J. gen, ViroL (1972), I6, 327-338
327
Printed in Great Britain
Properties of Virus and R N A Components of
Raspberry Ringspot Virus
By A. F. M U R A N T , M. A. MAYO, B. D. H A R R I S O N AND
R. A. G O O L D
Scottish Horticultural Research Institute, Invergowrie, Dundee, Scotland
(Accepted 8 May I972)
SUMMARY
Particles of raspberry ringspot virus were homogeneous in immunoelectrophoresis tests but sedimented as three components, T, M and B, with sedimentation
coefficients of 52, 92 and I3OS respectively, and containing o, 3o and about 44%
RNA. The three components were serologicallyindistinguishable.In CsC1gradients,
M and B were each homogeneous, with buoyant densities of ~.43 and 1.52 g./cm?
respectively. Infectivity was associated with B. RNA extracted from the virus
preparations was single-stranded and showed about 2o% hyperchromicity in
o-I M-sodium phosphate buffer at pH 7-0, indicating about 4o% base-pairing.
Analysis of RNA preparations by polyacrylamide gel electrophoresis revealed two
predominant RNA species with tool. wt of 2"4 × Io 8 (RNA-I) or 1.4 x IO6 (RNA-2);
RNA-2 was indistinguishable in mol. wt from the RNA-2 of tobacco ringspot
virus but RNA- I migrated slightly more slowly than the RNA-I of tobacco ringspot
virus. M particles of raspberry ringspot virus contain one molecule of RNA-2;
B particles contain either one molecule of RNA-r or, probably, two molecules of
RNA-2. Whereas B particles of raspberry ringspot virus were apparently homogeneous in CsC1 gradients, B particles of tobacco ringspot virus formed two
buoyant density classes.
INTRODUCTION
Plant viruses belonging to the nepovirus group (Cadman, I963; Harrison et al. I97 I)
have isometric particles 28 to 30 nm. in diameter containing RNA, are transmitted in soil
by free-living plant parasitic nematodes and resemble each other in physical properties.
They reach similar concentrations in plant sap, cause similar symptoms in a wide range of
host plants, and are seed-borne. The particles of different nepoviruses probably have the
same ultrastructure. They look identical in the electron microscope (Harrison & Nixon,
t96o) and the protein shells of three nepoviruses are probably composed of 60 polypeptide
subunits of about 54,ooo mol. wt (Mayo, Mutant & Harrison, I97I).
Some nepoviruses differ, however, in properties that might at first seem to be important.
For example, although preparations of nepoviruses typically can be separated in the ultracentrifuge into top, middle and bottom components, which differ in the amount of nucleic
acid they contain, this is not so for all members of the group; tomato ringspot virus seems
to lack a middle component (Stace-Smith, I966) and two putative nepoviruses, the golden
elderberry strain of cherry leaf roll virus (Hansen & Stace-Smith, 1971 ; Jones & Murant,
i97i ) and myrobalan latent ringspot virus (Dunez et al. I97I), apparently lack top
components.
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328
A . F . M U R A N T , M. A. MAYO~ B. D. H A R R I S O N
A N D R. A. G O O L D
Tobacco ringspot virus is the only nepovirus for which the size and number of the RNA
components has been reported. Diener & Schneider (1966) found two RNA species of
1.2 x Io6 and 2-2 × lO6 mol. wt and reported that only the heavier RNA was infective. To
extend the information available for nepoviruses we have studied another member of the
group, raspberry ringspot virus (Harrison, r958; Murant, 197o). This paper describes some
properties of its virus and nucleic acid components and the accompanying paper (Harrison,
Murant & Mayo, I972) deals with the role of these components in causing infection.
METHODS
Virus propagation and assay. The isolate of raspberry ringspot virus used in this work
was derived from the type culture of the common English strain, obtained from Himalaya
Giant blackberry, Rubusprocerus P. J. Muell. (Cadman, 196o). It was passed through three
successive single lesions in Chenopodium amaranticolor Coste & Reyn., and resembled the
parent culture in symptomatology and serological properties.
