J. gen. Virol. (1973), x8, 381-384
38~
Printed in Great Britain
Location of Vaccinia Virus Structural Polypeptides on the
Surface of the Virus Particle
( A c c e p t e d 28 N o v e m b e r
I972)
Vaccinia virus has a complex structure. Thin sections of virus particles show three structural components: the outer envelope, the lateral bodies and a central structure, k n o w n as
the ' c o r e ' , which contains the virus D N A (Dales, 1963). Easterbrook 0 9 6 6 ) showed that
treatment with the nonionic detergent N P 4 o , in the presence o f z-mercaptoethanol (2 ME)
leads to the dissociation of the outer membrane and release of the core.
By polyacrylamide gel electrophoresis, Holowczak & Joklik 0 9 6 7 ) identified at least
17 structural polypeptides, three of which were associated with the core. Katz & Moss (I97o)
showed that some o f these polypeptide peaks could be further separated into two to three
polypeptides, thus increasing the total n u m b e r of resolved structural polypeptides to 22.
Our knowledge concerning the arrangement of vaccinia virus polypeptides on the particle
surface is very limited. The present paper attempts to determine which o f the virus polypeptides is situated on the surface. We have used iodination of vaccinia virus polypeptides
lalb
2a2b.
4a 4b 4c
5
6a
6h
..,,.,
~,
~.~.
C3tochrome C
Bovine albumin
I.
l
o
x
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5
•
..
:
...,.
o
Q
10
20
I
I
I
P
I
I
30
4O
5O
60
7O
80
Slices (1 mm)
®
Fig. I. Polyacrylamide gel electrophoresis of vaccinia virus structural polypeptides. [ZH]-leucinelabelled virus was disrupted and the polypeptides were separated by electrophoresis on 7"5 % polyacrylamide gel at 3 mA for 19 h. A diagram of the Coomassie-brilliant-blue-stained gel (up) and the
[aH]-leucine-labelled polypeptides of the same gel (down) are shown. Marker proteins: bovine serum
albumin and cytochrome C were applied to a separate gel. Their positions, indicated by arrows were
determined by staining with Coomassie blue.
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382
I
1
1
I
I
1
I
I
I
I
I
4a
Virus particle
4b
6a
Envelope
6a
I
I
I
I
I
I
I
I
I
I
0
1
2
3
4
5
6
7
8
9
10
Centimetres
Fig. 2. Polyacrylamide gel electrophoresis of virus particle and envelope (released) polypeptides.
Followingelectrophoresis,the gelswere immersedin TCA (2o %), stained with Coomassiebtue (o. ~%
in Io % TCA), washed in acetic acid (7"5%), sliced longitudinally, dried and exposed to X-ray film
(Fairbanks, Levinthal & Reeder, I965). Tracings of the developed X-ray films were made with a
Joyce-Loebl microdensitometer.
by [12~i] and lactoperoxidase, an enzyme which because of its relatively high mol. wt.
(Rombants, Schroeder & Morrison, ~967) catalyses iodination of only the surface proteins of
intact red blood cells (Phillips & Morrison, ~97o). Lactoperoxidase has been shown to be
very efficient in specific iodination of surface proteins of intact influenza virus (Stanley &
Haslam, I971).
Vaccinia virus (strain wR) was grown on HeLa cell monolayers in Eagle's media supplemented with 5 % calf serum. Two days after infection the cells were homogenized and virus
particles were purified as described by Joklik (1962). Labelled virus was prepared by the addition of IO #Ci/ml of [aH]-leucine (52 Ci/m-mol, Amersham, England) to infected cells in
Eagle's media containing I/5o the regular concentration of leucine and 2 % dialysed calf
serum. The structural polypeptides of vaccinia virus were analysed by electrophoresis on
polyacrylamide gels after disruption of the virus with 2 % SDS and 1% 2-ME, for I rain at
Ioo °C (Katz & Moss, I97o).
A pattern of the structural polypeptides of vaccinia virus is shown in Fig. ~. The upper
part of the Fig. indicates the position of the Coomassie-blue-stained polypeptides and the
lower part the [aH]-leucine labelled polypeptides.
A modification of the method of Easterbrook 0966) was used for dissociating virus
particles into envelope and core components. This procedure was also used by Holowczak &
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I
I
I
I
I
383
t
I
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4a a n d 4b
A
4c
6a 6b
•
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t
C~
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.
,E
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t
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t
¢
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"'r-.
t
6b
B
4c
0
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I
!
