Journal of General Virology (1995), 76, 3125-3130. Printed in Great Britain
3125
The origin-binding domain of the herpes simplex virus type 1 UL9 protein
is not required for DNA helicase activity
Adrian P. Abbotts and Nigel D. Stow*
M R C Virology Unit, Institute o f Virology, Church Street, Glasgow G l l 5JR, UK
The UL9 protein of herpes simplex virus type 1 binds to
specific sequences within the viral origins of DNA
replication and also functions as a DNA helicase. The
C-terminal 317 amino acids of the 851 residue protein
specify sequence-specific binding to the viral origins and
the N-terminal 400 contain several motifs characteristic
of many DNA and RNA helicases. To investigate
whether the origin-binding domain is required for
helicase function we have expressed a truncated version
comprising amino acids 1-535 of UL9 using a recombinant baculovirus. Extracts were prepared from cells
infected with the recombinant virus and chromatographer over ATP-agarose. DNA helicase, DNAdependent ATPase and a novel single-stranded DNAbinding activity were present in fractions containing the
truncated UL9 protein but not in corresponding gradient
fractions from a control virus infection. These results
indicate that the DNA helicase function of UL9 does not
require the presence of the origin-binding domain and
suggest that an interaction between the N-terminal
domain and distorted or partially single-stranded regions
of DNA may play a role in unwinding the origin region.
Gene UL9 of herpes simplex virus type 1 (HSV-1)
encodes one of the seven viral proteins which perform
direct and essential roles in viral DNA synthesis
(reviewed by Challberg, 1991). The UL9 protein comprises 851 amino acids and has two important activities
which are likely to contribute during viral genome
replication. Firstly, UL9 is a sequence-specific DNAbinding protein which interacts with defined sites within
the viral origins of replication. These sequences are
recognized in double-stranded DNA with the C-terminal
317 amino acids being sufficient for binding (Olivo et al.,
1988; Koff & Tegtmeyer, 1988; Weir et al., 1989).
Secondly, UL9 exhibits DNA helicase and associated
DNA-dependent ATPase activities (Fierer & Challberg,
1992; Boehmer et al., 1993; Dodson & Lehman, 1993),
consistent with the presence of several motifs characteristic of a superfamily of DNA and RNA helicases
within the N-terminal 400 amino acids (Gorbalenya &
Koonin, 1993). Genetic evidence suggests that both
functions are necessary for genome replication (Martinez
et al., 1992; Stow et al., 1993) and that UL9 may
participate in the initiation of DNA synthesis by first
binding to and then subsequently facilitating the
unwinding of the origin regions. Although UL9 bound to
an HSV-1 origin is able to distort the DNA duplex,
energy-dependent, sequence-specific, DNA unwinding
has not been demonstrated (Koff et al., 1991; Fierer &
Challberg, 1992).
To investigate whether the DNA helicase activity of
UL9 is independent of its origin-binding function we
have characterized a truncated UL9 molecule expressed
by a recombinant baculovirus which comprises only the
N-terminal 535 amino acids but retains all the conserved
helicase motifs. The recombinant baculoviruses AcUL9
and AcUL9NT express full-length UL9 and amino acids
1-535 respectively (Stow, 1992; McLean et al., 1994). A
recombinant, AcRP231acZ (Possee & Howard, 1987),
which expresses fl-galactosidase was employed as a
control. Spodopterafrugiperda (St-) cells were propagated
and virus stocks prepared as previously described (Stow,
1992).
Nuclear extracts were prepared from approximately
2 × 108 Sf cells infected with AcUL9, AcUL9NT or
A c R P 2 3 l a c Z for 72 h as described by Calder & Stow
(1990) and diluted to 300 mM-NaC1 using Buffer A
(20 mM-HEPES-NaOH pH 7"6, 10% glycerol, 1 mMDTT, 1 mM-PMSF) supplemented with 0.1% NP40. The
diluted nuclear extract was applied to a 4 ml SP
Sepharose Fast Flow (Pharmacia) column preequilibrated with Buffer B (20 mM-HEPES-NaOH pH
7.6, 10% glycerol, l mM-EDTA, l mM-DTT, l mMPMSF) containing 300 mM-NaC1 and 0.1% NP40. The
columns were washed with 16 ml Buffer B containing
300 mM-NaC1 and 0"1% NP40. The eluate was retained,
further diluted to 250 mM-NaC1 using Buffer A and
* Author for correspondence. Fax +44 141 337 2236,
0001-3299 © 1995SGM
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lacZ
E
2
4
6
8
10
12
14
M
UL9NT
. . . . .
