J. gen. Virol. (1967), 1, 479-486
479
Printed in Great Britain
The Ribonucleic Acids of the Infective and Interfering
Components of Vesicular Stomatitis Virus
By F. B R O W N , S. J. M A R T I N ,
B. C A R T W R I G H T
AND J O A N
CRICK
Animal Virus Research Institute, Pirbright, Surrey
(Accepted 8 June ~967)
SUMMARY
The RNA-containing components of vesicular stomatitis virus were labelled
with asp by growing the virus in a phosphate-deficient medium containing
a2PO4 and Actinomycin-D. Centrifugation of the virus suspensions in a sucrose
gradient gave four radioactive fractions. The most rapidly sedimenting
fraction contained the infective component of the virus, whereas most of the
interfering activity was in the second fraction, which contained 'caps',
filaments and rosette-like structures. The interfering activity was associated
with the 'cap', which could be separated from the filaments and rosettes by
centrifuging in a potassium tartrate gradient.
The viral R N A sedimented predominantly in the 36 to 4o S position in
sucrose gradients prepared in o.I M-acetate buffer but frequently exhibited
a more heterogeneous profile. The percentage distribution of 32p in the four
nucleotides was A = 29"3, C = 2i.i, G = 2o'9 and U = 28"7. Ribonucleic
acid from the interfering component invariably gave a sharp profile with a
sedimentation coefficient of I8 to 2oS and the percentage distribution of
32p was A = 27"I, C = 2I'9, G = 2o'3 and U -- 30"7. Both nucleic acids
were hydrolysed to low molecular weight materials by ribonuclease o.m
/zg./ml.
INTRODUCTION
Suspensions of vesicular stomatitis virus grown in baby hamster kidney cells contain
several complement-fixing components with very different sedimentation rates. These
components can be separated into four distinct fractions of complement-fixing and
immunogenic activity by centrifugation of the virus suspensions in sucrose gradients
(Brown, Cartwright & Almeida, I966). The most rapidly sedimenting fraction contains only the infective virus, but the others contain mixtures of viral antigens. The
second contains a component which interferes with the growth of the virus (Crick,
Cartwright & Brown, ~966; Huang, Greenawalt & Wagner, I966; Huang & Wagner,
I966a; Hackett, Schaffer & Madin, I967). This interfering component has a structure
resembling the rounded end of the bullet-shaped infective component and for this
reason we refer to it as the ' cap'. It can be separated from the filamentous and rosettelike structures, which are also present in this sucrose gradient fraction, by centrifugation
in potassium tartrate gradients (Crick et al. 1966). By growing the virus in the presence
of 3~p and Actinomycin-D we have shown that the interfering component also contains RNA. In this paper some of the properties of this RNA are compared with those
of the viral RNA.
480
r . B R O W N , S. J. M A R T I N , B. C A R T W R I G H T
A N D J. C R I C K
METHODS
Virus growth and titration. Monolayers of B H K 2 I cells (Macpherson & Stoker,
I962) in Roux flasks were infected with 2o ml. of vesicular stomatitis virus (Indiana
strain) at a virus: cell ratio of approximately o.oi to minimize interfering effects. After
incubating for 2 hr at 37 ° the medium was removed and the monolayers washed three
times with a phosphate-deficient medium (Earle's saline with O'OIi-tris, p H 7"6
replacing the phosphate; the phosphate concentration was less than 4 × IO-SM)• The
monolayers were then incubated with gentle rocking in 2o ml. of the same medium
containing Actinomycin-D I/zg./ml. and a2PO4 until the cell sheet disintegrated. This
usually occurred in 18 to 24 hr.
Infectivity determinations were made either by intracerebral inoculation of 7-dayold mice with serial tenfold dilutions of the virus or by plaque titration on B H K
cell monolayers. The titres obtained by the two methods were in good agreement for
this strain of virus.
Measurement of interfering activity. Monolayers of baby hamster kidney cells in
2 oz prescription bottles were incubated for I hr at 37 ° with I ml. of the material to be
tested. Ten ml. of Eagle's medium containing IOa p.Lu. of virus were then added.
Incubation was continued for a further 24 hr period when the yield of virus was
measured by plaque assay and the interfering activity expressed as the depression of
virus yield produced by I ml. of the material under test.
Concentration of virus. Virus in IOO ml. of phosphate-deficient medium was concentrated by precipitation with 60 ~o saturated ammonium sulphate. The precipitate
was collected by centrifugation at 25oog for 15 min. and resuspended in 4 mL
o-o4i-phosphate buffer, p H 7"6. In some experiments this concentrate was applied
directly to sucrose gradients but the virus was usually filtered through Sephadex G-25
(2o x 2.0 cm.) in equilibrium with o'o4M phosphate buffer to remove low molecular
weight materials before centrifugation. Both the infective virus and the interfering
component were eluted from the G-25 column in the first radioactive peak.
