Download Phenotypic characterization of three temperature

Journal of General Virology (1992), 73, 3155-3167.
3155
Printed in Great Britain
Phenotypic characterization of three temperature-sensitive mutations in
the vaccinia virus early gene transcription initiation factor
Linda Christen, Meghan A. Higman and Edward G. Niles*
Department of Biochemistry, 140 Farber Hall, State University of New York, School of Medicine and Biomedical
Sciences, Buffalo, New York 14214, U.S.A.
Vaccinia virus gene D6R encodes the small subunit of
the virion early gene transcription initiation factor.
Three temperature-sensitive mutations have been
mapped to this gene. The biochemical phenotype
exhibited by each mutation was examined. All mutants
displayed altered viral protein synthesis in pulselabelling analyses at both the permissive and nonpermissive temperatures. The onset of early protein
synthesis was delayed, and the rate of early protein
synthesis was reduced in each case. Furthermore the
shut-off of both host and early protein synthesis was
delayed. In pulse-chase experiments, the stability of
the D6R protein in E93- or C46-infected cells was
shown to be reduced at 40 *C relative to that at 31 *C.
Early m R N A was quantified in cells at 2 h postinfection and shown to be reduced substantially. The
ability of each mutant virus to support transcription in
vitro was examined at both temperatures and, of the
three mutants, only $4 transcription was shown to
exhibit reversible temperature sensitivity.
Introduction
(Kates & McAuslan, 1967). Initiation requires a sequence-specific dimeric initiation factor that recognizes
and binds to the D N A both upstream and downstream
from the transcription initiation site (Broyles et al., 1988,
1991 ; Rohrmann et al., 1986). This factor possesses an
NTPase activity, and ATP hydrolysis is required for
high levels of transcription (Broyles & Moss, 1988). The
energy released is apparently employed in dissociating
the initiation factor from the template DNA, which, in
the presence of limiting initiation factor, would permit
transcription initiation of many genes (Broyles, 1991).
The two subunits of the early transcription initiation
factor are the products of genes A8L and D6R (Broyles &
Fesler, 1990; Gershon & Moss, 1990).
As part of the characterization of the genes in the VV
HindlII D fragment (Niles et al. 1986), we have
demonstrated that gene D6R is expressed late in the
virus life cycle and that two m R N A species can be
identified, differing at their 5' ends (Lee-Chen et al.
1988; Lee-Chert & Niles, 1988). Furthermore, three
temperature sensitive (ts) mutations have been mapped
to gene D6R (Seto et al., 1987; R. C. Condit & E. G.
Niles, unpublished data). Mutations C46 and E93 lie
within the N-terminal 15~ of the protein, whereas $4
maps within the remaining portion of the gene. A genetic
approach to investigating the complex mechanism of
transcription initiation would complement the biochemical studies currently under way. In this report, we
Poxviruses are large DNA-containing viruses that
replicate in the cytoplasm of infected host cells (Moss,
1990). Vaccinia virus (VV), the best characterized
poxvirus, has a dsDNA genome of 191686 bp (Goebel et
al., 1990) and encodes approximately 200 proteins. To
carry on their life cycle, poxviruses encode the enzymes
necessary to express their genes and replicate their
DNA. The enzymes required for early gene expression
are encapsidated in the virion core and include a multisubunit R N A polymerase (Baroudy & Moss, 1980;
Nevins & Joklik, 1977), a class-specific trans-acting
transcription factor (Broyles et al., 1988; Keck et al.,
1990; Vos & Stunnenberg, 1988; Yuen et al., 1987), an
early gene transcription termination factor (Rohrmann
et al., 1986; Shuman et al., 1987), an m R N A capping
enzyme (Martin et al., 1975; Wei & Moss, 1974) and a
poly(A) polymerase (Moss et al., 1975). Gene expression
is divided into three temporal categories: early genes are
transcribed immediately after infection; intermediate
genes are transcribed subsequent to the onset of D N A
replication and this requires at least two early gene
products (Vos et al., 1991); late genes are transcribed
after intermediate gene expression and this requires at
least three intermediate gene products (Keck et al., 1990).
Early gene transcription takes place in the virus core
0001-1089 O 1992 SGM
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3156
L. Christen, M . A . H i g m a n and E. G. Niles
d e s c r i b e the b i o c h e m i c a l p h e n o t y p e s o f these t h r e e D 6 R
mutations.
Results
Protein synthesis in virus-infected cells
Methods
Ceils and viruses. Wild-type VV WR and ts mutants C46 (Condit &
Motyczka, 1981; Condit et al., 1983) and E93 (Ensinger, 1982) have
been described. The parent of mutant $4 was isolated by Dr Richard C.
