J. gen. Virol. (I973), 2x, 469-475
469
Printed in Great Britain
Characterization and Mixed Infections of Three Strains of Vaccinia
Virus: Wild Type, IBT-resistant and IBT-dependent Mutants
By E. K A T Z , EVA M A R G A L I T H , B E L A W I N E R AND A. L A Z A R
Department of Virology, Hebrew University- Hadassah Medical School,
Jerusalem, Israel
(Accepted 3 July I973)
SUMMARY
The characteristics of IBT-dependent (IBT D) and IBT-resistant (IBT R) mutants
of vaccinia virus were compared with those of the wild type (wt) strain. The
mutants did not differ from the wild type strain by their sedimentation in sucrose
gradients. Minor differences in the polypeptide composition of the virus particles
and in the neutralization of the three virus strains by anti-vaccinia human
immunoglobulin were observed. Mixed infections of the viruses in HeLa cells
enabled the growth of the strains under their unfavourable conditions (wt in the
presence of IBT and IBT D in the absence of IBT).
INTRODUCTION
Isatin-fl-thiosemicarbazone 0BT) is an effective inhibitor of the multiplication of poxviruses (Sheffield, Bauer & Stephenson, ~96o). Its mode of action is not fully understood.
It was found that IBT does not interrupt early events such as the uncoating of the virus
particle, synthesis of early enzymes and early structural polypeptides (Appleyard, Hume &
Westwood, I965; Woodson & Joklik, ~965), DNA replication (Easterbrook, I962; Woodson & Joklik, ~965) and m-RNA transcription and association with ribosomes (Woodson
& Joklik, I965). Since polyribosomes, which are formed by late m-RNA, are broken down
soon after their formation, Woodson & Joklik 0965) suggested that late structural virus
polypeptide synthesis is the target for the block caused by IBT.
Mutants of vaccinia virus which are resistant or dependent on IBT may be very useful
in the clarification of the mechanism of action of IBT. In the present study we further
characterize an IBT-dependent and an IBT-resistant mutant of vaccinia virus and study
the interactions between these viruses and the wild type strain during multiplication in
mixed-infected cells.
METHODS
Compounds and reagents. [~H]-thymidine 08"9 Ci/m-mol), [l~C]-thymidine (520 mCi]mtool), [3H]-phenylalanine 0 8 Ci/m-mol), [l~C]-phenylalanine (15"3 mCi/m-mol) and pS]methionine (I44 Ci/m-mol) were obtained from the Radiochemical Centre, Amersham,
England; IBT (Mann Research Laboratories, New York, N.Y.) and anti-vaccinia human
immunoglobulin (Povite, Poviet Producten N.V. Amsterdam, Holland).
Cell cultures and viruses. HeLa S-3 cells were grown in monolayer cultures in Eagle's
medium (Eagle, I959), supplemented with Io % calf serum. Stocks of WR strain, IBTresistant mutant (IBT~) and IBT-dependent mutant (IBT~) of vaccinia virus were prepared
in HeLa cells and titrated on BSC I monolayers, as previously described (Katz et al.
I973).
470
z. K A T Z , E. M A R G A L I T H , B. W I N E R
A N D A. L A Z A R
Infection. Monolayers of HeLa S-3 cells were washed with saline and infected with virus
at an input multiplicity of 5 p.f.u.]cell. Following incubation for 45 rain at 37 °C, the
cultures were washed and Eagle's medium containing 2 % calf serum was added.
Preparation of labelled virus. Thymidine-labelled virus was prepared by the addition of
o'3 #Ci/ml of [~4C]-thymidine or 7o #Ci/ml of [3H]-thymidine to the infected cultures in
Eagle's medium supplemented with 2 % calf serum. Phenylalanine-labelled virus was prepared by the addition of I-2 #Ci/ml of [14C]-phenylalanine or 7 #Ci/ml of [3H]-phenylalanine
in Eagle's medium containing I[zo the regular concentration of phenylalanine. Methioninelabelled virus was prepared by the addition of 40 #Ci/ml of [asS]-methionine in Eagle's
medium containing i/2o the regular concentration of methionine. The cultures were harvested 2 days after infection, and virus was purified as described by Joklik (I962).
