Proc. Nati. Acad. Sci. USA Vol. 86, pp. 2186-2189, April 1989 Biochemistry Herpes simplex virus 1 helicase-primase: A complex of three herpes-encoded gene products (replication origin/DNA binding protein/DNA polymerase) JAMES J. CRUTE*, TATSUYA TsURUMI*, LIANG ZHUt, SANDRA K. WELLERt, PAUL D. OLIVOt, MARK D. CHALLBERGt, EDWARD S. MOCARSKI§, AND I. R. LEHMAN* Departments of *Biochemistry and §Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305; tDepartment of Microbiology, University of Connecticut Health Center, Farmington, CT 06032; and tLaboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, 9000 Rockville Pike, Bethesda, MD 20892 Contributed by I. R. Lehman, January 3, 1989

ABSTRACT In an earlier report, we described a DNA Cells and Viruses. A305, a thymidine kinase-deficient helicase that is specifically induced upon infection ofVero cells mutant of HSV-1[F], was used to infect roller-bottle cultures with herpes simplex virus 1. We have purified this enzyme to of Vero cells at a multiplicity of infection of 5 plaque-forming near homogeneity and found it to consist of three polypeptides units per cell. with molecular weights of 120,000, 97,000, and 70,000. Im- Buffers. The buffers used were described (14) except that munochemical analysis has shown these polypeptides to be the the concentration of glycerol in buffer A was increased to products of three of the genes UL52, UL5, and UL8 that are 20% (vol/vol) and the concentrations of leupeptin and pep- required for replication of a plasmid containing a herpes statin A were increased to 10 ,ug/ml. simplex 1 origin (oris). In addition to helicase activity, the Enzymatic Assays. DNA-dependent ATPase and GTPase enzyme contains a tightly associated DNA primase. Thus, the assays were performed as described (14) except that incuba- three-subunit enzyme is a helicase-primase complex that may tion was for 20 min at 34TC. DNA primase assays were carried prime lagging-strand synthesis as it unwinds DNA at the viral out essentially as described (15). The reaction mixture, in 30 replication fork. A.l, contained 50 mM Tris HCl (pH 8.7), 3.5 mM MgCI2, 10% (vol/vol) glycerol, bovine serum albumin at 100 jig/ml, 1.0 mM ATP, 13 ,uM [3H]dATP (10 Ci/mmol; 1 Ci = 37 GBq), 40 The 153-kilobase genome of herpes simplex virus 1 (HSV-1) ,uM (dT)6", and 1.0 unit ofthe large fragment ofEscherichia contains both cis- and trans-acting elements that function in coli DNA polymerase I obtained from United States Bio- viral DNA replication (1). The cis-acting elements corre- chemical. The reaction was started by the addition of 3.0 ,ul spond to the origins of DNA replication (oris and oriL) (2-4), of enzyme solution and, after 60 min at 34°C, was stopped by and the trans-acting elements very likely code for most and the addition of 10 ,ul of 0.5 M EDTA. Incorporation of possibly all of the enzymes required for HSV-1 DNA repli- [3H]dATP into acid-insoluble material was determined as cation. The nucleotide sequence ofthe entire HSV-1 genome described (16). One unit of primase activity