J. gen. Virol. (1983), 64, 983-995. Printed in Great Britain
983
Key words: HSV/DNA-binding proteins/ts mutants/protein function
Herpes Simplex Virus Non-structural Proteins. III. Function of the Major
DNA-binding Protein
By E D W A R D
LITTLER,~ DOROTHY PURIFOY, ANTHONY MINSON 1
AND K E N N E T H L. POWELL*
Department of Microbiology, University of Leeds, Leeds LS2 9JT, U.K. and ~Department of
Pathology, University of Cambridge, Laboratories Block, Addenbrooke's Hospital, Hills Road,
Cambridge CB1 2QQ, U.K.
(Accepted 30 December 1982)
SUMMARY
The herpes simplex virus type 2 major DNA-binding protein has been functionally
characterized using temperature-sensitive mutants in the complementation group 2-2.
The mutants were shown to be defective in the DNA-binding protein gene by mapping
the mutants to the area of the genome known to code for the protein, and by
demonstrating alterations in the major DNA-binding protein induced in mutantinfected cells. The mutants were shown to be defective in the replication of virus DNA.
The nature of this defect was examined by studying virus D N A synthesis in vitro and by
the examination of virus enzymes. An effect of mutation in the DNA-binding protein
was to destabilize both the D N A polymerase and the alkaline exonuclease.
INTRODUCTION
With the exception of a few virus-induced enzymes almost nothing is known of the function of
herpes simplex virus (HSV) non-structural proteins. This situation is largely caused by a lack of
well-characterized temperature-sensitive (ts) mutants with defects in known virus-specific
proteins. One exception to t h i s general situation is thc g r o u p of mutants in HSV-1
complementati0n group 1-2 in the immediate-early protein ICP 4 (Courtney & Powell, 1975;
Preston, 1979; Preston, 1981 ; Dixon & Schaffer, 1980; Halliburton, 1980). Studies of this group
of mutants have clearly implicated ICP 4 as a central protein in the early control of protein
synthesis in HSV-infected cells.
Many of the functions of the remaining HSV non-structural proteins are probably related to
D N A synthesis. Evidence for this comes from the distribution of complementation groups of
both HSV-1 and HSV-2 ts mutants between those with a DNA-positive and those with a DNAnegative phenotype, Mutants in about half of all the complemcntation groups so far identified
have a DNA-ncgative phenotype (Schaffer et al., 1978). Starting from this point it seemed
reasonable to identify proteins with possible roles in D N A synthesis and then to characterize
these in detail using ts mutants.
The major HSV DNA-binding protein ICSP 11, 12 in HSV-2 (Purifoy & Powell, 1976)or ICP
8 in HSV-1 (Wilcox et al., 1980; Conley et al., 1981) was purified by Powell et al. (1981) and
shown to have a possible role in the synthesis of virus DNA. Furthermore, the purified protein
was used to prepare a monospecific antiserum which could be used to detect the synthesis of
ICSP 11, 12 in infected cells. The serum was used to recognize a potential ts mutant in ICSP 11,
12 which was incapable of normal synthesis of the protein in infected cells (tsH9 in
complementation group 2-2). In the present study, we have extended the examination of
mutants in complementation group 2-2, we have demonstrated that they are indeed mutants in
the DNA-binding protein and we have used them to examine the role of ICSP 11, 12 in virus
replication and D N A synthesis.
t Present address: Department of Pathology, McMaster University, Hamilton, Ontario, Canada.
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E. LITTLER AND OTHERS
METHODS
Cells and viruses. The virus strains used were HSV-2 strain 186 and the ts mutants of HSV-2, tall9 (originally
isolated and kindly provided by Dr P. Schaffer) and tsl9 (originally isolated and kindly provided by Dr M.
Takahashi) and HSV-1, tsD9 (originally isolated and provided by Dr G. Aron). For all experiments young
confluent monolayers of HEp-2 cells were infected at an input multiplicity of infection of 20. The derivation of
various methods for cell and virus propagation and virus assay have been described previously (Purifoy &
Benyesh-Melnick, 1975). The derivation of virus-transformed cells is described below.
Production ofradiolabelled virus proteins. Where labelled proteins were required, infected cells were incubated
from 1 to 18 h post-infection in medium containing 2 ~LCi t*C-label!ed amino acid mixture (Amersham
International) per ml, the normal amounts of arginine and reduced amounts of the other amino acids as described
previously (Purifoy & Powell, 1976). Alternatively, a similar amount of [35S]methionine was used in which case the
concentration of unlabeUed methionine was reduced 10-fold in the medium and the concentration of other amino
acids remained the same.