I
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I
I
10
20
30
40
50
60
70
°'°'t-.'~
_
80
Slices (I mini
Fig. 3- Lactoperoxidase labelling of purified vaccinia virus. (A) Vaccinia virus was disrupted with
SDS (2 % final concentration) for 30 min at 37 °C, dialysed overnight against o'~5 M-NaC1,buffered
at pH 7"3 with o.2-tris (to remove most of the detergent) and iodinated using lactoperoxidase. The
iodinated polypeptides were treated with SDS and 2-ME and analysed by electrophoresis on IO %
polyacrylamide gel at 4 m A for [7 h. (B) Pure vaccinia virus was iodinated using lactoperoxidase,
then treated with SDS and 2-ME and analysed by electrophoresis as in (A). The gels were stained
with Coomassie-brilliant blue, in order to determine the position of the main virus polypeptides and
then sliced into I m m fractions which were counted in a Packard Tri-Carb Auto-Gamma spectrometer.
Joklik (I967) to localize core and membrane proteins. Purified virus was incubated for
3o min at 37 °C in a solution containing 5o mM-tris buffer (pH 8"6), o'5 % N P 4 o (Shell,
London) and 5o mM-dithiothreitol (dTT), The mixture was then treated with ultrasonic
vibrations and layered on to 36 % (w/v) sucrose in to m~-tris buffer (pH 8.6) containing
2 mM-dTT. After centrifuging in an SW 5oL rotor at 25 ooog for 8o rain, the top fraction
(released envelope) and the pellet (cores) were collected as previously described (Katz &
Moss, ]970). It can be seen (Fig. 2) that at least three polypeptides: 4c, 6a and 6b are
released from the virus by N P 4 o and dTT. Similar results were previously observed by
Katz & Moss (I97o) and by Garon & Moss ( [ 9 7 0 .
Purified vaccinia virus was iodinated using lactoperoxidase, which was prepared by
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384
Short communications
U. Olshevsky f r o m our l a b o r a t o r y , following the m e t h o d o f M o r r i s o n , H a m i l t o n & Stotz
(I957). T o 2 ml o f purified virus (75/~g protein) in o-15 M-NaC1, buffered at p H 7"3 with
o.2 M-tris, 4 #1 o f l a c t o p e r o x i d a s e (E2s0 = o'5; E~I~ = o'3), lOO #1 o f [1~5I] ( = 40o #Ci, free
f r o m reducing agents, A m e r s h a m , England), 2o#1 o f o.I mM-KI a n d 2o #1 o f I mM-HzO2
(Merck, G e r m a n y ) were added. The reagents were vigorously mixed a n d allowed to react for
Io min at r o o m temperature. I n a control r e a c t i o n mixture, f r o m which l a c t o p e r o x i d a s e was
omitted, p r o t e i n i o d i n a t i o n c o u l d n o t be detected. W h e n intact virus was iodinated, the
reaction was s t o p p e d by diluting the mixture in l o mM-tris, p H 9, and sedimenting the virus
in 5o L r o t o r at 13500 rev/min for I h.
The purified vaccinia virus i o d i n a t e d using lactoperoxidase, was dissociated with SDS a n d
z - M E a n d analysed by gel electrophoresis. A s a control, the virus was disrupted with S D S
before iodination. The two gel p a t t e r n s thus o b t a i n e d are shown in Fig. 3. Virus i o d i n a t e d
after d i s r u p t i o n (Fig. 3 A) gave a similar p o l y p e p t i d e p a t t e r n shown in Fig. I. Clearly, all the
m a i n virus p o l y p e p t i d e s c o n t a i n tyrosyl a n d / o r histidyl residues susceptible to iodination.
I o d i n a t i o n o f intact virus gave a m a r k e d l y different result (Fig. 3B). Peaks 4 c a n d 6 b were
the m a j o r electrophoretic c o m p o n e n t s to be labelled. T w o a d d i t i o n a l p o l y p e p t i d e s o f lower
mol. wt. t h a n 6 b were also i o d i n a t e d to great extent. This suggests t h a t these f o u r polypeptides have an external l o c a t i o n in the virus particle structure. The finding t h a t b o t h
6 a a n d 6 b are p o l y p e p t i d e s o f the envelope (Fig. z) b u t only 6 b is i o d i n a t e d (Fig. 3 B) suggests t h a t 6 b has an external p o s i t i o n as c o m p a r e d to 6 a in the envelope. The latter is a m a j o r
glycosylated p o l y p e p t i d e o f the virus ( H o l o w c z a k , I97o; G a r o n & Moss, I97 0 .
T o D r D o v K a r p a s - in m e m o r i a m .
W e should like to t h a n k D r N. G o l d b l u m for his useful advice in the course o f this work.
Department o f Virology
Hebrew University - Hadassah Medical School
Jerusalem
Israel
E. KATZ
EVA MARGALITH
REFERENCES
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(Received 8 September I972 )
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