Condit as a double mutant, that is both ts and resistant to the Sadenosylhomocysteine analogue sinefungin. The ts lesion in $4 was
separated from the drug resistance locus by back-crossing with wildtype virus. Viruses were propagated on BSC40 cells; wild-type virus
was grown at 31 °C or 37 °C, and the mutants at 31 °C (Condit &
Motyczka, 1981). Virus preparations were routinely tested for retention
of the ts phenotype by comparison of their plaque formation ability at
both 31 °C and 40 °C~ Each mutant phenotype remained stable. Large
scale preparation of wild type and mutant virus, propagated at 31 °C in
150 mm dishes, was carried out as described previously (Joklik, 1962).
The yield of virus propagated at 31 °C, was routinely 2 × 109 to 3 × 109
p.f.u./107 cells for wild-type virus, E93 and $4, and 5 × 108 p.f.u./107
cells for C46.
Immunological techniques. In pulse-labelling experiments, infected
cells were metabolically labelled, in 60 mm dishes, with 100 ~tCi/ml
[35S]methionine. After 30 rain the radioactivity was removed, the
monolayer was washed two times with medium, and the infected cells
were disrupted by the addition of 0-5 ml 1% SDS, 100 mM-2mercaptoethanol, 50 mM-Tris-HCl pH 8 and subjected to two cycles of
boiling and freezing.
In the pulse-chase experiments, cells were pulsed for 30 min with 100
IxCi/ml [35S]methionine at 6 h post-infection, washed as described
above, and the chase was initiated by the addition of 1 ml of medium
containing 1 mM-methionine. At various times after initiation of the
chase, infected cells were washed and disrupted as described above.
Immunoprecipitations were carried out as described (Niles et al.,
1989). Antisera were raised to fusion proteins containing a portion of
either the VV D1R or D6R protein. A fusion vector in which a segment
of gene D6R, from position 7111 (Niles et al., 1986) to the end of the
coding region, was linked to pATH 11 (Dieckmann & Tzagoloff, 1985)
was constructed. A portion of gene D1 R, between nucleotides 601 and
1959, was linked to the pATH 3 vector. Induction of Escherichia coli
containing these plasmids with indole acrylic acid resulted in the
synthesis of fusion proteins which were isolated by preparative gel
electrophoresis followed by electroelution. They were used as antigens
in rabbits, as described by Niles & Seto, (1988). The D6R fusion
protein contains the C-terminal 85% of D6R, and the DIR fusion
protein contains the middle region of D1R, from 19% to 76%. AntiD8L (Niles & Seto, 1988), and anti-D12L (Niles et al., 1989) antibodies
have been characterized. In separate control experiments, each
antibody was shown to precipitate at least 90% of its antigen from
infected cell extracts. In Western blot analysis, infected ceil proteins
were transferred to Transblot (Bio-Rad), incubated with one or more
antibodies, and either stained after treatment with an alkaline phosphatase-linked second antibody or autoradiographed after incubation
with 125I-protein A.
Nucleic acid techniques, mRNA was isolated from cells infected for
2 h at either 31 °C or 40 °C by the guanidinium thiocyanate method
(Pacha & Condit, 1985). Gene D 1R- (nucleotides 601 to 1959) or D 12L(nucleotides 12858 to 14323) specific dsDNA probes were employed in
the Northern blot analyses (Niles et al. 1989). In vitro transcription was
carried out in NP40-permeabilized virions prepared as described
(Shuman & Moss, 1989). Reactions were conducted at 31 °C or 40 °C,
0-05 absorbance units (at 260 nm) of virus being used in each reaction.
T h e synthesis a n d s t a b i l i t y o f v i r a l p r o t e i n s were assessed
in cells i n f e c t e d w i t h w i l d - t y p e or ts m u t a n t virus at the
permissive and non-permissive temperatures. Infected
cells were pulse-labelled at v a r i o u s t i m e s after i n f e c t i o n ;
cells pulsed at 6 h p o s t - i n f e c t i o n w e r e c h a s e d for up to
4 h, e x t r a c t s were p r e p a r e d , a n d p r o t e i n s were s e p a r a t e d
b y gel e l e c t r o p h o r e s i s a n d o b s e r v e d by a u t o r a d i o g r a p h y .
I n the case o f w i l d - t y p e virus at 31 °C (Fig. 1 a), the r a t e
o f early p r o t e i n s y n t h e s i s p e a k e d at 2 h p o s t - i n f e c t i o n
a n d t h e n d e c r e a s e d o v e r the n e x t 6 to 8 h. L a t e proteins,
p 4 a a n d p4b, were initially d e t e c t a b l e at 4 h b u t were
p r o m i n e n t at 6 h a n d t h e i r synthesis c o n t i n u e d t h r o u g h o u t the infection. H o s t p r o t e i n synthesis was shut off b y 4
to 6 h. A t 40 °C (Fig. 1 b), the rate o f e a r l y p r o t e i n
synthesis p e a k e d w i t h i n 1 h a n d t h e n d e c l i n e d r a p i d l y .