incorporated 1.0 has been determined (5), allowing assignment ofHSV-1 genes pmol of [3H]dATP in 60 min. Nuclease assays were per- and their products to specific open reading frames. Seven of formed as described (14). these open reading frames have been shown to be necessary Gel Electrophoresis and Immunoblot Analysis. SDS/poly- and sufficient for the replication in trans of plasmids con- acrylamide gel electrophoresis was performed and the gels taining either origin of DNA replication, oriL or oris (6). were silver-stained as described (17, 18). Immunoblot anal- These open reading frames also correspond to seven com- yses were performed (19, 20) using rabbit antiserum against plementation groups known to be essential for HSV-1 DNA the UL5 gene product expressed in E. coli (21) or rabbit replication (7-9). Of the seven open reading frames, three antisera raised against peptides whose sequence corresponds have thus far been identified and shown to encode the herpes to the predicted carboxyl-terminal 10 amino acid residues of DNA polymerase (Pol) (10), a single-stranded DNA-binding the HSV-1 UL8 or UL52 open reading frames (5, 22). protein (ICP8) (11), and the oris-binding protein (UL9) (12). Preparation of Nuclei. HSV-1-infected Vero cells were A double-stranded DNA-binding protein whose role in DNA harvested from roller bottles 14-15 hr after infection by replication is unknown is encoded by the fourth open reading vigorous shaking. The nuclei were prepared by the hypotonic frame (UL42) (13). lysis method (23), except that the nuclear wash was omitted In this report we show that the HSV-1-induced DNA and the concentrations of leupeptin and pepstatin A were helicase that we have identified (14) consists of three poly- increased to 10 ,g/ml. peptides encoded by the three remaining open reading frames Enzyme Purification. The HSV-1 DNA helicase was puri- UL5, UL8, and UL52. We have also found that a DNA fied as described (14) with several modifications. In immu- primase activity is tightly associated with the three-subunit noblotting experiments with UL5 antibody, we had found enzyme, establishing the presence of an HSV-1-encoded that the UL5 gene product corresponded to the HSV- helicase-primase complex in HSV-1-infected cells. 1-induced helicase, as assayed by its DNA-dependent ATPase (GTPase) activity (J.J.C., L.Z., S.K.W., and I.R.L., unpublished results). We also observed that the amount of MATERIALS AND METHODS DNA-dependent ATPase as well as the level of intact UL5 Materials. Reagents and substrates have been described gene product varied considerably among our nuclear prepa- (14), unless otherwise noted. (dT)6w0 was obtained from rations, very likely as a result of uncontrolled proteolysis. To Midland Certified Reagent (Midland, TX). ensure that nuclei containing undegraded helicase were used for enzyme purification, we immunoblotted small amounts (100 ,ul) of the high-salt nuclear extract as a means of The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Abbreviation: HSV-1, herpes simplex virus 1.

