Proc. Natl. Acad. Sci. USA Vol. 90, pp. 84R4-8448, September 1993 Biochemistry Physical interaction between the virus 1 origin-binding and single-stranded DNA-binding protein ICP8 (DNA replicton/origin unwIn /proteIn-affnIty chromatogaphy/proteifprotein interacdons) PAUL E. BOEHMER AND I. R. LEHMAN Department of Biochemistry, Beckman Center, Stanford University School of Medicine, Stanford, CA 94305-5307 Contributed by I. R. Lehman, June 15, 1993

ABSTRACT We had previously demonstrated that the functional stimulation is due to the tight interaction between herpes splex virus 1 (HSV-1) ngle-stranded DNA-binding ICP8 and UL9 protein. protein (ICP8) can specfly stimulate the actvity of the HSV-1 origin-binding protein (UL9). We show here that MATERIALS AND METHODS this functional stimulation is a mafesation of a tight inter- action between UL9 protein and ICP8. By using protein- Materials. media and reagents were obtained affalnty chromatography, we have demonstrated the specflc from GIBCO/BRL. Fetal calf serum was obtained from bndin of purified UL9 protein to immobized ICPS and vice Gemini Biological Products (Calabasas, CA). SDS/PAGE versa. Furthermore, ICP8 ispecalyis retained by a colum and immunoblot analysis were done as described (11, 12). on which the C-tenal 37-kDa DNA-binding domin of the The anti-ICP8-specific rabbit serum 3-83 was from D. Knipe UL9 protein was immobized. The interaction between ICP8 (Harvard University). The recombinant baculovirus AcUL29 and the DNA-binding domain of the UL9 protein was con- (13) was from N. Stow (Medical Research Council Virology firmed by cochrmatography ofthe two . These results Unit, Glasgow). The E. coli over-expression plasmid pET- suggest that the UL9 protein and ICP8 form a tight complex 3a/HisAOBP was from P. Elias (Gothenburg University). E. that functions in origin recognition and uninding. coli TOP10/pTrcHisCAT was obtained from Invitrogen. Protein standards for SDS/PAGE were obtained from Bio- The herpes simplex virus 1 (HSV-1) chromosome is a linear Rad. duplex DNA of "150 kbp, containing three origins of DNA Buffers. The molar concentration of NaCl in each buffer is replication (1). HSV-1 encodes seven gene products that are indicated separately according to use. Buffer A contained 20 both necessary and sufficient for the replication of origin- mM Hepes'NaOH (pH 7.5), 10% (vol/vol) glycerol, 1 mM containing plasmids in transient replication assays (forreview, dithiothreitol, and 0.1 mM EDTA. Buffer B was the same as see ref. 2). These seven genes encode a highly processive buffer A, except that the pH was adjusted to 7.6. Buffer C heterodimeric DNA polymerase (UL30/UL42), a heterotri- contained 10 mM sodium phosphate (pH 7.5), 10% glycerol, meric helicase-primase (UL5/UL8/UL52), a single-stranded 1 mM dithiothreitol, and 0.1 M NaCl. Buffer D was identical DNA-binding protein (SSB) (UL29), and an origin-binding to buffer C, except that the pH was adjusted to 7.0. Buffer E protein (UL9) (2, 3). contained 20 mM Hepes'NaOH (pH 7.5), 10% glycerol, and Studies ofthe replication in vitro ofplasmids containing the 1 mM 2-mercaptoethanol. Buffer F was identical to buffer A, Escherichia coli origin (oriC) and simian virus 40 origin have except that EDTA was omitted. Protease inhibitors (Sigma) shown that origin-specific initiation of DNA replication in- were added at the following final concentrations: leupeptin volves protein-mediated destabilization ofthe DNA duplex at (0.5 ,ug/ml), pepstatin (0.7 pg/ml), and phenylmethanesulfo- the origin, thereby allowing access of the