Download Interference in Infections of Tobacco Protoplasts with Two

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J. gen. Virol. (1983), 64, 1347-1354. Printed in Great Britain
Key words: mixed infection/bromovirus/interference/tobacco protoplasts
Interference in Infections of Tobacco Protoplasts with Two Bromoviruses
By F. S A K A I , J. R. O. D A W S O N
AND J. W . W A T T S *
John Innes Institute, Colney Lane, Norwich N R 4 7UH, U.K.
(Accepted 25 January 1983)
SUMMARY
Freshly prepared tobacco mesophyll protoplasts behave as if they contain two
classes, one of which is resistant to infection but the other is susceptible and can readily
be doubly infected with brome mosaic virus (BMV) and cowpea chlorotic mottle virus
(CCMV). BMV dominates in all double infections and although both types of virus
particles are produced, only those of BMV are infectious. The defective C C M V
particles contain normal amounts of R N A but only R N A 3 could be detected in them.
No CCMV R N A 2 could be detected in doubly infected protoplasts, showing that there
was only partial replication of the genome of CCMV. The proteins encoded by C C M V
R N A 3 were produced in mixed infections. It is possible that BMV may exploit the
gene products of CCMV while suppressing synthesis of infectious C C M V particles.
There was no evidence that BMV R N A was encapsidated in C C M V coat protein.
INTRODUCTION
Isolated plant protoplasts are well suited for the study of mixed virus infections (Dawson et al.,
1975; Otsuki & Takebe, 1976, 1978; Barker & Harrison, 1978; Watts & Dawson, 1980). They
can be efficiently infected with two or more viruses and virus development is synchronous. I n a
previous paper (Watts & Dawson, 1980) we described experiments with the two related
bromoviruses brome mosaic virus (BMV) and cowpea chlorotic mottle virus (CCMV) in tobacco
protoplasts. Mixed infections were readily produced and interference between the two viruses
was observed. W h e n both viruses developed in a protoplast, BMV always dominated and there
was little if any synthesis of infectious CCMV, although fluorescent antibody staining, specific
for intact virus (Bancroft, 1970), showed that C C M V capsids were present. Attempts to rescue a
temperature-sensitive (ts) strain of C C M V with BMV were unsuccessful and it was found that
BMV suppressed the synthesis of infectious C C M V R N A . We report here further studies of
interactions of C C M V and BMV in mixed infections designed to clarify these results.
METHODS
Protoplast preparation and infection
Protoplasts were prepared from leaves of Nicotiana tabacum (cv. White Burley or Xanthi) and cultured by the
methods used previously (Motoyoshi et al., 1974a, b). Type strain CCMV and V5 strain BMV were used
throughout. Protoplasts were doubly infected with viruses by one of the following methods.
Consecutive inoculation. Freshly pelleted protoplasts (2 × 106) were resuspended in 20 ml citrate-buffered
mannitol (CMB, 0.01 M-potassium citrate in 0.7 i-mannitol, pH 5-2) containing poly-L-omithine (tool. wt.
120000; 1~tg/ml)and either CCMV (1 Ixg/ml)orBMV (5 ~tg/ml).The inoculum was preincubated for l0 min before
use. After 10 min the protoplasts were collected by centrifugation (600 rev/min for 2 min), washed with 0.7 Mmannitol, and resuspended in a similar volume of inoculum containing the other virus. After a further 10 min the
protoplasts were washed twice with 0-7 M-mannitoland cultured.
Simultaneous inoculation. Freshly pelleted protoplasts (2 × 106)were resuspended in 20 ml CMB containing
poly-L-ornithine(1 ~tg/ml),CCMV (1 ~tg/ml)and BMV (5 ~tg/ml)for 10 min. The inoculum was preincubated for 10
min before use. The protoplasts were then washed twice with 0-7 M-mannitol and cultured.
Polyethylene glycol inoculation. Freshly pelleted protoplasts (2 x 106) were resuspended in 1 ml 40~ (w/v)
polyethylene glycol 1500 (PEG) containing 25 ~tg CCMV, 20 ~tg BMV and 3 mM-CaCl2 and immediately after
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F. SAKAI, J. R. O. DAWSON AND J. W. WATTS
mixing diluted to 40 ml with 0.7 i-mannitol. After 30 min the protoplasts were washed three times with 0.7 Mmannitol and cultured (Dawson et al., 1978).
