811
J. gen, Virol. (1985), 66, 811 815. Printed in Great Britain
Key words : RGD V/protein/core/immunoblotting
Location of Structural Proteins in Particles of Rice Gall Dwarf Virus
By T. O M U R A , 1. Y. M I N O B E , - ' M. M A T S U O K A , 2 Y.
T. T S U C H I Z A K I 1 AND Y. S A I T O 3
NOZU, 2
~National Agriculture Research Center, "-National Institute o / Agrobiological Resources and
3National Institute o]Agro-environmental Sciences, Tsukuba Science City, Ibaraki 305, Japan
(Accepted 19 December 1984)
SUMMARY
Protein from particles of rice gall dwarf virus was resolved into seven components by
polyacrylamide gel electrophoresis. The estimated molecular weights ( × 103) were 183,
165, 150, 143, 120, 56 and 45 (K). Cores prepared by treating intact particles with CsC1
contained the 183K, 165K, 120K and 56K proteins: the 150K, 143K and 45K proteins
were recovered from the top layer of gradients after CsC1 equilibrium centrifugation.
Antiserum against intact particles reacted mainly with 150K and 45K proteins and
antiserum against core particles reacted mainly with 183K and 120K proteins. The
results suggest that the 150K and 45K proteins are located on the surface of the outer
capsid, that the 183K and 120K proteins are on the surface of the core, that the 165K
and 56K proteins are inside the core, and that the 143K protein is either inside the outer
capsid or inside the core.
INTRODUCTION
Rice gall dwarf virus (RGDV) is a recently described virus of rice (Omura et al., 1980; Ong &
Omura, 1982; F a a n et al., 1983) that is transmitted in a persistent m a n n e r by five species of
leafhoppers (Inoue & Omura, 1982: Morinaka et al., 1982) and which has polyhedral particles
about 65 n m in diameter. R G D V is thought to be a Phytoreovirus (Omura et al., 1982), a
suggestion supported by the finding that the genome of R G D V consists of 12 segments of
double-stranded R N A (Hibi et al., 1984).
Protein components of the virus particles have been studied in some viruses belonging to the
reovirus group. The Phytoreoviruses such as wound tumour virus (WTV) (Reddy & Macleod,
1976) and rice dwarf virus (RDV) (Nakata et al., 1978) have six or seven similar-sized protein
components that differ in size from the six particle proteins of maize rough dwarf virus
(Boccardo & Milne, 1975) and the three particle proteins of core preparations of Fiji disease
virus (Van der Lubbe et al., 1979), both of which are Fijiviruses. We have examined the n u m b e r
and molecular weights of the protein components of R G D V to see if these are similar to those of
WTV and RDV. Also, outer capsids were removed from core particles by a CsC1 treatment and
the locations of the protein components were investigated by electrophoresis and immunoblotting using antisera to virus particles and to cores.
METHODS
Virus. RGDV was purified by the method reported by Omura et al. (1982), except that 1% Triton X-100 was
added to the extract containing polyethyleneglycol and sodium chloride. The final pellets were resuspended in 0.1
M-histidine, 0.01 M-MgCI2, adjusted pH to 7.0 with HCI (His-Mg) and stored at -70"C until use.
CsCI equilibrium centriJugation. A purified preparation of RGDV suspended in 0.1 ml His Mg was added to a
solution of CsCI to make the final concentration of CsCI 36°.o(w/w). The mixture was kept for 40 rain at room
temperature (approx. 20'~C) and centrifuged at 165000 g for 24 h at 4"~C in a Hitachi RPS-50 rotor. The tube
contents were fractionated with an ISCO model UA5 ultraviolet analyser and virus particles or core particles were
recovered by centrifugation (96000g) for 2 h at 4 °C in a Hitachi RP-40 rotor and resuspended in His-Mg. The
density of the fractions was calculated from their refractive indices using the method of Brakke (1967).
0000-6403 © 1985 SGM
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812
T. OMURA AND OTHERS
The fraction from the meniscus to about 3 mm below the meniscus was mixed with 0.2 vol, 50°~ trichloroacetic
acid and centrifuged. The pellet was washed twice with acetone, then dried and dissolved in His-Mg containing
2°o SDS and 2°.o 2-mercaptoethanol. The sample was frozen at - 2 0 ° C until use.
Electron microscopy. Preparations were negatively stained with either 2°o~ neutralized phosphotungstic acid
(PTA) or 2°~ uranyl acetate and were examined in a Hitachi H-500 electron microscope.
Antisera. A rabbit was immunized by intramuscular injection of purified preparations of core particles
emulsified with an equal volume of Freund's complete adjuvant followed by two similar intravenous injections at
10-day intervals. The titre of the antiserum in a precipitin ring interface test was 1/64 against homologous inner
core particles. Sera were stored at - 7 0 °C until use.
