes 3 and 4). Tenfold (lanes 7 and 8) and 100-fold (lanes 9 and 10) dilutions of samples from leaves inoculated with wild-type BMV were loaded to estimate the relative accumulation of CP in leaves inoculated with B3(C{Delta}42). The samples loaded in lanes 7 and 9 were derived from the same inoculated leaf, as were those in lanes 8 and 10. At 14 days p.i., inoculated and upper uninoculated leaves were harvested and ground in Laemmli sample buffer. Total proteins from leaves inoculated with buffer were loaded in lanes 5 and 6 (Mock). Two samples from different inoculated plants are shown for each inoculation. The relative amounts of samples loaded are indicated below the photograph. (I) and (U) indicate inoculated and upper uninoculated leaves, respectively.

 

   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
In this study, we have shown that the C terminus of BMV MP controls the requirement for CP in cell-to-cell movement (Table 4). Here, on the basis of this requirement for CP and the form of cell-to-cell movement, we classify the plant virus MPs as follows: MPs that do not require CP as ‘type I’; MPs that require CP but not virions as ‘type II’; MPs that require encapsidation-competent CP as ‘type III’. To date, BMV MP have been known as type III and BMV MP mutants classified as other types have not yet been reported (Callaway et al., 2001). However, we identified three BMV MP mutants, AS262, AS267 and C{Delta}42 that have the ability to mediate the cell-to-cell movement of BMV in the absence of CP (Table 4). These results indicate that BMV MP is altered from type III to type I by point mutations introduced into the C terminus of BMV MP. To our knowledge, this is the first report demonstrating type-III-to-type-I alteration of a plant viral MP and identifying the amino acid residues which determine the type of MP. In viruses belonging to the Bromoviridae family, type-II-to-type-I alterations have been demonstrated in CMV MP (Nagano et al., 2001) and type-I-to-type-II or -III alterations have been shown in CCMV MP (Osman et al., 1999). The features common to these two examples and the present study suggest that the C-terminal region of Bromoviridae MP is involved in the determination of MP type.

We have shown that the C terminus of BMV MP plays a role in long-distance movement (Fig. 4). In this study, to investigate the role of the C terminus of BMV MP in virus movement, we constructed B3(C{Delta}42) by introducing a premature termination codon into the MP gene rather than a deletion mutation, because RNA sequences encoding the C terminus of BMV MP are required for efficient encapsidation of RNA3 (Choi & Rao, 2003; Damayanti et al., 2002, 2003). As expected, the point mutations introduced into B3(C{Delta}42) did not affect the efficiency of encapsidation (Fig. 3), although B3(C{Delta}42) did not move systemically (Fig. 4). These results suggest that efficient virion formation is not sufficient to allow the long-distance movement of BMV, and that the C terminus of BMV MP plays a crucial role in long-distance movement. The inability of B3(C{Delta}42) to move over long distances may be explained by the putative interaction between MP and CP. BMV MP requires CP for virus movement (Rao, 1997; Sasaki et al., 2003) and this suggests that BMV MP interacts with BMV CP. Furthermore, a chimeric analysis of BMV and CMV suggested that BMV MP and its CP interact specifically (Nagano et al., 1999). From these data, we infer that the C terminus of BMV MP plays a role in the putative interaction with its CP. The lack or reduction of interaction between BMV CP and a BMV MP after truncation of the C-terminal 42 amino acids may cause the defect in long-distance movement of B3(C{Delta}42). This scenario is further supported by recent studies of CMV (Nagano et al., 2001), AMV (Sanchez-Navarro & Bol, 2001) and CPMV (Carvalho et al., 2003), which showed or suggested that the C termini of their MPs interact with their cognate CPs.