L a t e p r o t e i n synthesis was r e a d i l y o b s e r v e d b y 4 h a n d
c o n t i n u e d t h r o u g h o u t infection. T h e shut-off o f host
p r o t e i n synthesis was c o m p l e t e b y 4 h p o s t - i n f e c t i o n
( M o s s & S a l z m a n , 1968; S a l z m a n & S e b r i n g , 1967). A t
b o t h 31 °C a n d 4 0 ° C , the late s t r u c t u r a l p r o t e i n
precursors, p4a, p 4 b a n d p28 ( K a t z & Moss, 1970; M o s s
& R o s e n b l u m , 1973) were p r o c e s s e d into t h e i r s m a l l e r
products.
M o d e s t c h a n g e s in the synthesis o f host a n d E93 v i r a l
p r o t e i n s c a n be o b s e r v e d at b o t h t h e p e r m i s s i v e a n d n o n p e r m i s s i v e t e m p e r a t u r e s , (Fig. l c, d). E a r l y p r o t e i n
synthesis b e g a n w i t h i n 1 h b u t the shut-off o f early
p r o t e i n synthesis was d e l a y e d in c o m p a r i s o n to t h a t in
w i l d - t y p e v i r u s - i n f e c t e d cells. L i k e w i s e , the shut-off o f
host p r o t e i n synthesis was n o t i c e a b l y d e l a y e d in cells
i n f e c t e d w i t h E93. F u r t h e r m o r e , t h e r e was a d e l a y in the
a p p e a r a n c e o f late p r o t e i n s p 4 a a n d p 4 b at e a c h
t e m p e r a t u r e , a l t h o u g h t h e i r p r o c e s s i n g a p p e a r e d to be
unaffected. I n cells i n f e c t e d w i t h $4 at 31 °C, the p a t t e r n
o f p r o t e i n synthesis was s i m i l a r to t h a t o b s e r v e d for E93
(Fig. 1 e) a n d at 40 °C, t h e shut-off o f b o t h early v i r a l
p r o t e i n a n d host p r o t e i n synthesis, a n d t h e i n i t i a t i o n o f
late p r o t e i n synthesis were n o t i c e a b l y d e l a y e d (Fig. l f ) .
A s o b s e r v e d in E 9 3 - i n f e c t e d cells, the p r o t e o l y t i c
p r o c e s s i n g o f the structural p r o t e i n p r e c u r s o r s p r o c e e d e d
n o r m a l l y d u r i n g the $4 infection. I n the case o f C46, the
p h e n o t y p e was e v e n m o r e p r o n o u n c e d w i t h the shut-off
o f b o t h host a n d early viral p r o t e i n synthesis d e l a y e d at
e a c h t e m p e r a t u r e (Fig. 1 g, h). T h e o n s e t o f late viral
p r o t e i n synthesis was m a r k e d l y d e l a y e d , b e i n g a p p a r e n t
only at 8 to 10 h p o s t - i n f e c t i o n at the p e r m i s s i v e
t e m p e r a t u r e a n d a l m o s t u n d e t e c t a b l e at 40 °C.
T o o b t a i n a m o r e p r e c i s e m o d e l o f e a r l y a n d late
p r o t e i n synthesis a n d early gene e x p r e s s i o n shut-off,
a n t i b o d i e s d i r e c t e d a g a i n s t t h e early p r o t e i n D 1 R , the
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Vaccinia virus transcription ts mutations
late protein D8L (Niles & Seto, 1988) and the D6R
protein were used to precipitate the three proteins
simultaneously from cells pulse-labelled or chased at
31 °C or 40 °C (Fig. 2a to h). The anti-D6R antibody was
characterized by identifying the proteins precipitated
from cells pulsed at different times during infection. In
Fig. 3, there are three proteins in the immunoprecipitate,
a major component of about 72K and two minor proteins
of about 66K and 62K. The appearance of one or two
minor proteins varied from preparation to preparation
and probably resulted from a variable degree of
proteolysis of the 72K protein. Note that in Fig. 2 (a to h)
the rate of synthesis and the turnover of the two visible
D6R proteins are equivalent.
In the case of wild-type virus-infected cells, at 31 °C
early protein D1R appeared at 1 h and continued to be
synthesized throughout the infection. The late protein
D8L was first visible 4 h post-infection. D6R, previously
identified as a late gene (Lee-Chen et al., 1988; Lee-Chen
& Niles, 1988) encodes two proteins which appeared at
about 4 h. At 40 °C, D1R synthesis peaked at 1 h postinfection and its rate of synthesis declined rapidly. D8L
was first identified at 4 h and was synthesized throughout
infection. The two D6R bands were first seen at 2 h postinfection, before the appearance of D8L, suggesting that
D6R may be an intermediate gene.
In the case of E93, at 31 °C (Fig. 2c) the pattern of
synthesis of D1R, D6R and D8L was similar to that of
the wild-type viral proteins, but was reduced in rate. At
40 °C (Fig. 2 d ), D 1R synthesis was delayed in relation to
that of the wild-type viral protein, and its synthesis was
not shut off. The synthesis of both D6R and D8L was
delayed 2 h. In S4-infected cells, (Fig. 2e, f ) the synthesis
of the early protein D 1R was both delayed and reduced
in amount; shut-off did not occur at either temperature.
