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.