Alanine-scanning mutagenesis of BMV MP in this study gave novel information, as discussed below. First, the amino acid residues in the central region of BMV MP are important for cell-to-cell movement (Fig. 1b and Table 2). The central region of BMV MP is highly conserved among the four bromoviruses (Table 1) and shares sequence similarity with the MPs of viruses belonging to other genera (Koonin et al., 1991). In Red clover necrotic mosaic virus, CMV and AMV, the regions in the MPs corresponding to the central region of BMV MP are also important for infectivity (Giesman-Cookmeyer & Lommel, 1993; Li et al., 2001; Huang et al., 2001). Residues 189–242 in the central region of BMV MP constitute the RNA-binding domain (Fujita et al., 1998). The loss of infectivity of AS mutants with mutations in this domain might be due to a lack or reduction of RNA-binding activity.

Second, BMV MP plays a role in long-distance movement (Table 2, Figs 1b and 4). In inoculated leaves, viruses initially infect epidermal cells and then move into mesophyll and bundle sheath cells and into sequential vascular systems to infect the plants systemically (Carrington et al., 1996). Although six AS mutants and one C-terminally-truncated MP mutant moved locally, they did not move systemically in C. quinoa (Table 2, Figs 1b and 4). The accumulation of CP in leaves inoculated with AS201 and AS208 was significantly low (Fig. 1b). These results suggest that these two AS mutants do not reach the vasculature in the inoculated leaves, due to quite low efficiency in cell-to-cell movement. The other four AS mutants and one C-terminally-truncated MP mutant may have reached the vasculature, but may have specifically failed to move through the vascular system of C. quinoa because the accumulation of CP from the five MP mutants was equal to that of AS244 in inoculated leaves (Figs 1b and 4). AS244 moved systemically, although the accumulation of CP in the inoculated leaves was approximately 100-fold lower than that of wild-type BMV (Fig. 1b). These results suggest that the five BMV MP mutants may not have the ability to overcome a later intercellular barrier(s) (e.g. epidermal–mesophyll, mesophyll–vascular cells and so on) as reported for other virus MPs (Fujita et al., 2000; Wang et al., 1998).

Third, BMV MP could play a role in the ‘unloading’ process in upper uninoculated leaves. After moving into upper uninoculated leaves, plant viruses are sequentially unloaded into companion, phloem parenchyma, bundle sheath and mesophyll cells, and then move into the epidermal cells (Carrington et al., 1996). The lower accumulation of CP in the upper uninoculated leaves of plants infected with AS12, AS27 or AS244 (Fig. 1b) suggests that the MPs of these AS mutants may have a defect in the unloading process. Furthermore, the upper uninoculated leaves of plants infected with these AS mutants displayed mild or no leaf distortions (Table 2). These results suggest that the induction of severe leaf distortions in BMV-infected C. quinoa requires efficient cell-to-cell movement in upper uninoculated leaves.

As shown in this study, C-terminal truncation and point mutations in the C terminus of BMV MP alter the CP requirement in cell-to-cell movement. These findings suggest novel interpretations of two previous studies. First, Sanchez-Navarro & Bol (2001) showed that a chimeric AMV containing wild-type BMV MP (pGFP/B1-303/CP) did not move from cell to cell, whereas a chimeric AMV encoding BMV MP with a deletion of the C-terminal 48 amino acids (pGFP/B1-255/CP) did. We speculate that the BMV MP in pGFP/B1-255/CP has the ability to mediate cell-to-cell movement independently of CP. Second, Flasinski et al. (1995) has exceptionally reported that BMV has the ability to move from cell to cell in the absence of CP. In that report they may not have observed the ability for cell-to-cell movement of a CP-less mutant of BMV but may, instead, have observed that of its progeny viruses, in which point mutations that alter the CP-requirement in cell-to-cell movement had been occasionally introduced into the MP gene during virus replication. This possibility is supported by our recent findings that BMV MP mutants with the ability to move from cell to cell in the absence of CP were isolated in the leaves inoculated with a CP-defective BMV variant (W. Nakamura, A. Takeda, N. Sasaki, and K. Mise, unpublished results).


