In Vitro Transcription of Herpes Simplex Virus Genes PARTIAL PURIFICATION AND PROPERTIES OF RNA POLYMERASE I1 FROM UNINFECTED AND INFECTED HEp-2 CELLS*

(Received for publication, February 1, 1982)

Thomas W. Beck and Robert L. Millette From fhe Department of Immunology and Microbiology an.d the Department of Biochemistry, Wayne State University School of Medicine, Detroit, Michigan 48201

We have used partially purified preparations of RNA polymerase I1 from herpes simplex virus type 1 (HSV- 1)-infected (PoZII-H) and uninfected (PoZII) HEp-2 cells to study the transcription of HSV-1 DNA in vitro. These preparations are essentially free of nucleases and greater than 97% of the RNA polymerase activity is directed by RNA polymerase 11. The enzyme prepara- tions exhibit continued transcription and initiation of RNA chains from HSV DNA for at least 90 min. The two enzymes are similar with respect to their chromato- graphic properties, ionic strength optima, divalent cat- ion preference, and sensitivity to inhibitors. However, they show striking differences in their template pref- erences and in their selectivity of transcription from both whole and cloned HSV-1 DNA. The RNAs synthe- sized from whole HSV DNA by PoZII and PoZII-H have weight average chain lengths of 4.00 and 3.50 kilobases (kb), respectively. Both enzymes transcribe essentially all regions of the HSV genome; however, Pol11 prefer- entially transcribes the terminal and internal repeat regions and adjacent sequences, regions known to con- tain the immediate early genes. The two enzymes also produced different sets of RNA transcripts from the cloned BarnHI-Q fragment of HSV DNA containing the thymidine kinase gene. Pol11 produced major products of 2.12 and 1.53 kb whereas PoZII-H yielded products of 2.84, 1.62, and 1.47 kb. Only PolXI-H synthesized an RNA having the appropriate size (2.84 kb) for a run-off transcript from the thymidine kinase promoter. These transcriptional differences are independent of enzyme and DNA concentration. The results demonstrate that RNA polymerase I1 preparations from uninfected and HSV-1-infected cells differ in their in vitro kranscription of HSV DNA. This partially purified RNA polymerase I1 system should prove useful for studying the regula- tion of gene transcription in vitro.

Recently, two soluble RNA polymerase I1 systems have been described that give accurate transcription of eukaryotic genes in uitro (1, 2). The system of Weil et aZ. (1) contains a crude cellular extract (S-100) and purified RNA polymerase I1 from KB cells, whereas the preparation of Manley et aZ. (2) consists of a concentrated whole cell extract from HeLa cells. Both of these preparations have been shown to transcribe

* This work was supported by Grant CA-21065 from the National Cancer Institute, National Institutes of Health, and Biomedical Re- search Support Grant NIH 147-1 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.

RNA accurately and selectively from a variety of eukaryotic and viral gene promoters. These promoters include those for the adenovirus major late transcription unit (1,2), adenovirus early region lA, lB, 111, IV (2-4), globin genes (5-7), conal- bumin (8), ovalbumin (8,9), and simian virus 40 early and late regions (10-15). However, both of these preparations generally have failed to exhibit the transcriptional regulation that is observed in uiuo. For example, both systems utilize the em- bryonic, fetal, and adult globin gene promoters with nearly equal efficiencies even though they are prepared from cell lines that apparently do not express globin genes at all (6, 7). Similarly, the Manley system utilizes the early and late pro- moters of simian virus 40 DNA (10, 11, 13). Therefore, it appears that both systems contain general transcriptional control factors, but seem to lack tissue-specific or viral-specific transcriptional control activities in uitro.

HSV-1’-infected cells provide a useful system for studying transcriptional regulation in uitro. Throughout the infectious cycle, the bulk of viral transcription is mediated by the host cell RNA polymerase IT (16, 17). The synthesis of HSV-1 mRNA is coordinately and sequentially regulated. Immediate early (a) mRNA (i.e. RNA synthesized in infected cells in the absence of protein synthesis) is homologous to about 10 to 12% of the viral DNA (18,19). Hybridization of the immediate early mRNA to restriction fragments of HSV DNA demon- strated that it originates primarily from the repeat sequences and adjacent regions of the viral genome (19, 20). Early (/I) and late ( y ) RNA (i.e. RNA made before and after the onset of viral DNA synthesis, respectively) are homologous to about 20 to 30% and 41 to 50% of the viral DNA, respectively (19, 21-23). These RNAs hybridize to essentially all regions of the HSV genome (19, 20).

