83
Y. gen. Virol. (I977), 38, 83-95
Printed in Great Britain
Further Physicochemical Characterization
o f N o d a m u r a Virus. Evidence that the Divided Genome
Occurs in a Single Component
By J. F. E. N E W M A N
AND F. B R O W N
Animal Virus Research Institute, Pirbright, Surrey
(Accepted 5 August I977)
SUMMARY
Nodamura virus, a small non-enveloped R N A virus, contains two species of
R N A sedimenting at 22S ( R N A - 0 and I5S (RNA-2), a single major polypeptide
of mol. wt. 4o x Io 3 and two minor polypeptides, of tool. wt. 38 and 43 x io ~.
Evidence is presented that the two R N A species are in the same particle. Although
extraction of the virus with SDS-phenol yields the two species of R N A as separate
entities, gentle treatment of the virus with guanidine and low concentrations of
SDS releases the R N A as a z7S component which contains both RNA-I and
RNA-2 together with a trace of protein. It seems likely that the two R N A species
replicate separately because double stranded molecules corresponding to the
single stranded RNA-1 and RNA-2 molecules were present in B H K cells infected
with the virus.
INTRODUCTION
In a previous paper (Newman & Brown, I973) we showed that Nodamura virus, an
arthropod-transmissible small R N A virus, contains two R N A molecules (RNA-I and
RNA-2) which sediment at about 228 and 15S respectively in sucrose gradients (corresponding to tool. wt. c. I.O and 0"5 x 1@), and one major polypeptide, tool. wt. c. 35 x IoL The
two RNA molecules are present in equimolar amounts and both are required for infectivity. Although virus particles containing different R N A species could not be separated
by sucrose gradient centrifugation, extraction of RNA-I by treatment of the virus with
phenol alone compared with extraction of both RNA-I and RNA-z with phenol-SDS
led us to favour the idea that our virus preparations contained more than one kind of
particle. In addition, centrifugafion in caesium chloride gradients appeared to separate
the virus into two components, one of which contained RNA-I and the other RNA-2.
This work suggested that Nodamura virus might resemble many of the small R N A viruses
of plants in having multiple components, but our attention was constantly drawn to two
observations which conflict with this idea. Firstly, the two RNAs were always present in
equimolar amounts, which is inconsistent with the concept that they are in separate components. Secondly, infectivity titrations of Nodamura virus in mice have been typified by
the same degree of scatter at the end point as is observed with those of Mengo virus and
foot-and-mouth disease virus, each of which consists of a single infective component. It
would be expected that a virus producing two types of particle in equal amounts, each of
which is necessary for infectivity, should have a more abrupt end-point than a virus which
produces only one infective component. These observations suggested therefore that
Nodamura virus might consist of a single infective component.
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84
J. F. E. N E W M A N
A N D F. B R O W N
Further experiments have now shown that the virus is disrupted by chloride ions, thus
providing an explanation for our observations made when the virus was centrifuged in
CsC1. The work described in this paper shows that both the R N A molecules are present in
the virus particles as a single 27S R N A and may be linked by a labile, non-covalent bond.
METHODS
Except for those below, all the materials and methods used were described by Newman
& Brown (I973).
Preparation of radiolabelled virus. Virus labelled with 35S-methionine or 3H-leucine was
obtained as previously described for z2P-labelled virus (Newman & Brown, I973) except
that 5 to I o # C i of 35S-methionine or I5 to 25 #Ci 3H-leucine was inoculated into each
infected 7-day-old mouse three times during the incubation period.
lsoelectric focusing. A pH gradient from 3 to Io was prepared in a horizontal zone
convection-stabilized isoelectric focusing apparatus using LKB Ampholine (Talbot, i972).
A o.I ml sample of radioactively labelled virus was added at either pH 3 or 9 and a potential
difference of Iooo V applied for 72 h. The gradient was then fractionated and the p H of
each I ml fraction determined. Samples of each fraction were treated with Io o/o trichloracetic acid (TCA) and the precipitates collected on glass fibre discs. The dried discs were
then counted in a toluene-based scintillant in a Packard scintillation counter.