Protein extraction and purification
Major DNA-bindingprotein. The protein was purified as described previously (Powell et al., 1981). Briefly, the
protein was extracted in high salt from virus-infected cells grown at 34 °C, and then purified by chromatography
on DEAE-, phospho- and DNA-cellulose columns.
DNA polymerase. The method for the purification of tsH9 DNA polymerase was exactly that used previously
for isolation of mutant enzymes (Purifoy et al., 1977).
Enzyme assays
DNA polymerase assays and heat inactivation experiments. These were done exactly as described previously
(Purifoy & Benyesh-Melnick, 1975; Purifoy et al., 1977).
Alkaline exonuclease activity. This was assayed by a method described previously (Purifoy & PoweU, 1976).
The reaction mixture contained 50 mM-Tris-HC1 pH 9, 2 mM-magnesium acetate, 10 mM-2-mercaptoethanol and
10 ktg [3H]thymidine-labelled native HEp-2 cell DNA (10000 ct/min/p.g approx.).
DNA melting assays. The melting of a poly(dAT) helix by mutant or wild-type DNA-binding protein was
observed using the purified proteins and a method described previously (Powell et al., 1981).
Assays o f DNA synthesis. In infected cells DNA synthesis was measured by uptake of [3H]thymidine into DNA
of virus density. This was determined by CsCI equilibrium density gradient centrifugation. The method used has
been described previously (Purifoy & Benyesh-Melnick, 1975). Briefly, ceils were harvested, washed and then
lysed with SDS and Sarkosyl (SLS). The lysed cells were digested with Pronase at 37 °C, and then mixed with dense
CsC1 solution to a refractive index of about 1-415. The resulting mixture was overlaid with liquid paraffin and
centrifuged at 120000g at 20 °C for 40 h in the T865 rotor ofa Sorvall OTD-50 centrifuge: After centrifugation the
gradients were fractionated by bottom puncture and the refractive index and radioactivity in each fraction
measured.
In isolated chromatin, DNA synthesis was measured by incorporation of [3H]thymidine 5'-triphosphate using
the modification of the method of Yamada et al. (1978) described previously (Powell et al., 1981).
Genetic mapping techniques
Complementation ofts mutants grown on HSV-transformed cells. This was done using the D21 and D26 cell lines
originally described by Minson et al. (1982). These lines are derivatives of the Ltk- cell line transformed to a tk ÷
phenotype with sheared HSV-2 DNA. The cells were shown to contain different fragments of virus DNA as well
as the thymidine kinase gene (Minson et al. 1982). To detect complementation the mutant and wild-type viruses
were assayed for plaque formation at either permissive or non-permissive temperature on monolayers of either
Ltk-, D21 or D26 cells. The number Of virus plaques formed on each monolayer was recorded.
Marker rescue using cloned DNA fragments. Marker rescue experiments were done as described by Stow &
Wilkie (1978). BHK monolayers were seeded with 2 x 106 cells per 60 mm dish 1 day prior to transfection.
Mixtures of infectious DNA from the ts mutant (about 1 p.g) and plasmid DNA containing cloned HSV-2
restriction fragments were precipitated by the calcium phosphate technique (Graham & Van der Eb, 1973) for 30
min, adsorbed to monolayers for 40 min and incubated for 4 h at 34 °C prior to a 4 rain treatment with 25%
dimethyl sulphoxide. Infected monolayers were then incubated for 3 to 4 days at 34 °C until cytopathic effects were
complete, whereupon they were harvested and the yield of progeny was determined by assay at 34 and 38 °C.
Immunofluorescence tests. Antisera to the major DNA-binding protein were prepared as described previously
using the purified protein (Powell et al., 1981). The sera were used in the indirect immunofluorescence test (Powell
& Watson, 1975). Vero cells on coverslips infected for 6 h at either permissive or non-permissive temperature with
mutants or wild-type virus were fixed with methanol at - 70 °C overnight. The fixed coverslips were stained using
the antiserum to the major DNA-binding protein as the primary serum and fluorescent conjugated goat anti-rabbit
IgG serum as the secondary antibody. The coverslips were then observed with a GS conference research
microscope fitted with a Zeiss epifluorescent illuminator.
