J. gen. ViroL (1989), 70, 2279-2286. Printed in Great Britain
2279
Key words: CPMV/M RNA mutants/cell-to-cell transport
Cell-to-cell Transport of Cowpea Mosaic Virus Requires Both the 58K/48K
Proteins and the Capsid Proteins
By J O A N W E L L I N K * AND AB V A N K A M M E N
Department o f Molecular Biology, Agricultural University, Dreijenlaan 3, 6703 H A Wageningen,
The Netherlands
(Accepted 17 May 1989)
SUMMARY
Insertions and deletions have been introduced into an infectious c D N A clone of M
R N A of cowpea mosaic virus (CPMV), in the coding regions of the 58K/48K and
capsid proteins. Transcripts derived from these mutant clones appeared to be
replicated in cowpea protoplasts as detected by immunofluorescent staining and
Northern blotting. However in cowpea plants, mutations in either region restricted the
replication of the viral RNAs to the inoculated cells and thus prevented a successful
systemic infection of the plant. These results indicate that the M RNA-encoded
58K/48K proteins are involved in cell-to-cell transport of CPMV, and that the virus
can spread only if the R N A is encapsidated in particles.
INTRODUCTION
Cowpea mosaic virus (CPMV) has a genome consisting of two positive-sense ssRNA
molecules. Both RNAs (M, middle component; B, bottom component) are translated into
polyproteins which are cleaved by a B RNA-encoded protease into functional proteins
(Goldbach & van Kammen, 1985; Vos et al., 1988a). B R N A is able to replicate independently
of M RNA in isolated protoplasts and encodes all the functions necessary for replication of the
RNAs (Goldbach et al., 1980; Eggen & van Kammen, 1988). However for a successful infection
of plants, M R N A is essential. Therefore it probably encodes proteins involved in transport of
the virus (Rezelman et al., 1982).
M RNA is translated in vitro into two polyproteins, 105K and 95K (see Fig. 1), which have
overlapping amino acid sequences. The translation of the smaller polyprotein is initiated at an
internal A U G codon (Vos et al., 1984). Proteolytic processing of the polyproteins yields 58K and
48K proteins, and the 60K precursor to the capsid proteins (Fig. 1; Franssen et al., 1982).
Because M R N A expression is necessary for plants to become infected, it was postulated that the
58K and/or 48K proteins were involved in cell-to-cell transport of the virus (Rezelman et al.,
1982). The occurrence of the 48K protein in the membrane fraction of infected leaves supported
this notion (Wellink et al., 1987). Furthermore, the 105K, 95K and 58K proteins have been
detected in protoplasts inoculated with CPMV (G. Rezelman, A. van K a m m e n & J. Wellink,
unpublished results) which shows that the same proteins are produced in vivo as in vitro.
A full-length e D N A clone of M R N A has been obtained which produced infectious R N A by
in vitro transcription (Vos et al., 1988b; Eggen, 1989). To test the involvement of the M RNAencoded proteins in cell-to-cell transport of the virus, this c D N A clone has been used to create
insertion and deletion mutants in the coding regions of the 58K/48K proteins and the capsid
proteins. The effects of these mutations on infectivity was studied in cowpea protoplasts and
cowpea plants.
METHODS
Construction of mutant M eDNA clones. (See Fig. 2.) pTMABgl and pTMANco containing four nucleotide
insertions in the M eDNA region were constructed by filling in the protruding ends left after digestion of pTMIG
(Eggen, 1989)with BglII and NcoI respectively,followedby ligation of the blunt ends. pTMAXho and pTMAEco
0000-8941 © 1989 SGM
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J. W E L L I N K AND A. VAN KAMMEN
512/524
161 I
I
I
M RNA
3299
I
I
I
I
I-'-'A.
105K
•
X7
95K
•
V
58K
60K
48K
60K
VP37
Fig. 1. Model for the expression of CPMV M
RNA. The ORF on the RNA is indicated by an
open bar and the positions of the translational
start and stop codons are indicated on this bar.