2186 Downloaded by guest on September 28, 2021 Biochemistry: Crute et al. Proc. Natl. Acad. Sci. USA 86 (1989) 2187 screening for full-length UL5 gene product. Of five nuclear mally active at pH 8.5-9.0 and at Mg2+ concentrations from preparations tested, two were found suitable for further 1 to 3 mM. processing; the remainder showed considerable degradation. HSV-1 Helicase-Primase Is Composed of Three Subunits. Nuclei (60 g) containing intact UL5 product were thawed and Purification of the DEAE-Sephadex fraction by filtration centrifuged, and the pellets were resuspended in an equal through Superose 12 again yielded a single peak of DNA- volume of buffer A supplemented with 90 mM NaCI. One- dependent ATPase that coincided with primase activity. The eighth volume of buffer A containing 4.0 M NaCl was added step also removed a small amount of contaminating nuclease and the nuclear suspension was centrifuged at 90,000 rpm for (Fig. 2A). In agreement with the previous report (14), the 10 min at 20C in a TLA.100.3 rotor. The supernatant solution molecular weight of the helicase-primase complex was was dialyzed against buffer A containing 50 mM NaCl until -440,000. The specific activity of the primase was 2.2 X 104 the NaCl concentration was 100 mM. The extract was units/mg and that of the DNA-dependent ATPase was 2.5 X chromatographed on phosphocellulose and DEAE-Seph- 104 units/mg. The DNA-dependent ATPase and primase adex, and a portion (40%) of the DEAE-Sephadex fraction activities also coincided with a single peak of protein as was filtered through a Superose 12 column as described (14). measured by absorbance at 280 nm. When fractions across Approximately 20 jig of near-homogeneous enzyme was the peak of helicase and primase activity were analyzed by recovered. SDS/polyacrylamide gel electrophoresis, three polypeptides with molecular weights of 120,000, 97,000, and 70,000 were RESULTS found to increase and decrease concomitant with the two enzymatic activities as well as with the peak of protein (Fig. Association of Primase Activity with HSV-1 Helicase. The 2B). As estimated by silver staining, the three polypeptides HSV-1-induced DNA helicase, assayed by its DNA- accounted for >95% of the protein in the peak. dependent ATPase (GTPase) activity, can be effectively Identification ofthe Subunits ofthe HSV-1 Helicase-Primase separated from host DNA-dependent ATPase activity by as the Products of UL52, UL5, and UL8 Genes. We sought to chromatography on phosphocellulose (14). Further purifica- identify which, if any, of the three polypeptides associated tion of the herpes enzyme by chromatography on DEAE- with the helicase-primase were encoded by the seven HSV-1 Sephadex yielded two fractions, one that failed to adsorb to genes that are required for origin-dependent DNA replica- the column and a second that did adsorb and subsequently tion. The UL52, UL5, and UL8 genes, which encode proteins could be eluted (14). There was, however, considerable with predicted molecular weights of 114,000, 99,000, and variability in the relative amounts of the two fractions, and, 80,000, respectively, were the most likely candidates (6). We indeed, in a number ofpreparations, activity was not retained therefore immunoblotted fractions across the Superose 12 by the column at all. Immunoblot analysis with antibody peak with rabbit antiserum raised against the products of directed against the UL5 product showed that the bound each ofthese genes. As shown in Fig. 3, the polypeptides with enzyme contained full-length UL5 polypeptide (Mr, 97,000). molecular weights of 120,000, 97,000, and 70,000 were, in We presume that the unbound fraction of DNA-dependent fact, recognized by the antisera against the UL52, UL5, and ATPase represents a degraded form of the enzyme. By using UL8 products. In each case, immunoreactive material (i) nuclei from HSV-1-infected cells shown by immunoblotting co-eluted closely with the peak of DNA-dependent ATPase to contain the full-length UL5 product, we have obtained and primase activity, (ii) had a molecular weight near that preparations ofDNA-dependent ATPase (GTPase) that, after predicted from the DNA sequence ofthe corresponding open phosphocellulose chromatography, bound to and was eluted reading frame, and (iii) co-eluted with the three major from DEAE-Sephadex as a single symmetrical peak (Fig. 1). polypeptides observed after SDS/polyacrylamide gel elec- Furthermore, assay of the DEAE-Sephadex fractions for trophoresis of the purified enzyme (Fig. 2B). Furthermore, DNA primase activity showed that primase coeluted per- analysis of fractions containing the HSV-1 helicase-primase fectly with the herpes-induced DNA-dependent ATPase from earlier stages in the purification (phosphocellulose or (GTPase) (Fig. 1). Primase activity was not detectable in DEAE-Sephadex) showed a coincidence of the UL52, UL5, phosphocellulose fractions of the enzyme because of high and UL8 products with enzymatic activity (data not shown). levels of nuclease that very likely masked primase activity. We conclude that the HSV-1 helicase-primase is encoded by The primase associated with the HSV-1 helicase was opti- the UL52, UL5, and UL8 genes.