Sedimentation of virus components. Two ml. volumes of the virus concentrate were
layered on to 23"5 ml. of I5 to 45 ~o sucrose gradients in o.o4i-phosphate buffer and
centrifuged for 2 hr at 2o,ooo rev./min, in the SW25 rotor of the Spinco Ultracentrifuge. One ml. fractions were collected from the bottom of the tube into bottles conraining I drop I ~o bovine plasma albumin, using a device similar to that described
by Schaffer & Frommhagen (I965). The fractions were then examined for radioactivity,
infectivity and interfering activity. Samples (0"5 ml.) of the fractions containing the
interfering component were layered on to 4"2 ml. of preformed potassium tartrate
gradients (density range I-O2 to 1.25 g./ml.) and centrifuged for 2 hr at 35,000 rev./min.
in the SW 39 rotor. Single drop fractions were collected from the bottom of the tube, as
described above. Every third drop was used for density determination. The remaining
drops were collected into I ml. of phosphate buffer and o.I ml. of each dilute fraction
was used for radioactive counting. Appropriate fractions were then combined for
the estimation of interfering activity.
Extraction and sedimentation of RNA. Appropriate fractions from the gradients
were either diluted with or dialysed against o.oI M-phosphate buffer, p H 7"6. Sodium
dodecyl sulphate was then added to a final concentration of I ~o and the suspensions
extracted twice with an equal volume of phenol saturated with o.oIM-phosphate
R N A o f two V S V components
481
buffer. The RNA was then extracted twice with ether to remove phenol and mixed
with approximately 5oo/*g. RNA from baby hamster kidney cells before centrifuging
it in a sucrose gradient. In some experiments the mixture of viral RNA and cell R N A
was first precipitated with 2 vol. of ethanol and stored overnight at - 2 o °. The precipitate was then separated, redissolved in O'lM-acetate buffer, pH 5"o and centrifuged.
The RNA was centrifuged in 23"5 ml. o f a 5 to 25 ~o sucrose gradient in o.I M-acetate
buffer, pH 5"o at 2o,ooo rev./min, for 15 hr and I ml. fractions then collected from the
bottom of the tube. The radioactivity and absorbence at 26o m# were measured on
samples of each fraction and appropriate fractions were combined, mixed with 5o0 #g.
yeast RNA and precipitated with 2 vol. of ethanol at - 2o °.
Determination of base composition of RNA. The RNA precipitates were hydrolysed
in o'5 ml. o'3N-KOH at 37 ° for 18 hr. After cooling to o °, the solutions were adjusted
to pH 3"5 with HC104 and the precipitate of KC104 removed. The nucleotide solutions
were applied to Whatman 3MM paper strips (75 × 7"5 cm.) that had been moistened in
o'25i-citric acid trisodium citrate buffer, pH 3"5 and electrophoresis was continued
for I8 hr at an initial current of 2 mA per 7"5 cm. strip. The papers were dried and the
ultraviolet-absorbing regions eluted for counting in a 'Panax' automatic counter. In
some experiments the elect~ophoretograms were scanned along their entire length in
an EKCO Chromatogram Scanner (type No. N679A) to ensure that the radioactivity
was associated with the nucleotide regions only. There was no evidence that the
nucleotide fractions were contaminated with other 32P-containing substances (Davidson
& Smellie, 1952).
RESULTS
Fractionation of virus components by sucrose gradient centrifugation
A radioactive virus concentrate prepared by precipitation with 6o ~o saturated
ammonium sulphate was centrifuged for 2 hr at 2o,ooo rev./min, in a 15 to 45
sucrose gradient. Infectivity was associated with the most rapidly sedimenting fraction
(peak A) and interfering activity was predominantly in peak B (Fig. I a). Mixtures of the
three most active fractions from each of peaks A and B were precipitated with 6o ~o
saturated ammonium sulphate, dissolved in o.o4i-phosphate buffer pH 7"6 and
centrifuged through 15 to 45 ~o sucrose gradients under the same conditions as before.
Each sample sedimented homogeneously (Fig. I b, c) with a small proportion of the
radioactivity at the meniscus of each gradient. This slowly sedimenting material was
considered to have consisted of residual contaminants, principally inorganic phosphate,
which remained associated with the virus components in the first gradient. However,
some of the slowly sedimenting radioactivity was possibly derived from degradation
of the viral components.