The synthesis of both D8L and D6R was delayed 2 h at
each temperature.
Marked alterations in the synthesis of viral proteins
were observed at both temperatures in the case of C46
(Fig. 2g, h). D 1R synthesis was dramatically delayed and
greatly reduced, but shut-off did not occur. The
appearance of D6R was delayed at least 2 h and that of
D8L by 2 to 4 h.
There appears to be a gradient in the severity of the ts
phenotype for both the pattern of total virus protein
synthesis, and that of these three specific viral proteins.
E93 is most similar to the wild-type virus product, $4 is
more distinct and C46 is the most distinct. To quantify
more precisely the levels of early viral proteins at both 1
and 2 h post-infection, simultaneous immunoprecipitation using antibodies against the two early proteins
D1R and D12L (Niles et al., 1989) was carried out (Fig.
4a to d). It can be seen that at both temperatures each
mutant showed reduced levels of synthesis of both early
3157
proteins. At 40 °C, not only were the levels of each
protein reduced, but the time of synthesis was delayed in
comparison to that in wild-type virus-infected cells.
Stability of D6R in virus-infected cells
To assess the relative stability of wild-type and ts mutant
D6R proteins, pulse-chase analyses were carried out
(Fig. 2 a to h, lanes C 1 to C4). The rate of turnover of
D6R was compared to that of the stable late protein D8L,
which resides in the membrane of the intracellular form
of the virus (Niles & Seto, t988) or, in the case of C46, to
that of the early protein D 1R. In wild-type virus-infected
cells there was little degradation of D8L and modest
turnover of the D6R protein (Fig. 5). In the case of E93
and $4, D8L was stable at both temperatures, but in
E93-infected cells the D6R protein was dramatically destabilized at 40 °C (Fig. 5). In the case of C46, because
the onset of late gene expression was delayed, we
compared the stability of D6R to that of the early protein
D1R. In this case, although the D1R protein was stable
at both temperatures, the D6R protein was degraded
rapidly at the non-permissive temperature. Again, in
terms of relative stability, there was a gradation in
severity: wild type > $4 > E93 > C46.
Level of virion D6R protein
The reduced rates of early gene expression by the mutant
viruses could be explained by a reduced level of the D6R
protein being packaged into the progeny virus at 31 °C.
To obtain a qualitative assessment of the relative level of
D6R in wild-type and ts mutant virus, virus purified
through sucrose gradients was subjected to Western blot
analysis. Antibodies against both D6R and D8L were
simultaneously employed so that the D8L protein could
serve as an internal standard (Fig. 6). The level of D6R
relative to that of D8L was about 55%, 40% and 110%
for E93, $4 and C46, respectively, in comparison to the
wild-type virus. However, in the case of $4, D6R
appeared to be distributed over two protein bands. The
relationship between these two proteins is not clear.
Note that a low level of the upper protein is observable
for both wild-type virus and E93.
Quantification of the level of early mRNA
To determine whether the reduced rates of early protein
synthesis observed in the pulse-labelling analyses were
due to reduced levels of early m R N A , Northern blot
analysis was carried out on total R N A isolated from cells
infected with wild-type or mutant virus at 2 h postinfection. Total R N A (5 to 15 ~tg) was separated by
agarose gel electrophoresis and, after transfer, filters
were blotted with probes to either early gene D1R (data
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L. Christen, M. A. Higman and E. G. Niles
3158
(a)
(b)
M
1
2
4
6
8
10
C1
C2
C4
M
1
2
4
6
8
10
C1
M
1
2
4
6
8
10
C1
C2
C4
31
211
(a)
(c)
M
1
2
4
6
8
10
C1
C2
C4
C2
C4
Fig. 1. Protein synthesis in wild type and ts mutant VV-infected cells. BSC40 cells were infected at a multiplicity of 20, infected cells
were pulse-labelled for 30 min with 100 ~tCi/ml [3sS]methionine, washed and lysed in 2 ~ SDS and 100 mM-2-mercaptoethanol. An
aliquot of each lysate was separated by gel electrophoresis and observed by autoradiography. (a, b) Wild-type virus; (c, d ) E93, (e,f) $4
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3159
Vaccinia virus transcription ts mutations
(~
(f)
M
1
2
4
6
8
10
C1
C2
C4
~g)
M
1
2
4
6
8
10
C1
C2
C4
(h)
M
1
2
4
6
8
10
CI
C2
C4
M
I
2
4
6
8
10
Cl
C2
C4
p4a
p4b
and (g, h), C46 at 31 °C and 40 °C, respectively. Lanes M, mock-infected cells; lanes 1, 2, 3, 4, 6, 8 and 10, pulse at 1, 2, 4, 6, 8 and 10 h
post-infection; lanes C 1 to C4, chase of 1,2 or 4 h; H, host protein; E, early viral protein; p4a, p4b and p28, late viral protein precursors;
4a, 4b and 25K, viral protein processing products.