Immunoprecipitation tests. These were done as described by Honess & Watson (1974) except that the antigen
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Herpesvirus DNA-binding protein
98 5
used was a high salt extract of infected cells (Purifoy & Powell, 1976)and the immune precipitates were washed six
times with ice-cold phosphate-buffered saline (PBS) followed by resuspension in PBS. The precipitates were
allowed to re-form overnight and were then collected by sedimentation.
Polyacrylamidegel eleetrophoresis. Single-dimension SDS-polyacrylamide gels were run exactly as described
previously (Powell & Courtney, 1975). Two-dimensional polyacrylamide gels were run by an adaptation of the
method of O'Farrell (1975).
Briefly, immunoprecipitates were prepared as described for single-dimension gels and made 9 Mwith respect to
urea. They were then mixed with an equal volume of buffer containing 9-5 M-urea, 2% Nonidet P40 (NP40), 2%
Ampholines (LKB; 1.6% pH range 5 to 7 and 0-4% pH range 3 to 10) and 5% 2-mercaptoethanol. Isoelectric
focusing gels were prepared in glass tubes (130 × 2.5 ram) and consisted of 4% polyacrylamide, 2% NP40, 2%
Ampholines (1-5% pH range 5 to 7 and 0-5% pH range 3 to 10) and 9 M-urea. The gels were polymerized by adding
tetraethylenemethylenediamine (0-03%) and ammonium persulphate (0-07%). After polymerization had occurred
samples were loaded on to the top of the gels and were overlaid with 10 ptl9 M-ureaand 1% Ampholines (0-8~ pH
range 5 to 7 and 0-2% pH range 3 to 10). The upper electrode buffer was a degassed solution of NaOH (0.02 M)and
the lower electrode buffer was phosphoric acid (0-01 M). The gels were focused at 400 V for 12 h and at 800 V for 1
h. Gels were removed from the glass tubes used for the first dimension and soaked in four changes of the sample
buffer for the second dimension gel (10 ~ glycerol, 5 % 2-mercaptoethanol, 2.3 % SDS and 0-0625 M-Tris-HCI pH
6-8) over a total period of 2 h. The second dimension SDS-polyacrylamide gel was run exactly as described
previously (Powell & Courtney, 1975)except that the sample was applied in the tube gel laid horizontallyacross the
stacking gel and attached to it via 1~ agarose in stacking gel buffer. The gels were analysed using fluorography as
described by Bonner & Laskey (1974).
RESULTS
Identification of mutants in the HSV-2 major DNA-binding protein
As previously reported, the m u t a n t tsH9 o f the Houston HSV-2 mutants accumulates the
major D N A - b i n d i n g protein in the cytoplasm rather than the nucleus of infected cells (Fig. 1).
This mutant is in complementation group 2-2 of the HSV-2 mutants (Schaffer et al., 1978). A
second mutant in this group, tsl9, was tested to ascertain if it shared similar phenotypic
characteristics. It is clear from Fig. 1 that the major D N A - b i n d i n g protein accumulates in the
cytoplasm o f tsl9-infected cells at non-permissive temperature just as it does in cells infected
with tsH9 under the restrictive condition. The control wild-type virus-infected cells on the other
hand, were shown to accumulate the protein in the nucleus, as did ceils infected with either
m u t a n t at the permissive temperature.
Characterization of mutant DNA-binding proteins
In order to characterize the major D N A - b i n d i n g protein induced by complementation group
2-2 mutants, the protein was purified from mutant-infected cells using direct immunoprecipitation with specific antiserum to the protein. The resulting i m m u n e precipitates were
first characterized by SDS-polyacrylamide gel electrophoresis (Fig. 2a). The tsH9 and wild-type
virus proteins behaved in an identical m a n n e r using this m e t h o d ; therefore, the immunoprecipitates were re-analysed by two-dimensional electrophoresis and isoelectric focusing.
Using this technique (Fig. 2b) a clear difference in the polypeptides induced by tsH9 a n d the
wild-type virus was visible, and was emphasized by co-running the two i m m u n o p r e c i p i t a t e s on
the same gel. The alteration in the mobility o f the tsH9 I C S P 11, 12 was observed in both
dimensions of the gel.