Culture and labelling ofprotoplasts. Protoplasts were cultured at a density of 1 x 105 to 2 x 105/ml in conical
flasks. When radioactivity was present, 5 ml cultures in 25 ml flasks were used. 1*C-labelled compounds were
added at 2 ~tCi/ml, and 3H-labelled compounds at 25 ~tCi/ml. L-[U-14C]Leucine (300 mCi/mmol), L-[4,53H]leucine (50 Ci/mmol), [U-14C]uridine (500 mCi/mmol), and carrier-free [32p]orthophosphate were obtained
from Amersham International.
Assay of infectivity. The percentage of infected protoplasts was determined, after at least 24 h of culture, by
staining with fluorescent antibody; 300 protoplasts were scored (Motoyoshi et al., 1974 b). Biological infectivity
was assayed by the methods of Watts & Dawson (1980).
Separation of viruses by electrophoresis. To separate particles of BMV from those of CCMV, infected protoplasts
were homogenized in 0-1 M-acetate buffer (pH 5-0) and the clarified homogenate was fractionated by
electrophoresis in cylindrical 2.5~ polyacrylamide gels using the procedure of Hull & Lane (1973).
Extraction o f R N A . RNA was prepared by a method similar to that described by Aoki & Takebe (1975) and
fractionated by electrophoresis (Loening, 1969).
When the RNA in virus separated by gel electrophoresis was required, the relevant gel sections were
homogenized in the buffer used to extract whole RNA (bands from six gels in 1 ml buffer). The homogenates were
allowed to stand for 1 h at room temperature before being loaded onto polyacrylamide gels for electrophoresis.
Electrophoresis of proteins. Proteins were fractionated by the methods of Sakai et al. (1979).
RESULTS
Improving the efficiency of double infection
The susceptibility of protoplasts to infection depends on their physiological condition
(Motoyoshi et al., 1974 b). As experience was gained with double infections it became apparent
that even when the final level of infection was quite low, e.g. 25%, over 90% of these would be
doubly infected. That is to say, provided there was a minimum of delay between inoculations,
the protoplasts that became infected were doubly infected (Table 1). When there is a delay, even
of minutes, between inoculations, there is always an appreciable risk that singly infected
protoplasts will be present. The method of inoculation was therefore modified to reduce this
possibility. BMV and CCMV have opposite electrical charges under the conditions used for
inoculation (citrate-buffered mannitol, pH 5.2) and readily associate. Both viruses were
therefore preincubated together in the presence of poly-L-ornithine to produce mixed
aggregates, and the mixed inoculum was used for what was literally a simultaneous double
inoculation. In this way, variations caused by destruction of protoplasts, inactivation or
activation through washing etc. were reduced or eliminated. In practice this gave cultures in
which all infected protoplasts were reliably doubly infected and simplified analysis of the results,
even when the batch of protoplasts did not have a very high susceptibility to infection.
Attempts to recover infectious CCMV from mixed infections
When care was taken to ensure that virtually all infected protoplasts were doubly infected
with BMV and CCMV, as measured by fluorescent antibody staining, infectivity assays on
Chenopodium hybridum, barley and cowpea showed that there were large amounts of infectious
BMV but no detectable infectious CCMV. This result was in agreement with the previous
observations that a ts strain of CCMV could not be rescued with BMV at 35 °C because
synthesis of infectious CCMV R N A had been suppressed in mixedly infected protoplasts
(Watts & Dawson, 1980).
Production of CCMV particles in mixed infections
The absence of infectious CCMV from protoplasts that fluorescent antibody staining had
shown to be infected with both CCMV and BMV suggested that only CCMV coat protein might
be formed. Alternatively, the level of CCMV production might be below the limits of sensitivity
of the biological assays; this is however unlikely because assay directly onto the systemic host
cowpea was extremely sensitive. The differing charges on CCMV and BMV allow a simple
physical separation of the viruses by polyacrylamide gel electrophoresis. Under the conditions
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Interference in mixed virus infections
Table 1. Mixed infection of tobacco protoplasts
Method of
inoculation*
Consecutive
Simultaneous
PEG
Number of
experiments
7
7
2
Fluorescing protoplasts (%)
r
~
~
Total
BMV
CCMV
64
62
63
74
74
71
66
63
67
Range of
~ total
infection
47-85
50-94
65-67
* For experimental details, see Methods.