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L. Christen, M. A. Higman and E. G. Niles
3160
(a)
(b)
W
M
1
2
4
6
8
10
C1
C2
W
C4
1
M
97K
2
4
6
8
mm--~
10
C1
C2
C4
................
DIR
D6R
66K
43K
I
31K
II1
~
, ~ , ~ m ~
D8L
~
(d)
(c)
W
M
1
2
4
6
8
10
C1
C2
C4
W
M
1
2
4
6
8
10
C1
C2 C4
97K
DIR
66K
~w
D6R
43K
D8L
31K
--p
21K
Fig. 2 Immunoprecipitation of viral proteins from pulse-labelled virus infected cells. An aliquot of each extract, prepared as described
in Methods was incubated with antisera raised to fusion proteins containing a portion of the products of genes D1R, D6R and D8L.
Precipitates were collected, washed, solubilized in protein gel sample buffer, separated by gel electrophoresis and fluorographed. (a, b)
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Vaccinia
(e)
W
97K ~
M
1
2
4
6
8
::::
66K~
10
CI
C2
~
:
:::
.... ~ " ~
~:~lr.,
C4
(f)
W
M
1
:
~
virus
2
......
4
transcription
6
8
10
C1
C2
C4
~.,,.,..- ~m, , . , . ~ : ~
;~
~,: ~. , ~
~
:~
3161
ts mutations
:,.~ : D1R
,D.,,,: ~ ::~: : D6R
~
43K
~
:*-~ D8L
C2
C4
31K
21K
(g)
W
(h)
M
1
2
4
6
8
10
C1
C2
C4
W
M
1
2
~-"
4
6
8
.~...~.~.
•~
~
10
C1
~
~,,-i ~
~ ,~
,;~
:
~
:
D1R
:
D8L
31K
I
•
•
•
~o
21K
Wild-type virus-; (c, d), E93-; (e,f), S4- and (g, h), C46-infected cell extracts at 31 °C and 40 ° C , respectively. Lanes W, Mr markers;
lanes M, extract prepared from mock-infected cells; Lanes 1, 2, 4, 6, 8 and 10, pulse labelling 1, 2, 4, 6, 8 and 10 h post-infection; lanes
C 1, C2 and C4, cells pulsed at 6 h and chased for 1, 2 or 4 h respectively. P, A late viral protein unrelated to D6R precipitated by the D8L
preimmune serum.
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L. Christen, M. A. Higman and E. G. Niles
3162
W
M
2
4
6
10
12
PI
Fig. 3. Immunoprecipitation of viral proteins from infected cell
extracts, pulsed at 2, 4, 6, 10 and 12 h (lanes 2, 4, 6, 10 and 12) using the
anti-D6R antiserum; lane PI, extract from cells pulse-labelled at 6
hours post-infection was treated with preimmune serum. P', An early
viral protein unrelated to D6R precipitated with the preimmune serum.
Mrs of the D6R-related proteins are shown to the right. Lane W, Mr
markers; lane M, mock-infected cells.
(a)
wt
W
M
1
W
M
1
2
1
2
1
E93
2
$4
1
C46
1
2
2
(b)
wt
E93
$4
2
1
2
C46
1
2
--D1R
n o t shown) or D 1 2 L m R N A (Fig. 7). F o r E93 a n d $4, the
c o n c e n t r a t i o n o f m u t a n t D 12L m R N A was d r a m a t i c a l l y
r e d u c e d in c o m p a r i s o n to t h a t o f the w i l d - t y p e virus at
b o t h the p e r m i s s i v e a n d n o n - p e r m i s s i v e t e m p e r a t u r e ,
d e m o n s t r a t i n g t h a t the d e c r e a s e d rates o f p r o t e i n
synthesis were due to d e c r e a s e d levels o f early m R N A .
T h e results o b s e r v e d w i t h the gene D 1 R p r o b e were
i d e n t i c a l to those w i t h the gene D 1 2 L p r o b e .
Transcription in NP40-permeabilized virions
T o d e t e r m i n e w h e t h e r t r a n s c r i p t i o n is t e m p e r a t u r e sensitive in a n y o f the ts m u t a n t s , the rate o f t r a n s c r i p tion in N P 4 0 - p e r m e a b i l i z e d virions was assessed (Fig.