Physical mapping of the mutation in complementation group 2-2 mutants
Complementation studies
Since cell lines were available that contained various defined HSV-2 sequences (Minson et al.,
1982) these cells could be used as a quick method to obtain a m a p position for any m u t a n t
complemented by the cells. Fig. 3 shows the results of plaque assays o f c o m p l e m e n t a t i o n group
2-2 mutants at permissive and non-permissive temperatures on three different cell lines. The
tsH9 virus produced plaques on all the cell lines at permissive temperature but only on the D21
cell line at non-permissive temperature. As can be seen from the plaque numbers recorded in
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986
E. LITTLER AND OTHERS
Fig. 1. Indirect immunofluorescence antibody tests with anti-ICSP 11, 12 antiserum and cells infected
with (a) HSV-2 186 wild-type virus at 38 °C, (b) HSV-2 tsH9 at 34 °C, (c) HSV-2 tsl9 at 34 °C, (d) mockinfected cells, (e) HSV-2 ts-H9 at 38 °C and (f) HSV-2 tsl9 at 38 °C.
Table 1 the complementation was efficient. Similar results were seen with the tsl9 virus. In this
case the plaques were smaller at the permissive temperature (Fig. 3) and the complementation
was less efficient (Table 1).
Marker rescue studies
Complementation tests indicated that regions of the HSV-2 genome between about 0-31 and
0.41 code for a protein which can be expressed in the tk ÷ transformed ceils to c o m p l e m e n t the
defect in mutants of complementation group 2-2. W e then tested the ability of cloned D N A
fragments from this region for m a r k e r rescue of the ts mutations of these mutants. Tests were
done with tsl9, since infectious D N A preparations have not been obtained with tsH9. Table 2
shows rescue by fragments B a m H I b and HindlII h. These d a t a are consistent with the defect in
tsl9 m a p p i n g in the B a m H I b fragment o f HSV-2, i.e. about 0-34 to 0.41, a region encompassing
the previously determined m a p position for the major D N A - b i n d i n g protein of HSV-2 (Morse et
al., 1978). The mapping d a t a are summarized in Fig. 4.
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Herpesvirus DNA-binding protein
(a)
987
(b)
I
I
I
I
tsH9
wt
tsH9
wt and tsH9
Fig. 2. Electrophoresis of immunoprecipitates using anti-ICSP 11, 12 serum. ICSP 11, 12 was
precipitated from tsH9 or from wild-type virus-infected cell extracts (grown at 34 °C). In both cases the
immunoprecipitates were dissolved in disruption buffer (0-05 M-Tris-HC1 pH 6-7, 1 ~ SDS, l ~ 2mercaptoethanol, 0.5 M-urea) and electrophoresed on (a) one-dimensional or (b) two-dimensional gels as
described in Methods. The polypeptides were visualized by fluorography of the dried gels. The
molecular weights of the ICSP 11, 12 bands are 146K and 143K respectively (as determined by coelectrophoresis with standards on SDS-polyacrylamide gels).
T a b l e 1. Plaque production of HSV-2 wild-type and temperature-sensitive mutants at permissive
and non-permissive temperatures
Virus
A
¢
tsH9
HSV-2 wt
t'
Cell line
LtkD2t
D26
"~
34 °C
106*
130
119
38 °C
62
60
66
¢
tsl9
"1
34 °C
100
135
115
¢'
38 °C
0
96
0
"~
34 °C
42
45
41
38 °C
0
6
0
* Plaque numbers were obtained in the experiment shown in Fig. 3. Similar results were obtained in separate
experiments.
T a b l e 2. Marker rescue of HSV-2 ts19 by DNA fragments B a m H I b and HindlII h
Virus DNA
Cloned HSV-2
fragment
tsl9
tsl9
tsl9
BamHI b
HindIII h
r
34 °C transfeetion yield
(p.f.u./ml) assayed at
x
~
34 °C
38 °C
3-0 × 107
1.0 x 102
2.8 × 106
7.0 x 104
3.8 × 106
1-3 × 10a
Relative
38 °C titre/34 °C titre
( x l04)
0.033
250
34
Effects of the ts lesion in complementation group 2-2 mutants on DNA synthesis
Cells i n f e c t e d w i t h w i l d - t y p e v i r u s o r w i t h t h e m u t a n t tsH9 w e r e e x a m i n e d for t h e i r a b i l i t y to
s y n t h e s i z e D N A at t h e n o n - p e r m i s s i v e t e m p e r a t u r e a f t e r g r o w i n g for 6 h a t t h e p e r m i s s i v e
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988
E. LITTLER AND OTHERS
tk
i
D21
l
D26
g
n
i
l
I
wt
34 °C
tsH9
tsl9
m
i
wt
38 °C
tsH9
tsl9
Fig. 3. Complementation of the ts defect in mutants of complementation group 2-2 by HSV-2 tk ÷
transformed D1 cells. Plaque formation is shown in tk- cells, tk ÷ D21 cells and tk ÷ D26 cells infected
with HSV-2 186 wild-type, HSV-2 tsH9 and HSV-2 tsl9 at 34°C and 38°C.