VPg is represented by a black square and the
other proteins by single lines below. Both capsid
proteins are indicated by thick lines. Gin/Met
and Gln/Gly cleavage sites are indicated by solid
arrowheads and open arrowheads, respectively.
VP23
Coat proteins
VPg
_..115,','SK] 1",
',
ABgl
I
Ill
48K_ ...... 2 '
AB
AP
AXho
I1
] VP23 ~ " AAA
1
VP37
x
/
AEco
95K
ABgl
48K
1
2K
AXho
90K
~ 60K
80K
70K
AEco
88K
78K
AP
35K
25K
1
ANco
100K
60K
62K
AB
i
/
58K ~
48K
100K
90K
60K
60K
27K
27K
ANco
|
58K
~
48K
55K
55K
Fig. 2. Model for the expression of the mutant M RNAs. The positions of the cleavage sites used for the
construction of the mutants are shown above the open bar that represents the M R N A ORF. For each
mutant are shown the primary translation products and the cleavage products which are expected upon
processing.
containing deletions in the M cDNA region were created by digestion of pTMIG with XhoI or EcoRV
respectively, followed by ligation of the plasmids. This resulted in loss of the small XhoI or EcoRV fragments
respectively. Two other plasmids containing deletions in the M cDNA region, pTMAP and pTMAB, were
constructed by digestion of pTMIG with Accl and PvulI, or BgllI and BamHI respectively, filling in of the
protruding ends and subsequent ligafion of the blunt ends.
In vitro transcription and translation. In vitro transcription of the M cDNA clones with T7 RNA polymerase
(New England Biolabs) was as described by Vos et al. (1988a). The transcripts were translated in rabbit
reticulocyte lysates in the presence of[3sS]methionine for 1 h at 30 °C (Vos et al., 1988a). In addition, processing of
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Proteins required for transport o f C P M V
2281
the translation products was carried out with 3 volumes of non-labelled translation products of CPMV RNA for
6 h at 30 °C. The proteins were separated in a 10% polyacrylamide gel (Richards et al., 1989).
Inoculation ofcowpea protoplasts andplants. Cowpea protoplasts were isolated and inoculated with RNA using
polyethylene glycol as described by Eggen (1989).
For inoculation of cowpea plants the upper epidermis of primary leaves of 9 day-old plants were dusted with
carborundum powder. Five p_gof the pTBIG transcript together with 5 ~tgof the pTMIG or mutant M transcript
(in 30 ~tl HE0) were rubbed on the leaves with a gloved finger. The plants were grown at 28 °C under continuous
illumination.
Analysis ofprotoplasts. At 42 h post-inoculation (p.i.) protoplasts were collected for immunofluorescent staining
by centrifugation. Anti-24K serum was used to determine the amount of cells infected with B RNA, and antiCPMV or anti-48K serum (Wellink et al., 1987) was used to detect cells infected with M RNA and producing M
RNA-specific proteins as described by Vos et al. (1988b). For Northern blot analysis protoplasts were collected by
centrifugation at 68 h p.i. and the pellets were frozen in liquid nitrogen. The RNA was extracted from the
protoplasts with phenol and precipitated from 2 M-LiC1as described by de Vries et al. (1982). The RNAs were
glyoxalated and separated in a 1% agarose gel, blotted onto GeneScreen and hybridized with an M RNA-specific
32p-labelled probe according to instructions supplied by the manufacturer (New England Nuclear). The probe
comprised the SatI/BamHI fragment of pTMIG.
Analysis of plants. Six days p.i. the primary leaves were homogenized in 3 ml/g HB buffer (50 mM-Tris-acetate
pH 7.4, 10 raM-potassium acetate, 1 mM-EDTA, 5 mM-2-mercaptoethanol, 0.5 mM-PMSF) and filtered through
two layers of Miracloth (Calbiochem). The filtrate was subjected to a low-speed centrifugation to remove debris
and the supernatant fraction was assayed for CPMV-specific proteins by immunoblot analysis using anti-24K
serum and anti-VP23 (coat protein) serum (Richards et al., 1989).
