THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 265, No. 19, Issue of July 5, pp. 112!27-11232,199O 0 1990 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S. A.

Functional Interaction between the Herpes Simplex- 1 DNA Polymerase and UL42 Protein*

(Received for publication, October 30, 1989)

Thomas R. HernandezS and I. R. Lehman From the Department of Biochemistry, Beckman Center, Stanford University School of Medicine, Stanford, California 94305-5307

The herpes simplex virus 1 (HSV-1) UL42 protein, one of seven herpes-encoded polypeptides that are re- quired for the replication of the HSV-1 genome, is found in a 1:l complex with the HSV-1 DNA polym- erase (Crute, J. J., and Lehman, I. R. (1989) J. Biol. Chem. 264, 19266-19270). To obtain herpes DNA polymerase free of UL42 protein, we have cloned and overexpressed the Pol gene in a recombinant baculo- virus vector and purified the recombinant DNA polym- erase to near homogeneity. Replication of singly primed M13mp18 single-stranded DNA by the recom- binant enzyme in the presence of the herpes encoded single-stranded DNA-binding protein ICPS yields in addition to some full-length product a distribution of intermediate length products by a quasi-processive mode of deoxynucleotide polymerization. Addition of the purified UL42 protein results in completely pro- cessive polymerization and the generation of full- length products. Similar processivity is observed with the HSV-1 DNA polymerase purified from herpes-in- fected Vero cells. Processive DNA replication by the DNA polymerase isolated from HSV-l-infected Vero cells or the recombinant DNA polymerase-UL42 pro- tein complex requires that the single-stranded DNA be coated with saturating levels of ICPS. ICPS which binds single-stranded DNA in a highly cooperative manner is presumably required to melt out regions of secondary structure in the single-stranded DNA tem- plate, thereby potentiating the processivity enhancing action of the UL42 protein.

The genome of herpes simplex virus type 1 (HSV-1)’ con- sists of a linear duplex DNA, 153 kilobases in length (1). Of the approximately 15 genes that have been identified in the genome, seven have been shown to be both necessary and sufficient to promote replication of the HSV-1 genome (2-4). The products of the seven genes include a DNA polymerase, a single-stranded DNA binding protein (ICP8) (5-8), an origin binding protein (9, lo), the three subunits of a DNA helicase- primase (11, 12), and the UL42 protein, initially identified as a double-stranded DNA-binding protein (13-15). The 62-kDa

* This research was supported by Grant AI26538 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Supported by Medical Scientist Training Grant GM07365 from the National Institute of General Medical Sciences.

’ The abbreviations used are: HSV-1, herpes simplex virus, type 1; ssDNA, single-stranded DNA; SDS, sodium dodecyl sulfate; EGTA, [ethylenebis (oxyethylenenitrilo)]tetraacetic acid; HEPES, 4-(Z-hy- droxyethyl)-1-piperazineethanesulforic acid; TEMED, N, N, N’, N’- tetramethylethylenediamine.

UL42 protein copurifies with the HSV-1 DNA polymerase (X), and the HSV-2 analogue of the HSV-1 UL42 protein (lCSP34/35) exists in close association with the HSV-2 DNA polymerase (16, 17). More recently, a highly purified prepa- ration of the HSV-1 DNA polymerase was shown to consist of the UL42 protein in a 1:l complex with the 136-kDa polymerase polypeptide (18).

We report here the construction of a recombinant baculo- virus containing the HSV-1 DNA polymerase gene and its overexpression in an insect cell line. Interaction of the bacu- lovirus-expressed DNA polymerase with the purified UL42 protein results in a large increase in the processivity of deox- ynucleotide polymerization. The increase in processivity is, however, observed only when the ssDNA template is com- plexed with ICP8. Thus, the UL42 protein appears to be a polymerase accessory factor that interacts functionally with the herpes DNA polymerase to enhance its processivity in a manner analogous to the fl subunit of the Escherichia coli DNA polymerase III holoenzyme (19-22).