O 1.20 DNA-dependent 6000 ATPase O 1.00 _ 5000 - '-Primase

- co 0.80 DNA-dependent 4000 .' GTPase E Z 0.60 - * 3000 - #1 ~~~~~~~~~~~~~@1 004 2000 .E IU & z 0.20

FRACTION NUMBER FIG. 1. DEAE-Sephadex chromatography of the HSV-1 DNA helicase. The phosphocellulose fraction was applied to and eluted from a DEAE-Sephadex column. The indicated fractions were assayed for DNA-dependent ATPase (20 ILI; o), DNA-dependent GTPase (20 pl; 0), and primase activity (3 Il; o). Downloaded by guest on September 28, 2021 2188 Biochemistry: Crute et al. Proc. Natl. Acad. Sci. USA 86 (1989) A

la (WV 0.E 0 0 x 0. -o 12 8 U C :2 E 91 0. U 6 co I- z

35 40 45 50 55 60 65 70 FRACTION NUMBER

B 4 FRACTION NUMBER 49 50 51 52 53 54 55 56 57 58 I0

_' _lu 1 214102 - - 40m em. __ 97 i:owI. 4.0Iasosun _mb _P _ 70

DF

FIG. 2. Superose 12 gel filtration of the HSV-1 helicase-primase. The DEAE-Sephadex fraction was filtered through a Superose 12 gel filtration column. (A) The indicated fractions were assayed for DNA-dependent ATPase (5 IdL; o), primase (3 1I; e), and nuclease activity (5 plI; o). The positions of elution of proteins of known molecular weights are indicated: thyroglobulin (670,000), ferritin (440,000), aldolase (160,000), bovine serum albumin (67,000), and ovalbumin (43,000). Blue dextran was used to determine the excluded volume of the column. (B) SDS/polyacrylamide gel electrophoresis of 20 ,ul of Superose fractions. Polypeptides were identified by silver staining. The molecular weight (X 10-3) of each major polypeptide is indicated. DF, dye front. Fraction 53 contains the peak of helicase and primase activity. DISCUSSION also differ in their molecular weights: 440,000 for the heli- The DNA helicase induced upon HSV-1 infection of Vero case-primase described here, compared with 40,000 for the cells (14) has been purified to near homogeneity and found to enzyme reported by Holmes et al. (24). The relationship, if consist of three subunits with molecular weights of 120,000, any, between the two enzymes remains to be determined. 97,000, and 70,000. Immunochemical analysis of the enzyme The specific activity ofthe primase associated with the HSV- has shown these three subunits to be the products of the 1-induced helicase (2.2 x 104 units/mg) is considerably lower HSV-1 UL52, UL5, and UL8 open reading frames (5, 22). than that reported for the corresponding enzyme from cultured A DNA primase activity copurifies with the HSV-1 heli- mammalian cells [5 x 105 units/mg (25)]. We do not know the case through the final two chromatographic steps in the reason for this difference; however, it may reflect differences in purification and is clearly a component of the three-subunit the two enzymes that will become clear upon further investi- enzyme. We could not detect primase activity at the early gation of the herpes-induced helicase-primase complex. stages of purification (i.e., phosphocellulose chromatogra- The association of primase with helicase in HSV-1 has two phy), where contaminating nucleases very likely masked well-documented precedents among the prokaryotic viruses. activity by degrading DNA substrates and products. Thus, the products ofbacteriophage T4 genes 41 and 61 form Holmes et al. (24) have identified a herpes-induced DNA a complex with helicase and primase activities (26), and the primase using the coupled primase-polymerase assay de- T7 gene 4 product is both a helicase and primase (27). The scribed here. The two enzymes are similar in their alkaline association of these two activities is obviously a useful pH optimum. However, they do differ in their Mg2+ optima; arrangement that permits close coordination ofthe priming of the primase activity associated with the DNA helicase is lagging strand synthesis with unwinding of the DNA duplex optimally active at Mg2+ concentrations from 1 to 3 mM and at the replication fork. is essentially inactive at 8 mM Mg2+, the concentration used Although primase and helicase activities are both associ- by Holmes et al. (24) to assay their enzyme. The two enzymes ated with the three-subunit enzyme, we cannot at present Downloaded by guest on September 28, 2021 Biochemistry: Crute et al. Proc. Natl. Acad. Sci. USA 86 (1989) 2189 A FRACTION f zyme or by purification of the individual UL5, UL8, and NUMBER 49 50 51 52 53 54 55 56 57 58 UL52 gene