The Herpes Simplex Virus 1 Origin Binding Protein : A DNA Helicase ”

A recombinant herpes simplex 1 origin binding protein, the product of the herpes UL9 gene, has been overexpressed in mammalian cells and purified to near homogeneity. The origin binding protein shows DNAdependent nucleoside 5’-triphosphatase and DNA helicase activities in addition to its origin binding activity. The ability to hydrolyze nucleoside 5”triphosphates is influenced strongly by the structure and sequence of the DNA cofactor. The properties of the recombinant origin binding protein are identical to those of the protein synthesized in herpes simplex 1-infected mammalian cells.

structure and function. We have found that the recombinant OBP has both DNA-dependent nucleoside 5"triphosphatase and DNA helicase activities in addition to its origin binding activity.' The recombinant protein is identical to the OBP isolated from HSV-1-infected mammalian cells.

Materials-Plasmid
DNA, activated calf thymus DNA, and M13mp18 single-stranded DNA were prepared as described (37). The plasmid pON103 (32) was digested with EcoRI and PstI and 3' end labeled with [ L~-"~P ]~A T P a n d t h e Escherichia coli DNA polymerase I large fragment as described (37). The labeled 322-base pair Or& containing fragment was then separated from the vector by polyacrylamide gel electrophoresis and the DNA isolated from the gel (38). Plasmid pET3a (39) was obtained from Per Elias (Gothenberg University). Plasmid pOS-822 was obtained from Priscilla Schaffer and Scott Wong (Harvard University). Plasmid pDL411, containing ori,, (IO), was obtained from Denise Galloway (Fred Hutchinson Cancer Research Center). Oligonucleotides 3/4 and 7/8 were prepared as described (33) as were the DNA helicase substrates (3'-tailed, 5'tailed, and untailed) (29). EcoRI linkers were obtained from New England Biolabs, and (dA)4DO0 and (dT)Glao were from Midland Certified Reagent Co. (Midland, TX). Ribo-and deoxyribonucleoside 5'triphosphates were obtained from Pharmacia LKB Biotechnology Inc., and [a-"'PIdATP (3000 Ci/mmol) was from Amersham Corp. Restriction endonucleases were obtained from New England Biolabs and Bethesda Research Laboratories. T4 DNA polymerase and T4 DNA ligase were from New England Biolabs. DNA polymerase I large fragment, calf intestinal alkaline phosphatase, sodium persulfate, bisacrylamide, and TEMED were obtained from Boehringer Mannheim. Acetylated bovine serum albumin was obtained from Bethesda Research Laboratories. Phospbocellulose P-11 was obtained from Whatman. Heparin was from Sigma and was coupled to Sepharose CL-4B (Pharmacia) as described (40). A prepacked Superose 12 gel filtration column and molecular weight standards were obtained from Pharmacia. Sodium dodecyl sulfate was from BDH Chemicals (Poole, United Kingdom). Acrylamide was obtained from U. S. Biochemical Corp. Molecular weight standards for electrophoresis were obtained from Bio-Rad. CV-1 cells and 293 cells were from American Type Culture Collection. Sterile HEPES, glutamine, penicillin/streptomycin, and G418 were from GIBCO. Fetal calf serum was obtained from Irvine Scientific, Santa Ana, CA.
Cells and Viruses-Cells were grown a t 37 "C in Dulbecco's modified Eagle's medium containing 10% (v/v) fetal calf serum and supplemented with 2 mM glutamine, 100 units/ml penicillin, and 100 pg/ml streptomycin in an atmosphere of 6.5% CO2. For large scale growth, the medium was buffered with 10 mM HEPES, pH 7.5. Active Dr. Mark Cballberg (NIH) has observed independently that OBP purified from a baculovirus expression system has DNA-dependent ATPase and DNA helicase activity.  G418 (800 pg/ml) was added during selection as described (41). Ad5.SVR3 (42) was grown using 293 cells as a host.