8). I d e n t i c a l a m o u n t s o f virus were used in e a c h assay. I n
the case o f E93, t r a n s c r i p t i o n was at least as efficient as
t h a t o f w i l d - t y p e virus at e a c h t e m p e r a t u r e ; C46
e x h i b i t e d r e d u c e d t r a n s c r i p t i o n b u t no i n d i c a t i o n o f
t e m p e r a t u r e s e n s i t i v i t y was o b s e r v e d . H o w e v e r , $4,
Fig. 4. Simultaneous precipitation of early viral proteins D1R and
DI2L from wild type and mutant virus infected cell extracts. Extracts
prepared from infected cells as described in Methods were incubated
with saturating levels of antisera raised to the viral early gene D 1R and
D12L proteins. After collection of the precipitate, the proteins were
solubilized in gel sample buffer, separated by electrophoresis and
--D12L
4
(c)
~
(d)
~3
0 1 2
wt
1
2
1
E93
2
$4
1
2
l
C46
wt
Time (h)
2[1
12 I1
E93
2
$4
1
C46
fluorographed. (a, b) extracts prepared from cells infected at 31 °C and
40 °C, respectively. Lanes M, extract prepared from mock-infected
cells; lanes 1 and 2, pulse labelling 1 and 2 h post-infection. PI, D1R
and DI2L preimmune sera were used. (c, d). The fluorographs were
quantified by densitometry and the results are presented as the
absorbance of DIR (11) or D12L (D) precipitated from wild-type or
mutant virus-infected cell extracts at 31 °C (c) or 40 °C (d).
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3163
Vaccinia virus transcription ts mutations
10001~
I
I
I
I
I
I
I
I~1~
I
I
I
I
I
I
I
I
~
(a)
1
.~ 1000
t
~
II
II
i I i I ii
-
i
(b)
4o
1
3
T
1
3.4
'~
~11~-------~....~
--'-"-'-~
I
0
I
1
I
I
I
I
I
2
3
Time (h)
I
I
4 0
I
1
I
I
3
Fig. 5. Stability of D6R in wild-type (a) or E93 (b), $4 (c) or C46 (d)
virus-infected cells. Cells infected at 31 °C (open symbols) or 40 °C
(filled symbols) with wild-type or ts mutant virus were pulse-labelled at
6 h post-infection for 30 min. Medium containing an additional 1 m~methionine was returned to the washed monolayer and at various times
after the chase was started, cell extracts were prepared. The levels of
D1R (A, A) D6R (O, ~) and D8L (H, II) were quantified by
densitometry and plotted as a percentage of the protein present in the
6 h pulse.
M
:i~
1
:
:~
~.![~ ~ ~:~
2
:: : ~ ~ i
~:: '
:~~: ~:
:
3
~
~
:: ~3:' ¢"!7
4
:i:,~e~ :~i
::
:~:[3
4
0.07
40
1
S
3
0.3
40
7-3....
0.01
31
1
~
2
Time (h)
3
(d)
31
[ T-3
1
40
2
--1
5.9
(c)
100 ~
3
0.02
....
~
( S-3
~ ....... :
0-15
1-4
Fig. 7. Northern blot analysis of D12L m R N A levels in cells infected
for 2 h at 31 °C or 40 °C with wild type (a) or E93 (b), $4 (c) or C46 (d)
mutant virus. R N A was prepared from ceils infected at a multiplicity of
20 for 2 h at either 3t °C or 40 °C. Total R N A (5, 10 or I5 lag, lanes 1 to
3) was separated by gel electrophoresis and transferred to GeneScreen
Plus (NEN). Identical filters were incubated with radioactive D N A
probes complementary to D1R or D12L m R N A . After washing, the
filters were autoradiographed and the R N A levels were quantified by
densitometry. After densitometry, the relative level of DI2L m R N A
was estimated from the slope of a line derived from a plot of absorbance
versus R N A concentration and expressed as absorbance/10 lag total
R N A (values at bottom).
displayed not only a somewhat reduced rate of transcription at 31 °C, but also a marked temperature sensitivity
at 40 °C, transcription being 10% of the wild-type virus
rate. To determine whether the temperature sensitivity
exhibited by $4 was reversible, a shift-down experiment
was carried out. NP40-permeabilized virions were
incubated in transcription assay mix at 40 °C and R N A
synthesis was measured at intervals. After 10 min one
sample was shifted to 31 °C. In Fig. 8 (b), it can be seen
that the low rate of transcription observed at 40 °C was
increased to 44% of the 31 °C rate after the temperature
shift, demonstrating that the block in transcription in $4
is partially reversible.
In an attempt to determine whether the virion
transcription machinery in the ts mutant virions is more
sensitive to thermal inactivation than that of the wildtype virus, NP40-solubilized virus was incubated at
45 °C and at different times an aliquot was removed and
: : - - D8L
Fig.& Western blot analysis of D6R and D8L proteins present in wildtype (lane 1), E93, $4 and C46 (lanes 2 to 4) virions. Virus was purified
from cells infected at 31 °C, and 0-2 A 260 units of virus was disrupted by
boiling in 2 % SDS and 100 mM 2-mercaptoethanol and separated by gel
electrophoresis. The proteins were transferred to Transblot (Bio-Rad)
and incubated with a 1:100 dilution of antiserum raised against the
D8L and D6R proteins. The positions of the bound D6R and D8L
antibodies were determined by incubating with alkaline phosphataselinked secondary antibody and staining. Equivalent results were
obtained for incubations carried out with antisera diluted at either
l : 1 0 0 o r 1:200.