The virus was propagated in Nicotiana clevelandii Gray and assayed by counting the
lesions produced in inoculated leaves of C. amaranticolor. Treatments were distributed
among the upper three inoculable leaves, or among the upper two when half-leaf comparisons were made. The leaves of test plants, dusted either with 60o-mesh Carborundum
(Hopkin & Williams) or 'Corundum' (Bausch & Lomb, Rochester, N.Y.), were inoculated
by rubbing them with a cheesecloth pad dipped in inoculum. Virus inocula were prepared
in o-o6 M-sodium phosphate buffer, pH 7. When inoculating nucleic acid preparations,
plastic gloves were worn to guard against contamination with ribonuclease (RNase) from
the fingers; muslin pads, dipped once in inoculum, were used to rub the leaves. Nucleic acid
preparations were diluted in fresh o.o6 M-phosphate buffer, pH 8, which was boiled and
cooled before use.
Virus purification. Extracts from systemically infected N. clevelandii leaves were clarified
with. n-butanol by the method of Tomlinson, Shepherd & Walker (1959) and concentrated
either by three cycles of differential centrifugation (IO rain. at 1o,ooo g, then 4 hr at 78,000 g
or 2 hr at I5o,ooo g) or by two cycles, followed by exclusion chromatography in columns of
2 ~o agarose beads (Sepharose 2B; Pharmacia). Preparations were used immediately or kept
at 4° in the presence ofo-o2 ~o sodium azide. Virus yields were 4 to 8 mg./IOO g. leaf material,
about 2 to 4 times greater than those obtained by the butanol-chloroform technique of
Steere (I956).
Separation of virus components. Virus components were separated by centrifuging twice
in sucrose density gradients. These were prepared in 3"5 x I in. Beckman SW 27 tubes by
layering lO, IO, Io and 5 ml. respectively of lO, 2o, 3° and 40% (w/v) sucrose in 0.06 ~phosphate buffer, pH 7"o, and allowing diffusion at 4 ° overnight. Samples (o'5 to 1 ml.)
containing up to 3 mg. virus were floated on the gradients. After centrifuging, fractions
were collected by upward displacement of the gradients and analysed with an ISCO Model
UA-z ultraviolet analyser (Instrumentation Specialties Co., Lincoln, Nebraska). Fractions
containing any one virus component were pooled, diluted at least tenfold and centrifuged
for 2 hr at I5O,OOOg to sediment the virus.
Serological tests. Antiserum to the strain of raspberry ringspot virus used in this work
was prepared by injecting a rabbit once intravenously with about o'5 mg. virus and, after 3
weeks, once intramuscularly with about 5 rag. virus emulsified with Freund's complete
adjuvant. Antiserum was obtained 4 weeks after the intramuscular injection. Gel-diffusion
tests were done in 0. 5 % agarose gels containing o'I5 M-NaC1 and o.02% sodium azide,
using wells 7 mm. in diameter spaced 11 mm. apart between centres.
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Virus and RNA components of R R V
329
For immunoelectrophoresis, 3 x I in. microscope slides were coated with 1% I.D. agar
(Oxoid) containing barbitone-acetate buffer (ionic strength o'o25), p H 8.6. Virus preparations containing about o'5 to 1.o mg. virus/ml, were placed in the sample hole and electrophoresed at a constant current of 4o mA for 1 to 4 hr at 4 °. The starting voltage was 23o and
the final voltage about I7O. After electrophoresis the serum troughs were filled with antiserum at dilutions of 1/5o to 1/5oo.
Analytical ultracentrifugation. Virus preparations were examined in a Beckman Model E
ultracentrifuge, using Schlieren optics. Sedimentation coefficients were calculated as
described by Markham (196o), and buoyant densities in CsC1 solutions were determined by
the method of Szybalski (I968). Approximate estimates of the relative proportions of the
virus components were made from the areas under the Schlieren peaks.
Preparation of nucleic acid. Nucleic acid was extracted from purified virus preparations
by two methods:
I. The phenol method B of Loening & Ingle (I967) modified from that of Kirby (I965). In
this method the virus is suspended in buffer containing naphthalene sulphonate detergents
+ 4-amino salicylate and homogenized with a mixture of water-saturated phenol + m-cresol
(9: t, v]v)+ o'I % 8-hydroxyquinoline. Nucleic acid preparations were stored as precipitates
in 7o % ethanol at - 15°. Preliminary experiments showed that these nucleic acid preparations had much higher specific infectivities than those prepared by diluting virus in phosphate
buffer and extracting with water-saturated phenol.