DNA synthetic nyl fluoride (0.1 mM). machinery to the DNA (4). For simian virus 40, the initiator Cell Culture and Preparation of Cell Extracts. Spodoptera protein, large tumor , recognizes elements within the frugiperda (Sf9) cells and stocks ofAutographa californica and leads to the destabilization of the nuclear polyhedrosis virus (AcNPV) recombinant for the DNA duplex through its DNA helicase activity. This process HSV-1 UL9 and UL29 genes were maintained and propa- also requires the action ofthe cellular SSB, RP-A, to maintain gated as described (13-15). Sf9 cells were grown in 20 the DNA in a single-stranded conformation (2, 5, 6). 225-cm2 tissue culture flasks to a cell confluency of=480% and The ability of HSV-1 UL9 protein to recognize elements infected with recombinant AcNPV at a multiplicity of infec- within the HSV-1 origin ofreplication (2, 3, 7) and to function tion of ==15. Sixty hours after infection, the cells were as a DNA helicase (8, 9) suggests that it is involved in harvested, washed in phosphate-buffered saline, and frozen origin-specific DNA unwinding during HSV-1 DNA replica- at -80°C. Nuclear extracts were prepared as described (16). tion. Origin-unwinding and the maintenance of the single- Vero cells (African green monkey kidney fibroblasts) were stranded conformation should be aided by the helix- propagated at 37°C in Dulbecco's modified Eagle's minimal destabilizing properties of the HSV-1-encoded SSB ICP8 essential medium/10% fetal calf serum/0.1 mM nonessential (10). Consequently, efficient destabilization of the DNA amino acids/2 mM glutamine. HSV-1-infected cell extracts duplex at the origin might be expected to involve the con- were prepared by infecting 20 150-cm2 tissue culture flasks certed action of the UL9 protein and ICP8. with HSV-1 strain A305 (17) at a multiplicity of infection of We had reported (9) that ICP8 can specifically stimulate the 10. Ten hours after infection, the cells were harvested, DNA helicase activity ofUL9protein. We show here that this Abbreviations: SSB, single-stranded DNA-binding protein; AcNPV, Autographa californica nuclear polyhedrosis virus; HSV-1, herpes The publication costs ofthis article were defrayed in part by page charge simplex virus 1; BSA, bovine serum albumin; CAT, chloramphenicol payment. This article must therefore be hereby marked "advertisement" acetyltransferase; AUL9, C-terminal DNA-binding domain of UL9 in accordance with 18 U.S.C. §1734 solely to indicate this fact. protein. 8444 Downloaded by guest on October 2, 2021 Biochemistry: Boehmer and Lehman Proc. NatL Acad. Sci. USA 90 (1993) 8445 washed in phosphate-buffered saline, and frozen at -80°C. NaCI) and cleared by centrifugation at 29,000 rpm for 45 min All subsequent steps were done at 4°C. The cells, -1.3 g, in a Beckman 45 Ti rotor. Cleared extracts were mixed were thawed and resuspended in buffer A (1.2 M NaCI) batchwise with 4 ml of Ni-NTA resin (Qiagen, Chatsworth, containing 1 mM EDTA. The cells were transferred to a CA) equilibrated in buffer E (0.15 M NaCI). After 60 min with Dounce homogenizer and lysed with 10 strokes of the tight- gentle stirring, the mixture was poured into an Econo-Pac fitting pestle. The lysed cells were cleared by centrifugation column and washed extensively with buffer E (0.15 M NaCI). at 55,000 rpm for 60 min in a Beckman 70.1 Ti rotor. Nucleic Proteins were eluted with 10-ml steps of buffer E (0.15 M acid was precipitated from the supernatant by the drop-wise NaCa) containing 10, 20, 40, 60, 80, and 200 mM imidazole. addition of 0.05 vol of 0.3 M