EXPERIMENTAL PROCEDURES

Materiuki-All reagents were obtained from Sigma unless other- wise noted. Unlabeled deoxynucleoside 5’.triphosphates were from Pharmacia LKB Biotechnology Inc. [a-“*P]dTTP (80 Ci/mmol) was from Amersham Corp. Molecular weight markers, TEMED, and ammonium persulfate were from Bio-Rad. Acrylamide and bis-acryl- amide for SDS-polyacrylamide gel electrophoresis and prestained molecular weight markers (high range) were from Bethesda Research Laboratories. Anti-rabbit IgG (H+L) conjugated to horseradish per- oxidase was from ICN Immunobiologicals. The Sequenase dideoxy sequencing kit was from the U. S. Biochemical Corp. The reverse sequencing primer for M13mp18/19 was from New England Biolabs. E. coli DNA polymerase III* was a gift from Z. Tsuchihashi (Stan- ford), the p subunit was from P. Burgers (Washington University), and E. coli SSB was a gift from Z. Tsuchihashi and H. Chiang. Primer 1, 5’-GTT TTC CCA GTC ACG AC-3’, complementary to residues 6291-6307 of M13mp18, was synthesized and purified as described (12). Primer 2,5’-CGG AAA ACA TAT GGG TTG TTC CC-3’, was synthesized by Operon Technologies, Inc. M13mp18 and M13mp19 RF1 and single-stranded viral forms were prepared as described (23). The template for DNA polymerase reactions was prepared by using a lo-fold molar excess of primer 1 to viral M13mp18 ssDNA in 50 mM Tris-Cl, pH 8.0, 0.1 M NaCl. The solution was heated at 95 “C for 3 min, cooled to 80 “C and then allowed to cool to 42 “C over a period of 30 min. It was then incubated further at 37 “C for 45 min.

Antisera-Antiserum to the HSV-1 DNA polymerase was a gift from Donald Coen (Harvard University); it was produced against a P-galactosidase/HSV-1 DNA polymerase fusion protein (C-terminal domain). Antiserum to the UL42 protein was a gift from Deborah Parris (Ohio State University) or was generated against the purified UL42 protein in the following manner. The UL42 protein was sub- jected to 10% SDS-polyacrylamide gel electrophoresis (24), and the gel soaked in ice-cold 0.25 M KC1 until the SDS in the protein bands was detectable as a white precipitate (25). The UL42 protein band was excised from the gel, and the gel slices were emulsified and used as antigen. An initial injection using 75 pg of UL42 was performed on a virgin female New Zealand rabbit and three subsequent boosts,

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75 pg each, were given at 3-week intervals. Buffers-Buffer A is 20 mM HEPES-Na+, pH 7.6, 150 mM NaCl.

and 0.5 mM dithiothreitol. Buffer B is 20 rni HEPES-Na+, pH 7.6, 0.5 mM dithiothreitol. 10 mM NaHSOs. DH 7.0. 0.5 mM uhenvlmeth- ylsulfonyl fluoride, 2 ig/ml leupeptin, and 2 fig/ml pepstaiin A Buffer C is buffer B containing 1 mM EDTA, 1 mM EGTA, and 10% glycerol, but without NaHS03. Buffer D is 20 mM HEPES-Na+, pH 7.6,2 mM MgC12, 4% glycerol, 40 pg/ml bovine serum albumin, 1 mM dithio- threitol, and 0.1 mM EDTA.

Cells and Viruses-RA305 (26), a thymidine kinase deletion mutant of HSV-1 (strain F), was used to infect roller-bottle cultures of Vero cells or U35 cells (an ICPB overproducing cell line, kindly provided by Priscilla Schaffer) at a multiplicity of infection of 5-10. Spodoptera frugiperda cells (Sf9 cells), kindly provided by M. Summers (Texas A and M), were grown at 27 “C in Grace’s medium (Hazelton) supple- mented with 0.33% TC Yeastolate (Difco), 0.33% la&albumin hy- drolysate (Difco), and 10% fetal bovine serum (Irvine Scientific) as described (27). Wild-type and recombinant Autogrupha californica nuclear polyhedrosis viruses (AcMNPV), a gift of M. Summers, were maintained in St9 cells (27).