HSV-1 Origin Binding Protein
Electrophoresis-Agarose gel electrophoresis was performed as described (37). DNA was isolated from the gel by binding to glass beads (Geneclean, Bio 101, La Jolla, CA). SDS-polyacrylamide gel electrophoresis was performed and the gels silver-stained as described (43,44).
DNA-dependent ATPase Assay-The reaction mixture (25 pl) in Buffer B contained 1 pg of (dT)-or other DNA cofactor as indicated. After the addition of OBP the mixture was incubated at 37 "C for 30 min. Acidic molybdate containing Malachite green (45) was added, and after 10 min of color development A,,, was determined. One unit of ATPase hydrolyzes 1.0 nmol of ATP in 30 min.
Filter Binding Assay-OBP (fraction IV, 460 ng/pl) was incubated with 17 ng of labeled Oris fragment and a 100-fold molar excess of the nucleotides 3/4 or 7/8 (33) in 25 pl of Buffer B [0.1] for 5 min at 23 'C followed by 10 min at 0 "C. Ice-cold Buffer B [0.1] (950 p l ) was added and the mixture immediately filtered through Millipore 0.45-Fm type HA filters. The filter was washed with 1 ml of ice-cold Buffer B [0.1] and dried. Radioactivity was determined by scintillation counting.
DNA Helicase Assay-DNA helicase activity was measured as described for the HSV-1 helicase-primase (29) using 170 ng of OBP purified through Superose 12 and 4.0 /LM (nucleotide) helicase substrate. ATP (3.0 mM) was added where indicated. Incubation was at 37 "C for 15 min.
Nuclease and Protein Assays-Exonuclease activity was assayed as described (29). Protein concentrations were determined by the method of Bradford (46), using bovine serum albumin as a standard.
Insertion of UL9 Gene into Expression Vector p91023B"The plasmid pET3a is an E. coli expression vector that contains the HSV-1 UL9 gene with an NdeI restriction site at the start codon of UL9 (39). The DNA segment containing UL9 was excised by digestion with NdeI and Hind111 and the single-stranded ends filled in with T4 DNA polymerase. The DNA fragments were separated by agarose gel electrophoresis, and the fragment containing UL9 was isolated from the gel and ligated to EcoRI linkers. After removing excess Iinker DNA by agarose gel electrophoresis, the ligated product was excised from the gel and digested with EcoRI. The expression vector p91023B (47) was digested with EcoRI and treated with calf intestinal phosphatase (37). The two DNA fragments were ligated and transformed into E. coli HB101 (37). A clone containing UL9 inserted into the vector in the proper orientation, pRCB25, was then selected.
Construction of a Recombinant Cell Line-A recombinant CV-1 cell line was generated by cotransfection of CV-1 cells with pRCB25 and pRSVneo. G418-resistant cell lines were isolated and expanded (41). Large Scale Growth and Induetion-One of the cell lines, CV-1 UL938, was expanded into six 850-cm2 roller bottles. Cells web grown to half-confluence and infected with Ad5.SVR3 (200-251 plaque-forming units/cell). They were then grown for 4 days with on1 change of medium. Approximately 4 g of infected cells was obtained Nuclear and cytosolic fractions were prepared as described (32) anc then frozen in liquid nitrogen and stored at -80 "C.
Purification of OBP from CV-l/UL9:38 Cell Line-The purificatiol of OBP through the phosphocellulose step was modified from thl method of Elias et al. (32). All steps were carried out at 4 "C. Nucle prepared from approximately 4 g of CV-l/UL9:38 cells were thawec and centrifuged for 15 min at 750 X g. The nuclear pellet wa: resuspended to a volume of 7. containing fractions were frozen in liquid nitrogen and stored al -80 "C. OBP at this stage of purification was greater than 95% purc and was free of detectable exo-and endonuclease activity.