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3164
L. Christen, M. A. Higman and E. G. Niles
120
I
I
I
I
I
I
I
I
I
100,
I
- (a)
100
80
60
40 ¸
o.o
20
×
m
0-"
(aO
- (c)
100
>
80
<
60
20
0
1C
0
10
14
I
20
I
I
I
I
I
I
I
20
!
0
Time (min)
10
I
I
I
I
I
I
I
I
20
I
I
I
I
30
I
40
I
60
Time (min)
Fig. 9. Thermolability of the virion transcription machinery. NP40solubilized wild-type (©), E93 (O), $4 (D) or C46 (11) mutant virus was
incubated at 45 °C in transcription assay mix lacking rNTPs. At
various times an aliquot was transferred to an in vitro transcription
mixture. After incubation at 31 °C for 30 min, an aliquot was removed,
and the product R N A was precipitated with TCA and quantified by
liquid scintillation counting. The results are presented as the
percentage activity remaining in comparison to that of virus not
incubated at 45 °C.
~ lO
41
i
20
1
5
10
15
Time (min)
20
25
Fig. 8. Transcription activity in NP40-permeabilized wild-type (a),
E93 (b), S4 (c) or C46 (d) virions. Purified virus (0-05 A260 units) was
permeabilized by treatment with 0-05~ NP40 and incubated in the
presence of rNTPs including [32p]GTP at either 31 °C (O) or 40 °C (0).
At various times after initiating the reaction, an aliquot was removed,
and R N A was precipitated with TCA and counted in a liquid
scintillation counter. (e) The transcription rate in $4 virions was
measured in a temperature shift-down experiment. A transcription
assay mixture was first incubated at 40 °C and R N A synthesis was
measured at the times indicated. After 10 minutes (~), one sample was
shifted to 31 °C and R N A synthesis was quantified. (O) 31 °C; (0)
40 °C; (A) 40 °C shifted to 3 1 °C.
the rate of RNA synthesis was measured at 31 °C (Fig.
9). It can be seen that the ts mutant virus transcription
machinery is not significantly more thermolabile than
that of the wild type virus.
Discussion
The ability of each mutant to synthesize mature viral
proteins was assessed by pulse-chase analysis. In each
case, the mutants exhibited altered viral gene expression
in the form of reduced rates of early and late protein
synthesis at both the permissive and the non-permissive
temperatures. In addition, a marked delay in the
expression of both early and late genes is seen at 40 °C.
Shut-off of early viral protein synthesis is relatively slow
at 31 °C in both the mutant- and the wild-type virusinfected cells. At 40 °C, shut-offproceeds rapidly in wildtype virus-infected cells, but is markedly delayed for each
mutant. There is a gradient in severity of the observed
phenotype, in which E93 is most similar to wild-type
virus, $4 is more severely inhibited and C46 is the most
markedly affected.
In several VV ts mutants, the altered protein exhibits
an enhanced rate of turnover at the non-permissive
temperature (Evans & Traktman, 1987; Hooda-Dhingra
et al., 1990). We assessed the rate of turnover of the D6R
protein by pulse-chase analysis. E93 and C46 display an
elevated rate of degradation at 40 °C, relative both to the
wild-type D6R protein and to an internal control, either
D8L or D1R. A gradient of stability was observed in
which wild-type protein is most stable followed by those
of $4, E93 and C46. One possible explanation for the
reduced rate of gene expression observed at 31 °C is that
owing to a low level of proteolysis of D6R at 31 °C, fewer
D6R molecules are packaged into the virus. To obtain a
qualitative measurement of the amount of D6R in wildtype and mutant virus grown at 31 °C, the level of D6R
was estimated relative to that of another virion protein,
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Vaccinia virus transcription ts mutations
D8L. C46 was shown to possess a level of D6R
equivalent to that of the wild-type virus, whereas that of
both E93 and $4 was reduced by about 50~o. It should be
noted that in $4 there are two D6R bands and it is not
clear what the relationship between the two components
is.
To determine whether the reduced rates of early viral
protein synthesis observed in mutant virus-infected cells
is reflected in altered mRNA concentrations, the level of
early D1R and D12L mRNA was measured by Northern
blot analysis at 2 h post-infection and compared to the
rate of synthesis of D1R and D 12L determined by pulselabelling. In each case, the mutant virus-infected cells
possess less early mRNA then wild-type virus-infected
cells. A gradient in severity was again observed in which
wild-type > C46 > E93 > $4. For both wild-type virusand E93-infected cells, the rate of D 1R and D 12L protein
synthesis is roughly proportional to the level of message.
In C46-infected cells, the rate of protein synthesis is
somewhat less than expected for the amount of early
mRNA observed. In the case of $4 infected cells, early
mRNA is barely detectable, less than 1 ~ of normal,
whereas early proteins are made at 10 to 30 ~oof the wildtype rate. This unusually low level of early mRNA found
in S4-infected cells may be due either to a reduced rate of
transcription or to an enhanced rate of degradation. It is
surprising that in S4-infected cells such a low level of
mRNA can allow the synthesis of early proteins at a
relatively high rate. This may indicate that early mRNA
is produced in excess in vaccinia virus-infected cells.