Sucrose gradient electrophoresis. Electrophoresis was performed in the apparatus described by Pringle (I969). Linear gradients of 5 w/v) to 2o % (sucrose in o.oI M-phosphate,
pH 7"8, were prepared in 55 x I cm jacketed electrophoresis tubes which had been sealed at
the lower end with dialysis membrane. On top of this gradient, 2 ml of z'5 % sucrose were
layered and then 2 ml of phosphate buffer to form a steep o to 5 ~/o gradient. The tubes were
then placed in the vertical electrophoresis apparatus with both ends making contact with
the o.oI M-phosphate buffer, pH 7"8, in each electrode bath. The virus sample (o.I ml
virus, o.I ml I5°/O sucrose and o.I ml 0.05% phenol red) was layered on to the top of the
gradient and an initial current of 1.5 mA/column applied for I7 h. During this time, cooled
water was passed through the outer jacket of each gradient tube to maintain a temperature
of IO °C. Normally, after I7 h, the phenol red had almost traversed the length of the
column. When electrophoresis had been completed the gradient was fractionated from the
bottom of the tube. Samples from each fraction were dried on to glass fibre discs and
counted in a scintillation counter.
U.v. irradiation. Freshly prepared z~P-virus was diluted fivefold with 0"o4 M-phosphate
buffer, p H 7"6, and placed in a Io cm plastic Petri dish. The open dish was then irradiated
at a distance of IO cm, with a Hanovia Chromatolite portable lamp with a u.v. emission
wavelength of 254 nm. At selected intervals aliquots were removed from the dish for infectivity determination and R N A extraction.
Polyacrylamide gel electrophoresis of virus proteins. Preparations of virus particles labelled
with aH-leucine were mixed with 14C-amino acid labelled polypeptides induced by rhinovirus IA, made 1% with respect to SDS and mercaptoethanol in 0"5 M-urea and heated
for 2 min at IOO °C. The samples were then dialysed for I7 h in o'oI M-sodium phosphate,
pH7"2, 0"5 M-urea, o.1% SDS, o . I % mercaptoethanol and I mM-sodium azide. After
reheating for I min at IOO °C the samples were electrophoresed on columns (0.6 x 25 cm)
of m°/o polyacrylamide containing 0"3 % (v/v) ethylene diacrylate as cross-linker, o.1%
SDS and o.I M-sodium phosphate, pH 7"2. The electrophoresis buffer was o.t M-sodium
phosphate, pH7"2, containing o'I~o SDS and o.1 M-sodium 3-mercaptopropionate.
Electrophoresis was normally at 8 mA/gel for 20 h.
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R N A s of Nodamura virus
85
+
m
I
×
ff
1
r~
I
tt
t
I
/
9
bzl
I
I
2O
4~)
Fraction
Fig. I. Electrophoresis of 82P-labelled Nodamura virus in a 5 to zo % sucrose gradient
in o-ox M-phosphate, pH 7"8.
RESULTS
Attempts to separate different virus particles by physical methods
In our previous work (Newman & Brown, I973) we were unable to separate two components by sucrose gradient centrifugation and the R N A extracted from individual fractions
of the single virus peak sedimenting at I35S contained the same proportions of RNA-I
and RNA-z. Additional experiments with individual fractions from the sucrose gradient
peak of purified virus have confirmed these earlier observations.
Electrophoresis of radioactively labelled virus on sucrose gradients, using the method
described by Pringle 0969), gave a single sharp peak (Fig. I). A single peak, at pH 4,
was also obtained when the virus was electrophoresed in an ampholyte pH gradient (Fig. 2).
Similarly, no separation of different virus components could be achieved using ion exchange
resin chromatography. These results show that, if more than one component is present in
the virus, they must have strikingly similar sedimentation and charge characteristics.
Attempt to link RNA-I and RNA-z by ultraviolet irradiation
Mayo et al. (I973) have shown that u.v. irradiation of the bottom component particles
of raspberry ringspot virus causes dimerization of the r.4 x io ~ tool. wt. R N A to give a
tool. wt. of 2.8 x io 6. They have suggested that this observation provides evidence that two
molecules of the I-4 x io 6 RNA are present in the same particle. We have used u.v. irradiation in an attempt to determine whether RNA-I and RNA-2 of Nodamura virus are
present in the same or different particles. If they are in the same particle an R N A with a
mol. wt. of e. I-5 x io 6 should be obtained.