2. The pronase/SDS method, based on that described by Ringborg et al. (I 97o). A solution
containing o-oi5 M-sodium citrate, o'15 M-NaCI, o.I to o'I5 % pronase (Calbiochem) and
o'5 % sodium dodecyl sulphate (SDS) was kept at 37 ° for 30 min. to digest any contaminating
nuclease. It was then used to resuspend virus pellets to give a virus concentration of about
o.t %. The mixture was kept at 37 ° for I6 hr. When the nucleic acid was used immediately,
the digest was applied directly to the gels, but otherwise it was stored as a precipitate in
70% ethanol at - I5 °.
Spectrophotometry. Solutions of virus and nucleic acid were examined in a Pye Unicam
SP 18oo double beam recording spectrophotometer. Concentrations were estimated using
assumed extinction coefficients \rE°~%~
l c m . ] at 26o nm. of 25 for nucleic acid, 7 for purified virus,
1 for virus top component, 7 for virus middle component and lO for virus bottom component; these assumptions were based on the known percentage compositions of nucleic
acid and protein and by analogy with other well studied viruses.
The 'melting' profile of nucleic acid solutions was determined using a Unicam SP 876
temperature programme controller and a Unicam SP 877 electrically heated cell-holder.
The cell block was heated at o'25 ° or o.5°/min, and temperature readings were taken in a
duplicate cell adjacent to the sample cell. Extinction readings were corrected for the thermal
expansion of water, and plotted against temperature.
Polyacrylamide gel electrophoresis. Polyacrylamide gels (2.2 to a'5 %) were prepared from
purified monomer as described by Loening (I967). Gels were prepared in Perspex tubes of
6 mm. internal diameter and approximately Io cm. long. They were supported on I cm.
plugs of Io % acrylamide gel placed in the bottoms of the tubes. The electrophoresis buffer
was o.o2 M-tris, 0-02 u-sodium phosphate, pH 7"8, containing o.oo2 M-EDTA; in most
experiments, it also contained o.2 % SDS. Current was passed through gels at 6 mA/gel,
7 v/cm. for 1 to 2 hr before the samples, containing Io to 25 #g. nucleic acid, were applied.
Electrophoresis was then continued for 2 to 3 hr at room temperature. After electrophoresis,
gels were scanned with an ultraviolet densitometer (UV Scanner, Joyce-Loebl). Washing
gels for 2 to 24 hr in buffer after electrophoresis removed a diffuse u.v.-absorbing zone near
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330
A.F. MURANT~ M. A. MAYO, B. D. H A R R I S O N AND R. A. GOOLD
e
Fig. I. Immunoelectrophoresis of raspberry ringspot virus in I ~ Oxoid I.D. agar containing 0"025
ionic strength barbitone-acetate buffer, pH 8.6. Electrophoresis was for 4 hr at constant current
(40 mA). Starting voltage 230, final voltage t7o.
Fig. 2. Schlieren diagrams of raspberry ringspot virus preparations. (a) Components T, M and B,
after analytical centrifugation of a preparation containing about 7'5 mg./ml., Schlieren angle 60°.
Photograph taken after I2 min. at 3o,ooo rev./min. Sedimentation is from left to right. (b) After
centrifuging for 15"5 hr at 44,000 rev./min, in a CsC1 solution of initial density I"463 g./cm?,
Schlieren angle 70°. Note M and B components; T is at the meniscus. (c) After centrifuging for 18 hr
at 44,ooo rev./min, in a CsCI solution of initial density 1.5o8 g./cm., ~Schlieren angle 60°. T and'M
components are at the meniscus.
the top o f the gel. Mol. wt of R N A s were estimated by comparing their mobilities with those
o f ribosomal R N A s o f k n o w n mol. wt f r o m pea tissue (Loening, 1968). Estimates of mobility
were made before washing to avoid errors caused by non-linear stretching o f gels.
RESULTS
Components of purified virus preparations
Purified preparations o f raspberry ringspot virus were homogeneous in immunoelectrophoresis tests (Fig. I), producing a single zone which migrated slowly towards the a n o d ( i n
barbitone-acetate buffer, p H 8.6. However, in the analytical ultracentrifuge (Fig. 2a), the
preparations separated into the three components characteristic o f nepoviruses, t o p (T),
middle (M) and b o t t o m (B). Their sedimentation coefficients $2o' w (at infinite dilution) were
5 z, 9 2 and 13 ° s respectively, and their relative proportions were usually about 3: I : 2- 5 with
the strain o f raspberry ringspot virus used in this work.
Fig. 3 shows the distribution o f u.v.-absorbing material in a sucrose density gradient.