spermidine HCI/1 M NaCI/0.31 Fractions, 1 ml, containing near-homogenous (>95% pure) vol of 5% (wt/vol) streptomycin sulfate, both in buffer A. histidine-tagged AUL9 and CAT proteins eluted in the 80 and After a 30-min incubation, the nucleic acid precipitate was 200 mM imidazole fractions and were stored at -80°C. removed by centrifugation at 25,000 rpm for 30 min in a E. coli SSB was from E. H. Lee (this department); its Beckman 70.1 Ti rotor. The supernatant was dialyzed three concentration was determined by the method ofBradford (20). times against 500 ml of buffer A (0.1 M NaCl). After dialysis, Bovine serum albumin (BSA) was obtained from Pharmacia. the precipitate was removed by centrifugation at 55,000 rpm Protein concentrations were determined by using extinc- for 30 min in a Beckman 70.1 Ti rotor, and the supernatant tion coefficients of 82,720, 89,220, and 43,626 M-1 cm-1 at was frozen at -80°C. 280 nm for ICP8, UL9 protein, and BSA, respectively. Proteins. All chromatographic steps were done at 4°C, Concentrations of the histidine-tagged AUL9 and CAT pro- using a Pharmacia fast protein liquid chromatography sys- teins were determined by assuming that one A280 unit corre- tem, at a flow rate of 0.5 ml/min. At each stage of the sponds to a 1 mg/ml solution. purification, purity was assessed by SDS/PAGE followed by Protein-Affinity Chromatography. One milliliterof1 mg/ml Coomassie blue staining and densitometry. solutions of UL9 protein, ICP8, or BSA was dialyzed exten- The UL9 protein was purified from nuclear extracts pre- sively against 0.2 M NaHCO3/0.5 M NaCl, pH 8.3. The pared from Sf9 cells infected with an AcNPV recombinant for dialyzed proteins were then immobilized on 1 ml of N-hy- the UL9 gene. Cleared nuclear extracts, dialyzed against droxysuccinimide (NHS)-activated HiTrap columns (Phar- buffer B (0.1 M NaCl), were loaded onto a5-ml HiTrap heparin macia), as recommended by the manufacturer. column (Pharmacia) equilibrated with the same buffer. The HiTrap protein-affinity column chromatography was done column was washed, and proteins were eluted with a 60-ml with a Pharmacia fast protein liquid chromatography system. linear gradient of NaCl (0.1-0.8 M), and 1-ml fractions were The columns were run at a flow rate of 0.05 ml/min, and collected. The peak of UL9 protein, eluting at -0.7 M NaCl, 0.35-ml fractions were collected. Protein mixtures containing was applied onto a 5-ml Econo-Pac HTP cartridge (Bio-Rad) 100 jig ofeach protein species were dialyzed against buffer F equilibrated with buffer C, and proteins were eluted with a (0.1 M NaCI) and loaded onto the HiTrap protein-affinity 50-ml linear gradient of sodium phosphate (0.01-0.3 M). Peak columns equilibrated with the same buffer. After sample fractions, 1 ml each, eluting at =0.09 M sodium phosphate, application, the columns were washed with 2 ml of buffer F were dialyzed against buffer B (0.1 M NaCI) and loaded onto (0.1 M NaCI) and developed with 10-ml linear gradients of a Mono S HR 5/5 column (Pharmacia) equilibrated with the NaCl (0.1-1.0 M) followed by elution with 1 ml at 1.0 M NaCl same buffer. The column was washed, and proteins were and 4 ml at 0.1 M NaCl. eluted with a 15-ml linear gradient of NaCl (0.1-1.0 M). Purified histidine-tagged AUL9 and CAT proteins (1 mg) Fractions, 0.25 ml, containing near-homogeneous (>95% were dialyzed against buffer E (0.05 M NaCl) and adsorbed pure) UL9 protein eluting at -0.55 M NaCl, were divided into batchwise to 100 1d of Ni-NTA resin (Qiagen) equilibrated aliquots and stored at -800C. The yield of