A preparation of purified 32P-virus was irradiated and samples removed at 'different
times for the determination of infectivity and for the extraction and analysis of the virus
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86
J. F. E. N E W M A N AND F. B R O W N
"••
12
,
I
8
8
" ~
o-q
i
4
6
b
t
'7
~
'
x
'
.o'
~
E
(a)
4
q
. 0"0"0- 0_0_0_0_0_0_0.0_0. 0.0. O- O- 0-0"0" 0~0.0"0"
~.0_0.0_ 0
~I~
(b)
~2
8
8
~
6
O
4
-
/
0-.0-0- 0-0- O-n- o-ooo~-O-t",~J-n-n.o-
~
_
4
o--0- O- O- d - o -o- o"
10
20
30
Fraction
Fig. 2. Isoelectric focusing of 32P-labelled Nodamura virus. (a) Inserted at pH 9; (b) inserted at
pH 3, O - - - O ,
a2p; •
0 , pH gradient.
I
I
I
100
50
"~
[]
..~
~ t ~
43
2-=
10
20
U.v. irradiation (rain)
30
Fig. 3. Effect of u.v. irradiation on the infectivity ([~--V~) of Nodamura virus and on the amount
of RNA (O--©) that could be extracted with phenol-SDS.
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87
R N A s o f N o d a m u r a virus
I
(a)
3
o/o
/
2
!
I
0
i
1
9
o I'o
% %
k
s
%%000000
i
3
(h)
×
"6
2
~2
d
1
O
'o
,i
•
',o.j~, o,
(c)
2-
,o°q~
1
20
Bottom
40
Top
Fraction
Fig. 4. Effect of u.v. irradiation on the sedimentation profiles of Nodamura virus R N A in 5 to
25 ~ sucrose gradients in o'I M-acetate-o.~ ~ SDS, pH 5"o. Centrifugation was for ~7 h at 3oooog.
Virus was irradiated for (a) o; (b) I6 and (c) 32 min.
RNA. Infectivity was lost according to single hit kinetics, suggesting that the RNAs were
contained in a single component (Fig. 3).
We could find no evidence for R N A molecules larger than the individual R N A species
and the profiles in the sucrose gradients remained unchanged qualitatively (Fig. 4). However, the amount of R N A that could be extracted from the preparations decreased as
irradiation proceeded (Fig. 3). This was presumed to be caused by interaction of the R N A
with the virus protein. Additionally, the R N A extracted from u.v. irradiated virus labelled
in both the R N A and protein components contained traces of the isotope associated with.
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88
J . F . E . NEWMAN AND F. BROWN
the protein component. Miller & Plagemann (t974) have also shown in u.v. irradiation
experiments with Mengo virus that linkage of the RNA and protein occurs.
Equilibration in gradients of caesium salts
When purified 3zP-virus which had been stored for a few days at 4 °C was centrifuged
in a pre-formed caesium chloride gradient, pH 7"5, for 6 h at 6oooog, conditions which
allow small RNA viruses to reach equilibrium, two peaks of radioactivity were obtained
(Newman & Brown, I973). The faster sedimelxting peak contained RNA-I and the slower
peak contained RNA-2. This observation led us to the conclusion that the virus probably
comprised two particle types, each of which contained a different R N A species. However,
we have now found that purified virus particles are unstable in the presence of C1- ions
(see below) which indicates that our previous observations in the CsC1 centrifugation
experiments were due to release of R N A from the virus particles. The two peaks of z2p
observed in the CsCl gradients were obtained because the conditions of centrifugation,
although sufficient for the equilibration of small R N A virus particles (Rowlands, Sangar &
Brown, I97~), do not allow the released R N A molecules to reach equilibrium.