   ACKNOWLEDGEMENTS
 
We are grateful to Dr Paul Ahlquist for pB3RS2 and the cDNA clones of the BMV M1 strain. We wish to express our appreciation to Dr Iwao Furusawa for valuable suggestions. We thank Dr Hideaki Nagano, Dr Nobumitsu Sasaki and Dr Koki Fujisaki for helpful discussion. This work was supported in part by a Grant-in-Aid (12052201) for Scientific Research on Priority Area (A) from the Ministry of Education, Culture, Sports, Science and Technology, Japan, a Grant-in-Aid (JSPS-RFTF96L00603) from the ‘Research for the Future’ program, a Grant-in-Aid (15380035) for Scientific Research (B) and a Grant-in-Aid (13306005) for Scientific Research (A) from the Japan Society for the Promotion of Science.


   REFERENCES
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Ahlquist, P. (1999). Bromoviruses (Bromoviridae). In Encyclopedia of Virology, 2nd edn, vol. 1, pp. 198–204. Edited by A. Granoff & R. G. Webster. San Diego, CA: Academic Press.

Ahlquist, P., Luckow, V. & Kaesberg, P. (1981). Complete nucleotide sequence of brome mosaic virus RNA3. J Mol Biol 153, 23–38.[Medline]

Allison, R. F., Janda, M. & Ahlquist, P. (1989). Sequence of cowpea chlorotic mottle virus RNAs 2 and 3 and evidence of a recombination event during bromovirus evolution. Virology 172, 321–330.[Medline]

Blackman, L. M., Boevink, P., Santa Cruz, S., Palukaitis, P. & Oparka, K. J. (1998). The movement protein of cucumber mosaic virus traffics into sieve elements in minor veins of Nicotiana clevelandii. Plant Cell 10, 525–537.[Abstract/Free Full Text]

Callaway, A., Giesman-Cookmeyer, D., Gillock, E. T., Sit, T. L. & Lommel, S. A. (2001). The multifunctional capsid proteins of plant RNA virus. Annu Rev Phytopathol 39, 419–460.[CrossRef][Medline]

Canto, T., Prior, D. A., Hellwald, K. H., Oparka, K. J. & Palukaitis, P. (1997). Characterization of cucumber mosaic virus. IV. Movement protein and coat protein are both essential for cell-to-cell movement of cucumber mosaic virus. Virology 237, 237–248.[CrossRef][Medline]

Carrington, J. C., Kasschau, K. D., Mahajan, S. K. & Schaad, M. C. (1996). Cell-to-cell and long-distance transport of viruses in plants. Plant Cell 8, 1669–1681.[Free Full Text]

Carvalho, C. M., Wellink, J., Ribeiro, S. G., Goldbach, R. W. & van Lent, J. W. M. (2003). The C-terminal region of the movement protein of Cowpea mosaic virus is involved in binding to the large but not to the small coat protein. J Gen Virol 84, 2271–2277.[Abstract/Free Full Text]

Choi, Y. G. & Rao, A. L. N. (2003). Packaging of brome mosaic virus RNA3 is mediated through a bipartite signal. J Virol 77, 9750–9757.[Abstract/Free Full Text]

Cormack, B. P., Valdivia, R. H. & Falkow, S. (1996). FACS-optimized mutants of the green fluorescent protein (GFP). Gene 173, 33–38.[CrossRef][Medline]

Cunningham, B. C. & Wells, J. A. (1989). High-resolution epitope mapping of hGH-receptor interactions by alanine-scanning mutagenesis. Science 244, 1081–1085.[Medline]

Damayanti, T. A., Nagano, H., Mise, K., Furusawa, I. & Okuno, T. (1999). Brome mosaic virus defective RNAs generated during infection of barley plants. J Gen Virol 80, 2511–2518.[Abstract/Free Full Text]