Polyacrylumide Gel Electrophoresis and Protein Determination- SDS-polyacrylamide gel electrophoresis was performed as described (24). Gels were fixed and stained with silver (28) or Coomassie Blue. Immunoblotting was performed as described (29). Protein concentra- tions were determined by the Bradford method with bovine serum albumin (Miles Scientific, fraction V) as a standard (30).

Enzyme Assays-DNA polymerase and DNase activities were as- sayed as previously described (12, 18). RNase H activity was assayed using the (dT)6,. [3H]oligo(rA) substrate (18). Measurements of the effect of ICP8 on DNA polymerase activity were performed in buffer D (50 ~1) containing 125 GM NaCI, 60 & dATP, dGTP, dCTP, 25 uM [cu-32PldTTP. 4 UM (in nucleotide) viral M13mn18 ssDNA hvbrid- ized to primer 1 ‘and 15 ng of HSV-1 DNA polymerase. ICP8”(3 ~1) was added, and the reaction mixtures were preincubated on ice for 5 min. Reactions were initiated by bringing the reaction mixtures to 37 “C. After 50 min, 15-pl aliquots were removed and quenched in 25 ~1 of 40 mM EDTA. The DNA was precipitated and filtered on GFC filters (Whatman). For measurement of the time course of DNA polymerase activity, 15-~1 aliquots were removed at lo-, 30., and 50- min intervals and processed as above.

Measurements of the effect of UL42 protein on DNA polymerase activity were performed under the same conditions used to determine the effect of ICP8, except that the reaction volume was 60 ~1, and 15 ng of HSV-1 DNA polymerase from infected Vero cells or the recom- binant enzyme was used. After addition of ICPS or buffer C containing 200 mM NaCl, the reaction mixtures were preincubated on ice for 3- 5 min. Five ~1 of UL42 protein was added, and preincubation was continued for an additional 4 min. Reactions were initiated by bring- ing the reaction mixtures to 37 “C. After 50 min, 15-~1 aliquots were removed and quenched in 25 ~1 of 40 mM EDTA. The DNA was precipitated and filtered on GFC filters as above. When the products were analyzed by neutral agarose gel electrophoresis, 10-J aliquots were removed after quenching with EDTA, and 3 ~1 of sample buffer (0.18% bromphenol blue, 22% glycerol, and 5% SDS) was added. Reactions were subjected to 1% agarose gel electrophoresis as de- scribed (31). The gels were dried on Whatman chromatography paper under vacuum, and the dried gels were exposed to Kodak XAR x-ray film using an intensifying screen at -80 “C.

Purification of the HSV DNA Polymeruse, ICP8, and lJL.42 Pro- tein-The DNA polymerase, ICP8, and the UL42 protein were puri- fled from HSV-1 infected Vero cells by the following procedure. Nuclei from 60 g (wet weight) of cells were extracted with an equal volume of buffer C containing 3.4 M NaCl as previously described (9, 32). After dialysis against buffer C containing O.lO- M NaCl, the cleared lysate was applied to a phosphocellulose column (30 ml) equilibrated with buffer C containing 0.10 M NaCl. A linear gradient (360 ml) was applied from 0.10 to 0.80 M NaCl in buffer C. The fractions (7 ml) were analyzed by 10% SDS-polyacrylamide gel elec- trophoresis followed by staining with Coomassie Blue to identify ICPS and the UL42 protein. The UL42 protein was also monitored by immunoblotting of the fractions following electrophoresis with antiserum to UL42 protein. ICPS the UL42 protein, and DNA polymerase eluted at 6.15, 0.24, and 0.30 M NaCl, respectively. The HSV-1 DNA polymerase was further purified as described previously (18, 32). ICP8 was purified as previously described (33) except that gel filtration on Superose 12 rather than glycerol gradient sedimen- tation was used as the final step. ICP8 was also purified by this procedure from the ICPB overproducing cell line U35.