t Error (standard deviation) for results with consecutive or simultaneous inoculation were + 3~; for PEG
inoculation + 5~.
used routinely for electrophoresis of the bromoviruses (Hull & Lane, 1973) BMV travelled
towards the cathode and C C M V towards the anode. When homogenates of doubly infected
protoplasts were fractionated on polyacrylamide gel, substantial amounts of material that comigrated with BMV or C C M V were detected. These bands corresponding to the viruses were
cut out and assayed on C. hybridum, barley and cowpea leaves. The band migrating with BMV
behaved as BMV uncontaminated by C C M V but that co-migrating with C C M V possessed no
infectivity, either of BMV or CCMV. Electrophoresis of the proteins in these bands in S D S polyacrylamide gels (Weber & Osborn, 1969) showed that they corresponded to the respective
coat proteins. There was therefore production of non-infectious C C M V particles and no
transcapsidation either of C C M V R N A into BMV capsids or vice versa.
The presence of RNA in non-infectious CCMV particles
C C M V coat proteins spontaneously reassociate to form 'empty' capsids (Bancroft et al.,
1968). The u.v. absorption spectrum of the particles separated on polyacrylamide gels showed,
however, that they contained nucleic acid. The precise ratio of coat protein to nucleic acid was
not easily determined directly because of the difficulties of preparing sufficient purified virus to
allow analysis. Doubly infected protoplasts were therefore cultured in medium containing
[3H]leucine and [14C]adenine to label proteins and R N A . The protoplasts were homogenized
and the viruses separated on cylindrical polyacrylamide gels. The gels were cut into slices and
the ratio of 3H : 14C determined in each slice. If the specific radioactivities of the pools of protein
and R N A precursors are assumed to be the same for both viruses, then the ratio should give
some indication as to whether the C C M V particles were rich or poor in R N A relative to BMV.
The results (Fig. 1) showed that the ratios were similar for both infectious BMV and noninfectious CCMV. The samples indicated by the arrows contained in the case of BMV (Fig. 1 a)
905 ct/min in 3H (protein) and 291 ct/min in 14C ( R N A ) giving a ratio of 3H : 14C of 3-1 ; in the
case of C C M V (Fig. 1 b) the corresponding figures were 429 and 188 ct/min, giving a ratio of 2.3.
There was therefore no less R N A in the ' C C M V ' than in BMV particles.
The nature of the RNA in non-infectious CCMV particles
Protoplasts that had been infected with both BMV and C C M V were cultured in medium
containing [32p]orthophosphate, homogenized and the viruses separated on cylindrical
polyacrylamide gels as before. The C C M V band was then subjected to electrophoresis on 2-5 ~o
gels in the presence of SDS and autoradiographs of the gels were prepared. The result is shown in
Fig. 2. The autoradiograph of the total virus before fractionation showed bands characteristic of
BMV R N A , i.e. R N A s 1 and 2 running close together and poorly resolved and R N A 3 running
somewhat faster and well resolved from the other two; R N A 4 was obscured by labelled material
of unknown origin, perhaps degradation products, and this section of the gel has been omitted.
In contrast, the autoradiograph of the ' C C M V ' showed one band corresponding to R N A 3 ; the
amount of material recovered was too small to determine whether R N A 4 was also present.
Synthesis of RNA and protein in doubly infected protoplasts
Doubly infected protoplasts were cultured in medium containing [~4C]uridine, the total (i.e.
host and viral) R N A was prepared and separated on 2-5~ polyacrylamide gels. An
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F. SAKAI, J. R. O. DAWSON AND J. W. WATTS
1350
,sL
10
x
~
~
/1
o
o
J3
o
x x
e-. ¢xl
5
1 ~ x
o
m
o
o
o
~)~,~.