purified UL9 with the same buffer. The resin was pipetted into a pierced, protein was =2.5 mg from 12 225-cm2 tissue culture flasks. long 400-I4 Eppendorf microcentrifuge tube, plugged with ICP8 was purified either from HSV-1-infected U35 cells, glass beads (212-300 ,m; Sigma) and tipped with a 0.5 x 16 essentially as described by Hernandez and Lehman (18), or mm syringe needle. The columns were washed with buffer E from nuclear extracts prepared from Sf9 cells infected with (0.05 M NaCl/20 mM imidazole). A mixture of proteins in AcUL29. Cleared nuclear extracts, dialyzed against buffer B buffer E (0.05 M NaCl/20 mM imidazole), consisting of50 pg (0.1 M NaCI), were loaded onto a 5-ml HiTrap heparin each of ICP8, BSA, and E. coli SSB in -100 I1, was applied column (Pharmacia) equilibrated with the same buffer. The to the columns, and the flow-through fraction was collected. column was washed, and proteins were eluted with a 50-ml The columns were washed with 500 jd ofbuffer E containing linear gradient of NaCl (0.1-0.8 M), and 1-ml fractions were increased concentrations ofNaCl and imidazole as indicated collected. The peak of ICP8, eluting at -0.35 M NaCl, was in Fig. 3, and the eluant at each step was collected. applied onto a 5-ml Econo-Pac HTP cartridge equilibrated Gel-Filtration Chromatography. A Superose 12 HR 10/30 with buffer D, and proteins were eluted with a 50-ml linear gel-filtration column (Pharmacia) was equilibrated with gradient of sodium phosphate (0.01-0.2 M). Peak fractions, buffer F (0.2 M NaCl) at 0.2 ml/min. Protein samples, -1 1 ml each, eluting at "0.08 M sodium phosphate, were nmol, were dialyzed into chromatography buffer and injected dialyzed against buffer A (0.1 M NaCI) and loaded onto a onto the column. The column was developed at 0.2 ml/min, Mono Q HR 5/5 column (Pharmacia) equilibrated with the and 0.2-ml fractions were collected. same buffer. The column was washed, and proteins were eluted with a 16.5-ml linear gradient of NaCl (0.1-0.75 M). RESULTS Fractions, 0.25 ml, containing near-homogeneous (>95% Protein-Affinity Chromatography Demonstrates a Tight pure) ICP8 eluting at =0.4 M NaCl were divided into aliquots Physical Interaction Between ICP8 and UL9 Protein. Protein- and stored at -800C. The yield of purified ICP8 was -20 mg affinity chromatography was used to detect a specific inter- from 20 225-cm2 tissue culture flasks. action between ICP8 and the UL9 protein. Purified ICP8, The histidine-tagged chloramphenicol acetyltransferase UL9 protein, and BSA were covalently attached to activated (CAT) protein and C-terminal DNA-binding domain of the agarose beads. A mixture of purified proteins was then UL9 protein (AUL9) were purified from E. coli TOP10/ chromatographed on the three protein-affinity columns. The pTrcHisCAT and BL21(DE3)pLysS/pET-3a/HisAOBP, re- ability of an ICP8-agarose column to specifically bind UL9 spectively. Extracts from 1 liter ofisopropyl 3-D-thiogalacto- protein is shown in Fig. 1A. SDS/PAGE analysis of the side-induced cells grown at 30°C were prepared as described column fractions indicated that the control proteins, BSA and (19). Cell extracts were diluted to 50 ml with buffer E (0.15 M E. coli SSB, failed to adsorb to the column, whereas the UL9 Downloaded by guest on October 2, 2021 8446 Biochemistry: Boehmer and Lehman Proc. Natl. Acad. Sci. USA 90 (1993)