RESULTS
Purification of Recombinant OBP-To obtain quantities oi the HSV-1 origin binding protein sufficient for the study of its structure and associated enzymatic activities, UL9 was cloned into the SV40 origin-containing expression vectoI p91023B (47) (Fig. 1). After cotransfection of CV-1 cells with pRSVneo, G418-resistant cell lines capable of expressing high levels of OBP upon infection with the T-antigen expressing adenovirus Ad5.SVR3 were selected (41, 42).
Nitrocellulose filter binding had been used initially to measure OBP activity (32,33). However, we observed that addition   Table  I) were subjected to 10% SDS-polyacrylamide gel electrophoresis and then silver stained. of ATP significantly increased filter retention," suggesting that OBP may have an ATP binding site and hence an associated ATPase activity. Examination of OBP preparations purified from HSV-1-infected CV-1 cells showed that they did indeed possess a single-stranded DNA-dependent ATPase. DNA-dependent hydrolysis of ATP was therefore used to quantitate the recombinant OBP during purification. As shown in Table I the level of OBP expression was sufficient to allow purification to near homogeneity after only two chromatographic steps. Although UL9 encodes a protein with a predicted molecular mass of 92 kDa, OBP from both HSV-1-infected CV-1 cells and the mammalian expression system migrated as an 83-kDa protein during SDS-polyacrylamide gel electrophoresis (Fig. 2). The proteolysis products (65-70 kDa) that often represent a major fraction of OBP purified from HSV-1-infected CV-1 cells are only a minor contaminant in the recombinant OBP preparation.
The DNA-dependent ATPase activity of the OBP was coincident with origin binding activity at each step in the purification (data not shown), suggesting that it is an intrinsic property of OBP. To provide further evidence for this association, fraction IV was subjected to gel filtration on Superose 12. A single peak of protein was observed which coincided perfectly with the DNA-dependent ATPase activity and the 83-kDa OBP (Fig. 3, A and B). The peak of protein and ATPase eluted just after aldolase (160 kDa), suggesting that OBP may exist as a homodimer. However, the shoulder on the trailing edge of the protein peak, eluting with bovine serum albumin (68 kD), indicates that some of the OBP may also be in a monomeric form.
Recombinant OBP Binds Specifically to Oris-The minimal  Ori.9 sequence contains two sequences, designated Box I and Box 11, that have been shown to bind OBP (Fig. 4B) (33-36). A third sequence that shares significant homology with Boxes I and I1 but has not been shown to bind OBP is contained within the minimal Oris sequence and has been designated Box I11 (39, 48). An A-T spacer sequence separates Boxes I and 11. T o investigate the interaction of the recombinant OBP with Oris, OBP was added to reaction mixtures that included a labeled restriction fragment containing Oris and the unlabeled competing (7/8) or noncompeting (3/4) oligonucleotide (33). The two unlabeled oligonucleotides share 17 base pairs of homology, but only 7/8 contains a complete OBP binding site (box I). Interaction of OBP with the Oris sequence in the presence of the two oligonucleotides was determined by nitrocellulose filter binding. As shown in Fig. 4A, the binding curve obtained with the noncompeting oligonucleotide 3/4 was hyperbolic and reached binding saturation when 94% of the label was retained on the filter. The binding curve in the presence of the competing oligonucleotide 7/8 was sigmoidal, and 50% retention required a 6.5-fold greater amount of OBP than with oligonucleotide 314. Saturation also required higher amounts of OBP. The ability of the 7/8 but not 314 to compete effectively with Oris for binding indicates that binding of the recombinant OBP to Oris is sequence specific.

HSV-1 Origin Binding Protein
DNA and Nucleotide Specificity of OBP-OBP was incubated with a variety of DNA cofactors, and ATP hydrolysis was measured. As shown in Fig. 5, OBP has a weak ATPase activity even in the absence of DNA. Addition of supercoiled DNA with or without the Oris sequence failed to stimulate ATPase activity. However, addition of activated calf thymus DNA, which has short single-stranded regions, stimulated ATPase activity 1.4-fold whereas single-stranded M13mp18 DNA increased ATP hydrolysis %fold over the basal level.
The greatest effect was seen with (dT)6000, which produced a 6-fold stimulation of ATPase activity. In contrast, (dA)4000 failed to stimulate ATPase activity even at high concentrations (>1 pg). These results suggest that the sequence and possibly the structure of the single-stranded DNA influence its ability to serve as a cofactor for the ATPase associated with OBP.
OBP was incubated with a variety of nucleoside 5"triphosphates in the presence of (dT)6000, and the amount of hydrolysis after a 30-min period was compared with that observed with ATP. As shown in Table 11, dATP was hydrolyzed at nearly the same rate as ATP (80%). In contrast, GTP and dGTP were very poor substrates compared with ATP (12 and 6%, respectively). Both CTP and dCTP were good substrates (75 and 61%). UTP was also hydrolyzed (37%), but dTTP was a relatively poor substrate (15% of that observed with ATP). The efficiency of hydrolysis of the various nucleoside 5'-triphosphates by the recombinant OBP was not significantly different from the enzyme isolated from HSV-l-infected CV-1 cells (Table 11).