RESULTS
Construction o f mutant M R N A s
W e have used the full-length c D N A clone p T M I G to introduce deletions and small insertions
into the open reading frame ( O R F ) of M R N A . In vitro transcription o f these c D N A clones
resulted in several mutant M R N A s . M A N c o R N A and M A E c o R N A contained mutations in
the coding region of the capsid proteins created by an insertion of four nucleotides into the
coding region of VP23 and a deletion of most of the coding region of VP23 and part of VP37,
respectively (Fig. 2 and 5). MAXho R N A lacks the coding region for the glutamine/methionine
cleavage site between the 58K/48K proteins and the 60K precursor to the capsid proteins as a
result of the deletion of a XhoI fragment. MAP, MAB and MABgl R N A s have mutations in the
coding region of the 58K/48K proteins created respectively by a 486 nucleotide deletion in the
48K protein encoding region, a 1311 nucleotide deletion in the 58K protein encoding region and
a four nucleotide insertion disrupting only the coding region o f the N-terminal end o f the 58K
protein (Fig. 2 and 4).
To examine whether the O R F s in the deletion mutants were disrupted and frameshifts were
present at the correct position in the insertion mutants, the m u t a n t R N A s were first analysed by
in vitro translation in rabbit reticulocyte lysates. As expected the m u t a n t that lacked the 48K
protein encoding region (MAB) produced only one polyprotein (Fig. 2 and 3). MABgl R N A ,
containing an insertion in the 58K protein encoding region, was expected to produce only the
95K polyprotein; however small amounts of a 110K protein were also generated (Fig. 3, lane
10). To explain the production of this protein it is necessary to assume that a frameshift occurs
during translation, because the two A U G codons preceding the frameshift at positions 115 and
161 are out of frame with the MABgl R N A O R F (Van Wezenbeek et al., 1983). As expected, the
other m u t a n t R N A s produced two polyproteins.
All polyproteins could still be cleaved by the B R N A - e n c o d e d protease in an in vitro
processing assay, except for the two polyproteins produced from MAXho R N A which lack the
cleavage site (Fig. 3). The N-terminal cleavage product of the 88K polyprotein of MAP R N A
was present in smaller amounts than expected (Fig. 3, lane 13). This truncated protein is
probably very unstable and degraded rather rapidly.
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J. W E L L I N K
M
I
--
7oK- m
ANco
+
II
--
+
A N D A. VAN K A M M E N
AEco AXho
11
--
+
II
ABgl
II
-
+ --
+
AP
It
AB
+
II
-
I
+
.......
105K-- ' ° " ~
%
58K--:~
:
/2?: ;:~):
:;+,
.
.
.
,....
.
.
i
32K--~I
1
--
62K
--
55K
~ ?:i?:
::: : : ~
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Fig. 3. In vitro translations of the M RNA mutants. The translations were analysed in a 10%
polyacrylamide gel. A plus sign (+) on top of each lane indicates that unlabelled translation products of
CPMV RNA were added followed by incubation for 6 h at 30 °C. Lane 1 contains the translation
products of CPMV RNA.
Infectivity o f the mutant M R N A in cowpea protoplasts
To determine whether the different M R N A mutants had lost their ability to be replicated,
cowpea protoplasts were inoculated with transcripts from the full-length c D N A clone of B
R N A , pTBIG, plus the M R N A mutant transcripts. The percentage of infected cells 42 h p.i.
containing B and M RNA-encoded proteins was determined by immunofluorescent staining. The
anti-24K serum was used to detect the protoplasts infected with B R N A ; both anti-CPMV
serum and anti-48K serum were used to detect protoplasts infected with M R N A . When 25 gg of
the p T B I G transcript and 25 gg of the p T M I G transcript were used to inoculate 2.5 x 106
protoplasts, about 20% of the living protoplasts became infected with B R N A and about 10%
became infected also with M R N A . In Fig. 4 and 5 the percentage of infected cells found with
the mutant M R N A s are shown, relative to the 10% found from infection with the nonmutagenized p T M I G transcripts. Almost all mutants were replicated as judged by the
production of M RNA-encoded proteins in the protoplasts, with the exception of the MABgl
mutant. Cells in which MAEco R N A was replicated could be identified only with anti-48K
serum, and not with the anti-CPMV serum. The anti-CPMV serum probably cannot react with
the truncated VP37 that is expressed in these cells. On the other hand, the severely truncated
58K protein that is expressed in MAB RNA-infected cells is still recognized by the anti-48K
serum. In the case of the MABgl mutant, no fluorescent cells were detected in spite of the fact
that in the previous in vitro translations the production of the 95K protein was not affected by the
mutation.