Fractionation of peak B by tartrate gradient centrifugation
Previous electron microscopic studies had shown that peak B of the sucrose
gradients contained three components, 'caps', filaments and rosettes (Brown et al.
1966). The interfering activity was associated with the 'cap' which could be separated
from the other two constituents by centrifugation in a preformed potassium tartrate
gradient for 2 hr at 35,ooo rev.]min. (Crick et al. 1966). In the present experiments
the radioactively labelled fraction B from two cycles of centrifugation in sucrose
gradients was centrifuged in a tartrate gradient ranging from I.O2 to 1.25 g./ml.
482
F. BROWN, S. J. MARTIN, B. C A R T W R I G H T AND J. C R I C K
(Fig. 2). Three peaks o f radioactivity were obtained with densities o f 1.2[, H 4 and
[.oz g./ml, respectively. The interfering activity was associated with the peak having
a density of z ' I 4 g./ml, and contained the ' c a p ' (Crick et al. [966). The peaks at t.2I
and [.oz g./ml, are under investigation, but are presumed to correspond to the filamentous and rosette structures present in peak B o f the sucrose gradient.
9.0
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Fig. I. Centrifugation in a 15 to 45 % sucrose gradient of 32P-labelledvesicular stomatitis virus.
(a) Shows the distribution of infectivity, interfering activity and radioactivity when the virus
was sedimented for z hr at 20,000 rev./min, in a SW25 rotor; (b) and (c) showthe distribution
of radioactivity when peaks A and B shown in (a) were re-centrifuged through separate
gradients. O--O, infectivity; O - - I , interfering activity; x - - - ×, 3zp.
Sedimentation characteristics of RNA from the infective and interfering components
The virus in peak A o f the sucrose gradient (Fig. [ b) was mixed with 3 vol. o f [ %
sodium dodecyl sulphate and extracted twice with phenol saturated with o.oIMphosphate buffer, p H 7"6. A b o u t zo % of the radioactivity was left in the aqueous
layer and was associated with R N A . The R N A was then mixed with 5oo/~g. R N A
f r o m baby hamster kidney cells and centrifuged in a 5 to 25 % sucrose gradient in
o-I M-acetate buffer, p H 5. The distribution o f s2p and absorbence at 26o m # indicated that the viral R N A had a sedimentation coefficient o f about 38 S. Although the
RNA of two VSV components
483
typical profile of the viral R N A was sharply defined occasionally it was less so, when
the asp was distributed in the bottom Io ml. of the gradient as a heterogeneous peak
(Fig. 3a). The possibility that this was caused by aggregation is being investigated.
In some experiments the mixture of viral and cell R N A was first precipitated with
2 vol. of cold ethanol and stored overnight at - 2o °. The precipitate was then separated
by centrifuging at IOOOg , dissolved in 2 ml. o.IM-acetate buffer and centrifuged in
a 5 to 25 ~o sucrose gradient as before. This treatment did not alter the sedimentation
characteristics of the R N A . Treatment of the viral R N A with ribonuclease o.oI #g./ml.
1.3
1.2
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Fig. 2. Centrifugation in a potassium tartrate gradient of 32P-labelled peak B from sucrose
gradients of vesicular stomatitis virus (see Fig. O. The isolated peak B was centrifuged for 2 hr
at 35,000 rev./min, in a SW39 rotor. - - ,
interfering activity; x - - - x, 32p.
before centrifugation in the sucrose gradient converted the 38 S peak into slowly
sedimenting material (Fig. 3 b). When the interfering component was extracted with
sodium dodecyl sulphate and phenol, approximately I5 ~ of the 32p was found in the
R N A . In contrast to the viral R N A , the interfering component R N A invariably gave
a sharp peak at I8 to 2 o S under the same conditions of centrifugation (Fig. 3c).
This R N A also was hydrolysed by ribonuclease o-oI/zg./ml, to slowly sedimenting
material (Fig. 3 d).
Base composition of the RNA from the infective and interfering components
The R N A preparations were centrifuged in sucrose gradients and the fractions
corresponding to the two peaks sedimenting at 36 to 40 S and 18 to 2o S were mixed
with yeast R N A and precipitated with 2 vol. o f ethanol at - 2o °. The base compositions
484
F. B R O W N ,
S. J. M A R T I N ,
B. C A R T W R I G H T
AND
J. C R I C K
o f t h e R N A s w e r e d e t e r m i n e d b y 20 a n a l y s e s o f five p r e p a r a t i o n s o f t h e i n f e c t i v e
c o m p o n e n t a n d ~ ~ a n a l y s e s o f five p r e p a r a t i o n s o f t h e i n t e r f e r i n g c o m p o n e n t ( T a b l e 0 T h e d i s t r i b u t i o n o f z2p a m o n g t h e f o u r n u c l e o t i d e s in t h e i n f e c t i v e c o m p o n e n t R N A
~
a)
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x..i~ ,x..x .,x .,,~..x..x.. x..x..X,.X..x..x,. ~.,x.