products from cells in which they have been overexpressed, followed by the appropriate reconstitution experiments, should provide an answer to this question. With the finding that UL5, UL8, and UL52 code for a helicase-primase complex and the identification of the UL9 ANTI-U L52 l l~120 gene product as the oris binding protein (12), all of the seven HSV-1-encoded gene products required for HSV-1 DNA replication have been identified and obtained in near homo- geneous form. It remains now to be determined whether they can, in the proper combination and under the appropriate conditions, catalyze the origin-specific replication of HSV-1 DNA. We thank Gloria Chui for technical assistance in the early phases of this work. This research was supported by Grants A126538 and GM06196 to I.R.L., A120211 to E.S.M., and A121747 to S.K.W. from -OF the National Institutes of Health. S.K.W. is the recipient of an American Cancer Society Junior Faculty Research Award. J.J.C. is B FRACTION f a Postdoctoral Fellow of the California Division of the American NUMBER 49 50 51 52 53 54 55 56 57 58 Cancer Society. 1. Roizman, B. & Batterson, W. (1985) in Virology, ed. Fields, B. N. (Raven, New York), pp. 497-526. 2. Spaete, R. R. & Frenkel, N. (1982) Cell 30, 295-304. 3. Stowe, N. D. (1982) EMBO J. 1, 863-867. 4. Weller, S. K., Spadaro, A., Schaffer, J. E., Murray, A. W., ANTI-U L5 Maxam, A. M. & Schaffer, P. A. (1985) Mol. Cell. Biol. 5,930- 942. 5. McGeoch, D. J., Dalrymple, M. A., Davison, A. J., Dolan, A., Frame, M. C., McNab, D., Perry, L. J., Scott, J. E. & Taylor, P. (1988) J. Gen. Virol. 63, 1531-1574. 6. Wu, C. A., Nelson, N. J., McGeoch, D. J. & Challberg, M. D. (1988) J. Virol. 62, 435-443. 7. Schaffer, P. A., Aron, G. M., Biswal, N. & Benyesh-Melnick, M. (1973) Virology 52, 57-71. 8. Weller, S. K., Aschman, D. P., Sacks, W. R., Coen, D. M. & D F Schaffer, P. A. (1983) Virology 130, 290-305. 9. Marchetti, M. E., Smith, C. A. & Schaffer, P. A. (1988) J. C FRACTION Virol. 62, 715-721. NUMBER 49 50 51 52 53 54 55 56 57 58 10. Purifoy, D. J. M., Lewis, R. B. & Powell, K. L. (1977) Nature (London) 269, 621-623. 11. Weller, S. K., Lee, K. J., Sabourin, D. J. & Schaffer, P. A. (1983) J. Virol. 45, 354-366. 12. Olivo, P. D., Nelson, N. J. & Challberg, M. D. (1988) Proc. Natl. Acad. Sci. USA 85, 5414-5418. 13. Gallo, M. L., Jackwood, D. H., Murphy, M., Marsden, H. S. ANTI-U L8 -I 70 & Parris, D. S. (1988) J. Virol. 62, 2874-2883. 14. Crute, J. J., Mocarski, E. S. & Lehman, I. R. (1988) Nucleic Acids Res. 16, 6585-6596. 15. Conaway, R. C. & Lehman, I. R. (1982) Proc. Natl. Acad. Sci. USA 79, 2523-2527. 16. Crute, J. J., Wahl, A. F. & Bambara, R. A. (1986) Biochem- istry 25, 26-36. 17. Laemmli, U. K. (1970) Nature (London) 226, 680-685. 18. Marshall, T. (1984) Anal. Biochem. 136, 340-346. 19. Tijssen, P. & Kurstak, E. (1984) Anal. Biochem. 136, 451-457. 20. Knecht, D. A. & Dimond, R. L. (1984) Anal. Biochem. 136, 180-184. EDF 21. Zhu, L. & Weller, S. K. (1988) Virology 166, 366-378. 22. Olivo, P. D., Nelson, N. J. & Challberg, M. D. (1989) J. Virol. FIG. 3. Immunological analysis of Superose 12 fractions. The 63, 196-204. Superose 12 fractions shown in Fig. 2B were immunoblotted. The 23. Elias, P., O'Donnell, M. E., Mocarski, E. S. & Lehman, I. R. antisera and dilution used were anti-UL52 (1:50 dilution) (A), (1986) Proc. Natl. Acad. Sci. USA 83, 6322-6326. anti-UL5 (1:50 dilution) (B), and anti-UL8 (1:25 dilution) (C). The 24. Holmes, A. M., Weitstock, S. M. & Ruyechan, W. T. (1988) J. signal was enhanced in C by blocking the nitrocellulose membrane Virol. 62, 1038-1045. after the transfer step at 65°C for 2 hr prior to the addition of the 25. Wang, T. S.-F., Hu, S.-Z. & Korn, D. (1984) J. Biol. Chem. anti-UL8 antiserum (20). The molecular weight (x 10-3) ofthe major 259, 1854-1865. immunoreactive polypeptide is indicated. Arrow denotes peak of 26. Nossal, N. G. & Alberts, B. M. (1983) in Bacteriophage T4, helicase and primase activities. DF, dye front. eds. Mathews, C. K., Kutter, E., Mosig, G. & Berget, P. (Am. Soc. Microbiol., Washington, DC), pp. 71-81. assign these activities to specific subunits. Isolation of the 27. Nakai, H. & Richardson, C. C. (1988) J. Biol. Chem. 263, 9818- individual subunits either by dissociation of the intact en- 9830. Downloaded by guest on September 28, 2021