Further purification of the UL42 protein was accomplished as follows. After phosphocellulose chromatography, the peak fractions containing UL42 protein were pooled and dialyzed against buffer C containing 0.09 M NaCl. The protein was then applied to a heparin- agarose column (7-ml bed volume) equilibrated with buffer C con- taining 0.10 M NaCl, and a linear gradient (90 ml) from 0.10 to 0.60 M NaCl in buffer C was applied. The fractions (1.8 ml) containing the peak of UL42 protein, which eluted at 0.30 M NaCl, were pooled and dialyzed against buffer C containing 150 mM ammonium sulfate. The dialyzed protein was concentrated to 250 ~1 on a Centricon 30 Microconcentrator (Amicon), and applied to a prepacked bed of Superose 12 (25 ml) equilibrated in buffer C containing 150 mM ammonium sulfate. The column was eluted at a flow rate of 0.20 ml/ min, and 0.2-ml fractions were collected. SDS-polyacrylamide gel electrophoresis of the Superose 12 fractions showed that the peak of protein visualized by silver staining was coincident with the peak of UL42 protein as judged by immunoblotting with UL42 antiserum (Fig. 1).

Cloning of HSV-1 DNA Polymerase Gene-Plasmid pDP1 contain- ing the HSV-1 (strain KOS) DNA polymerase open reading frame (a gift from D. Coen, Harvard University) was used to prepare a recom- binant plasmid that would permit overexpression of the HSV-1 DNA polymerase. A unique restriction site for NdeI was introduced into the region overlapping the first codon of the polymerase open reading frame by site-directed mutagenesis. Introduction of the restriction site permitted cleavage of the open reading frame so as to leave a minimal untranslated 5’ sequence while maintaining the entire HSV- 1 DNA polymerase gene intact.

The oolvmerase eerie was excised from DDP~ as a Hind111 to XbaI fragment and intriduced into M13mplSARFI at the polylinker by standard methods (31). The resulting construct, M13mp19/UL30, was used to prepare M13mp19 ssDNA according to the method of Kunkel (34). Site-directed mutagenesis was performed using primer 2 which contains three consecutive mismatches (34). A clone, M13mn19/UL30.1. was identified that contained the desired muta- tions. Although this clone contained the 5’ portion of the polymerase gene, the 3’ portion had been deleted as determined by restriction analysis. It was therefore sequenced using the reverse sequencing primer for M13mp18/19 (New England Biolabs) and the Sequenase dideoxy sequencing method to ensure that only the desired mutations had been introduced. This construct was found to contain the first 220 bases of the DNA polymerase open reading frame; the remainder of the polymerase sequence had been deleted. M13mp19/UL30.1 did, however, contain the desired base substitutions (producing an NdeI restriction site) and no other mutations. To generate the full-length gene. the DNA nolvmerase sequence from M13mp19/UL30.1 was excised and ligated to the remainder of the open read&g frame excised from pDP1. This fragment was introduced into pT7-7 (a gift from S. Tabor, Harvard University), generating the plasmid pTH1. pTH1 was constructed for expression of the HSV-1 DNA polymerase in E. coli driven by the T7 RNA polymerase as described (35). However, overexpression of the HSV DNA polymerase using this system was unsuccessful. The HSV-1 DNA polymerase gene was therefore intro- duced into the baculovirus high expression vector, pVL941 (a gift from M. Summers). The DNA polymerase open reading frame was excised from pTH1 at the NdeI and the XbuI sites; BumHI linkers were added, and the fragment was inserted into pVL941 at the unique BumHI-cloning site. Competent E. coli HBlOl cells were transformed with this construct by the CaCh method (31). The resulting plasmid, pVL941/UL30, was isolated and used to generate the recombinant virus AcMNPV/UL30 containing the HSV-1 DNA polymerase gene (27).

Overexpression of the Recombinant DNA Polymcraae-Tweiity ~50- cm2 flasks of Sf9 cells at 75% confluence were infected witl: Ac- MNPV/ULSO at a multiplicity of infection of 5. The infected cells were incubated at 27 “C for 60 h. After dislodging the cells from the flasks, the cells were chilled on ice, then centrifuged at 1400 X g for 20 min at 4 ‘C. The cells were washed with 50 ml of buffer A at 4 “C and centrifuged at 1400 x g for 10 min. The cells were resuspended in 40 ml of buffer B, lysed in a Dounce homogenizer with 10 strokes of a tight-fitting pestle, and centrifuged as above. The supernatant fluid was removed, and the pelleted nuclear fraction was resuspended in 10 ml of buffer B containing 10% sucrose. It was frozen in liquid nitrogen and stored at -80 “C.

