Journal of General Virology (1991), 72, 2853-2856. Printed in Great Britain
2853
Detection of the movement protein of red clover necrotic mosaic virus in a
cell wall fraction from infected Nicotiana clevelandii plants
T. A. M. Osman and K. W. Buck*
Department of Biology, Imperial College o f Science, Technology and Medicine, London S W 7 2BB, U.K.
The movement protein of red clover necrotic mosaic
virus (RCNMV) was expressed in Escherichia coli as a
fusion with a maltose-binding protein using the vector
pMAL-cRI and used to produce an antiserum. The
R C N M V movement protein was detected in a cell wall
fraction obtained from infected Nicotiana clevelandii
leaf tissue by immunoblotting using the movement
protein antiserum. The movement protein could be
detected 6 h after inoculation and reached a maximum
after 24 h. In contrast, the virus capsid protein,
detected in a soluble fraction by immunoblotting using
a capsid antiserum, continued to increase for 72 h after
inoculation.
Red clover necrotic mosaic dianthovirus (RCNMV) has
a genome of two positive-sense ssRNA components
(Gould et al., 1981). R N A 1 (3.9 kb) has three open
reading frames (ORFs) with the potential to encode (in
the order 5' to Y) proteins of Mr 27000 (27K), 57K and
37K (Xiong & Lommel, 1989). The 57K protein has
amino acid sequence motifs characteristic of nucleic acid
polymerases (Argos, 1988; Xiong & Lommel, 1989) and
helicases (Habili & Symons, 1989; Buck, 1990), suggesting a role in RNA replication; it may be expressed as an
88K fusion protein resulting from a ribosomal frameshift
near the end of the ORF for the 27K protein (Xiong &
Lommel, 1989). The capsid protein (Mr 37K to 40K) is
translated from a subgenomic R N A derived from R N A
1 (Morris-Krsinich et al., 1983; Marriott & Buck, 1988;
Osman & Buck, 1990). The proteins encoded by R N A 1
are consistent with its ability to replicate and produce
virus particles in plant protoplasts in the absence of
R N A 2 (Osman & Buck, 1987; Paje-Manalo & Lommel,
1989).
Production of lesions on inoculated leaves and
systemic invasion of plants requires both R N A 1 and
RNA 2 (Gould et al., 1981 ; Okuno et al., 1983; Osman et
al., 1986), suggesting that R N A 2 encodes a protein
required for cell-to-cell movement of the virus. R N A 2
(1.45 kb) contains a single ORF with the potential to
encode a protein of Mr 35K to 36K (Lommel et al., 1988;
Osman et al., 1991a), which has limited amino acid
sequence homology with the movement proteins of
brome mosaic virus (Lommel et al., 1988) and cowpea
chlorotic mottle virus (Allison et al., 1989). Although a
protein of 35K to 36K has been produced by in vitro
translation of RNA 2 (Lommel et al., 1988; Xiong &
Lommel, 1991; Osman et al., 1991 b), this protein has not
hitherto been detected in vivo. We now report the
detection of the R C N M V movement protein in a cell
wall fraction from infected Nicotiana clevelandii plants.
To produce an antiserum to the R C N M V movement
protein, a fusion protein consisting of a maltose-binding
protein fused to the whole of the movement protein
(excluding the first methionine) was first produced in
Escherichia coli. A full-length cDNA clone of R N A 2 of
R C N M V Czech isolate TpM-34 in pUC19 (Osman et al.,
1991b) was excised from the vector with HindlII and
PstI, and cloned into the corresponding sites of pEMBL9
(+). An EcoRI site was introduced at the start of the
coding region of the movement protein by in vitro
mutagenesis (Kunkel et al., 1987) using an oligonucleotide, A G G T A G G T T T G A A T T C G C T A T T C , which
corresponds to nucleotides 66 to 88 of R N A 2 (Osman et
al., 1991 a). The coding region of the movement protein,
together with the 3' untranslated region, was then
excised with EcoRI and PstI and cloned into pMAL-cRI
(New England Biolabs), a derivative of pMAL-c (Maina
et al., 1988), an expression vector designed to express
proteins as fusions to the C-terminal end of the E. coli
maltose-binding protein. At the fusion junction, the
vector encodes the recognition sequence for Factor Xa
protease, which allows the cleavage of the fusion protein
into its two constituents.