:
:
:
:i?:;
............
?
E
2
4
6
8
10
12
14
M
UL9
E
2
4
6
8
10
12
14
M
Fig. 1. S D S - P A G E analysis of ATP-agarose column fractions. The SP Sepharose flow-through (E) and ATP agarose column fractions
(1-14) obtained from cells infected with AcRP23/acZ (lacZ), AcUL9NT (UL9NT) or AcUL9 (UL9) were analysed on 9%
polyacrylamide gels stained with Coomassie blue. Lane M, protein molecular mass markers (205, 116, 97.4, 66, 45 and 29 kDa). *,
U L g N T and UL9 proteins; 0 , an example of an AcNPV or host protein present in each extract.
applied directly onto a 1,5 ml ATP-agarose (Sigma)
column pre-equilibrated with Buffer B containing
250 mN-NaC1. The column was washed with 6 ml Buffer
B containing 250 mM-NaC1 and proteins were eluted
with a 14 ml gradient containing 250-1250 mM-NaC1 in
Buffer B. Fractions o f 1 ml were collected and stored at
- 70 °C.
D N A helicase assays were performed with a substrate
consisting of a labelled 45 base oligonucleotide annealed
to M 13mpl 8 single-stranded D N A and containing a 23
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base 3' tail as previously described (Calder & Stow, 1990)
except that reactions contained 5 lal column fraction in a
final volume of 50 lal and were incubated for 1'5 h at
37 °C. The oligonucleotide lacks sequences corresponding to the UL9 origin-binding sites.
DNA-dependent ATPase activity was assayed in 50 lal
reactions containing 5 lal enzyme fraction, 40 m~t-EPPSNaOH pH 8"3, 3 mM-MgCI~, 100 lag/ml BSA, 10%
glycerol, 0.03 mM-ATP, 0.1 lag activated calf thymus
DNA (Sigma) and 0.9 laCi [7-32P]ATP (Dodson &
Lehman, 1993, Calder & Stow, 1990). After incubation
at 37 °C for 1.5 h, 150 lal activated charcoal in 50 mt~HC1, 5 mN-H3PO ~ was added, the mixes vortexed,
allowed to stand for 2 min and centrifuged at 13 000 g for
1 min. Inorganic phosphate liberated during the reaction
was detected by counting the radioactivity in 100 lal of
the supernatant and the amount of ATP hydrolysed was
determined.
Single-stranded DNA-binding activity was determined
in a gel retardation assay. Mixtures (30 lal) containing
20 mM-HEPES-NaOH pH 7.6, 5 mM-MgCI~, 1 mMDTT, 10 % glycerol (Buffer M), 5 lal column fraction and
125 pg of a 32p 3'-end-labelled 45 base oligonucleotide
(see above) were incubated for 20 min at 18 °C; 6 lal
running dye [1 ×TBE (90 mM-Tris base, 90 raM-boric
acid, 1 mM-EDTA), 10 mM-DTT, 25 % glycerol, 0.05 %
bromophenol blue] was added and complexes were
resolved by electrophoresis through a 10% nondenaturing polyacrylamide gel in 1 × TBE. To verify that
complexes contained UL9-related protein, 5 lal of a
monoclonal antibody against the UL9 protein (13938;
McLean et al., 1994) was added to similar reactions and
incubation continued at 4 °C overnight. To collect the
immune complexes, 20 lal of a 6"6 % (v/v) preparation of
fixed Staphylococcus aureus cells (Pansorbin, Calbiochem) in Buffer M was added and mixing continued
for 1 h. Finally, each reaction tube was supplemented
with 100 lal Buffer M, vortexed and the S. aureus cells
were pelleted by centrifugation at 13 000 g for 1 min; 32p_
labelled single-stranded D N A in the pellet was determined in a liquid scintillation counter.
Fig. 1 shows the results of ATP-agarose chromatography of extracts from Sf cells infected with AcUL9,
AcUL9NT or the control virus, AcRP231acZ. Several
proteins, presumably representing host- or AcNPVencoded species, were eluted by an NaC1 gradient from
all three extracts, but additional major proteins corresponding in size to full-length UL9 and the truncated
form, UL9NT, were also detected in the appropriate
extracts. The identities of UL9 and UL9NT were
confirmed and their relative yields compared by electroblotting various dilutions of column fractions and
detecting the proteins with monoclonal antibody I3938.