Light-scattering zones, corresponding to the T, M and B components, formed at 8 to IO,
I5 to I7 and 22 to 24 mm. f r o m the meniscus. Infectivity was associated with c o m p o n e n t B.
Samples f r o m each zone were centrifuged a second time in density gradients. Material
recovered f r o m the T, M or B zones o f the second density gradient tube contained no
measurable amounts o f the other components when centrifuged in a third density gradient.
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Virus and R N A components o f R R V
33 r
B
[.0
200
v,..,
100
0.5
.R
,..a
\
M
T
~
o
0
i
t
=
i
2
I
I
i•
.,
o
3
Depth, cm.
Fig. 3. Distribution of virus components T, M and B (continuous line = extinction at 254 nm.) and
infectivity (columns), after sucrose density gradient centrifugation of a preparation of raspberry"
ringspot virus (0"5ml. ; E26o = 5) for 90 min. at 26,o00 rev./min. Consecutive fractions (o'85 ml.)
were diluted 5oo-fold and inoculated to four half-leaves of Chenopodiumamaranticolor.
Preparations of each component were therefore considered suitable for further study after
centrifuging twice in density gradients. They were used as soon as possible after preparation
because gradual degradation occurred even after I week at 4 ° in o.06 M-phosphate buffer,
p H 7"2: B preparations developed increasing proportions of particles that sedimented like
T in density gradients, as well as low tool. wt protein and nucleic acid degradation products
that remained near the tops of the gradients. However, no particles sedimenting like M
were defected in B preparations, even after 6 weeks.
Properties of the separated virus components
U. v.-absorption spectra
The u.v.-absorption spectra of the separated components (Fig. 4 a) suggest that T (mean E26o/
E28o = o.68), like the top component of tobacco ringspot virus (Stace-Smith, Reichmann
& Wright, 1965), is the nucleic acid-free protein shell of the virus; M (mean Ez6o/E28o = I'48)
and B (mean E26o]E28o = I "69) are nucleoproteins. Using Reichmann's 0965) formula and
the sedimentation coefficients given above, Mayo et al. ( I 9 7 0 calculated that M and B contain
about 28 and 43 % nucleic acid respectively.
Serology
N o spurs formed when T or B preparations, each at concentrations of 1 -o and 2.o mg]ml.
were placed in adjacent wells in gel-diffusion plates and undiluted antiserum (titre in geldiffusion tests = I/4OOO) placed in the antiserum well (Fig. 5). The same result was obtained
in a second experiment, in which T, M and B preparations were used at o'5 mg./ml. This
indicates that all three components are serologically indistinguishable.
Buoyant density
When an unfractionated virus preparation was centrifuged to equilibrium in CsC1 solutions in the analytical ultracentrifuge, M and B components each formed a single zone at
positions corresponding to buoyant densities of 1-43 and 1.52 g./cm? respectively (Fig. 2b).
We found no evidence that b o t t o m component was itself heterogeneous (Fig. 2 c) although
22
VIR
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16
332
A. F, MURANT, M. A. MAYO, B. D. HARRISON AND R. A. GOOLD
t.0
1-0
! i I I(b) 1
0-8
0-8
0"6
0"6
0.4
04
0.2
0-2
E
0.0
220
260
300
0.0
220
Wavelength. nm.
I
I
260
I
I
300
Fig. 4. U.v.-absorption spectra of (a) separated T, M and B components from raspberry
ringspot virus, (b) RNA extracted from an unfractionated virus preparation.
P it
b
)
Fig. 5. Immunodiffusionwith raspberry ringspot virus components and antiserum. Well S: undiluted
antiserum (titre I/4OOO). Outer wells: (a) component T, z mg./ml. ; (b) component T, x mg./ml. ;
(e) component B, 2 mg.]ml. ; (d) component B, I mg./ml. High antigen concentrations were necessary
to compensate for the high antiserum titre.
i n tests with tobacco ringspot virus we, like Schneider (I97o), f o u n d two classes of b o t t o m
c o m p o n e n t particles with b u o y a n t densities of I "5I a n d i .52 g./cm, a. However, the Schlieren
diagrams given b y raspberry ringspot virus suggest that some aggregation o f particles had
occurred.
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Virus and RNA components of R R V
I
I
I
|
|
I
20
333
~
•
]
g
15
N 10
5
0 --
30
50
70
Temperature (°)
i
90
Fig. 6." Melting' profile of raspberry ringspot virus RNA in o"I M-sodiumphosphate buffer, pH 7"o.