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described. of ICP8 and UL9 protein. Proteins were chromatographed on the indicated columns, as Fia. 1. Protein-affinity chromatography (A) blue lines represent linear gradients of NaCI. Column fractions were analyzed by SDS/PAGE followed by Coomassie staining. Diagonal and E. coli SSB are as indicated. M, MF ICP8-agarose. (B) UL9 protein-agrose. (C) BSA-aarose. Positions of ICP8, UL9 protein, BSA, standards; L, load fraction; FT7, flow-through fraction. Downloaded by guest on October 2, 2021 Biochemistry: Boehmer and Lehman Proc. Natl. Acad. Sci. USA 90 (1993) 8447 protein was specifically retained, eluting at -%0.6 M NaCl. A .M M NaCt This result indicates a tight physical interaction between ICP8 and the UL9 protein. Similarly, immobilized UL9 0.2 M Imidazole 0.0 ()0.21 ()X0.08 protein could specifically retain ICP8 (Fig. 1B). When a M 1. FT '0 0.2. M mixture ofproteins consisting ofICP8, BSA, and E. coli SSB was applied to the UL9 protein column, the control proteins, t.i. BSA and E. coli SSB, were not retained, whereas ICP8 ICP8-_- adsorbed to the column and was eluted as a broad peak at BSA-'P -0.6 M NaCl. In contrast, there was no significant retention of ICP8, UL9 protein, or E. coli SSB by a column consisting of immobilized BSA (Fig. 1C). -AUL9 To determine whether the interaction between ICP8 and the UL9 protein could be detected in a crude cell extract, an HSV-1-infected cell extract was chromatographed on a col- umn containing immobilized UL9 protein. ICP8 in the col- umn fractions was monitored by immunoblotting with an SSB-- anti-ICP8-specific rabbit serum. Fig. 2 shows that ICP8 was retained by the immobilized UL9 protein and then eluted upon increased ionic strength, with a peak appearing at 0.5 M NaCl. These results show that the tight interaction seen between purified ICP8 and UL9 protein can also be detected when ICP8 is present in a crude cell extract. ICPS Binds to the 37-kDa C-Terminal DNA-Binding Domain ofUL9 Protein. The C-terminal DNA-binding domain ofUL9 B 1.0 M NaCI protein was expressed in E. coli as a histidine-tagged 37-kDa 0. M Imidazole protein. The purified AUL9 protein was immobilized on 0.1.08 Ni-NTA resin because ofits histidine tag. Fig. 3A shows that M I FT 00 0.(12004 M the immobilized AUL9 protein could specifically retain ICP8; most of the ICP8 eluted in the 1.0 M NaCl steps. Control proteins BSA and E. coli SSB, on the other hand, both lCP8-*O appeared in the flow-through and wash fractions. No inter- BSA action between ICP8, BSA, or E. coli SSB was seen with Al~ ~ ~ CA resin alone (data not shown) or with immobilized, histidine- tagged CAT protein (Fig. 3B). These results show that the specific interaction between ICP8 and UL9 proteins is con- fined to the C-terminal DNA-binding domain ofUL9 protein. Cochromatography of ICP8 and the DNA-Binding Domain ~~~~~~~~~~...... ; ofUL9 Protein. To confirm the interaction between ICP8 and SSB-* the AUL9 protein detected by protein-affinity chromatogra- phy, the behavior ofthe two proteins during gel filtration was ..i.it~~~~~~~~~~~~~~~~~~~~~~~~~~~~~..,...... examined. The UV-absorbance profiles of proteins eluting ...... from a Superose 12 HR 10/30 column, as well as SDS/PAGE analysis of the peak protein fractions, are shown in Fig. 4. Fig. 4A and B show the elution proffles ofICP8 and the AUL9 protein, eluting after 11.6 and 14.3 ml ofbuffer, respectively. FIG. 3. ICP8 binds specifically to the 37-kDa AUL9. Proteins Fig. 4C depicts the elution profie of a mixture of ICP8 and were chromatographed on immobilized AUL9 and CAT proteins, as AUL9 protein. The UV-absorbance profile indicates that the described. Elution conditions at each step were as indicated. Proteins elution position of the AUL9 protein shifted to that of ICP8. were identified by SDS/PAGE followed by Coomassie blue staining. Coelution of AUL9 protein and ICP8 was confirmed by the (A) Immobilized AUL9 protein. (B) Immobilized CAT protein. Positions of ICP8, AUL9 protein, CAT, BSA, and E. coli SSB are as indicated. M, Mr standards; L, load fraction; FT, flow-through 2.0 fraction. 