For some mutants the replication of the M R N A was also examined by Northern blotting. At
68 h p.i., R N A was isolated from protoplasts inoculated with these mutants and analysed on a
Northern blot using an M RNA-specific probe. This analysis showed that for these mutants,
R N A of the expected size was found to hybridize with the M RNA-specific probe (Fig. 6). Also
it was found that MABgl R N A was still replicated, although at a low level (Fig. 6, lane 2).
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Proteins required for transport of CPMV
,
MIG
='{sSKI
2283
Fluorescent cells (%) with
Anti-48K
Anti-CPMV
, Infectivity
on plants
+
4gK
I
VP37 I v v 2 3 ~
1oo
10o
48K
I
VP37
I VP23~V
0
0
[
VP37
I VP23~VV
100
100
VP37
[ VP23~"~V
0'5
0'5
+4
MABgl Y [
-
MAP
486
Y
d35Ki 25K
-1311
Y
MAB
-~l
2K
Fig. 4
r
MIG
~15~!
48K
1
vP37
I vP231.-~
Fluorescent cells (%) with
Anti-48K
Anti-CPMV
1 Infectivity
on plants
ioo
100
+
ND
25
ND
50
50
0
- 174
MAXho
Y
--185KI
75K
I VP23 ~
+4
MANco
-158K!
48K
l
VP37 I I S K ~
-784
\ /
MAEco
"-'{5SKi
48K
] 27K 1~-"-"~
-
Fig. 5
Fig. 4. Infectivity of M RNA mutated in the 58K/48K protein encoding region in cowpea protoplasts
and plants. The infectivity of the pTMIG transcript in protoplasts is defined as being 100K. The
position and the size of the deletion or insertion is shown for each mutant M RNA.
Fig. 5. InfectivityofM RNA mutated in the capsid proteins encoding region in cowpea protoplasts and
plants. Details as for Fig. 4. ND, Not determined.
Infectivity of the mutant M RNAs in plants
H a v i n g established that the mutant M R N A s had not lost their capacity to be replicated in
infected cells, the mutants were tested for their ability to spread in cowpea plants. P r i m a r y
cowpea leaves were inoculated with 5 ~tg of the p T B I G transcript together with 5 p.g of the
p T M I G transcript or with a m u t a n t M R N A . After 3 days, symptoms were visible on these
leaves infected with p T M I G R N A ; however, symptoms could not be detected with any of the
m u t a n t M R N A s , even after 6 days. To test whether virus-specific proteins were synthesized in
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J. W E L L I N K AND A. VAN K A M M E N
5
Fig. 6. Northern blot analysis of the M R N A mutants: ABgl, lane
2; AEco, lane 3; AP, lane 4; MIG, lane 5. Total R N A was
extracted from protoplasts at 68 h p.i. and about 10 p.g R N A
was analysed on a glyoxal gel, except for lane 3 which contains
about 5 ~tg RNA. Lane 1 contains R N A from non-infected
protoplasts, and lane 6 contains R N A from protoplasts
inoculated with 1 ktg C P M V RNA. Lanes 1 to 4 were exposed
for 16 h (with a screen) and lanes 5 and 6 for 3 h to a Kodak
X A R film.
these plants crude extracts were prepared of the primary leaves 6 days p.i. and these extracts
were analysed by Western blotting. In the leaves inoculated with the mutant M RNAs virusspecies proteins were not detected (data not shown), whereas in leaves inoculated with nonmutant p T M I G RNA, virus-specific proteins were detected, and virus could be recovered from
these leaves, as described by Eggen (1989).