E. coli DH5~ cells containing the recombinant pMALcRI/RCNMV plasmid were grown to a n O D 6 o o of 0"5
and expression of the fusion protein was induced with
IPTG (0.3 mM). After disruption of the cells by sonication, the fusion protein was purified by binding to an
amylose resin, followed by elution with maltose (Maina
0001-0412 0 1991 SGM
2854
Short communication
(a)
1
2
3
4
MBP-MP---
(b)
MBP
MP--
Fig. 1. (a) SDS-PAGE. Lanes: 1, fusion protein, purified by
chromatographyon the amyloseresin; 2, fusion protein cleaved with
Factor Xa protease; 3, maltose-binding protein; 4, RCNMV movement protein. The gel was stained with Coomassie blue R-250. (b)
Immunoblot of a gel similar to that in (a) using the movement protein
antiserum. The positions of the maltose-binding protein (MBP) and
movement protein (MP), and the Mr values of protein standards
(bovine serum albumin, 66K; ovalbumin, 45K; glyceraldehyde-3phosphate dehydrogenase,36K; carbonic anhydrase,29K) are shown.
et al., 1988). In S D S - P A G E (Laemmli, 1970), followed
by staining with Coomassie blue, a band was detected in
the approximate position expected for the fusion protein
(predicted Mr 78K), together with a slightly faster
moving band (apparent Mr 70K) and a doublet in the
approximate position expected for the E. coli maltosebinding protein (Mr 42K) (Fig. l a, lane 1). After
cleavage with Factor Xa protease, only two bands were
detected, one in the position of the maltose-binding
protein and the other which migrated with an apparent
Mr of 36K as expected for the R C N M V movement
protein (Fig. 1a, lane 2). The reason why the movement
protein stained more intensely than the maltose-binding
protein is not known. These two proteins were separated
by chromatography on the amylose resin (Maina et al.,
1988). The maltose-binding protein bound to the column
and was eluted with maltose (Fig. l a, lane 3), whereas
the R C N M V movement protein did not bind to the
column (Fig. 1 a, lane 4).
An antiserum to the fusion protein was prepared in a
rabbit by one intravenous injection [1 mg protein in 1 ml
of 10 mM-sodium phosphate, 0.5 M-NaC1, pH 7.2 (PBS
buffer)] followed by four intramuscular injections (1 mg
protein in PBS buffer, emulsified with an equal volume
of Freund's incomplete adjuvant) given at intervals of 4
weeks. The antiserum was mixed with an equal volume
of glycerol and stored at - 20 °C. A gel, similar to that
shown in Fig. l(a), was blotted onto Hybond-C
membrane and probed with this antiserum (further
diluted 1:200 in PBS). Proteins to which the antibodies
had bound were then detected by incubation with
Protein A-peroxidase and chloronaphthol (Sherwood,
1987) (Fig. 1b). It is clear that the antiserum contained
antibodies both to the maltose-binding protein and to the
R C N M V movement protein. The antiserum did not
react with the virus capsid protein (not shown).
N. clevelandii seedlings were grown for 3 to 4 weeks
after seed germination at 22 to 25 °C and then inoculated
with sap from bean leaves infected with R C N M V - T p M 34. At various time intervals after inoculation, samples
of the inoculated leaf tissue were processed either for
movement protein detection or for coat protein detection
(see below). For the former, a modification of the method
of Albrecht et al. (1988) was used. One g leaf tissue was
ground with a pestle and mortar in 1 ml ice-cold grinding
buffer (GB; 10 mM-KC1, 5 mM-MgC12, 0.4 M-sucrose,
10Yoo v/v glycerol, 10 mM-2-mercaptoethanol, 100 mMTris-HC1 pH 7.5). The mixture was filtered through
muslin and one-third volume of 200 mM-Tris-HC1 buffer
pH 6.8, containing 40 mM-dithiothreitol, 8 ~ (w/v) SDS
and 10~o glycerol was added to the filtrate, followed by
heating at 100°C for 5 min, and centrifugation at
10000g to remove insoluble material. This soluble
fraction was designated S.
The material retained by the muslin was washed twice
with GB buffer containing 2Yoo (v/v) Triton X-100 by
stirring and filtration again through muslin each time.
The final residue was heated for 10 min at 100 °C with
0.2 ml SU buffer (4-5Yo SDS, 9 M-urea, 10 mM-2-mercaptoethanol, 75 mM-Tris-HC1 pH 6-8) and then centrifuged
at 10000 g to remove insoluble material. This cell wall
extract was designated CW.