Polyacrylamide gel electrophoresis. Gel electrophoresis was performed essentially as
described by Summers, Maizel & Darnell 0965). Gels (Io by o.6 cm) were prepared of
IO % acrylamide, 0.27 % N,N'-methylene bisacrylamide in o.t M-sodium phosphate (pH 7"Q
and o.t % sodium dodecyl sulphate (SDS). Prior to use, excess catalyst was removed from
the gels by electrophoresis at 5 mA/gel for I h. Purified virus was dissociated with 2 % SDS
and 1% mercaptoethanol (ME) for I rain at too °C. Sucrose was added to the solubilized
virus to l o % , and 2oo#1 were applied to each gel. Electrophoresis was carried out at
3"5 mA/gel for I7 h. After electrophoresis, the gels were placed in IO % trichloroacetic acid
(TCA), stained with o-1% Coomassie blue in Io % TCA and washed in 7"5 % acetic acid.
The gels were sliced into I mm slices, dissolved in H202 and radioactivity was determined,
or were sliced longitudinally, dried and placed in contact with X-ray film (Fairbanks,
Levinthal & Reeder, I965).
Electron microscopy. Infected cultures were scraped from the glass and the cells were
centrifuged at low speed for 5 rain. Small pellets were fixed, stained, dehydrated, infiltrated
and embedded, according to the technique developed by Hayat & Giaquinta (I97o). The
blocks were sectioned using an LKB ultrotome III and stained with uranyl acetate and lead
citrate. The stained samples were examined in a Philips EM 3oo electron microscope.
RESULTS
Physical and chemical characterization of the virus strains
Sedimentation in sucrose gradients and the polypeptide composition of the virus strains
were compared.
Sedimentation of wt, I B T ~ and I B T D in sucrose gradients
Mixtures of [l~C]-thymidine-labelled wt strain of vaccinia virus, with either one of
[aH]-thymidine-labelled IBT R or IBT D, were prepared and layered on 25 to 40 % sucrose
gradients. Fractions were collected after centrifuging in an SW 5° . I rotor at I3 ooo rev/min
for 35 rain and radioactivity was determined. IBT ~ (Fig. I a) andIBT D(Fig. ~ b) co-sedimented
in the two sucrose gradients with the wt strain.
Comparison of the polypeptides of wt, I B T ~ and I B T D by polyacrylamide gel electrophoresis
Mixtures of [14C]-phenylalanine-labelled wt strain with either one of [3H]-phenylalaninelabelled IBT ~ or IBT D mutants were prepared, sotubilized and the polypeptides then
separated by polyacrylamide gel electrophoresis. No significant differences were observed
between the polypeptides of the wt strain and those of IBT R or IBT D.
An improved resolution of the different polypeptides of vaccinia virus can be obtained
M i x e d vaccinia virus infections
1
I
(a)
~" 12
m
I
(t,)
I
I
471
I
24~
O
-20 x
_
.=E
16~
x 10
.E
.g
o
e~
.~ 6
>, 4
:~ 2
•,Q
-
-
8 >"
Y,
r..)
4"
1
5
10
15
20
0
5
Fraction number
10
15
20
25
Fig. I. Sedimentation of thymidine-labelled vaccinia virus strains in sucrose gradients. Mixtures of
[14C]-thymidine-labelled wt strain (©--©) with [aH]-thymidine-labelled mutant ( 0 - - - 0) ((a),
IBT~; (b), IBTD) were sedimented through 25 to 4o ~ sucrose gradient in I mM-tris, pH 9, in an
SW 50. I rotor at I3OOOrev/min for 35 rain. Fractions (ten drops), collected after piercing the
bottom of the tubes, were dissolved in toluene scintillator solvent containing 4o ~ Triton X-IOO
and counted in a TriCarb scintillator counter.