We had shown that freshly purified virus gave only one peak in a CsC1 gradient at pH 8
(Newman & Brown, I973). We have now found that, even with freshly purified virus, two
peaks are obtained if the gradients are below pH 7"5 or above pH 8"5. Between p H 7"5
and 8"5, a single peak at a buoyant density of I'34 g/ml was obtained. Fixation of the virus
particles with o'1% formaldehyde for I7 h at zo °C prevented their disruption in CsC1
gradients in the range pH 4 to 9 and these equilibrated at a density of ~.34 g/ml. However,
even unfixed particles gave a single peak at a density of 1.34 g/ml when Cs2SO4 was used
instead of CsC1.
Instability of virus particles in chloride ions
In our previous paper (Newman & Brown, ~973) we found that virus purified by sucrose
gradient centrifugation in solutions containing tris-HC1 was extremely unstable and
frequently lost more than 9o % of its infectivity when stored overnight at 4 °C. Similarly,
centrifugation of purified virus particles in CsC1 gradients caused their disruption and
there was very low recovery of infectivity. The loss of infectivity in the CsC1 gradients was
not due to the shear forces involved in centrifugation because storage of the virus in a CsC1
solution with a density of ~.34 g/ml also caused loss of infectivity. The instability of virus
particles in CsC1 compared with Cs2SO~ suggested that C1- ions were responsible for the
disruption of the particles. To test this possibility, purified virus was mixed with several
different salts at a concentration of o.I M in O'I M-acetate buffer, pH 6, and incubated for
I5 rain at 2o °C before dilution in o'o4 M-phosphate and titration in mice. The results in
Table I show that, of the anions tested, only C1- and I- ions had any effect on infectivity
and that the effect of C1- ions was considerably greater than that of I- ions. It is surprising
that F - and Br- ions had no effect on the infectivity of the virus. Unfractionated mouse
muscle extract or purified virus with 1% bovine serum albumin showed no loss of infectivity after similar treatment with C1- ions.
It has been reported that the infectivity of the cardioviruses is decreased significantly
by certain halide ions (Speir, I962; Rueckert, I 9 7 0 and the inactivation is dependent on
pH. In view of this, the influence of C1- ions on the infectivity of Nodamura virus over the
range pH 3 to 9 was tested. Purified virus particles in o.I M-acetate buffer at different pH
values were made o-I M with respect to NaC1 and incubated for ~5 rain at 2o °C before
titration in mice. At all the p H values tested infectivity was lower except at p H 3 to 4
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R N A s o f Nodamura virus
89
Table I. Effect of different salts on the infectivity of Nodamura virus in mice
Infectivity (log LDs0/o'o3 ml)
m
r
Salt
Unfractionated
mouse muscle
extract
Purified virus
None
NaC1
NaF
NaBr
Na2SO4
KI
KNO~
I
7"3
4"5
7"3
7"5
7"7
5"3
7"7
I
E 8 a-
6"9
6"7
7-I
6'5
6'7
6"9
6'5
I
I
n
[]
[
g,
.E
2
E
I
4
5
6
7
8
pH
Fig. 5. Effect of different pH values on the infectivity of Nodamura virus in the presence of chloride
ions. []--[], Virus in o-I N-acetate; ©--©, virus in o-I M-acetate, o'I M-NaC1.
(Fig. 5) which is the isoelectric point of N o d a m u r a virus as shown in our attempts to separate
different components of the virus by electrophoresis on an ampholyte p H gradient (Fig. 2).
The mechanism by which C1- ions inactivate the virus was investigated by centrifuging in
a sucrose gradient a sample which had been stored in a CsC1 solution 0"34 g/ml) for 15 rain
at 20 °C. Components sedimenting at 8oS or less were obtained and no intact virus particles
were seen in the electron microscope (Fig. 6). However, as much infective R N A could be
extracted from the disrupted preparation as from the untreated virus, indicating that loss
of infectivity was due to loss of particle integrity and not destruction of the RNA. However,
the product obtained by CI- disruption was insensitive to pancreatic RNase.