Several types of evidence indicate that a positive transcrip- tional control mechanism activates the switch from immediate early to early gene expression in HSV-1-infected cells. Exper- iments of Leung (24) suggested that an immediate early protein synthesized by a pre-infecting thymidine kinase minus virus could act in trans to activate transcription of the early thymidine kinase gene of a superinfecting virus. Studies by Preston (25,26) with a temperature-sensitive mutant of HSV- 1 (tsK) which synthesizes mainly immediate early gene prod- ucts at the nonpermissive temperature, indicated that the immediate early polypeptide affected by the tsK lesion is required for transcription of the thymidine kinase gene and other early and late genes. Watson and Clements (27) have shown by temperature shift experiments that the protein affected by the tsK lesion is required continuously for the synthesis of early and late mRNAs. More recently, Post et al.

‘ The abbreviations used are: HSV, herpes simplex virus; SDS, sodium dodecyl sulfate; kb, kilobase.

12780

In Vitro Transcription of HSV DNA by RNA Polymerase 11 12781

(28) elegantly demonstrated, by substituting the natural thy- midine kinase promoter with an immediate early promoter, that the thymidine kinase gene can be expressed and regulated as an immediate early gene. This indicates, not surprisingly, that the 5'-flanking DNA sequences are involved in this tran- scriptional regulation.

In this paper, we describe the purification and properties of RNA polymerase I1 from HSV-1-infected and uninfected HEp-2 (human) cells. These preparations contain negligible levels of RNA polymerases I and 111 and nucleases and give continued transcription and initiation of RNA chains for at least 90 min. Finally, we present evidence that certain tran- scriptional controls are operative in this system in that differ- ences in transcriptional selectivity between RNA polymerase 11 from uninfected and HSV-1-infected cells can be demon- strated in the in vitro transcription of HSV-1 DNA.

EXPERIMENTAL PROCEDURES

Cells and Virus-HEp-2 (human epidermoid carcinoma) cells and HSV-1 (F1 strain, obtained from B. Roizman, University of Chicago) were propagated and harvested as described previously (29).

Purification of RNA Polymerase-For the preparation of RNA polymerase I1 from uninfected cells (PolII), HEp-2 cells were prop- agated in 690-cm' glass roller bottles in medium 199 + 5% calf serum until 90-95% confluent and harvested by scraping in phosphate- buffered saline and centrifuging in 250-ml centrifuge bottles at 2000 rpm for 20 min. For RNA polymerase I1 from infected cells (PolII- H), 90-95% confluent HEp-2 cells in roller bottles were infected with HSV-1 (10 plaque-forming units/cell) for 1 h in medium 199 + 1% inactivated calf serum. The infecting medium was removed, and cells were overlayed with medium 199 + 1% inactivated calf serum and incubated at 37 "C for 8 h and then harvested by scraping in phos- phate-buffered saline. As a control, mock-infected cells were prepared in a similar manner but without virus in the infecting medium. One volume of packed cells was washed by suspension in a one-half volume of phosphate-buffered saline and centrifugation at 2000 rpm for 20 min. The cells were resuspended in a volume of TGMED (50 mM Tris-HC1, pH 7.9, 258 glycerol (v/v), 5 mM MgCl,, 0.1 mM EDTA, 1 mM dithiothreitol) equal to the wet weight of the cells and stored a t -70 "C until use.