The molar amount of UL9NT in peak fractions was
3127
generally 4-8 times greater than UL9, reflecting both its
higher expression level and also the loss of a proportion
of the full-length protein during SP Sepharose chromatography. The binding of UL9NT to ATP-agarose is
consistent with the presence of a functional NTP-binding
site as predicted from its amino acid sequence (helicase
motifs I and II; Gorbalenya & Koonin 1993).
To determine whether UL9NT retained helicase
activity, fractions from columns were assayed. In
agreement with previous results, Fig. 2(a) shows that
fractions containing full-length UL9 protein exhibited
DNA helicase activity significantly greater than corresponding fractions from the A c R P 2 3 l a c Z gradient.
Moreover, a similar peak of D N A helicase activity was
present in the fractions containing UL9NT protein.
Dilution of the UL9NT fractions showed that their
increased activity in comparison with the corresponding
UL9 fractions correlated closely with the increased
amount of UL9-related protein present (data not shown).
Fig. 2 (b) shows the results of ATPase assays performed
on the AcUL9NT and A c R P 2 3 l a c Z column fractions in
the presence of activated DNA. Greatly increased
ATPase activity was present in fractions containing
UL9NT, and further experiments showed that this
activity was reduced 5 10-fold when activated DNA was
omitted from the reaction.
Since DNA helicase and associated DNA-dependent
ATPase activities depend on the ability of a protein to
interact with single-stranded DNA, we also assayed
column fractions for single-stranded DNA-binding activity. Fig. 3 (a) shows an experiment in which binding to
the labelled 45 base single-stranded oligonucleotide was
assessed by gel retardation. An activity which resulted in
the generation of complexes exhibiting about one-third
the mobility of the free probe was present in fractions
6-12 of both the A c R P 2 3 l a c Z and UL9NT gradients
(labelled B). An additional activity resulting in the
formation of a much slower migrating complex (A) was
detected only with the UL9NT fractions, suggesting that
this might be due to binding of the truncated UL9
protein. To confirm the presence of UL9NT protein in
these fractions, protein-DNA complexes were immunoprecipitated with monoclonal antibody 13938, which
recognizes the truncated UL9 protein. The counts present
in immune complexes were determined following binding
to fixed S. aureus cells. Fig. 3 (c) shows that although
fractions from both gradients contained single-stranded
DNA-binding activity, labelled complexes were immunoprecipitated only from fractions containing UL9NT
protein. Addition of 0-25 lag unlabelled single-stranded
circular M13mpl8 D N A (Gibco BRL) to the binding
reactions prevented the formation of retarded complexes
A and B (Fig. 3 b) and reduced the counts in the immune
complexes formed with fractions containing UL9NT to
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(a)
UL9NT
4
5
6
7
8
UL9
9 10
4
5
6
7
lacZ
8
9 10
4
5
6
7
8
9 10
S
BS
-~--- S
"~"- O
1200
(b)
1000
800
600
,<
400
200
•
T
.
14
Fraction no.
Fig. 2. DNA helicase and ATPase activities of ATP-agarose column fractions. (a) Fractions 4~10 from each of the gradients shown
in Fig. 1 were assayed for DNA helicase activity. The reaction products were analysed on a 10 % non-denaturing polyacrylamide gel.
Marker lanes contain the starting undenatured helicase substrate (S) or the same substrate following heat denaturation (BS) to displace
the 45 base labelled oligonucleotide (O). (b) The AcRP23lacZ and AcUL9NT ATP agarose column fractions shown in Fig. 1 were
analysed for ATPase activity in the presence of activated calf thymus D N A . . , AcUL9NT fractions; O, AcRP231aeZ fractions.
1
5
the b a c k g r o u n d levels observed for the corresponding
fractions o f the A c R P 2 3 1 a c Z gradient. The D N A binding activity therefore appears to result f r o m a
genuine interaction with single-stranded regions o f D N A
as opposed to ends o f molecules.
The presence o f D N A - d e p e n d e n t ATPase, D N A
helicase and single-stranded D N A - b i n d i n g activities in
fractions containing U L 9 N T protein but not in corresponding fractions f r o m a control gradient strongly
suggests that U L 9 N T is directly responsible for these
activities. The possibility exists that a c o n t a m i n a t i n g
protein that sticks to U L 9 N T might be involved but it
would have to remain associated at NaC1 concentrations
up to 600mM. Moreover, all three activities co-
10
fractionated with U L 9 N T when an extract was purified
sequentially on A T P - a g a r o s e , SP Sepharose and
heparin-agarose. This procedure yielded greater than
90 % pure U L 9 N T and has the advantage o f not relying
u p o n A T P - b i n d i n g for the final stage (data not shown).