Results are from three experiments and are expressed as the percentage increase in extinction at
26o rim. at each temperature relative to that at 3o°.
Properties of nucleic acM preparations
The pronase]SDS method gave about 7 ° % of the theoretical m a x i m u m yield of nucleic
acid, calculated by assuming that about 80 to 88 % of the Ez6o of the virus was attributable
to the nucleic acid. This was about twice the yield obtained with the phenol method. The
infectivities, expressed on an extinction basis, of nucleic acids prepared by the two methods
were similar and were about IO to 2o % of the infectivity of the parent virus preparations,
diluted to contain the same concentration of nucleic acid. The pronase/SDS method, besides
giving better yields of nucleic acid than the phenol method, was also more convenient,
especially with small amounts of virus, and was therefore used for nearly all experiments.
When comparative tests were made, no qualitative differences were detected between nucleic
acid preparations made by the two methods.
Effect of RNase and DNase
The infectivity of nucleic acid preparations in o.o6 M-sodium phosphate buffer, p H 7"2,
or in the same buffer containing o ' I 5 M-NaCI, was abolished by treatment at o ° for Ioo rain.
with pancreatic R N a s e (Koch-Light, 5 × cryst., o-25 #g./i.). Treatment with D N a s e I (Sigma,
RNase-free, 5 rag./1.) in the presence o f z mM-Mg ~+ had no effect. RNase at o'25/zg./l, did
not alter the infectivity of virus preparations. These results indicate that raspberry ringspot
virus, like tobacco ringspot virus, contains single-stranded RNA.
U.v.-absorption
The R N A preparations had typical u.v.-absorption spectra (Fig. 4 b) with E26o]E28o
values around 2.0.
Melting curves obtained by heating R N A preparations in o-I M-sodium phosphate buffer
22-2
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334
A . F . M U R A N T , M. A. M A Y O , B. D. H A R R I S O N
A N D R. A. G O O L D
(a)
l
(b)
\
--
+
Fig. 7. Electrophoresis of R N A from an unfractionated preparation of raspberry ringspot virus in
2.2 ~ acrylamide gels. Before electrophoresis, R N A samples (z5 #g.) were either (a) dissolved in
electrophoresis buffer containing o.2 ~ SDS and kept at room temperature for IO min., or (b)
dissolved in electrophoresis buffer containing o.2 ~ SDS + 8 M-urea and heated at 60 ° for Io rain.
Electrophoretic migration is from left to right.
(pH 7) and measuring their extinctions at 26o nm. (Fig. 6) were characteristic of singlestranded RNA. On cooling, the extinction returned close to its original value. The thermal
denaturation mid-point was 56 ° and the hyperchromicity at 26o nm. (maximum increase in
extinction relative to that at 3o°) was 20 ~o indicating that in this buffer system about 40 %
of the nucleic acid bases are paired (Doty et al. 1959). This compares with an estimate of
40 to 6o ~o base-pairing made by these authors for tobacco mosaic virus RNA. By comparing
melting curves obtained at 26o nm. and at 28o nm. it can be calculated, using equation 5
of Cox (1966), that the guanine-cytosine content in the ordered regions of the RNA is about
55 %- A similar value (54~o) was obtained for the ribosomal RNA of Escherichia coli
(Edelman, Verma & Littauer, I97o).
Polyacrylarnide gel electrophoresis
Loening (r969) reported that the relation between the mol. wt of tobacco mosaic virus
R N A and its electrophoretic mobility in polyacrylamide gels was different from that for
E. coil ribosomal RNA and attributed this to differences between the two R N A species in
secondary structure. The hyperchromicity data presented above show that raspberry ringspot virus RNA resembles E. coli ribosomal RNA in secondary structure. Although the
buffers used in hyperchromicity experiments were different from those used in polyacrylamide gel electrophoresis, the hyperchromicity data suggest that useful estimates of the
mol. wt of raspberry ringspot virus RNA can be obtained by comparing its electrophoretic
mobility with that of ribosomal R N A species from pea, previously calibrated against E. coli
ribosomal RNA (Loening, 1968).
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Virus and R N A components o f R R V
335
(a)
(b)
(c)
/x.,,, f
--
)_
+
Fig, 8. Electrophoresis of R N A from M and B components of raspberry ringspot virus in z'5
acrylamide gels, (a) z5/zg. R N A from M component, (b) 25 #g. R N A from B component, (c) ~2.5 #g.