1.0 coincidence ofboth proteins upon SDS/PAGE. The ability of M 00. MMNoCI the two proteins to cochromatograph verifies that they as- sociate to form a tight complex. ICPS-. DISCUSSION ...~~~~~~~~~~~~~~~~~~~~~~~~~~.j ..: ...... We have investigated the physical interaction between the HSV-1 UL9 protein and ICP8 in an attempt to elucidate their mode ofaction in promoting origin-specific DNA unwinding. We had reported (9) that ICP8 can specifically stimulate the FIG. 2. Retention of ICP8 from HSV-1-infected cell extracts on DNA helicase activity ofthe UL9 protein. Here we have used UL9 protein-agarose. An HSV-1-infected cell extract was chromato- protein-affinity chromatography to show directly a tight and graphed on a HiTrap UL9 protein-affinity column, essentially as physical interaction between these two proteins. described for the purified proteins, except that buffer A was used, specific and gradient elution was followed by elution with 1 ml at 1.0 M NaCI, Purified UL9 protein was specifically retained by immobi- 1 ml at 2.0 M NaCl, and 3 ml at 0.1 M NaCl, as indicated. Column lized ICP8 and was eluted only at high ionic strength (0.6 M fractions were analyzed by SDS/PAGE, transferred to nitrocellu- NaCl). Similarly, ICP8 was specifically retained by immobi- lose, and immunoblotted with an anti-ICP8 serum. The position of lized UL9 protein. Furthermore, immobilized UL9 protein ICP8 is as indicated. M, M, markers. could retain ICP8 from an HSV-1-infected cell extract. These Downloaded by guest on October 2, 2021 8448 Biochemistry: Boehmer and Lehman Proc. Natl. Acad. Sci. USA 90 (1993) A Start ICP8 We have localized the site of interaction between the UL9 protein and ICP8 to the C-terminal 37-kDa DNA-binding domain of UL9 protein (AUL9). ICP8 was specifically re- m tained by immobilized AUL9 protein and was released only :. at high ionic strength (>0.5 M NaCI). The interaction be- iICP8 tween ICP8 and AUL9 proteins was confirmed by their * coelution upon gel filtration. The primary sequence of the UL9 protein reveals two leucine zipper motifs (21), one near the N terminus and the other closer to the C terminus, that may be involved in protein-protein interactions (22). In fact, Elias et al. (23) have shown the N-terminal portion ofUL9 protein to be required for cooperative binding to its recognition se- quences within ori. These cooperative protein-protein in- B teractions between UL9 protein monomers may be mediated through the N-terminal leucine zipper. Our findings are consistent with the possibility that the heterologous UL9 protein-ICP8 interaction is mediated through the C-terminal AUL9 leucine zipper. Further studies are needed to localize more Start precisely the site of interaction between ICP8 and the UL9 protein and to address the stoichiometry of the ICP8-UL9 protein complex...... mm Specific interactions between proteins involved in origin- specific initiation ofDNA replication are not unprecedented. For example, in simian virus 40 DNA replication, specific protein-protein interactions have been shown to exist be- tween the viral initiator protein, large tumor antigen, and the AUL9 cellular SSB RP-A (24), as well as with DNA polymerase a-primase (25). This paper is dedicated to the memory of Hatch Echols, who was among the first to point out the importance of protein-protein C interactions at a replication origin. This work was supported by Grant AI 26538 from the National Institutes of Health. 