2
10
10
20
Fraction number
30
Fig. 1. Relative amounts of protein and RNA in virus particles isolated from doubly infected
protoplasts. Protoplasts doubly infected with CCMV and BMV were cultured for 24 h in medium
containing [3H]leucine (10 ~tCi/ml) and [14C]adenine (2 ~tCi/ml). Virus particles were separated on
cylindrical gels, the gels were sliced, and the amounts of 3H and 14C in BMV (a) and CCMV (b) were
determined. Unlabelled carrier virus was added to the samples to assist location of the virus band. A,
Counts in 14C (RNA); 0 , counts in 3H (protein); O, ratio 3H :14C. The arrows show the virus band.
autoradiograph of gels of R N A from singly and doubly infected protoplasts is shown in Fig. 3;
R N A from uninfected protoplasts was unlabelled. There was over 80 ~ infection (fluorescent
antibody staining) with less than 5 ~ of protoplasts singly infected with C C M V (not significant
experimentally) and about 1 0 ~ singly infected with BMV. U n d e r these conditions C C M V
R N A runs rather differently from BMV R N A : R N A s 1, 2 and 3 are clearly resolved for C C M V ,
but R N A s 1 and 2 of BMV run very close together. The gels show that C C M V R N A 2 was
absent in the mixed infection but BMV R N A s 1, 2 and 3 co-migrate with, and so obscure, any
C C M V R N A s 1 and 3 present; the suppression of synthesis o f C C M V R N A 2 would in itself
account for the production of non-infectious C C M V (Bancroft, 1971). The relatively heavy
labelling of R N A 4 in the mixed infection may be an artefact o f p r e p a r a t i o n and has not been
studied further.
It is possible that failure to detect C C M V R N A 2 was due to lack o f sensitivity of the
experimental procedures. The corresponding results for protein synthesis are therefore of some
interest (Fig. 4). This is the same experiment illustrated in Fig. 3 but the protoplasts were
cultured in m e d i u m containing [~*C]leucine and the proteins were separated on S D S polyacrylamide gels (Sakai et al., 1979) using a Tris-glycine running buffer, U n d e r these
conditions BMV and C C M V coat proteins and virus-specific proteins of mol. wt. 35 000 can be
completely resolved. It can be seen that in the doubly infected protoplasts both coat proteins
were present, although there was considerably more BMV coat protein than C C M V coat
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Interference in m i x e d virus infections
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+~
I
(b)~/(a)
/
Migration
Fig. 2. RNA isolated from CCMV particles in doubly infected protoplasts. Protoplasts infected with
CCMV alone or BMV and CCMV together were cultured for 24 h in medium containing
[32p]orthophosphate (10 ~tCi/ml). Virus particles were separated by electrophoresis into 2.5%
polyacrylamide gels, RNA was isolated from CCMV-like particles and run in 2.5 % polyacrylamide gel.
Traces of autoradiograms of (a) total virus and (b) CCMV from a mixed infection are shown. Numbers
indicate RNA species 1 to 3.
(b)
~._..._.._..~.
(a):-
)'1"2
3
i
Migration
Fig. 3. RNA synthesis in doubly infected protoplasts. Protoplasts were irradiated with u.v. to reduce
host protein synthesis, inoculated with BMV, CCMV or both viruses, and cultured for 20 h in medium
containing actinomycin D (10 ~tg/ml)to suppress host RNA synthesis, and chloramphenicol (100 ~tg/ml)
to reduce host protein synthesis (Sakai et al., 1977), and [14C]uridine (2 ~tCi/ml) to label nucleic acids.
Traces of autoradiograms of nucleic acids separated on 2-5% polyacrylamide gels are shown. (a)
CCMV-infected; (b) BMV-infected; (c) doubly infected. Numbers indicate RNA species 1 to 4.
Uninfected controls showed no incorporation of label.
protein. I n contrast, this relation was reversed for the 35000 mol. wt. p r o t e i n s ; there was
considerably more of the C C M V - c o d e d t h a n of the B M V - c o d e d protein. N o c o n c l u s i o n could be
d r a w n about the proteins of mol. wt. a b o u t 105 other t h a n that two at least were present, one of
w h i c h m u s t be coded by B M V (Sakai et al., 1977, 1979); the C C M V - c o d e d p r o t e i n co-migrated
with the faster of the two B M V - c o d e d proteins (mol, wt, 105).