DISCUSSION
The B RNA of CPMV is able to replicate independently in isolated protoplasts without M
R N A being present, but for a successful infection of a whole cowpea plant, M R N A is
indispensable (Goldbach et al., 1980; Rezelman et al., 1982). The availability of both the
protoplast system and the plant to study the molecular biology of CPMV makes it possible to
separate functions such as replication and transport. In this paper we have directed our attention
to the latter function.
Although M R N A contains only one large ORF, two sets of proteins can be easily
distinguished. These are the 58K and 48K putative transport proteins at the N terminus and both
capsid proteins VP23 and VP37 at the C terminus of the 105K and 95K polyproteins. In this
paper we have shown that both sets of proteins are necessary for a successful infection of a whole
cowpea plant. With respect to the 58K and 48K proteins, this result was not unexpected. These
proteins show a limited homology with the 30K transport protein of tobamoviruses (Meyer et al.,
1986; Deom et al., 1987; Meshi et al., 1987). Furthermore the 48K protein is found in the
membrane fraction of infected leaves (Wellink et al., 1987) and recent results with immunogold
labelling of sections of infected leaves with anti-48K serum have revealed an association of this
protein with tubular structures protruding from the cell wall (J. van Lent, unpublished results).
These structures are found only in infected cells and are probably involved in ceil-to-cell
transport of the virus. The need for an intact coding region of the capsid proteins for successful
infection of a plant is a strong indication that during a normal infection CPMV is transported as
particles and not as naked RNA. This is supported by the observation that until now no coatless
mutants of CPMV have been identified.
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Proteins required for transport o f C P M V
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Malyshenko et al. (1988) have reported that it is possible to complement the transport of the B
R N A of red clover mottle comovirus (RCMV) with sunn-hemp mosaic tobamovirus (SHMV).
At first sight this result seems contradictory with our results because the B R N A of RCMV will
probably not be encapsidated by the SHMV coat protein and therefore must be transported as
naked RNA in this mixed infection. However a simple explanation would be to assume that the
CPMV transport protein(s) transports (or facilitates the transportation of) encapsidated RNA
only, whereas the tobarnovirus transport protein can transport (or perhaps can transport only)
naked RNA; tobacco mosaic tobamovirus coat protein is indeed dispensable for transport from
cell to cell (Takamatsu et al., 1987).
The results with the deletion mutants of M R N A show that substantial parts of the coding
region are dispensable for replication in cowpea protoplasts. The numbers of fluorescent
protoplasts obtained by inoculation with the mutants cannot be taken as an exact measure of the
replicability of the RNAs. The reason for this is that the truncated proteins may be less stable, or
less reactive than wild-type proteins are with the antisera. Also with Northern blot analysis,
differences between the mutants are caused not only by the differences in replicability of the
RNAs but also by differences in stability of the RNAs and whether they are still encapsidated.
Ideally, differences in replicability of the RNAs should be determined in an in vitro replication
system, but so far all attempts to develop such a system for CPMV have failed (Eggen & van
Kammen, 1988).
In CPMV-infected protoplasts, the 48K protein was found to be present in the membrane
fraction (Wellink et al., 1987). Recently the 105K, 95K and 58K proteins have also been
detected in protoplasts (G. Rezelman, A. van Kammen & J. Wellink, unpublished results). If
M R N A is translated into the same polypeptides in vitro and in vivo, it is very likely that, in vivo,
separate A U G codons are used to produce the 105K and 95K polyproteins as was shown to
occur in vitro by Vos et al. (1984). In this respect the result with the MABgl mutant in protoplasts
is difficult to explain. In in vitro translations the 105K protein is no longer produced because of a
four nucleotide insertion behind the start codon of this protein. As expected, the production of
the 95K protein was not affected in vitro. In spite of this, translation products could not be
detected in protoplasts inoculated with this mutant, although the R N A was replicated as
revealed by Northern blotting.
We thank Rik Eggen and Jan Verver for providing us with infectious c D N A clones, Piet Madern and Peter van
Druten for artwork and photography and Gr6 Heitk6nig for preparation of the manuscript.
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