In an attempt to determine whether transcription is
directly affected by any of the mutations, the rate of viral
transcription was measured in vitro in NP40-permeabilized virions (Shuman & Moss, 1989). At 31 °C, E93
displayed a transcription rate 25~o greater than that of
wild-type virus, whereas C46 showed 43~o of the wildtype virus transcription rate. For wild-type virus, E93
and C46, the transcription rate is increased at 40 °C
demonstrating that RNA synthesis is not temperaturesensitive. However, in the case of $4 the rate of
transcription, which is reduced by 25 ~ at the permissive
temperature, is decreased to 10~ of the wild-type virus
level at the non-permissive temperature. The temperature sensitivity of transcription exhibited by $4 was
shown to be partially reversible. $4 is the first VV
mutation displaying marked viral RNA synthesis temperature sensitivity (Ensinger, 1987; Hooda-Dhingra et
al., 1989, 1990).
Within this complementation group there may be two
mechanisms conferring temperature sensitivity. The E93
and C46 D6R protein is rapidly turned over in cells
infected at 40 °C. This increased rate of degradation
would cause reduced levels of D6R to be packaged into
progeny virus. As a result, in the next round of
3165
replication, early gene expression would be reduced and
the infection would be inhibited, preventing plaque
formation. Alternatively, since D6R is a component of
the virus core, a low level of D6R may result in the
formation of an unstable structure. However, the
proteolytic processing of the virion structural protein
precursors p4a, p4b and p28 proceeds normally in E93infected cells. Since the cleavage of these precursors
is a late step in the virion assembly pathway, it is likely
that the alteration in the D6R protein does not affect
virion assembly. However, in S4-infected cells the D6R
protein is relatively stable at 40 °C. In this virus, in vitro
transcription is temperature-sensitive in permeabilized
virions and the level of early mRNA in infected cells is
dramatically reduced. In the case of $4, ts plaque
formation is likely to be due to the presence of an early
transcription factor compromised in its ability to support
transcription. It is clear from a comparison of the level of
early D 12L mRNA to the observed rate of D 12L protein
synthesis that the phenotypes exhibited by these mutations are not straightforward in any case. As a result, the
proposed models of temperature sensitivity should be
viewed with a degree of scepticism until further
experimentation is carried out.
In the case of mutations in the D6R complementation
group, as has been shown for ts mutants in the large viral
RNA polymerase subunits (Ensinger, 1987; HoodaDhingra et al., 1989, 1990), there is a wide range of
biochemical phenotypes. The level of viral gene products
produced during infection and the timing of their
synthesis is affected at both the permissive and the nonpermissive temperature. However, there is little obvious
correlation between the rate of protein synthesis and the
plaque forming ability of the virus. In fact, for C46 and
$4 a more dramatic alteration in gene expression is
observed at the permissive temperature than is seen for
E93 at 40 °C. However, C46 and $4 form plaques at
31 °C, but E93 does not at 40 °C. It is clear that the ability
of a virus to form plaques at either temperature is not
dependent solely on the amount of viral protein
synthesized within the first 10 h of infection. Perhaps,
rather than the absolute amount of viral protein
synthesized during the infection, it is the timing of gene
expression that may be more critical. At 40 °C, the virus
life cycle proceeds much more rapidly than at 31 °C. At
the higher temperature, alteration of the time of
synthesis of one or more viral gene products may be
crucial to the successful completion of the life cycle,
resulting in ts plaque formation. At 31 °C, the virus life
cycle proceeds more slowly and a delay in the appearance
of a viral protein may not be critical.
The three mutations provide genetic access to different biochemical features of early gene expression.
Although the in vitro transcription rate of E93 is
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3166
L. Christen, M. A. Higman and E. G. Niles
equivalent to that of wild-type virus, in vivo early m R N A
levels are substantially lower than those in wild-type
virus-infected cells. C46 exhibits reduced transcription in
vitro, but transcription is clearly not temperaturesensitive. Again, in C46-infected cells, D12L m R N A is
substantially reduced in vivo. The mechanism responsible
for the reduction in early m R N A in E93- and C46infected cells should be defined. $4 is the most interesting
mutation. Since transcription in vitro is reversibly
temperature-sensitive, an evaluation of the mechanism
of temperature sensitivity would provide insight into the
pathway of early gene transcription initiation. Furthermore, the level of early $4 m R N A in vivo is at the limit of
detection, less than 1 ~ of that in wild-type virus-infected
cells. An analysis of m R N A metabolism in $4 infected
cells may provide a unique insight into the pathway of
early viral m R N A synthesis and degradation.
We would like to thank Mr James Myette for his helpful comments.
This work was supported in part by grants from the National Science
Foundation (DMB 8613181) and the U.S. Public Health Service (NIH
AI28824).
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