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.~ 1.0
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Fraction
Fig. 3. Centrifugation in a 5 to 25 % sucrose gradient of a2P-labelled R N A isolated from the
infective and interfering components of vesicular stomatitis virus. Ribosomal R N A from
BHK2I cells was added as marker. (a) The two types of 32p profile obtained for R N A from
the infective component; (c) the a~P profile for R N A from the interfering component; (b) and
(d) the profiles when the R N A from the infective and interfering components respectively
were treated with ribonuclease o.o[/~g./ml, before centrifugation. • 0 , absorbence
at 260 m#; x - - x , 32p; O
O, 32p profile of heterogeneous RNA.
Base composition of RNA from the infective and interfering
components of vesicular stomatitis virus
T a b l e I.
% a2p activity in
~
Source of
RNA
Virus (2o)*
c
A
U
G
C
29"3 + o'3 ?
28"7 + o.2
2o'9 + o.2
z I.I + 0"3
Interfering
27" I __+o-2
3o'7 + o'4
2o-3 + o'3
21 "9 + 0.2
Calculated corn-
30"7
27"I
2I-9
20.3
plementary strand
Calculated duplex
28. 9
28. 9
2i.i
21.i
component (I I)*
* No of determinations.
"~Mean _+S.E.
i n d i c a t e d t h a t A = U a n d G = C. T h e b a s e c o m p o s i t i o n o f t h e i n t e r f e r i n g c o m p o n e n t
R N A was slightly b u t significantly different f r o m t h a t o f t h e viral R N A . T h i s difference
RNA of two VSV components
485
was most marked in the lower value for A and higher value for U in the interfering
component RNA. The calculated duplex form of the interfering component RNA has
a base composition similar to that of the RNA from the infective component.
DISCUSSION
The results presented in this paper demonstrate three properties of the RNA from
the infective component of vesicular stomatitis virus. First the base composition
analysis indicates the complementarity of the two pairs, AU and GC. Secondly, the
RNA normally has a sedimentation coefficient of 36 to 4o S, although it frequently
shows a heterogeneous profile with S values ranging from 5° to 25 S. Finally the
RNA is hydrolysed by very low concentrations of ribonuclease. The sedimentation
coefficient and sensitivity to ribonuclease suggest that the RNA is single-stranded, with
a molecular weight of about 3 × xo~ and that the apparent complementarity of the
base composition is fortuitous.
The demonstration that the interfering component RNA has an S value of I8 to 2o S
and is sensitive to low concentrations of ribonuclease, together with its base composition, point to it being a single-stranded structure. This result is in agreement with
that recently obtained by Huang & Wagner (I966b) for the RNA isolated from interfering component which had been separated by sucrose gradient centrifugation. In
these experiments the sucrose gradient fraction containing the interfering component
(i.e. fraction B) has been fractionated further into rosettes, filaments and 'caps' by
centrifugation in a tartrate gradient and RNA then extracted from this preparation of
caps. The base composition of the infective component RNA is different from that
of the infective component RNA but the two may be related as shown in Table I.
Whether or not this reflects a sequential relationship between the two RNA molecules
remains to be elucidated.
In the meantime it may be useful to speculate on the possible structural relationships between the two RNAs described here, especially in view of the hypothesis
proposed by Huang & Wagner (I966a) that the RNA in the interfering component
contains only a fragment of the virus genome. The sedimentation behaviour of the
two RNA molecules suggests that they are single-stranded and the infective component RNA appears to be about four times the size of the RNA from the interfering
component. The observation that the latter has a base composition which is related
to the viral RNA by AU and GC base pairing could be interpreted as indicating that
the intact viral RNA is made up of a linear sequence of smaller subunits which have
complementary compositions. Only one of these subunits would become incorporated
into the interfering component. These speculations seem to be consistent with certain
features of the RNAs of influenza virus and Newcastle disease virus. A linear complex
of RNA subunits has been suggested for the structure of influenza virus RNA (e.g.
Hirst, I96Z; Davies & Barry, I966) and excessive amounts of minus strands are
found in cells infected with Newcastle disease virus (Kingsbury, I966; Bratt &
Robinson, I967).
The authors are grateful to Mrs P. Hill and Miss C. A. Pearce for considerable
assistance with this work.
3I
J . Virol. i
486
F. B R O W N , S. J. M A R T I N , B. C A R T W R I G H T
A N D J. C R I C K
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