Fractions S and CW, from healthy leaf tissue or leaf
tissue at different time intervals after inoculation, were
subjected to S D S - P A G E , blotted onto Hybond-C membrane and probed with the movement protein antiserum. Proteins to which the antibodies had bound were
then detected by incubation with Protein A-peroxidase
and chloronaphthol (Sherwood, 1987). No protein was
detected in either fraction from healthy leaf tissue,
confirming the specificity of the antiserum. A protein
with the same mobility as the movement protein was
detected in the CW fraction from leaf tissue 6 h after
inoculation, reached a maximum 24 h after inoculation
and thereafter declined, although it could still be
detected 96 h after inoculation (Fig. 2a). The movement
protein, detected in the cell wall fractions, formed a
closely spaced doublet in S D S - P A G E . Whether this
indicates that there are two forms of the movement
Short communication
(a)
1
2
3
4
5
6
7
8
9
(b)
1
2
3
4
5
6
7
8
Fig. 2. (a) Immunoblotusing the movementprotein antiserum. Lane 1,
fusion protein cleaved with Factor Xa protease. Lanes 2 to 9, cell wall
fractions (CW) from: healthy leaf tissue (lane 2); leaf tissue
immediatelyafter inoculation(lane 3); leaf tissue 6 0ane 4), 12 (lane 5),
24 (lane6), 48 (lane 7), 72 (lane 8) and 96 h (lane9) after inoculation.(b)
Immunoblot using the capsid protein antiserum. Lanes 1 to 8, soluble
fractionsfrom: healthyleaf tissue (lane 1); leaf tissue immediatelyafter
inoculation (lane 2); leaf tissue 6 (lane 3), 12 (lane 4), 24 (lane 5), 48
(lane 6), 72 (lane 7) and 96 H (lane 8) after inoculation.The positionsof
the maltose-binding protein (MBP), movement protein (MP) and
capsid protein (CP), and the Mr values of protein standards (as in Fig.
1) are shown.
protein will require further investigation. It is
noteworthy that the movement protein produced in E.
coli, using the pMAL-cRI vector, also formed a closely
spaced doublet. The decline in the level of the movement
protein after 24 h suggests that degradation of the
movement protein had occurred, since expansion of the
N. clevelandii leaves was insufficient to account for the
observed reduction.
No movement protein was detected in fraction S from
leaf tissue up to 96 h after infection. However, the
possibility that some movement protein could have been
present in a membrane subfraction of fraction S
(pelletable at 30000g; Pe-30) cannot be discounted,
since pelleting this subfraction would have concentrated
the movement protein (if present) and hence increased
the sensitivity of detection. It is noteworthy that some of
the putative movement protein of alfalfa mosaic virus
was located in Pe-30, although the majority was found in
the cell wall fraction (Godefroy-Colburn et al., 1986).
For comparison, the time course of accumulation of
the R C N M V capsid protein was also measured. Twohundred mg leaf samples were ground in 50 mM-TrisHC1, 2 ~ SDS, 10mM-dithiothreitol, 1 0 ~ glycerol,
2855
pH 6.8, heated at 100 °C for 5 min, cooled, centrifuged to
remove insoluble debris and subjected to S D S - P A G E .
After electrophoresis, the gel was blotted onto Hybond-C
membrane and the blot was incubated with an antiserum to the R C N M V capsid protein (diluted 1 : 1000 in
PBS) (Osman & Buck, 1987). Proteins to which
antibodies had bound were detected as before (Sherwood, 1987). The amount of coat protein detected
increased with time reaching a maximum 72 h after
inoculation.
There is accumulating evidence that cell-to-cell movement of plant viruses occurs through intercellular
connections, the plasmodesmata, and that such movement is mediated by virus-encoded movement proteins
(Hull, 1989; Robards & Lucas, 1990; Atabekov &
Taliansky, 1990). Immunocytological studies have
shown that the movement protein of tobacco mosaic
virus (TMV) is localized in the plasmodesmata, both in
infected plants and in uninfected transgenic plants
expressing the movement protein from a chromosomally
inserted gene (Tomenius et al., 1987; Atkins et al., 1991),
and that the protein is found in a cell wall-enriched
fraction after subcellular fractionation of tissue from
infected plants (Moser et al., 1988; Lehto et al., 1990).
Putative movement proteins of a number of other
viruses, e.g. alfalfa mosaic virus (Godefroy-Colburn et
al., 1986; Stussi-Garaud et al., 1987) and cauliflower
mosaic virus (Albrecht et al., 1988; Linstead et al., 1988)
have also been localized to the cell wall or plasmodesmata. The finding of the R C N M V movement protein in
a cell wall fraction therefore is consistent with the
possibility that this virus also moves between cells via the
plasmodesmata. However, immunocytological studies
will be needed to determine the subcellular location of its
movement protein more precisely.
The finding that the R C N M V movement protein
reaches a maximum 24 h after inoculation, whereas the
capsid protein continues to accumulate up to 72 h is
consistent with similar studies of the accumulation of the
T M V movement protein (Lehto et al., 1990) which
indicated that most of the movement protein accumulated during the early phase of virus replication. If only
single-stranded nucleic acids complexed with viral
movement proteins can be transported efficiently
through plasmodesmata, as speculated recently by
Citovsky et al. (1991), a temporal separation of movement protein and capsid protein production would
reduce competition for binding to the same RNA.
Further studies will be needed to determine whether the
R C N M V movement protein binds to single-stranded
nucleic acids.
We thank the Agricultural and Food Research Council for a grant.
RCNMV was held under licence no. PHF1250A/53(l10) obtained
from the Ministry of Agriculture, Fisheries and Food.
2856
Short communication
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(Received 5 June 1991; Accepted 23 July 1991)