Purification of the Recombinant HSV-1 DNA Polymerase-The recombinant DNA polymerase was purified by the same procedure used to purify the DNA polymerase expressed in HSV-l-infected Vero cells. The chromatographic behavior on phosphocellulose was

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1 L 10 11 12 13 14 15 16 17 18 19 20 22 23 24 25

1164

97+

4 L 101112 131415161716192022 232425

2044

103-+

67--W II

42-+

c

.

284

rn8mmme---

DF-b-em DF-+

FIG. 1. Superose 12 gel filtration of UL42 protein. A, aliquots (1 ~1) of the Superose 12 fractions were subjected to 10% SDS-polyacrylamide gel electrophoresis and silver stained. Lane L is the heparin-agarose fraction applied to the Superose 12 column (load) (0.5 ~1). DF is the dye front. The positions of the molecular weight standards (M, x lo-,‘) are indicated. The nrro~l denotes the peak of UL42 protein. B, immunological analysis of the Superose 12 fraction using polyclonal antiserum as described under “Experimental Procedures.” Aliquots (1 11) of the fractions were analyzed as above. The molecular weight standards (M, x lo-“) are indicated. The nrrou denotes the peak fractron

the same; however, the recombtnant enzyme eluted from the heparin- agarose column at a lower rontc strength than Vero cell enzyme (0.33 L’~TSU.~ 0.43 M NaCl). The molecular weight of the recombinant DNA polymerase as judged by Superose 12 gel filtration was 128,000, m contrast to the DNA polymerase purified from HSV-1 infected Vero cells whose molecular weight was 192,000. The latter consists of the DNA polymerase pobpepttde tn a 1.1 complex with the 62-kDa UL42 protem (18)

As shown in Ftg. 2A, the DNA polymerase and RNase H activtties of the recombtnant enzyme coeluted during Superose 12 gel filtration. Thetr specific activrties were tdentmal to those of the DNA poly- merase purtfied from HSV-1 mfected Vero cells. Analysis of the Superose 12 fractions by SDS-polyacrylamide gel electrophoresis followed by silver staimng showed them to consist of two closely spaced polypeptides with molecular weights of 133,000 and 126,000 (FIN. 2H). In contrast, the DNA polymerase purified from HSV-l- infected Vero cells contained the UL42 protem m addrtion to the doublet of polymerase polypeptrdes (data not shown). When the Superose 12 fracttons of the recombinant DNA polymerase were subjected to SDS-polyacrylamtde gel electrophoresis, then rmmuno- blotted with HSV-1 DNA polymerase antiserum, two bands that comctded wtth the DNA polymerase activity appeared wrth molecular weights Identical to those observed following silver staining of the gel (Ftg. :i). One or both of these bands may represent a parttally degraded or otherwise modified form of the DNA polymerase pal.ypeptide.

RESULTS

Stimulation of HSV-1 DNA Polymerase by ICPR-ICPS binds tightly and cooperatively to ssDNA (36, 37). Although its exact role in herpes DNA replication is still uncertain, its cellular abundance and its mode of binding to ssDNA make it likely that ICPS is functionally analogous to the E. coli SSB and the T4 gene 32 proteins, both of which bind to the ssDNA segments formed transiently at a replication fork during DNA replication (38-41).

In earlier experiments we had observed that replication of

singly primed @X174 ssDNA by the DNA polymerase purified from HSV-l-infected Vero cells was inhibited by ICPS; inhi- bition was maximal at a ratio of 1 ICPS to 12 nucleotides of ssDNA, a value that corresponded closely to saturation of the ssDNA with ICPS as determined in nuclease protection stud- ies (33). We have found that this inhibition is characteristic of ICPS preparations purified by the procedure that we had described earlier (33) which involves chromatography of nu- clear extracts of HSV-l-infected Vero cells on phosphocellu- lose and heparin-agarose. Gel filtration of these preparations through Superose 12 yielded ICP8 preparations that no longer inhibited the action of the herpes DNA polymerase on singly primed templates (data not shown). Presumably an inhibitor was present in the earlier ICPS preparations that was removed by gel filtration. ICPS purified from HSV-l-infected Vero cells or from the U35 cell line by our current procedure (see “Experimental Procedures”) showed no inhibition and, in fact, stimulated replication significantly.