la l b 2 a 2 b 4a
i
I
I
I
I
I
4b
N
4c
1/
I
\
I
6a 6b
I
I
I
t
/
wt
IBT R
IBT D
Fig. 2. Autoradiography of the labelled polypeptides of the three virus strains. [asS]-methioninelabelled wt, IBTR and IBTD strains of vaccinia virus were solubilized by SDS and ME. The polypeptides were separated by polyacrylamide gel electrophoresis, as described in Methods. The gels
were sliced longitudinally, dried and exposed to X-ray film. Photographs of the autoradiographs
are shown. The main wrus polypeptides are marked.
by a u t o r a d i o g r a p h y of the labelled-polypeptides, separated in polyacrylamide gels. W h e n
the [35S]-methionine-Iabetled viruses were compared, a few m i n o r differences, particularly
in the relative a m o u n t s of several of the polypeptides o f the viruses, could be observed
(Fig. 2). The lack of the polypeptide m i g r a t i n g in front of 6 b of [BT R virus (Fig. z) is the
m a i n qualitative difference observed.
Neutralization o f wt, I B T R and I B T D strains of vaeeinia virus by immunoglobulin
The degree of neutralization of wt, IBT R a n d IBT D strains of vaccinia virus by antivaccinia h u m a n i m m u n o g l o b u l i n was compared. Dilutions of the virus suspensions were
i n c u b a t e d at 37 °C for 3o rain with a c o n s t a n t a m o u n t of anti-vaccinia h u m a n i m m u n o globulin. A l t h o u g h all the three virus strains were neutralized by the i m m u n o g l o b u l i n ,
there were m i n o r differences in the degree of the neutralization (Table I).
31
VIR
2I
472
E. K A T Z ,
E. M A R G A L I T H ,
B. W I N E R
AND
A. L A Z A R
Table I. Neutralization of wt strain o f vaccinia virus and of the mutants
I B T ~ and I B T • by anti-vaccinia human immunoglobulin
r
Virus strain
wt
[BT R
IBT D
Virus titre (p.f.u.]ml)
'
~
Neutralization
Control
+ Immunoglobulin
(~)
6"2x
IO 9
9"I x Io 9
2"2 x IOs
I ' I x lO8
3"7 x IO7
3"7 x IO5
98-1
99"6
99"8
o'5 m l o f dilutions of the three vaccinia virus strains wt, IBT R a n d I B T " were i n c u b a t e d w i t h 0-5 ml ( = 17 rag)
of an ti-v accinia h u m a n i m m u n o g l o b u l i n for 3o min at 37 °C. Virus di l ut i ons w i t h o u t i m m u n o g l o b u l i n were
i n c u b a t e d as well. T h e virus was then titrated on BSC i monol a ye rs .
Electron microscopy
The effect of IBT on the intracellular development of the three virus strains was studied
by electron microscopy. Wild type-infected cells, in the absence of IBT (Fig. 3a), contained
a great majority of condensed forms with low electron-dense centres which appeared to be
fully mature infectious virus (Dales & Siminovitch, I96I). However, in cells exposed to
IBT (Fig. 3 b) there was a great number of large, almost spherical immature forms, most of
which appeared to have complete m e m b r a n e s - findings in agreement with those reported
by Easterbrook 0962). When IBT~-infected cultures, in the absence (Fig. 3 c) or in the presence of IBT (Fig. 3d), were examined, most of the virus particles were mature. In I B T ' infected cultures the results seem to be the opposite to that observed with the wt strain;
mature particles were very rare in the absence of IBT (Fig. 3 e) but, in the presence of IBT
most of the forms observed, were mature virus particles (Fig. 3f). It can be concluded that
the immature forms which appeared with wt in the presence of IBT look similar to those
which appeared with IBT D in the absence of IBT, suggesting a similar block in virus
development.
M i x e d infections between the different strains o f vaccinia virus,
in the presence and absence o f I B T
The multiplication of the wild type strain is inhibited in the presence of IBT, while the
growth of the mutant IBT D depends on a constant supply of IBT in the culture media. The
growth of these viruses under their non-favourable conditions, in cells co-infected with
another strain of vaccinia virus, was studied in order to find out whether the viruses interact
during their multiplication.