Damayanti, T. A., Nagano, H., Mise, K., Furusawa, I. & Okuno, T. (2002). Positional effect of deletions on viability, especially on encapsidation, of Brome mosaic virus D-RNA in barley protoplasts. Virology 293, 314–319.[CrossRef][Medline]

Damayanti, T. A., Tsukaguchi, S., Mise, K. & Okuno, T. (2003). cis-acting elements required for efficient packaging of brome mosaic virus RNA3 in barley protoplasts. J Virol 77, 9979–9986.[Abstract/Free Full Text]

Dawson, W. O., Bubrick, P. & Grantham, G. L. (1988). Modifications of the tobacco mosaic virus coat protein gene affecting replication, movement, and symptomology. Phytopathology 78, 783–789.

de Felipe, P., Hughes, L. E., Ryan, M. D. & Brown, J. D. (2003). Co-translational, intraribosomal cleavage of polypeptides by the foot-and-mouth disease virus 2A peptide. J Biol Chem 278, 11441–11448.[Abstract/Free Full Text]

De Jong, W., Chu, A. & Ahlquist, P. (1995). Coding changes in the 3a cell-to-cell movement gene can extend the host range of brome mosaic virus systemic infection. Virology 214, 464–474.[CrossRef][Medline]

Dohi, K., Mori, M., Furusawa, I., Mise, K. & Okuno, T. (2001). Brome mosaic virus replicase proteins localize with the movement protein at infection-specific cytoplasmic inclusions in infected barley leaf cells. Arch Virol 146, 1607–1615.[CrossRef][Medline]

Emini, E. A., Hughes, J. V., Perlow, D. S. & Boger, J. (1985). Induction of hepatitis A virus-neutralizing antibody by a virus-specific synthetic peptide. J Virol 55, 836–839.[Medline]

Flasinski, S., Dzianott, A., Pratt, S. & Bujarski, J. J. (1995). Mutational analysis of the coat protein gene of brome mosaic virus: effects on replication and movement in barley and in Chenopodium hybridum. Mol Plant Microbe Interact 8, 23–31.[Medline]

Fujisaki, K., Hagihara, F., Kaido, M., Mise, K. & Okuno, T. (2003). Complete nucleotide sequence of spring beauty latent virus, a bromovirus infectious to Arabidopsis thaliana. Arch Virol 148, 165–175.[CrossRef][Medline]

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.[CrossRef][Medline]

Fujita, M., Mise, K., Kajiura, Y., Dohi, K. & Furusawa, I. (1998). Nucleic acid-binding properties and subcellular localization of the 3a protein of brome mosaic bromovirus. J Gen Virol 79, 1273–1280.[Abstract]

Fujita, Y., Fujita, M., Mise, K., Kobori, T., Osaki, T. & Furusawa, I. (2000). Bromovirus movement protein conditions for the host specificity of virus movement through the vascular system and affects pathogenicity in cowpea. Mol Plant Microbe Interact 13, 1195–1203.[Medline]

Giesman-Cookmeyer, D. & Lommel, S. A. (1993). Alanine scanning mutagenesis of a plant virus movement protein identifies three functional domains. Plant Cell 5, 973–982.[Abstract/Free Full Text]

Gillespie, T., Boevink, P., Haupt, S., Roberts, A. G., Toth, R., Valentine, T., Chapman, S. & Oparka, K. J. (2002). Functional analysis of a DNA-shuffled movement protein reveals that microtubules are dispensable for the cell-to-cell movement of Tobacco mosaic virus. Plant Cell 14, 1207–1222.[Abstract/Free Full Text]

Huang, M., Jongejan, L., Zheng, H., Zhang, L. & Bol, J. F. (2001). Intracellular localization and movement phenotypes of Alfalfa mosaic virus movement protein mutants. Mol Plant Microbe Interact 14, 1063–1074.[Medline]

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.