S
I
:%"..,...
"'".....
:
0-30
I
D2~
I
!
!
!
0.35
I
D2,
0.40
I
""'...0.45
"'J
I ......
)
omplementation
HindllI h
BamHl b
! ) Marker rescue
Fig. 4. Summary ot the physical map locations derived for tsl9 and tsH9 using the data shown in Fig. 3
and Tables 1 and 2. The solid lines marked D21 or D26 indicate the differences in HSV DNA content in
these two cell lines (Minson et al., 1982). The vertical dashed lines indicate the sequences shared by
restriction enzyme fragments HindlII h and BamHI b and hence those required to rescue the tsl9 virus.
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989
Herpesvirus DNA-binding protein
100
I
e~
l
I
I
I
10
15
20
E
50
'~
1.2
~
e~
1.1
<
Z
._~
I
5
-
:
6
7
8
Time post-infection (h)
•
9
5
Time (rain)
Fig. 6
Fig. 5
Fig. 5. RateofsynthesisofHSV-2 wild-type and mutant virusDNA during replication as determined
by 1 h pulse labelling with [3H]thymidine and analysis by CsC1density gradient centrifugation. Results
are presented as a percentage of the HSV-2 wild-type incorporation at 8 to 9 h post-infection at 34 °C.
Infected cultures were either incubated continuously at permissive temperature (34 °C) (*, wild-type
186; A, tsH9; A, tsH9 in the presence of cycloheximide), or shifted from permissive to non-permissive
temperature (38 °C) at 6 h post-infection (O, wild-type 186; O, tsH9; 0 , wild-type 186 in the presence
of cycloheximide; II, tsH9 in the presence of cycloheximide).
Fig. 6. DNA-melting ability of ICSP 11, 12 derived from mutant virus-infected cells. ICSP 11, 12 was
purified from cells infected at 34 °C with tsH9 (Q) or wild-type virus (ll). The procedure used has been
published in detail previously (Powell et al., 1981). Purified protein and poly(dAT) were then mixed at a
10 : 1 (w/w) ratio. The mixture was incubated at 40 °C and the reaction monitored at 260 nm.
temperature. D N A synthesis was measured by labelling cells with [3H]thymidine and by
separating virus and cell D N A using CsC1 gradients. The results of these experiments (Fig. 5)
demonstrated the ability of wild-type virus or the mutant tsH9 to continue synthesis of virus
D N A at 34 °C; however, on shift to non-permissive temperature (39 °C) D N A synthesis ceased
abruptly in tsH9-infected cells but continued normally in wild-type virus-infected cells. Thus,
the temperature-sensitive protein produced by tsH9 is required continuously for D N A synthesis
to occur. It should also be noted that addition of cycloheximide to wild-type virus-infected cells
at the time o f shift to 39 °C only slightly inhibited the synthesis o f virus D N A .
Assay of major DNA-binding protein from tsH9 (group 2-2)-infected cells
Few assays of the activity of the major DNA-binding protein's functions are yet available.
One is its ability to melt a double-stranded D N A helix of poly(dAT) (Powell et al., 1981). In
order to examine the tsH9-induced protein in more detail, it was purified from both wild-type
and mutant virus-infected cells. Fig. 6 clearly shows the inability of tsH9-induced major D N A binding protein to melt a double-stranded D N A helix which the wild-type enzyme is capable of
denaturing.
Induction of DNA polymerase and alkaline exonuclease in mutant-infected cells
In view of the foregoing results, it was of interest to observe the induction of D N A synthetic
enzymes in both mutant and wild-type virus-infected cells. T h e results of an experiment where
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990
E. L I T T L E R AND OTHERS
I
100
(a)
(b)
(c)
~ 8o
60
E
•~ 40
......
12
.
.
.
.