R N A from M component plus 12"5 #g. R N A from B component. The R N A was not treated with
urea. Electrophoretic migration is from left to right.
When R N A preparations made from unfractionated raspberry ringspot virus were electrophoresed in polyacrylamide gels, the u.v.-absorbing material was distributed in two major
R N A bands (Fig. 7a). One appeared symmetrical, containing material of mol. wt about
I"4 x io6; the other appeared asymmetrical, containing material ranging in mol. wt from
2.4 x Io 6 to 3.o x ~o~. Lane & Kaesberg (197I) and Bancroft ( I 9 7 0 found that heating
R N A preparations before electrophoresis for Io rain. at 5°o in electrophoresis buffer containing I M-urea, o.2 % SDS and o'o5 M-z-mercaptoethanol decreased the number of bands
obtained in polyacrylamide gels, and concluded that the treatment removed artifacts. In
our experiments, the band containing the higher mol. wt material was sharpened by heating
raspberry ringspot virus R N A preparations for I5 rain. at 60 ° in electrophoresis buffer
containing 8 M-urea and 0-2 % SDS. Two major R N A species were thus resolved with mol.
wt of 2"4 x io 6 and I. 4 x io ° (Fig. 7b); these were called species I and 2 respectively. No
further change in the relative distribution of R N A components was obtained when 8 M-urea
was also present in the gels during electrophoresis. Although the band produced by RNA-r
always appeared more symmetrical in heated than in unheated preparations, a small band
of higher mol. wt material was sometimes partially resolved.
Heating the R N A in urea caused no detectable loss of infectivity. This suggests that the
polydisperse high mol. wt R N A in untreated preparations was an artifact caused
by aggregation or secondary structure. Moreover, it is unlikely that RNA-I is composed of
two pieces of RNA-2 linked by hydrogen bonds, which should have been disrupted by
treating with urea. Further evidence for this was obtained by heating preparations at 9°0
for 5 min. in half-strength electrophoresis buffer containing 9o % dimethyl sulphoxide and
o.2% SDS. N o change in the proportions of RNA-I and RNA-2 was produced by this
treatment. No R N A components smaller than I-4 x IO6 mol. wt and contributing more than
5 % of the total extinction were detected in tests with urea-treated preparations. These tests,
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336
A . F . M U R A N T , M. A. M A Y O , B. D. H A R R I S O N
(a)
AND
(h)
R. A. G O O L D
I
Fig. 9- Electrophoresis of RNA preparations from tobacco ringspot virus and raspberry ringspot
virus in 2-2 ~ acrylamide gels, (a) tobacco ringspot virus RNA, (b) a I : 2 mixture of RNA preparations from tobacco ringspot virus and raspberry ringspot virus respectively, (c) a 2:I mixture of
RNA preparations from tobacco ringspot virus and raspberry ringspot virus respectively. Before
electrophoresis RNA was treated as in Fig. 7b. The gel shown in (a) was not washed before scanning,
those in (b) and (c) were. Electrophoretic migration is from left to right.
in which pea leaf 4 s R N A was used as marker, would have detected R N A as small as
2"5 x i O mol. wt.
One explanation of the origin of R N A species I and 2 is that they occur respectively
inside the B and M nucleoprotein components. However, R N A preparations derived from
unfractionated virus always contained about 3 to 5 times as much RNA-2 as R N A - I by
weight, whereas virus preparations contained only a third to a half as much M component
as B component. Electrophoresis of R N A derived from separated nucleoprotein components (Fig. 8a, b) showed why this was so. All the R N A from M component was RNA-2
but about two-thirds to three-quarters of the R N A from B component was also RNA-2,
the remainder being R N A - I . As expected, extracts made from T component contained no
detectable RNA. N o difference in mobility was detected between RNA-2 f r o m M component and that from B component (Fig. 8 c).
Tobacco ringspot virus RNA, prepared in the same way as raspberry ringspot virus R N A ,
behaved in much the same way in polyacrylamide gels (Fig. 9a). However, when R N A
preparations from the two viruses were mixed in various proportions the larger R N A of
tobacco ringspot virus migrated slightly more rapidly than that of raspberry ringspot virus
(Fig. 9b, c); the smaller R N A species of the two viruses could not be distinguished from
each other, suggesting that they have very similar mol. wt. In these experiments the mol. wt
of tobacco ringspot virus R N A - r and RNA-2 were estimated to be 2. 3 × IO6 and 1.4 x IO6
respectively.