Maximal stimulation by ICPS of the replication of primed ssDNA by the DNA polymerase isolated from HSV-l-infected Vero cells occurred at pH 7.5 in the presence of 2 mM MgClr and 125 mM NaCl (data not shown). The level of ICPS that gave optimal stimulation corresponded to a weight ratio of ICP8 to ssDNA of 18:l (an approximate stoichiometry of 1 ICP8 to 22 ssDNA nucleotides) (Fig. 4A), in agreement with the stoichiometry of 1 ICPS to 20 ssDNA nucleotides deter- mined by nuclease protection (data not shown). At this level of ICPS the reaction was linear for up to 50 min (Fig. 4B).

As shown in Fig. 4A, stimulation of DNA polymerase by ICP8 is highly cooperative, consistent with the cooperativity of binding of ICPS to ssDNA (36). The level of stimulation observed with ICPS (5.fold) was still below the IO-fold stim-

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- 80

- 60

6 C L 4749515355575961 6365

204 +

103+

67-b

42+

28-w

C 47 49 5153 55 57 5961 6365

18-b

FIG. 3. Immunological analysis of Superose 12 fractions. The fractions shown in Fig. 2B were immunoblotted as described under “Experimental Procedures.” Two-p1 aliquots of the Superose 12 fraction were used as described in Fig. 2B. Lone C is the Vero cell HSV-1 DNA polymerase (4 ~1). The molecular weight standards 04, x lo-“) are indicated. The arrow denotes the peak of DNA polymerase and RNase H activities.

DF + ---

FIG. 2. Coelution of DNA polymerase and RNase H activi- ties of the recombinant HSV-1 DNA polymerase during Su- perose 12 gel filtration. A, The indicated fractions were assayed for DNA polymerase (W) and RNase H activity (0) as described under “Experimental Procedures.” The results are expressed as pmoles of nucleotide incorporated or released per microliter of fraction in 20 min at 34 “C. B, aliquots (2 ~1) of Superose 12 gel fractions were subjected to 10% SDS-polyacrylamide gel electrophoresis and stained with silver. Lane C is the Vero cell HSV-1 DNA polymerase (4 ~1) purified through the Superose 12 step. Lane L is the heparin-agarose fraction (0.5 ~1) of the recombinant HSV-1 DNA polymerase that was applied to the Superose 12 column (load). DF is the dye front. The positions of the molecular weight standards (M, X iOW3) are indicated. The arrow denotes the peak of DNA polymerase and RNase H activities.

ulation seen at saturating concentration of E. coli SSB (data not shown; see also Ref. 33). Maximal stimulation occurred over a narrow range of ICP8 concentrations; amounts in excess of this range were no longer stimulatory. Primer dis- placement cannot account for the lack of stimulation since a “‘P-labeled primer remained associated with the ssDNA tem- plate at these ICP8 concentrations. Possibly the effect is related to the tendency of ICP8 to polymerize into long filamentous structures (33).

Interaction of HSV-1 DNA Polymerase with UU2 Pro- tein-The DNA polymerase used in the experiments de- scribed thus far was the enzyme purified from HEW-l-infected Vero cells. This enzyme consists of two polypeptides, the products of the HSV-1 Poland UL42 genes (18). Investigation

of the influence of the UL42 protein on the DNA polymerase polypeptide required that it be obtained free of the UL42 protein. To accomplish this, the Pol gene was cloned in a baculovirus recombinant plasmid and overexpressed in Sf9 insect cells. The purified recombinant DNA poly- merase possessed RNase H activity (Fig. 2A), providing ad- ditional evidence that the RNase H activity of the herpes DNA polymerase is an intrinsic feature of the polymerase polypeptide (18).