,
--
24
12
24
12
24
Time post-inaction (h)
Fig. 7. Induction o f D N A polymerase and alkaline exonuclease in cells infected by (a) wild-type virus
or mutants (b) tsH9 or (c) tsl9 in complementation group 2-2. Cells infected at permissive or nonpermissive temperature were harvested at various times after infection and extracts were made (Powell
& Purifoy, 1977). The extracts were then assayed for both D N A polymerase activity ( 0 , 34°C; O ,
38 °C) and alkaline exonuclease activity (plotted as a bar for 38 °C results). The maximum activities
used to construct the diagram were as recorded below. For D N A polymerase (ct/min incorporuted/10
rain/10 s cell equivalents): wild-type, 6 x 104; tsH9, 6.6 x 104; tsl9, 1-3 x l0 s. For the alkaline
exonuclease (ct/min solubilized/10 min/10 s cell equivalents): wild-type, 2.2 × 10s ; tsH9, 2.2 x 10 s ;
ts19,2*l x 10s.
I
I
"~ 150
100
g
<
Z
5o
I
6
I
7
Time post-infection (h)
Fig. 8. D N A polymerase activity in cells infected with wild-type HSV-2 or tsH9 virus after shift up to
non-permissive temperature. HEp-2 cells were infected with either virus at an input multiplicity o f
infection of 10 and incubated at 34 °C for 6 h. Cells were harvested at intervals at 34 °C or after a shift to
non-permissive temperature and analysed for D N A polymerase activity. Cycloheximide was added to
replicate culture at 6 h. Results are expressed as a percentage of the HSV-2 wild-type 186 D N A
polymerase specific activity at 6 h post-infection for wild-type 186 at 38 °C (f-q), 186 at 38 °C in the
presence of cycloheximide (O), tsH9 at 34 °C (.A), tsH9 at 34 °C in the presence of cycloheximide (ZX),
tsH9 at 38 °C ( 0 ) and tsH9 at 38 °C in the presence of cycloheximide ( I ) .
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Herpesvirus DNA-binding protein
tO0
100
I
991
(a)
A
-~ 75
50
~
25
E 100
.~ 50
E
<
Z
r~
O
e~
<
Z 25
(b)
75
25
I
1
Time of preincubation (min)
I
2
-
I
5
Time (min)
÷
10
Fig. 9
Fig. 10
Fig. 9. In vitro inactivation of purified DNA polymerase. DNA polymerase was purified from either
wild-type virus-infected cells (O) or from tsH9-infected cells ( 0 ) using our standard procedure (Purifoy
et al., 1977). The purified enzymes were then heated at 39 °C in DNA polymerase reaction mixture
without DNA (Purifoy et al., 1977). At the times indicated DNA was added and the surviving
polymerase activity was measured.
Fig. 10. Complementation of DNA synthetic defects in chromatin from mutant-infected cells using
purified proteins. The preparation of chromatin extracts and the measurement of DNA synthesis
therein have been described previously (Powell et al., 1981). In the experiment shown in (a) chromatin
extracted from tsD9-infected cells grown at 34 °C (O), 39 °C (A) or in cells grown at 34 °C and shifted to
39 °C for 2 h prior to harvest (Z~) were compared to the chromatin prepared from the shifted culture
with DNA polymerase (about 0-1 ixg; Purifoy & Powell, 1977) added(B). In the experiment shown in
(b) similar chromatin preparations were prepared from tsH9-infected cells grown at 34 °C (O), 39 °C
(A) or 34 °C shifted to 39 °C for 2 h prior to harvest (Z~)- These preparations were then compared with
the shifted culture with DNA polymerase added (11, see above), with purified DNA-binding protein
(about 0.5 lag; Powell et al., 1981) added (V]) or with both added (O). Addition of control protein
(bovine serum albumin) caused no increase in activity. Similarly, addition of virus proteins to mockinfected cell chromatin showed no increase over the very low activity found in these preparations. Each
chromatin sample contained material from about 106 cells; 100% activity was about 1-5 x 104 ct/min in
each case.
the i n d u c t i o n o f b o t h the virus D N A p o l y m e r a s e and alkaline exonuclease was c o m p a r e d for
wild-type virus-infected cells and for cells i n f e c t e d w i t h tsH9 or tsl9 are s h o w n in Fig, 7. It can
clearly be seen that the i n d u c t i o n o f b o t h the D N A p o l y m e r a s e and the a l k a l i n e deoxyribonuclease was severely restricted in t s H 9 - i n f e c t e d cells at n o n - p e r m i s s i v e t e m p e r a t u r e . O n
the o t h e r hand, although the i n d u c t i o n o f b o t h e n z y m e s was clearly less restricted in tsl9infected cells, m u c h less of either e n z y m e was induced in these cells t h a n in those i n f e c t e d w i t h
the wild-type HSV-2.