Rescue of wt in the presence o f I B T during mixed infection with either I B T ~ or I B T D
The ability of IBT R and IBT D vaccinia mutants to rescue wt virus in the presence of IBT
was studied. Simultaneous infections were carried out with equal amounts of the virus
strains in the presence of II3qr. The virus titres of wt, IBT~ and IBT D were determined by
plaquing in the presence and absence of IBT. The increase in virus titre is shown in Table 2.
As expected, when IBT-treated cells were infected with wt alone, no increase in virus titre
was detected. However, when either IBT ~ or IBT D were used, an increase in virus titre was
observed. After a mixed infection with wt and IBT D (no. 4), growth of both wt (plaqued in
cultures without IBT) and IBT D (plaqued in cultures with IBT) occurred. After a mixed
infection with wt and IBT ~ (no. 5) the titre in the absence of IBT was more than three
times as high as in the presence of IBT. The latter result suggested that the progeny virus
from a mixed infection consisted of both genotypes. As proof, eight plaques, formed in the
Mixed vaccinia virus infections
473
Fig. 3- Electron microscopy. Infected ceils were harvested 22 h after infection and prepared for
electron microscopy as described in Methods. (a) w t without IBT; (b) w t with IBT; (c) IBT R
without IBT; (d) IBT g with IBT; (e) IBT D without IBT; (f) IBT D with IBT.
3T-~'
474
z. K A T Z ,
E. M A R G A L I T H ~
B. W I N E R
AND
A. L A Z A R
Table 2. Virus yield after single and mixed infections in the presence of I B T
Virus y i e l d t
c
Sample
no.
Virus i n o c u l u m *
,
'
~
wt
IBT D
IBT ~
z~.
Titre
~
c
- IBT
I
5
--
--
<
I0 ¢
2
--
5
--
<
lO4
3
4
5
--
--
5
5
5
--
-5
5
2. 3 x
C a l c u l a t e d p r o g e n y virus
~
~
+ IBT
<
7"5
1o 7
6-0 X I O 6
6-9 x Io 6
104
<
X IO s
1"9 x
IO 7
I'3
IO s
x
IB T D
wt
104
l-8x is ~
- -
--
7"5 X
--
--
6,o X I 0 6
5"t x IOs
IB T R
I'3
- -
IO a
-2'3
X I0 s
--
x
10 7
--
I - 8 x Io ~
* H e L a cells in 60 m m plastic Petri dishes (Nunc, D e n m a r k ) were infected w i t h 5 p.f.u./cell of either wt,
I B T 9, IBT ~ or with c o m b i n a t i o n s of two of these virus strains. F o l l o w i n g 45 m i n of i n c u b a t i o n at 37 °C,
5 m l of E a g l e ' s m e d i u m , s u p p l e m e n t e d with z 700 calf serum, a n d IB T 0 4 #M), were added.
t T h e virus yield (p.f.u./ml) was d e t e r m i n e d f r o m the increase in p.f.u, be t w e e n I a n d 22 h after infection.
The p l a q u e titre was m e a s u r e d in BSC I cells, in the absence a n d i n the presence of I B T 0 4 #M) in the a g a r
overlay. I n s a m p l e no. 4 the p r o g e n y IBT D virus is the p l a q u e titre w i t h IB T in the ove rl a y while the p r o g e n y
of w t is the p l a q u e titre w i t h o u t IBT in the overlay. I n s a m p l e no. 5 the proge ny IB T R is the p l a q u e titre w i t h
IBT in the overlay a n d the p r o g e n y w t is the difference be t w e e n t he titres w i t h o u t a n d w i t h IB T in the overlay.