Disruption of virus particles with guanidine
Treatment of 82P-virus particles with 1.5 M-guanidine followed by centrifugation in sucrose gradients in o.~ M-acetate, o.I % SDS, p H 5"o, releases the two R N A species (Newman
& Brown, I973). However, centrifugation of the guanidine-treated particles in sucroseacetate gradients lacking SDS demonstrated breakdown probably similar to that described
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9O
J. F. E. NEWMAN AND F. BROWN
Fig. 6. Electron micrographs of Nodamura virus, (a) before and (b) after storage for 15 min in
CsC1 solution (1"34 g/ml) in o.I M-tris-HCl, pH 7.
above using NaC1 or CsC1. Subsequent centrifugation of the guanidine treated virus in
gradients containing o-1% SDS released the two R N A species.
To investigate this observation further, purified virus was mixed with 1.5 M-guanidine
for I5 min at 20 °C and samples were then centrifuged in 5 to 25 % sucrose gradients in
o-I M-acetate containing different concentrations of SDS. When the concentration of SDS
was o.oI %, most of the radioactivity was associated with a broad peak which sedimented
at c. 27S and was infective (Fig. 7a). With o.1% SDS the two R N A s sedimenting at 22S
and I5S were released (Fig. 7b) and only a mixture of the 22S and I5S R N A s was infective
(Newman & Brown, I973). Subsequent centrifugation of the 27S component in sucrose
gradients containing o.1% SDS disrupted it into the 22S and I5S R N A species. Heating
the 27S component at 6o °C for I min or extracting with phenol also caused the release of
the 22S and I5S R N A species. These experiments provide strong evidence that the two
R N A species are present in the same virus component.
In experiments with virus labelled in the protein moiety with 35S-methionine some radioactive label was always associated with the 27S R N A (Fig. 7a) but none could be found in
the 22S and I5S R N A species.
Virus R N A and proteins
F r o m their sedimentation coefficients and electrophoretic mobility in polyacrylamide
gels, the mol. wt. of the two R N A s were estimated to be I × Io n and 0"5 × Io 6 (Newman &
Brown, I973). A theoretically more accurate estimate was obtained by electrophoresing a
mixture of 32P-Nodamura virus R N A and 3H-uridine labelled B H K ribosomal R N A in
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RNAs of Nodamura virus
91
i
(a)
,?.o
/
o" ',,
/ q'\
"~
\
2
nn.nn_nn~ - o .
Y /
/
°'°, _ /
\
o
i
×
m-rim--n-m--H-
E
n-m-m
(b)
?,
I %
~0
o
0
|
I
!
I ~
v
0
I
I
I
l
I
=
I ¢S
0
0
I
I
I
I
I
,!
mL,..~mm.,-mm,..mu"" ram--he'- I
10
Fraction
i_11
U~
I
O
"~.~'
\
1 u
20
Fig. 7. Centrifugation in 5 to 25 % sucrose gradients at 30 ooo g for 17 h of Nodamura virus that had
been treated with I"5 M-guanidine. The sucrose gradients contained different concentrations of
SDS. (a) Virus labelled with 32p and 85S-methionine in o-oI % SDS; (b) asP-virus in o.t % SDS;
O - - - O , a~p; 0 - - 0 , 85S; I - - I , infectivity. The arrows show the positions of the 28S and t8S
BHK cell RNA markers centrifuged in the same gradients.
4 % polyacrylamide gels in the presence o f barbitone buffered formamide at 2o °C (Pinder,
Staynov & Gratzer, I974). F r o m a plot o f log mol. wt. against migration, using ribosomal
R N A s as standards (28S R N A , 1"64 x lO6; I8S, o'67 x zo~; Peterman & Pavlovec, I966),
the z2S and I5S virus R N A s were estimated to have mol. wt. o f I.i 5 and o'46 x io 6,
respectively.
The R N A content o f the virus was estimated by a modified orcinol method (Hurlbert
et al. I954) and the protein content o f the same virus preparation was determined by the
L o w r y method (Lowry et al. 1951). Virus that had been given a second cycle o f centrifugation in a sucrose gradient was pelleted and the washed pellet was used for the estimations.
F r o m five separate determinations, the R N A and protein contents were estimated to be
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92
J . F . E . NEWMAN AND F. BROWN
3
5
1
4
5
10
15
Migration (cm)
20
Fig. 8. Polyacrylamide gel electrophoresis of Nodamura virus proteins labelled with 3H-leucine.