Electrophoresis. Slab gels consisting of 10°'o polyacrylamide (acrylamide :bisacrylamide = 30:0.8) containing
0.1 ° o SDS were prepared by the method of Laemmli (1970). Purified materials were solubilized by heating for 2
min at i00 ~C in 2°.-~SDS and 2 ~ 2-mercaptoethanol. Molecular weights of proteins were determined using SDSPAGE Marker l (molecular weights were 180, 140, 100, 42 and 39, all × 103: Biochemicals, Tokyo, Japan) as
standards. Gels were stained with Coomassie Brilliant Blue R (CBB) and scanned using a Shimadzu
chromatoscanner CS-910. The area under each absorbance peak was taken as a measure of the amount of protein
in the band.
Immunoblottmg. Polypeptides were transferred from polyacrylamide gels to nitrocellulose sheets using a
Transfer Blotting Apparatus (Model ETB-15 ; Toyo Science, Japan) as described by Towbin et al. (1979). Transfer
of the polypeptides to the nitrocellulose sheet was confirmed by staining with amido black. Immunological
detection of polypeptides on nitrocellulose sheets using fluorescein-conjugated sheep anti-rabbit IgG (Cappel
Laboratories) was performed by the method of Towbin et al. (1979) modified by Matsuoka & Asahi (1983).
RESULTS
Fractionation of virus particles using CsCI
Reddy & Macleod (1976) and Nakata et al. (1978) separated the outer capsid from the core of
WTV and RDV, respectively, by centrifuging particles in CsC1 density gradients; this method
was applied to R G D V particles. The pH of the buffer has been reported to have a marked effect
on CsC1 equilibrium sedimentation of bluetongue virus (Verwoerd et al., 1972) and WTV
(Reddy & Macleod, 1976). Therefore, we studied the effect of centrifuging R G D V particles in
CsC1 in His-Mg buffer at different pH values. The results are shown diagrammatically in Fig. 1.
Particles that were penetrable by stains and thatappeared empty were always found in a zone
with a density of 1.285 g/ml. At pH 6-0 a broad band formed with a density of 1.406 g/ml. It was
less obvious at higher pH and was not observed at pH > 7.5. This band contained virus particles
from which part of the outer capsid had been lost. At pH > 6, a clear band appeared which had a
density of 1.496 g/ml and which contained polyhedral particles with spike-like structures about
50 nm in diameter (Fig. 2, right). These particles were designated as core particles, From these
results, it appears that the outer capsid proteins were removed by CsC1 when the pH was higher
than 7-5 and His-Mg-buffered CsCl, pH 7.5, was adopted to separate the outer capsid proteins
from the core particles.
The ratios of A z 6 o / A 2 8 o for purified intact virus and core particles were 1-42 and 1.63,
respectively. As expected, the core had a higher ratio than intact particles, presumably because
the loss of the outer capsid had increased the proportion of nucleic acid.
Viral proteins
Protein from intact R G D V particles was resolved by potyacrylamide gel electrophoresis into
two major [120 x 103 and 45 × 103 (K)], three intermediate (l 65K and 143K, and 56K) and two
minor (183K and 150K) protein components (Fig. 3a, Fig. 4a), The relative amounts of each
protein were 2 6 : 5 9 : 5 : 5 : 4 : 1 : < 1 for the 120K, 45K, 165K, 143K, 56K, 183K, and 150K
proteins, respectively. Although the 183K and 56K proteins each sometimes appeared to be two
close bands, we have considered them as being single proteins in this report. When the core
particles were subjected to electrophoresis, the relative amounts of the 183K, 165K, 120K and
56K proteins were like those of intact particles (Fig. 3b), but the amounts of 150K, 143K and
45K proteins were much less than in intact particles. The 150K, 143K and 45K proteins did not
sediment in CsC1 gradients and were recovered from the tops of the gradients. The relative
amounts of the 183K, 165K, 120K and 56K proteins in this fraction were negligible.
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813
Proteins of RGD V
Density
(g/ml)
1.267
i
I
1.285
m
m
m
m
1.406
1.496
\
pH
6.0
6.5
7.0
7.5
8.0
7.5
Fig. l. Diagram of the light-scattering bands formed by equilibrium centrifugation of RGDV in CsCI
gradients buffered with His-Mg at various pH values and photograph of a gradient at pH 7.5.
Fig 2. RGDV particles negatively stained with PTA Left, intact particles: right, core particles. Bar
markers represent 100 nm.
Immunoblotting
Immunoblotting showed that antiserum against intact R G D V reacted mainly with the 150K
and 45K proteins (Fig. 4b), whereas the antiserum against R G D V core particles reacted mainly
with the 183K and 120K proteins (Fig. 4c).
DISCUSSION
The R G D V capsid was found to be composed of two layers, the outer capsid and the inner
capsid surrounding the R N A (Omura et al., 1982). The inner capsid plus R N A is described here
as the core. Separation of the outer capsid layer from the inner core enabled us to locate the
proteins in the respective components of the virus. The 183K, 165K, 120K and 56K proteins
could always be detected in almost equal amounts in core particles and intact virus particles
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814
T. O M U R A
(a)
183K
(b)
AND
OTHERS
(a)
(e)
(b)
(c)
•
165K
150K
143K
120K~
56K ~
P1111111111111111~- -
~
~
-
'
183K
165K
150K
143K
120K
~
~
J"
•"
56K
•
45K
•
....