HSV-1 Origin Binding Protein
OBP Is a DNA Helicase-Inasmuch as the SV40 large Tantigen that binds specifically to the SV40 origin is both a DNA-dependent ATPase and helicase (49, 50), we examined the ability of the HSV-1 OBP to act as a DNA helicase. Three different substrates were used. The first is a 22-residue oligonucleotide annealed to M13mp18 single-stranded DNA. The other two substrates share the same 22 base pairs of duplex DNA but have 23 noncomplementary nucleotides at either the 5'or 3' end. As shown in Fig. 6, OBP does have an associated DNA helicase activity. The helicase showed some activity with the untailed substrate; however, the presence of either a 5' or 3' tail increased activity significantly.

DISCUSSION
Overexpression of the HSV-1 origin binding protein in a mammalian cell system has made possible the detailed analysis of its structure and associated enzymatic activities. OBP free of detectable nuclease can be obtained in nearly homogeneous form and in high yield after only two chromatographic steps. The yield of 0.8 mg of OBP from approximately 4 g of CV-l/UL9:38 cells represents an approximately 150fold increase over the amount that can be purified from a comparable amount of HSV-1-infected mammalian cells (33). Moreover, the proteolytic products that often form a major part of OBP preparations purified from HSV-1-infected cells are only a minor contaminant in the recombinant enzyme.
The UL9 open reading frame predicts a protein with a mass of 92 kDa. However, the OBP from a variety of cells (HSV-1-infected CV-1 cells (33), the mammalian expression system described here, and a baculovirus expression system (34)) migrates during SDS-polyacrylamide gel electrophoresis as an 83-kDa protein. This altered mobility could be due to posttranslational modification. However, any modifications that occur in HSV-1-infected cells would also be expected to occur in the CV-l/ULS cell line.
The recombinant OBP is indistinguishable from the protein purified from HSV-1-infected CV-1 cells. Like the latter, the recombinant enzyme binds specifically to Oris and has the same DNA-dependent nucleoside triphosphatase. The nucleoside triphosphatase activity of OBP shows some specificity for its DNA cofactor. Duplex DNA, even with an HSV-1 origin of replication, is inactive. Surprisingly, (dT)GW shows a strong stimulation whereas is inactive. Thus, either the sequence or structure of the single-stranded DNA plays a role in its ability to serve as a cofactor.
The DNA-dependent ATPase activity of OBP is clearly a manifestation of its DNA helicase activity. In the presence of ATP, OBP can effectively unwind short regions of duplex DNA. Unwinding is most efficient with tailed substrates. However, 3' or 5' tails are equally effective. Thus far we have been unable to detect specific unwinding of supercoiled DNA containing an HSV-1 origin of replication, either Oris or OrG?
The N-terminal portion of OBP contains the amino acid sequence APMGSGKT (3), which in turn contains the consensus sequence G/AXXXXGKS/T found in many ATP binding proteins (51). Comparison of the amino acid sequences of known helicases has shown that they contain seven conserved regions of homology and could on this basis be divided into three superfamilies. These superfamilies contain the abridged ATP binding site GXGKS/T. The HSV-1 OBP falls into one of these three superfamilies (52).
SV40 T-antigen forms a nucleoprotein complex at the SV40 origin in the presence of ATP, and the helicase unwinds the adjacent A-T-rich sequence (50,53,54). This unwound region is stabilized by the addition of a single-stranded DNA binding protein and can be propagated by the addition of a topoisomerase to relieve torsional strain. The opening up of the DNA duplex at the origin then permits entry of the remainder of the replication machinery. The properties of OBP, i.e. specific binding to an origin sequence (Oris) and DNA helicase activity, suggest that OBP plays a role in HSV-1 replication analogous to that of T-antigen in SV40 replication. However, in contrast to SV40 T-antigen, we have not detected either the formation of a stable origin-specific nucleoprotein structure or, as noted above, the unwinding of the DNA duplex at Oris by OBP. The interaction of OBP with Oris is considerably weaker than the interaction of T-antigen with the SV40 origin. Thus, incubation at 0 "C is required to observe binding to Oris; at 23 "C OBP dissociates rapidly (33), even in the presence of ATP: The effect of various nucleoside 5-triphosphates, origin structure, and possibly host protein(s) on the OBP-Oris interaction clearly requires further investigation. We anticipate that elucidation of the conditions that allow stable binding and unwinding at the Oris sequence by OBP should provide clues that will permit the development of a system in which HSV-1 replication is specifically initiated at Oris in vitro.