Addition of UL42 protein to the recombinant DNA polym- erase resulted in an approximately 2-fold increase in the rate of DNA synthesis measured with the singly primed M13mp18 ssDNA template (Fig. 5). Maximal stimulation occurred at a molar ratio of DNA polymerase/UL42 protein of 1.6:1 and required that the ssDNA be coated with saturating levels of ICP8. There was no effect of added UL42 protein on the DNA polymerase isolated from HSV-l-infected Vero cells. The specific activity of the latter enzyme appears to be approxi- mately half that of the 1:l complex of recombinant DNA polymerase and UL42 protein. The reason for this difference is not clear and may relate to the presence of some inactive DNA polymerase in the preparation of Vero cell enzyme.

The increased rate of DNA synthesis observed upon addi- tion of UL42 protein to the recombinant DNA polymerase in the presence of ICP8 was due to the greatly increased proc- essivity of deoxynucleotide polymerization (Fig. 6B). Al- though some full-length product was observed after 50 min of incubation in the absence of the UL42 protein, a large pro- portion appeared as chains of variable length. In contrast, in the presence of even substoichiometric levels of UL42 protein

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oz 0.00 0.01 0.02 0.03 0.04 0.05 0.06 0.07

0.0 1.0 2.0 3.0

mol UL42 protein/m01 Pol mol ICPBlmol nucleotide

Time (min)

FIG. 4. Effect of ICPS on replication of primed ssDNA by herpes DNA polymerase. A, assays of labeled deoxynucleoside triphosphate incorporation were performed as described under “Ex- perimental Procedures.” Reaction mixtures contained 15 ng of HSV- 1 DNA polymerase, 66 ng of primed M13mp18 ssDNA, and the indicated amounts of ICPS, incubation was for 50 min at 37 “C. B, Time course of DNA polymerase activity with primed M13mp18 ssDNA in the presence (1.2 fig) (0) or absence of ICP8 (0). Samples were removed from the reaction mixture at 10, 30, and 50 min and acid-insoluble deoxynucleoside triphosphate incorporation was deter- mined.

(0.3 mol/mol of DNA polymerase), most of the product was in the form of RF11 (Fig. 6B). However, at a molar ratio of 0.1 only 10% of the product appeared as RF11 (data not shown). ICP8 was essential for this effect. Even at the highest level of UL42 protein added, only small amounts of RF11 could be detected in the absence of ICP8 (Fig. 6A). Similar results were observed in samples taken after 10 min of incu- bation (data not shown).

In the case of the DNA polymerase purified from HSV-l- infected Vero cells, processive DNA synthesis was observed even in the absence of added UL42 protein and there was no effect of additional UL42 protein. Like the recombinant DNA polymerase-UL42 protein complex, the processivity of the Vero cell enzyme was dependent upon the presence of ICP8 (Fig. 6).

DISCUSSION

The HSV-1 DNA polymerase is a heterodimer consisting of a trifunctional136-kDa polypeptide with DNA polymerase, 3’-&’ exonuclease and 5’+3’ RNase H activities and the 62-

FIG. 5. Stimulation of the recombinant herpes DNA polym- erase by UL42 protein. Reaction mixtures (60 ~1) contained 15 ng of Vero cell HSV-1 DNA polymerase (circles) or 15 ng recombinant HSV-1 DNA polymerase (squares), 1.4 pg of ICP8, 80 ng of primed M13mp18 ssDNA, and the indicated amounts of LJL42 protein; in- cubation was for 50 min at 37 “C. Acid-insoluble deoxynucleoside triphosphate incorporation was determined as described under “Ex- perimental Procedures.” Closed symbols represent reactions per- formed in the presence of ICP8; open symbols are reactions in the absence of ICP8.

kDa product of the UL42 gene (14,18). We have shown here that the UL42 protein serves to enhance the processivity of deoxynucleotide polymerization by the polymerase subunit. It therefore resembles the /3 subunit of the E. coli DNA polym- erase III holoenzyme (19-22). Unlike the latter which is easily dissociated from the polymerase, the UL42 protein binds very tightly to the polymerase subunit, and we were unable to dissociate the complex except under conditions that lead to denaturation of the protein.’