2o'5 % and 79"5 % respectively, assuming that any constituents other than R N A and protein
are either absent or present in only trace amounts.
The polypeptides of virus grown in the presence of 3H-leucine were analysed by polyacrylamide gel electrophoresis as described in Methods. After electrophoresis the gels
were cut latitudinally into I mm slices for the determination of radioactivity. One major
and two minor peaks were found with each preparation (Fig. 8). Using polypeptides induced
by rhinovirus IA as tool. wt. markers (McLean & Rueckert, ~973), the major peak was
estimated to have a tool. wt. of 4o × IO3 and the two minor polypeptides had tool. wt. of
38 and 43 × ~o~. The proportion of radioactivity in each of the three polypeptides of the
~H-leucine labelled preparation indicated that the minor polypeptides comprised about
6 ~o of the total protein. This probably represents a fairly accurate assessment because
there is a high proportion (about 7 %) of leucine in Nodamura virus protein (Newman
et aI. ~977). Experiments with unlabelled virus in which the proportion of protein in each
peak was estimated by the amount of staining with Coomassie blue gave a similar result.
The amounts of the minor proteins suggested that they might be contaminating host cell
material. However, similar polyacrylamide gel electrophoresis profiles were obtained with
disrupted virus particles purified from wax moth larvae infected with the virus.
A model f o r the virus
The present work has shown that the major polypeptide of the virus has a mol. wt. of
4o x io 3, compared with the value of 35 × Io 3 obtained previously (Newman & Brown, I973).
An independent determination of the mol. wt. of the major protein by chromatography on
Sepharose 6B in the presence of 6 M-guanidine gave a value of 39 × IO3 (Newman, I975).
Similarly, a mol. wt. of 4o × Io 3 was obtained from the amino acid composition of the
major protein (Newman et al. I977). Taking the mol. wt. of the R N A as H 5 +o'46 = I-6I x
Io 6 (from the formamide-polyacrylamide gel electrophoresis experiments) and the R N A
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RNAs of Nodamura virus
93
|
(a)
2O
8
?
×
101t
,o
4 P'
~
"r'2':":,"""-r"c'-"
4
"/'"
2
i/
',
I
l
J
4
8
Migration (cm)
Fig. 9. Polyacrylamide gel electrophoresis of Nodamura virus induced RNAs on 2"4 ~ gels containing 4 ~ agarose: (a) RNA extracted from cells at 3o h post infection following a 6 h labelling
period with 8H-uridine; (b) a sample of the same RNA incubated for I5 min at 37 °C with I #g/ml
pancreatic ribonuclease before electrophoresis. O---O, 3H; O - - O , 3~P-labelled BHK cell RNA.
c o n t e n t as 20"5 % , the mol. wt. o f the virus can be calculated to be 8 x i o 6, similar to t h a t
o b t a i n e d for the v e r t e b r a t e picornaviruses (Brown & Hull, ~973).
F r o m a t o t a l mol. wt. o f 6"4 x i o 6 for the virus protein a n d a p o l y p e p t i d e mol. wt. o f
c. 40 x ~o 3, it can be calculated t h a t there are p r o b a b l y a b o u t 16o p r o t e i n units in each
virus particle. A l l o w i n g f o r the inaccuracies in the d e t e r m i n a t i o n o f the a m o u n t a n d mol. wt.
o f the R N A a n d protein, these d a t a are n o t inconsistent with a I8O sub-unit structure for
the virus (Brown & Hull, I973).
Replication of the virus RNA
N o d a m u r a virus has been shown to replicate in B H K cells (Bailey, N e w m a n & Porterfield, 1975). T h e virus i n d u c e d R N A s were labelled with 3H-uridine between 24 a n d 30 h
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94
J. F. E. N E W M A N
AND
F. B R O W N
after infection at a multiplicity of I in the presence of actinomycin D (I #g/ml) and examined by polyacrylamide gel electrophoresis. The R N A was extracted from the infected
cells with phenol and precipitated twice with 2 vol. ethanol at - 2 o °C. The gel profile in
Fig. 9 (a) shows that the two RNAs found in virus particles and some species migrating
more slowly than RNA-I were present. Treatment of the induced R N A with pancreatic
ribonuclease at I #g/ml before electrophoresis gave the pattern shown in Fig. 9 (b). Two
distinct peaks were found at positions expected for the double stranded forms of RNA-I
and RNA-2 but there was no evidence for a peak at a position corresponding to a double
strand of the 27S R N A isolated by I'5 M-guanidine disruption of the virus and treatment
with o.oI % SDS.