Jansen, K. A., Wolfs, C. J., Lohuis, H., Goldbach, R. W. & Verduin, B. J. (1998). Characterization of the brome mosaic virus movement protein expressed in E. coli. Virology 242, 387–394.[CrossRef][Medline]

Kao, C. C. & Sivakumaran, K. (2000). Brome mosaic virus, good for an RNA virologist's basic needs. Mol Plant Pathol 1, 91–97.[CrossRef]

Kaplan, I. B., Zhang, L. & Palukaitis, P. (1998). Characterization of cucumber mosaic virus. V. Cell-to-cell movement requires capsid protein but not virions. Virology 246, 221–231.[CrossRef][Medline]

Kasteel, D., Wellink, J., Verver, J., van Lent, J., Goldbach, R. & van Kammen, A. (1993). The involvement of cowpea mosaic virus M RNA-encoded protein in tubule formation. J Gen Virol 74, 1721–1724.[Abstract]

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. J Gen Virol 78, 2089–2093.[Abstract]

Koonin, E. V., Mushegian, A. R., Ryabov, E. V. & Dolja, V. V. (1991). Diverse groups of plant RNA and DNA viruses share related movement proteins that may possess chaperone-like activity. J Gen Virol 72, 2895–2903.[Abstract]

Kroner, P. & Ahlquist, P. (1992). RNA-based viruses. In Molecular Plant Pathology: a Practical Approach, vol. 1, pp. 23–34. Edited by S. J. Gurr, M. J. McPherson & D. J. Bowles. Oxford: IRL Press.

Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.[Medline]

Lazarowitz, S. G. & Beachy, R. N. (1999). Viral movement proteins as probes for intracellular and intercellular trafficking in plants. Plant Cell 11, 535–548.[Free Full Text]

Li, Q., Ryu, K. H. & Palukaitis, P. (2001). Cucumber mosaic virus–plant interactions: identification of 3a protein sequences affecting infectivity, cell-to-cell movement, and long-distance movement. Mol Plant Microbe Interact 14, 378–385.[Medline]

Miller, W. A., Dreher, T. W. & Hall, T. C. (1985). Synthesis of brome mosaic virus subgenomic RNA in vitro by initiation on (–)-sense genomic RNA. Nature 313, 68–70.[Medline]

Mise, K. & Ahlquist, P. (1995). Host-specificity restriction by bromovirus cell-to-cell movement protein occurs after initial cell-to-cell spread of infection in nonhost plants. Virology 206, 276–286.[Medline]

Mise, K., Allison, R. F., Janda, M. & Ahlquist, P. (1993). Bromovirus movement protein genes play a crucial role in host specificity. J Virol 67, 2815–2823.[Abstract]

Nagano, H., Mise, K., Okuno, T. & Furusawa, I. (1999). The cognate coat protein is required for cell-to-cell movement of a chimeric brome mosaic virus mediated by the cucumber mosaic virus movement protein. Virology 265, 226–234.[CrossRef][Medline]

Nagano, H., Mise, K., Furusawa, I. & Okuno, T. (2001). Conversion in the requirement of coat protein in cell-to-cell movement mediated by the cucumber mosaic virus movement protein. J Virol 75, 8045–8053.[Abstract/Free Full Text]

Okinaka, Y., Mise, K., Suzuki, E., Okuno, T. & Furusawa, I. (2001). The C terminus of brome mosaic virus coat protein controls viral cell-to-cell and long-distance movement. J Virol 75, 5385–5390.[Abstract/Free Full Text]

Osman, F., Schmitz, I. & Rao, A. L. N. (1999). Effect of C-terminal deletions in the movement protein of cowpea chlorotic mottle virus on cell-to-cell and long-distance movement. J Gen Virol 80, 1357–1365.[Abstract]

Rao, A. L. N. (1997). Molecular studies on bromovirus capsid protein. III. Analysis of cell-to-cell movement competence of coat protein defective variants of cowpea chlorotic mottle virus. Virology 232, 385–395.[CrossRef][Medline]