To obtain the polymerase subunit free of the UL42 protein, we cloned and overexpressed the Pol gene in the baculovirus- insect cell system and obtained near homogeneous enzyme. The DNA polymerase and RNase H activities of the recom- binant DNA polymerase appear to be identical to the enzyme purified from HSV-infected Vero cells (18, 42, 43). Further- more, the recombinant DNA polymerase reacts with antibod- ies raised against the Vero cell enzyme and has the same molecular weight as the Vero cell polymerase polypeptide as judged by SDS-polyacrylamide gel electrophoresis.

Similarly, the 1:l complex of the recombinant polymerase polypeptide and UL42 protein show the same high degree of processivity as the enzyme isolated from HSV-l-infected Vero cells, suggesting strongly that the UL42 protein is interacting directly with the recombinant DNA polymerase. As expected, addition of UL42 protein to the Vero cell enzyme had no effect on the rate or processivity of deoxynucleotide polym- erization. Presumably the appropriate complex of DNA po- lymerase and UL42 protein had been assembled in uiuo.

Enhancement of the processivity of the HSV-1 DNA polym- erase by the UL42 protein in its replication of singly primed ssDNA templates requires that the ssDNA be coated with ICPS. Under these conditions full-length products are ob- tained. In the absence of ICP8, a series of discrete less than full-length products are generated that presumably reflect regions of secondary structure within the M13mp18 ssDNA that block the progression of the DNA polymerase-UL42 protein complex.

In earlier studies of the interaction of ICP8 with the HSV-

* T. R. Hernandez and I. R. Lehman, unpublished observation.

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A. No ICP8 Added

Vero Cell HSV-1 Recombinant HSV-1 DNA Polymerase DNA Polymerase

0 3 6 9 15 2330 0 3 6 9 1523 30

tures impede the DNA polymerase and must be removed to permit sustained processive replication of the ssDNA even in the presence of a processivity enhancing factor such as the UL42 protein.

(x10’) mol UL42 proteinlmol Pol

RFII +

primed + ssDNA

lCP8 Added

(x10’) mol UL42 protein/m01 Pol

RFll+

primed .+ ssDNA

Vero Cell HSV-1 Recombinant HSV-1 DNA Polymerase DNA Polymerase

0 3 6 91523300 3 6 9152330

FIG. 6. Processive replication of primed M13mp18 ssDNA by HSV DNA polymerase in the presence of UL42 protein. Aliquots (10 ~1) of the 50.min time points of the reactions shown in Fig. 5 were subjected to agarose gel electrophoresis and autoradiog- raphy as described under “Experimental Procedures.” A, reactions performed in the absence of ICP8. B, reactions in the presence of ICP8 (1.4 pg). The RF11 M13mp18 ssDNA standards were prepared by annealing M13mp18 ssDNA primed to a 5’-“‘P primer 1 (see “Experimental Procedures”) and incubating with 0.1 fig (240 units) of E. coli DNA polymerase III*, 52 ng of p subunit, and 0.8 pg of E. coli SSB for 10 min at 37 “C.

1 DNA polymerase, we had observed that ICP8 markedly inhibited the replication of primed ssDNA templates by the herpes DNA polymerase isolated from infected Vero cells (33). Maximal inhibition occurred at the point at which the ssDNA was fully coated with ICP8. This inhibition appears to be related to contamination of the ICP8 preparation used in those studies by an inhibitor that is removed upon Superose 12 gel filtration. Our current preparations of ICP8 whether purified from HSV-l-infected Vero cells by Superose 12 gel filtration or from the overproducing cell line U35 through heparin-agarose chromatography appear to lack the presump- tive inhibitor. Instead, they stimulate the replication of ssDNA templates by the herpes DNA polymerase, very likely by acting as a classical helix destabilizing protein that can melt out hairpins or other hydrogen-bonded structures that segments of the M13mp18 ssDNA can assume. Such struc-

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T R Hernandez and I R Lehmanprotein.

Functional interaction between the herpes simplex-1 DNA polymerase and UL42

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