DISCUSSION
Nodamura virus is unlike any o f the other small R N A viruses infecting vertebrates in
possessing a divided genome. Extension of our earlier work (Newman & Brown, 1973)
has led us to conclude that both R N A species found in the virus are present in the same
particle and not in two separate types of particle as we had thought previously. Our earlier
view stemmed largely from the observation that 32P-virus preparations gave two peaks in
caesium chloride gradients, each of which yielded a single R N A species on extraction with
SDS-phenol. It has become apparent from our present work that the virus is unstable in
C1- ions and when centrifuged in a CsC1 gradient is disrupted into its R N A and protein
components. Under the centrifugation conditions used in our previous work, namely 6 h
at 60ooo g in a pre-forrned CsC1 gradient, the two species of R N A were found as separate
peaks. If the virus is first fixed with o.I % formaldehyde before centrifuging in CsC1 or is
centrifuged in Cs~SO~ without fixing, only one radioactive peak is obtained, at 1.34 g/ml.
Our inability to separate different components, the occurrence of the two RNAs in
equimolar proportions and the pattern of decrease of infectivity on dilution all favour the
contention that the two RNAs are in the same virus particle. Probably the most compelling
evidence, however, is the isolation of a 27S component which comprises one molecule of
each of the 22S and I5S species. In all experiments to produce the 27S component we have
found traces of protein associated with this RNA. However, it is unclear whether this
protein represents a linker molecule between the two RNA species or is merely present as
a contaminant. This problem requires further study. The extraction of the 22S R N A
alone from the virus particle when phenol was used in the absence of SDS (Newman &
Brown, 1973) may mean that the I5S R N A is more closely associated with the virus protein
than is the 22S RNA; the reason for this R N A entering the phenol phase is not clear but
this difference is not due to the presence of poly (A) which is known to cause some RNAs
to enter the phenol phase (Brawerman, Mendecki & Lee, i972; Eaton & Faulkner, t972)
because neither species contains such a tract (Newman & Brown, I975). The u.v. irradiation
experiments did not provide evidence regarding the location of the two R N A species, in
contrast to the results obtained by Mayo et al. (1973) with raspberry ringspot virus. However,
the u.v. irradiation inactivation characteristics suggest a one-hit curve and support our
contention that the two R N A species are present in the same particle.
The loss of infectivity and disruption of the virus particles by C1- ions has a parallel
in the disruption of the cardioviruses by chloride and bromide ions (Speir, 1962; Mak,
O'Callaghan & Colter, I97O; Rueckert, 1971). Chloride ions disrupt Nodamura virus
particles at all pHs except c. 4, the isoelectric point of the virus. The cardioviruses also
appear to be stable at their isoelectric points (Chlumecka, D'Obrenan & CoRer, I973).
The fact that Nodamura virus is stable between pH 7"5 and 8"5 in CsC1 (I'34 g/ml) is
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RNAs of Nodamura virus
95
unexplained but it could be because the virus has two isoelectric points, that at pH 4
predominating in o.I M-NaCI. It is also possible, however, that the high concentration of
salt may be affording some protection.
Double stranded molecules corresponding to the 2zS and I5S RNAs were found in
infected cells, but we were unable to detect a double stranded RNA corresponding to a
27S RNA. This suggests that the two RNAs replicate separately, but we cannot rule out
the possibility that a double stranded RNA corresponding to the 27S RNA is present but
is disrupted by the extraction procedure. However, the isolation of a 27S component from
the virus, comprising one molecule of each of the two RNA species and possibly a linker
protein molecule, invites speculation regarding the replication of the RNA. Nodamura
virus could serve as a simple model for elucidating the mechanism by which RNA species
are selected in the assembly of segmented genome viruses. This problem is being investigated.
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