D N A polymerase activity in complementation group 2-2 mutant-infected cells
As the m u t a t i o n in the tsH9 m u t a n t h a d such a severe effect on virus D N A synthesis a n d the
m u t a n t was defective in D N A p o l y m e r a s e induction, it was o f interest to o b s e r v e the effects o f
this m u t a t i o n on the stability o f the v i r u s - i n d u c e d D N A polymerase. T h e stability o f the e n z y m e
w i t h i n infected cells was m e a s u r e d by shifting cells infected w i t h e i t h e r the wild-type virus or ts
m u t a n t to the n o n - p e r m i s s i v e t e m p e r a t u r e . T h e results o f this e x p e r i m e n t (Fig. 8) d e m o n s t r a t e
the e x t r e m e t h e r m o l a b i l i t y o f the tsH9 polymerase. W i t h i n 1 h o f shift to the n o n - p e r m i s s i v e
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E. LITTLER AND OTHERS
temperature essentially all the virus-specific D N A polymerase activity was lost. Under the same
conditions the wild-type virus enzyme was perfectly stable. At the permissive temperature the
level of D N A polymerase enzyme was similar in both mutant and wild-type virus-infected cells.
The addition of cycloheximide at the time of temperature shift had little effect on the subsequent
levels of D N A polymerase activity.
In vitro inactivation o f the D N A polymerase o f a complementation group 2-2 mutant
In order to examine the thermolability of the mutant-infected cell enzyme in vitro it was
essential that the enzyme be purified. This was done with both mutant and wild-type virusinfected cell enzymes and they were then tested for thermolability. The results of this experiment
are shown in Fig. 9. In the case of both mutant and wild-type enzymes, more than half of the
enzyme activity was lost within a minute at 39 °C. No difference was observed in the loss of
enzyme activity with mutant and wild-type enzyme indicating that the ts mutant D N A
polymerase per se was not temperature-sensitive.
Defects in D N A synthesis in vitro by DNA-binding protein mutants
In vitro D N A synthesis in isolated chromatin offers an ideal method for the assay of mutant
proteins by in vitro complementation. In order to evaluate this system we have taken mutants
known to be deficient in the virus D N A polymerase (Purifoy & Powell, 1981) and grown cells
infected with the mutants or wild-type virus at permissive temperature. At selected times postinfection, cultures were shifted to the non-permissive temperature and after a suitable time
period were then harvested. Isolated chromatin systems were prepared from mutant and wildtype virus-infected cells grown under these conditions and from control cultures grown under the
standard permissive and non-permissive conditions. The level of D N A synthesis in each
chromatin preparation was then determined with and without the addition of purified D N A
polymerase (Fig. 10a). Under these conditions D N A synthesis in polymerase-depleted
chromatin was restored. This experiment was then repeated using cells infected with mutants
from complementation group 2-2. Once again a system could be prepared (by a shift in the
incubation temperature) which lacked normal D N A synthesis. In this case D N A synthesis
could be recovered only on addition of ICSP 11, 12 and not by adding D N A polymerase. However, addition of both proteins resulted in a further increase in D N A synthesis (Fig. 10b).
DISCUSSION
Currently the use of conditionalqethal mutations in known virus gene products has been the
most productive approach to discerning the function of virus-induced non-structural proteins.
Little success has been achieved to this end in studies of HSV gene function with the notable
exceptions of the virus-induced proteins mentioned in Introduction. The principal problem has
been the lack of mutants defective in defined virus products. In this report we have identified
mutants whose lesion affects the major virus-specific DNA-binding protein. The mutants which
are in complementation group 2-2 of HSV were isolated independently by P. Schaffer (Chu &
Schaffer, 1975) in the U.S.A. and M. Takahashi (Takahashi & Yamanishi, 1974) in Japan. The
characterization of these two mutants reported here demonstrates the central role of the D N A binding protein in virus D N A synthesis.
The map position we have obtained for the mutants in the major DNA-binding protein is to
the left (on the prototype map) of that for the D N A polymerase gene (Chartrand et al., 1980).