1. McGeoch, D. J. (1989) Annu. Rev. Microbiol. 43, 235-265. lCP8 + Start 2. Challberg, M. D. & Kelly, T. J. (1989) Annu. Rev. Biochem. 58, 671-717. 3. Crute, J. J., Elias, P. & Lehman, I. R. (1990) Molecular Mecha- nisms in DNA Replication and Recombination (Liss, New York), .....- pp. 327-341. 4. Kornberg, A. & Baker, T. (1992) DNA Replication (Freeman, New ICP8 York). 5. Stillman, B. (1989) Annu. Rev. Cell Biol. 5, 197-245. ii. 6. Erdile, L. F., Collins, K. L., Russo, A., Simancek, P., Small, D., Umbricht, C., Virshup, D., Cheng, L., Randall, S., Weinberg, D., Moarefi, I., Fanning, E. & Kelly, T. (1991) Cold Spring Harbor AUL9 Symp. Quant. Biol. 56, 303-313. 7. Elias, P., Gustafsson, C. M. & Hammarsten, 0. (1990) J. Biol. Chem. 265, 17167-17173. 8. Fierer, D. S. & Challberg, M. D. (1992) J. Virol. 66, 3986-3995. FIG. 4. Coelution ofICP8 and AUL9 on Superose 12. Gel 9. Boehmer, P. E., Dodson, M. S. & Lehman, I. R. (1993) J. Biol. protein Chem. 268, 1220-1225. filtration was done as described. The UV-absorbance profiles are 10. Boehmer, P. E. & Lehman, I. R. (1993) J. Virol. 67, 711-715. shown as well as Coomassie blue-stained SDS/polyacrylamide gels 11. Laemmli, U. K. (1970) Nature (London) 227, 680-685. of peak protein fractions from each chromatogram. (A) ICP8 alone. 12. Johnson, D. A., Gautsch, J. W., Sportsman, J. R. & Elder, J. H. (B) AUL9 protein alone. (C) ICP8 and AUL9 protein. The start of (1984) Gene Anal. Tech. 1, 3-8. each chromatogram is as indicated, as are the positions of ICP8 and 13. Stow, N. D. (1992) J. Gen. Virol. 73, 313-321. AUL9 protein. M, Mr standards. 14. Dodson, M. S. & Lehman, I. R. (1993) J. Biol. Chem. 268, 1213- 1219. findings suggest that the UL9 protein and ICP8 associate to 15. Summers, M. D. & Smith, G. E. (1987) A Manual ofMethodsfor a stimulation of the UL9 Baculovirus Vectors andInsect CellProcedures, Bulletin 1555 (Tex. form tight complex and that the Agric. Exp. Stn., College Station, TX). protein helicase by ICP8 results from this association. 16. Rabkin, S. D. & Hanlon, B. (1990) J. Virol. 64, 4957-4967. Previous work has shown that the UL9 protein is an 17. Post, L. E., Mackem, S. & Roizman, B. (1981) Cell 24, 556-565. origin-binding protein (2, 3, 7) as well as a DNA helicase (8, 18. Hernandez, T. R. & Lehman, I. R. (1990) J. Biol. Chem. 265, 9). These properties are well-suited to a role in unwinding the 11227-11232. 19. Boehmer, P. E. & Emmerson, P. T. (1991) Gene 102, 1-6. HSV-1 chromosome at an origin of DNA replication. ICP8, 20. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254. by virtue of its ability to destabilize duplex DNA (10) and to 21. Deb, S. & Deb, S. P. (1991) J. Virol. 65, 2829-2838. stimulate the DNA helicase activity of UL9 protein (9), 22. Alber, T. (1992) Curr. Opin. Genet. Dev. 2, 205-210. should also be suited for a role in origin activation. Our 23. Elias, P., Gustafsson, C. M., Hammarsten, 0. & Stow, N. D. (1992) J. Biol. Chem. 267, 17424-17429. present studies show that the UL9 protein and ICP8 exist as 24. Dornreiter, I., Erdile, L. F., Gilbert, I. U., von Winkler, D., Kelly, a complex that might function in activating the HSV-1 origin T. J. & Fanning, E. (1992) EMBO J. 11, 769-776. of DNA replication. 25. Smale, S. T. & Tjian, R. (1986) Mol. Cell. Biol. 6, 4077-4087. Downloaded by guest on October 2, 2021