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F. SAKAI, J. R. O. D A W S O N AND J. W . WATTS
P3
t
)i
Migration-----~
Fig. 4. Protein synthesis in doubly infected protoplasts. The experiment was as described in Fig. 3
except that [14C]leucine (2 I~Ci/ml) was used to label proteins. Traces of autoradiograms of proteins
separated on 9 ~ polyacrylamide gels are shown. (a) Uninfected control; (b) BMV-infected; (c) CCMVinfected; (d) doubly infected. PI, Coat proteins; P2, mol. wt. 35000; P3, P4, tool. wt. 105.
T a b l e 2.
Relative amounts of virus-specific proteins in doubly infected protoplasts*
Radioactivity in protein (ct/min)
Experiment
no. (label)
1 (14C)
2 (~4C)
3 (3H)
•
CCMV
324
288
650
Coat proteins
~
~
BMV
CCMV : BMV
1544
0-21
904
0.32
11600
0.06
•
CCMV
2731
417
950
35000 mol. wt. proteins
x
BMV
CCMV : BMV
426
6-4
248
1.7
900
1.1
* Protoplasts were inoculated first with CCMV followed by BMV and cultured for 24h in medium containing 3Hor 14C-labelled leucine. Proteins were fractionated on 9 ~ polyacrylamide gels which were sliced and counted.
Table 2 illustrates similar experiments in which doubly infected protoplasts were cultured in
medium containing [14C]leucine or [3H]leucine and the proteins were prepared, separated on
gels and the gels were sliced and counted. Again it can be seen that the amount of BMV coat
protein was always greater than that of CCMV but that the reverse was true for the 35 000 mol.
wt. proteins. There was therefore appreciable but unbalanced expression of the CCMV genome
in the mixed infection.
DISCUSSION
A number of points emerge from the results. The data on inoculation shown in Table 1 suggest
that a population of protoplasts consists of two classes, one of which is susceptible to infection,
so that a sequential or simultaneous inoculation infects only one part of the population and gives
few singly infected protoplasts.
Turning to the phenomenon of mixed infections it can be seen that BMV dominance is a
complex process which permits only limited expression and replication of the CCMV genome,
suppressing production of infectious particles of CCMV and perhaps even exploiting those gene
products of CCMV that are produced. In the results presented here BMV consistently produced
infectious particles but those of CCMV were defective and may have lacked RNAs 1 and 2.
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Interference in mixed virus infections
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There are technical problems against recovering 100% of all species of R N A from CCMV and
the infection and other assays may be too insensitive to detect small amounts of RNAs 1 and 2 in
the presence of R N A 3; therefore, some caution is necessary in accepting this interpretation.
Other work (Trim et al., 1977) has shown that RNAs 1 and 2 are limiting factors in infection,
requiring only trace amounts of RNA 3 to cause infection. What is clear, however, is that there is
no mixing of R N A and capsid proteins : CCMV particles isolated from mixed infections never
gave BMV infections, and vice versa. It is known that transcapsidation can be produced in vitro
(Bancroft, 1970) so that virus assembly in protoplasts must be restricted to specific regions of the
cytoplasm, presumably within the vesicles that are observed to develop during infection
(Burgess et al., 1974).
The pattern of protein synthesis in doubly infected protoplasts shows that although no
infectious CCMV is obtained, the proteins that are coded by R N A 3 are both produced.
Remarkably, the quantitative data show that the amounts of the 35 000 mol. wt. protein coded
by CCMV in some cases exceeded that coded by BMV. There is therefore the suggestion of an
unbalanced translation of the genomes of both viruses in mixed infections; BMV coat protein
was always produced in larger amounts than that of CCMV. It is possible that BMV does not
simply suppress production of CCMV but exploits the machinery to assist its own synthesis.
Bancroft (1972) has shown that a hybrid of CCMV and BMV can be constructed in one direction
only, CCMV R N A 3 + BMV RNAs 1 and 2. It is thus possible that in the mixed infection the
35000 mol. wt. protein of CCMV may be exploited by BMV. It is, however, just as likely that the
effect is a consequence of unbalanced translation and replication of the CCMV genome.
The function of the proteins, other than coat, synthesized in bromovirus infections remains
unclear and differing opinions have been expressed (Hariharasubramanian et al., 1973; Sakai et
al., 1979; Bujarski et al., 1982). Until this problem has been resolved it will be difficult to explain
why there is so much expression of the CCMV infection without production of infectious virus.
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