Table 3- Virus yield after single and mixed injections in the absence of IBT
Virus y i e l d t
Virus i n o c u l u m *
Sample
no.
r
wt
IBT D
,
IBT R
Titre
~
~
- IBT
I
5
--
--
3'0
2
--
5
--
<
3
4
5
-5
--
-5
5
5
-5
X
IO 7
10 4
2"3x lO 7
6.6 × IOs
7"5 X 106
~
C a l c u l a t e d p r o g e n y virus
'
wt
IB T D
IB T ~
c
+ IB T
<~ 1 0 4
I' 7 X 10 5
1"9× IO7
1.8 × IS s
I' 6 X 107
3'0
X
I0 7
--
-6.6 × Io s
--
--
1.7 x
--
io s
-1.8 × IO°
8"5 X IO6
--
2"3× IO7
-7"5 X I O 6
* AS in T ab le 2 except for the absence of IBT in th e m e d i u m a d d e d fol l ow i ng the 45 m i n i n c u b a t i o n at
37 °C.
t As in T a b l e 2. I n s a m p l e no. 5 the p r o g e n y I B T g is t he p l a q u e titre w i t h o u t IB T in the ove rl a y a n d
the p r o g e n y IBT D is the difference between the titre w i t h a n d w i t h o u t IB T in t he overlay.
absence of IBT (no. 5), were picked and reptaqued in the presence and absence of IBT.
Of the eight, seven grew only in the absence of IBT, indicating that they were wt, and the
remaining one grew in both the presence and absence of IBT, indicating that it was an
IBT ~ mutant. N o t only does IBT R rescue wt, but wt also inhibits the growth of IBT R by
9o ~o (nos. 3 and 5) as previously was noted by Appleyard & Way (I966).
Rescue of I B T D in the absence of I B T during mixed infection with either wt or I B T R
The ability of wt and IBT R vaccinia virus strains to rescue IBT D virus in the absence of
IBT was tested. The increase in virus titre is shown in Table 3. As expected, when the cells
were infected ~,ith IBT" alone, only a slight increase in virus titre was detected; when wt
or IBT R were used alone, an increase in virus titre was observed. After a mixed infection
with IBT D and wt (no. 4), growth of both IBT D (plaqued in cultures with IBT) and wt
(plaqued in cultures without IBT) was observed. Following a mixed infection with IBT D
and IBT ~ (no. 5), the plaque titre was more than twice as high in the presence of IBT. The
latter result suggested that the progeny virus from a mixed infection consisted of the two
genotypes. As proof, nine plaques which formed in the presence of 1BT (no. 5) were picked
M i x e d vaccinia virus infections
475
and replaqued in the presence and absence of IBT. Of the nine, eight grew only in the
presence of IBT, indicating that they were IBT D, and the remaining one grew in both the
absence and presence of IBT, indicating that it was an IBT g.
DISCUSSION
The two mutants of vaccinia virus differ from the wild type strain in their resistance
or dependence on IBT, which is an effective inhibitor of the wild type strain. Physical and
biochemical characteristics of the mutants, morphology of the virus particle (revealed by
electron m i c r o s c o p y - our unpublished results), and sedimentation in sucrose gradient are
similar to those of the wild type strain. All three strains were neutralizable to approximately
similar extents by the antiserum used. The proportion of several polypeptides of the virus
strains are different and the apparent absence of one of them in the IBT R strain was noted.
Depending on the target of IBT in the vaccinia-infected H e L a cells and the extent to
which the products of the different genomes are capable of interacting in double-infected
cells, the following possibilities can be considered: (I) the effect of IBT on an event in the
development of one virus strain might interfere with the production of all particles; (2) a product coded by the genome of one of the strains might enable all particles to be made; (3) the
gene products of the two virus strains might not interact at all, so that some particles would
form while the formation of others would still be blocked. Measurements of the growth of
vaccinia virus strains showed that the second possibility exists, indicating assistance between
either IBT D or IBT ~ towards the growth of wt strain, in the presence of IBT, and also assistance between either wt or IBT ~, to the growth of IBT D in the absence of IBT. These findings
suggest that there are common events in the multiplication of these three virus strains and
that they are capable of sharing an event or a factor(s) involved in their multiplication.
We would like to thank Dr N. Goldblum for his useful advice in the course of this work.
This work was supported generously by a grant from the Frances and Ted Chanock
Foundation.
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