Rao, A. L. N. & Grantham, G. L. (1995a). A spontaneous mutation in the movement protein gene of brome mosaic virus modulates symptom phenotype in Nicotiana benthamiana. J Virol 69, 2689–2691.[Abstract]

Rao, A. L. N. & Grantham, G. L. (1995b). Biological significance of the seven amino-terminal basic residues of brome mosaic virus coat protein. Virology 211, 42–52.[CrossRef][Medline]

Rao, A. L. N. & Grantham, G. L. (1996). Molecular studies on bromovirus capsid protein. II. Functional analysis of the amino-terminal arginine-rich motif and its role in encapsidation, movement and pathology. Virology 226, 294–305.[CrossRef][Medline]

Romero, J., Dzianott, A. M. & Bujarski, J. J. (1992). The nucleotide sequence and genome organization of the RNA2 and RNA3 segments in broad bean mottle virus. Virology 187, 671–681.[Medline]

Ryan, M. D. & Drew, J. (1994). Foot-and-mouth disease virus 2A oligopeptide mediated cleavage of an artificial polyprotein. EMBO J 13, 928–933.[Abstract]

Sacher, R. & Ahlquist, P. (1989). Effects of deletions in the N-terminal basic arm of brome mosaic virus coat protein on RNA packaging and systemic infection. J Virol 63, 4545–4552.[Medline]

Sanchez-Navarro, J. A. & Bol, J. F. (2001). Role of the alfalfa mosaic virus movement protein and coat protein in virus transport. Mol Plant Microbe Interact 14, 1051–1062.[Medline]

Sanchez-Navarro, J., Miglino, R., Ragozzino, A. & Bol, J. F. (2001). Engineering of alfalfa mosaic virus RNA 3 into an expression vector. Arch Virol 146, 923–939.[CrossRef][Medline]

Sasaki, N., Fujita, Y., Mise, K. & Furusawa, I. (2001). Site-specific single amino acid changes to Lys or Arg in the central region of the movement protein of a hybrid bromovirus are required for adaptation to a nonhost. Virology 279, 47–57.[CrossRef][Medline]

Sasaki, N., Arimoto, M., Nagano, H., Mori, M., Kaido, M., Mise, K. & Okuno, T. (2003). The movement protein gene is involved in the virus-specific requirement of the coat protein in cell-to-cell movement of bromoviruses. Arch Virol 148, 803–812.[CrossRef][Medline]

Schmitz, I. & Rao, A. L. N. (1996). Molecular studies on bromovirus capsid protein. I. Characterization of cell-to-cell movement-defective RNA3 variants of brome mosaic virus. Virology 226, 281–293.[CrossRef][Medline]

Schmitz, I. & Rao, A. L. N. (1998). Deletions in the conserved amino-terminal basic arm of cucumber mosaic virus coat protein disrupt virion assembly but do not abolish infectivity and cell-to-cell movement. Virology 248, 323–331.[CrossRef][Medline]

Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22, 4673–4680.[Abstract]

Toth, R. L., Pogue, G. P. & Chapman, S. (2002). Improvement of the movement and host range properties of a plant virus vector through DNA shuffling. Plant J 30, 593–600.[CrossRef][Medline]

Wang, H.-L., Wang, Y., Giesman-Cookmeyer, D., Lommel, S. A. & Lucas, W. J. (1998). Mutations in viral movement protein alter systemic infection and identify an intercellular barrier to entry into the phloem long-distance transport system. Virology 245, 75–89.[CrossRef][Medline]

Wellink, J. & Van Kammen, A. (1989). Cell-to-cell transport of cowpea mosaic virus requires both the 58K/48K proteins and the capsid proteins. J Gen Virol 70, 2279–2286.

Received 15 January 2004; accepted 6 February 2004.