'
W
45K
Fig. 3
Fig. 4
Fig. 3, Electrophoresis of RGDV protein in 10% polyacrylamide gel, (a) Protein from intact particles.
(h) Protein from core particles. (c) Protein from the top fraction of gradients after CsCI equilibrium
centrifugation.
Fig. 4. Immunoblotting of RGDV proteins. RGDV proteins were separated by electrophoresis in
polyacrylamide gel and stained (a) or blotted onto nitrocellulose paper and treated with antiserum to
intact particles (h) or antiserum to cores (c), and then stained with fluorescein-conjugated second
antibody.
T a b l e 1.
Molecular weight and location of component proteins of RGD V
Detection by
antiserum to
Presence in
A.
Protein species
Mol. wt.
( × 10 -3)
Virus
particle
183
165
150
143
120
56
45
_
_
A
.
_
_
Core
Fraction
removed
from core
Virus
particle
Core
+
+
+
+
+
+
-
+
+
+
-
+
-
+
+
+
+
+
-
+
+
+
-
Deduced
location
Surface of core
Inner part of core
Surface of outer capsid
Inner part of either
outer capsid or core
Surface of core
Inner part of core
Surface of outer capsid
(Fig. 3b). O n the o t h e r h a n d , the 150K, 143K a n d 4 5 K p r o t e i n s w e r e f o u n d at t h e t o p s o f t h e
g r a d i e n t s a f t e r CsC1 e q u i l i b r i u m c e n t r i f u g a t i o n (Fig. 3 c). T h e s e results suggest t h a t 150K, 143K
a n d 4 5 K p r o t e i n s w e r e r e m o v e d f r o m t h e i n n e r core particles.
T h e 150K a n d 4 5 K p r o t e i n s r e a c t e d s t r o n g l y w i t h a n t i s e r u m to i n t a c t p a r t i c l e s (Fig. 4b),
w h e r e a s w h e n a n t i s e r u m a g a i n s t c o r e p a r t i c l e s was used, t h e 183K a n d 120K p r o t e i n s r e a c t e d
s t r o n g l y (Fig. 4c). T h e s e results suggest t h a t t h e o u t e r c a p s i d p r o t e i n s are p r e f e r e n t i a l l y
r e c o g n i z e d as i m m u n o g e n s in the i n t a c t p a r t i c l e s a n d t h a t p r o t e i n s o f the core are n o t r e c o g n i z e d
unless t h e o u t e r c a p s i d is r e m o v e d . O u r results t h e r e f o r e suggest t h e following l o c a t i o n s for the
p r o t e i n s : the 150K a n d 4 5 K p r o t e i n s are in t h e s u r f a c e o f t h e o u t e r c a p s i d , the 183K a n d 120K
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Proteins o f R G D V
815
proteins are in the o u t e r part o f the core, the 165K and 56K proteins are inside the core, and the
143K protein is either on the inside of the outer capsid or within, but not on, the surface of the
core (Table 1).
T w o m a j o r protein bands h a v e been o b s e r v e d in particles of three viruses that belong to the
P h y t o r e o v i r u s subgroup, i.e. 120K and 45K (Fig. 3a, 4a) in R G D V , 131K and 46K + 4 5 K in
R D V ( N a k a t a et al., 1978) and 118K and 36K + 35K in W T V ( R e d d y & M a c l e o d , 1976). It is
interesting that the larger proteins are located in the cores and the smaller proteins are found in
the capsid (Table 1 ; N a k a t a et al., 1978 ; R e d d y & M a c l e o d , 1976). T h e 4 5 K protein of R G D V
did not separate into two bands in our e x p e r i m e n t , but the c o r r e s p o n d i n g bands of R D V and
W T V separated into two (46K and 45K in R D V , and 36K and 35K in W T V ) .
T r e a t m e n t of particles with organic solvents r e m o v e s the o u t e r capsids o f Fijivirus s u b g r o u p
viruses but not those of viruses in the P h y t o r e o v i r u s subgroup ( M i l n e & Lovisolo, 1977; O m u r a
et al., 1982). As was shown by R e d d y & M a c l e o d (1976) for W T V and by N a k a t a et al. (1978) for
R D V , the outer capsid of R G D V was r e m o v e d by CsCI t r e a t m e n t at an a p p r o p r i a t e pH. T h e s e
results suggest that the b i n d i n g forces b e t w e e n the outer capsid proteins and the core proteins of
virus particles in the P h y t o r e o v i r u s subgroup and the Fijivirus subgroup are different. It is
possible that studies o f the r e m o v a l o f the outer capsid proteins by a c o m b i n a t i o n o f CsCI
t r e a t m e n t and variations in the p H o f the buffer m a y assist the study of the c o m p l e x structure of
these virus particles.
This work was supported in part by Special Coordination Funds for Promoting Science and Technology from
the Science and Technology Agency of Japan.
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DER
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(Received 26 September 1984)
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