The above results therefore indicate that the Cterminal sequence-specific origin-binding d o m a i n o f U L 9
is dispensable for both its D N A helicase and singlestranded D N A - b i n d i n g activities. A l t h o u g h the helicase
assay utilizes model substrates, this finding m a y nevertheless be relevant to the way in which U L 9 functions at
viral replication origins. Previous studies have indicated
that sequence-specific binding o f U L 9 to the viral
replication origins results in the f o r m a t i o n o f higher-
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(a)
UL9NT
4
6
lacZ
I0
i2
8
i4
i0
12
14
P
A-'-~
B ~
~--B
M
:, : :
P
:
: : : :i:
<:!ii!iiiii;ii!l,
i:
,~.--. p
:
{,i
''
,,'
(b)
1
2
3
4
5
10000
i
i
n
!
(c)
8O00
6000
4000
d
2000
!
1
I
I
5
10
ll4
Fraction no.
Fig. 3. Single-stranded DNA-binding activity of AcRP23/acZ and AcUL9NT ATP agarose column fractions. (a) Fractions shown in
Fig. 1 were incubated with a 45 base single-stranded DNA probe and retarded complexes resolved on 10% non-denaturing
polyacrylamide gels. P, indicates free probe and A and B retarded complexes. Fractions 1 5 contain significant levels of an activity
which degrades the labelled probe. (b) Fraction 7 from each of the two gradients was tested for DNA-binding activity in the presence
or absence of 0.25 lag M13mpl8 single-stranded DNA. Lanes 1 and 2, UL9NT gradient fraction without and with M13mpl8 DNA,
respectively; lanes 3 and 4, AcRP23lacZ gradient fraction without and with M l 3 m p l 8 DNA, respectively; lane 5, no added extract.
(c) Complexes formed as in (a) were immunoprecipitated with monoclonal antibody 13938 and the radioactivity determined in a liquid
scintillation counter. II, AcUL9NT fractions; O, AcRP23lacZ fractions.
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order nucleoprotein structures (Rabkin & Hanlon, 1991)
and that distortion and wrapping of the D N A occurs
under these conditions (Elias et al., 1990; Koff et al.,
1991; Hazuda et al., 1992; Fierer & Challberg, 1992).
The helicase-associated DNA-binding activity of UL9
molecules within the nucleoprotein complex may then
allow interaction with such regions of DNA and
subsequent energy-dependent unwinding of the DNA
duplex. Since origin-specific unwinding by UL9 has not
been demonstrated it is likely that other viral and/or
host proteins participate in modifying the DNA conformation of the origin region to promote UL9 helicase
activity. One such candidate, the viral single-stranded
DNA-binding protein, ICPS, may contribute to helix
destabilization and is able both to interact with UL9 and
to stimulate its helicase activity (Fierer et al., 1992;
Boehmer et al., 1993; Boehmer & Lehman, 1993). A
further specific interaction of origin-bound UL9 with the
UL8 component of the viral helicase-primase complex is
possibly also important for initial unwinding of the
origin region. Interestingly, the interaction with UL8
involves sequences which lie within the functional
helicase domain we have identified (McLean et al., 1994),
whilst sequences at the C terminus of UL9 that are
dispensable for origin binding contribute to the interaction with, and stimulation of helicase activity by,
ICP8 (Boehmer et al., 1994).
Finally, our data are consistent with the sequencespecific origin-binding and DNA helicase activities of
UL9 being independent functions carried out by distinct
domains of the protein. That these domains may even
have separate evolutionary origins is suggested by the
observation that whilst the N-terminal helicase domain
shares homology with a large number of putative
helicases from a variety of eukaryotes, prokaryotes and
viruses, sequences with significant homology to the
origin-binding region have been described only in other
members of the alphaherpesvirus subfamily. Distinct
additional domains have also been identified flanking a
core helicase domain in a number of other proteins and
these have been associated with endonuclease, primase
or protease activities (reviewed by Gorbalenya &
Koonin, 1993).
We thank Gordon McLean and Vivien Mautner for constructive
discussions and are grateful to Andrew Davison for helpful comments
on the manuscript.
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