The map coordinates we are using are slightly different from those used by Chartrand et aL
(1980) to define the polymerase locus, but it is clear that the HindlII site just to the left of 0-4 on
our map (Fig. 4) defines the right-hand end of the DNA-binding protein gene and the left-hand
end of the polymerase locus (Chartrand et al., 1980). The most refined map position for the
nuclease gene (0.145 to 0-185; Preston & Cordingley, 1982) is quite distinct from this site. The
DNA-binding protein polypeptides induced by the mutants have only been characterized in
relation to material extracted from cells grown at 34 °C, since extracts from ts mutant cells grown
at 38 °C were depleted in the ICSP 11, 12 protein. Whenever the protein was purified from crude
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Herpesvirus DNA-binding protein
993
extracts we no longer saw the double-band form after electrophoresis. This observation suggests
that other components of the cell extract are responsible for this phenomenon rather than
straightforward oxidation/reduction of the protein as suggested previously by Knipe et al.
(1982). We are as yet unable to explain fully the mobility differences observed using twodimensional gels between tsH9 and wild-type ICSP 11, 12. This should be possible when studies
of the structure of these proteins are complete.
In mutant virus-infected cells D N A synthesis was severely restricted upon the shift of cultures
to the non-permissive temperatures. This result suggested that the role of ICSP 11, 12 in virusinfected cells is in D N A replication. Such results support our previous hypothesis based on
observations of the effects of anti-ICSP 11, 12 serum on virus D N A replication in vitro (PoweU et
al., 1981) and similar observations made by Conley et al. (1981) on ts mutant HA1. The rapid
effects of temperature shift on D N A synthesis in cells infected with group 2-2 suggest that ICSP
11, 12 is required continuously for D N A synthesis. It also seemed possible that ICSP 11, 12
might have multiple effects on the synthesis of D N A in infected cells. For this reason the
induction of D N A synthetic enzymes was observed in virus-infected cells. The induction of both
the D N A polymerase and alkaline exonuclease was found to be affected by the tsH9 lesion in the
DNA-binding protein as was the stability of the D N A polymerase. Thus it seems likely that the
inhibition of D N A synthesis in mutant virus-infected cells is due to an effect of the lesion in the
DNA-binding protein on the stability of the D N A replication complex. The results on the
stability of the tsH9 D N A polymerase have practical implications for those attempting to
identify mutants in specific virus enzymes by in vitro thermal inactivation experiments. The only
reliable way to attempt this is the complete purification of the enzyme. Any other method would
allow interactions between virus D N A replication proteins to affect the stability of the enzyme
in question. Thus, previous work on the DNA-negative mutants in the Houston collection of
I-ISV-2 mutants, for example, would indicate that several mutants showing thermolabile D N A
polymerase may not actually be defective in that enzyme (Purifoy & Benyesh-Melnick, 1975).
They may have defects in other components of the D N A replication complex. The same
stricture would apply to studies of HSV-2 mutants in the Glasgow series (Hay et al., 1976) and to
work on other virus-induced enzymes.
The data obtained using in vitro complementation of ts mutant virus chromatin offer direct
evidence of a specific role of ICSP 11, 12 in D N A synthesis quite apart from its role of
stabilizing the replication complex. Since both ICSP 11, 12 and the D N A polymerase have been
mapped to the same region of the virus genome the finding that ICSP 11, 12 has an important
role in D N A replication suggests that a functional grouping of HSV proteins involved in D N A
synthesis may occur around the 0-4 map unit position (Morse et al., 1978; Marsden et al., 1978;
Chartrand et al., 1980; Conley et al., 1981). Additional interest in this observation comes from
the findings of several groups (Kaerner et al., 1979; Vlazny & Frenkel, 1981) that certain
defective strains of HSV appear to include an origin of D N A replication that maps at around
this site. In view of the likely importance of the structure of the D N A within this area of the
genome it will be of interest to observe the ability of ICSP 11, 12 to bind to specific fragments of
D N A derived from it.
It is apparent from the differences in behaviour of tsl 9 and tsH9 that they probably differ with
respect to the site of their respective mutations. In view of the likely importance of the protein to
virus D N A replication it is important to investigate fully the mechanisms of the mutations
involved with these mutants. It is also important to obtain many more mutants in the gene to
ensure that a full range of phenotypes are observed. Experiments to this end are in progress.
The combined biochemical, genetic and immunological approach we have used in this study
of the major DNA-binding protein of HSV-2 is applicable to any of the many non-structural
herpesvirus proteins whose functions are unknown. We are currently applying this approach to
several other HSV-2 DNA-binding proteins.
This work was supported by grants from the Medical Research Council and the Cancer Research Campaign.
The Authors are grateful to Professor D. H. Watson for reading the manuscript prior to its submission. We are
indebted to G. Hayward and G. Reyes for their gift of cloned H/ndliI h fragment.
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E. LITTLER AND OTHERS
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