DISCUSSION
The experiments described in this paper show that purified preparations of raspberry
ringspot virus contain three components with sedimentation coefficients of 52, 92 and 13o s;
one, called T, is the RNA-free protein shell; the others, M and B, are nucleoproteins. R N A
extracted from such preparations is of two predominant species, RNA-x and RNA-2, with
tool. wt of 2"4 × IO8 and i "4 × IO6 respectively. We found that RNA-2 occurs in M particles
and that both types of R N A occur in B particles.
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Virus and R N A components of R R V
337
Using the mol. wt of RRV particles calculated from the Svedberg equation (3"3 x io 6 (T),
4"8 x lO6 (M) and 6-zx lO6 (B); Mayo et al. I97I ), and assuming that all three types of
particle contain the same total weight of protein, it follows that M particles each contain
only one molecule of RNA-2. In B particles, the figures allow for one molecule of RNA-I
or two of RNA-z, but not for one molecule of each type of RNA. The possibility that some
B particles contain a 'makeweight' composed of one or more shorter R N A strands, seems
unlikely because we could detect no discrete class of R N A molecules of mol. wt between
2. 5 x lO4 and 1"4× lO6. The presence of 'makeweight' R N A of heterogeneous size is not
excluded by our results but there was no evidence in our density gradient experiments for
any substantial number of particles sedimenting at rates between those of M and B particles.
Thus the B component of raspberry ringspot virus must consist of particles of two types
containing different kinds of R N A molecules.
Calculations based on the mol. wt of the R N A and protein constituents of the particles
give mol. wt estimates for the virus particles of 3"3 × lO6 and 4"7 x lO6 for the T and M particles, and 5"7 × 1o 6 and 6. I x t o 6respectively for the B component particles containing RNA- I
and RNA-2. The corresponding percentage R N A compositions are o, 3o, 42 and 46.
Our data closely resemble those found for tobacco ringspot virus, the type member of
the nepovirus group, by Diener & Schneider (1966); these authors reported that the c o m
ponents of this virus sedimented at 52, 94 and IZ8 s and the R N A components had mol.
wt of I-Z x io 6 and 2-2 × lO6. They too concluded that middle component particles contained
one molecule of the smaller RNA, whereas bottom component particles contained either
one molecule of the larger R N A or two molecules of the smaller RNA.
Although our results suggest that two molecules of RNA-2 weigh more than one
molecule of RNA-I, B component was apparently homogeneous when centrifuged to equilibrium in CsC1 gradients. Aggregation of the particles may be one reason why no heterogeneity was detected. Another possibility is that there are slight errors in our estimates of the
tool. wt of the two R N A species so that the difference in density between the two types of
particle in B component is less than expected. It is noteworthy that two types of particle of
different buoyant density were resolved in the bottom component of tobacco ringspot virus
and that its RNA-I migrated slightly more rapidly than that of raspberry ringspot virus in
polyacrylamide gels; this suggests that, whatever the actual mol. wt of the R N A species,
the difference in weight between the two types of bottom component particles is greater
with tobacco ringspot virus than with raspberry ringspot virus, and might result in a more
easily detected difference in buoyant density. However, other explanations are possible for
the apparent homogeneity of the B component of raspberry ringspot virus, and this is the
subject of experiments still in progress.
Diener & Schneider (1966) considered the possibility that in tobacco ringspot virus the
smaller R N A was formed by breakage of the larger RNA; however, they preferred the
hypothesis that the larger R N A was formed by the joining of two molecules of the smaller
RNA. We found that the RNA-I of raspberry ringspot virus cannot be disrupted by heating
in 8 M-urea or in 9o % dimethyl sulphoxide, which suggests that, should such a linkage occur,
it is not through hydrogen bonds. Recent work has shown that many plant viruses have
more than one functional RNA component (see Matthews, I97O), and it would seem that a
more plausible explanation o f the origin of the two classes of R N A in raspberry ringspot
virus preparations is that they are both necessary and functional pieces of the virus genome.
This is considered in more detail in the accompanying paper (Harrison et al. I97z).
We thank D. Donald and Rhonda MacLagan for technical assistance.
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338
A.F. MURANT,
M. A. M A Y O , B. D. H A R R I S O N
AND
R. A. G O O L D
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