T o prepare RNA polymerase, approximately 1.05 X 10"' cells (from 30 roller bottles) were thawed and the enzyme was solubilized essen- tially as described by Schwartz et al. (30). Briefly, the cells were diluted to approximately 2 X 10" cells/ml in TGMED containing 0.1 mM phenylmethylsulfonyl fluoride and 0.5 trypsin inhibitor unit/ml Trasylol (Sigma); 4 M ( N H M O , (pH 8) was added to 0.3 M, and the suspension was sonicated on ice in 5-6-ml aliquots with a Branson Sonifier W-350 at setting 2 for two 5-s intervals 1 min apart. The sonicate was centrifuged at 200,000 X g for 60 min in a Spinco SW 41 rotor. The resulting supernatant was dialyzed for 5 h a t 4 "C against two changes of 500 ml of TGED (TGMED minus MgC12) containing 0.1 mM phenylmethylsulfonyl fluoride and 25 mM (NH4)eS04 and centrifuged a t 20,000 X g for 15 min. The clarified supernatant liquid was loaded onto a DEAE-cellulose column (1.2 X 25 cm) (Whatman DE52) previously equilibrated with the same buffer. The column was washed with 3 column volumes of buffer and RNA polymerase activity was eluted stepwise as described by Bagshaw (31) with TGED con- taining 160 mM (NH,)2SOa to elute RNA polymerases I and 111 and 500 mM (NHg)eSO, to elute RNA polymerase 11. Fractions of 1.0 ml were collected and those containing greater than one-half of the peak polymerase I1 activity were pooled, brought to 50% (v/v) in glycerol, quick frozen in a dry ice-acetone bath, and stored a t -70 "C.

RNA Polymerase Assay-RNA polymerase assays were performed in a final volume of 50 pl essentially as described by Schwartz et al. (30). Briefly, the incubations contained 50 mM Tris-HCI, pH 8.0, 2 mM MnCI,, 0.6 mM ATP, GTP, and CTP, 50 p~ [5,6-"H]UTp at 400 mCi/mmol (Amersham), 100 pg/ml calf thymus DNA (Sigma), and (NH,LSOa a t 60 mM (for polymerases I and 111 assay), 135 mM (for polymerase I1 assays), or as specified. In some experiments, a-aman- itin was added to either 0.2 or 1 pg/ml. Transcription was initiated by the addition of 5-10 p1 of RNA polymerase I1 preparation and allowed to proceed for 30 min a t 37 "C. Reactions were terminated by chilling the reaction tubes on ice and adding 0.25 ml of H,O, 0.25 ml of 0.1 M sodium pyrophosphate, pH 7, and 0.5 ml of ice-cold 10% trichloroace- tic acid. After holding the tubes on ice for 5 min, the precipitates were

collected onto nitrocellulose filters (Schleicher and Schuell, Grade BA85) by suction filtration and washed five times with 2-4 ml of ice- cold 5% trichloroacetic acid containing 0.05 M sodium pyrophosphate, two times with 1-2 ml of cold 5% trichloroacetic acid, and finally two times with 1-2 ml of cold 70% (v/v) ethanol. The filters were dried and the incorporated radioactivity was determined by liquid scintil- lation spectroscopy. One unit of enzyme activity represents the incor- poration of 1 pmol of UMP in 30 min a t 37 "C.

Isolation of HSV DNA-HSV DNA was prepared as described previously (32), except that RNase treatment was omitted. The virus was pelleted by centrifugation a t 11,OOO rpm for 90 min in a Sorvall GSA rotor and suspended in virus buffer (0.15 M NaCl, 0.02 M Tris- HCI, pH 7.5). Nonionic detergent P-40 was added to 0.75%> (v/v) and the mixture was homogenized with a Dounce homogenizer. The mixture was centrifuged at 2,000 X g for 5 min and the supernatant was layered onto a 10-50% (w/v) sucrose gradient in virus buffer and centrifuged a t 25,000 rpm for 50 min at 4 "C in a Spinco SW 27 rotor. The nucleocapsid bands were collected with a syringe, diluted %fold with virus buffer, and centrifuged as above for 2 h. The pelleted nucleocapsids were suspended in virus buffer and EDTA was added to 5 mM, Sarkosyl to 2% (v/v), and SDS to 0.5% (w/v) with mild vortexing. The DNA was extracted three times with phenol saturated with NET (0.1 M NaC1, 1 mM EDTA, 10 mM Tris-HCI, pH 7.5) and two times with chloroform containing 2% (v/v) isoamyl alcohol. The DNA was then precipitated at -20 "C with 2 volumes of ethanol, resuspended in 10 mM Tris-HC1, pH 8, 1 mM EDTA, 0.12 M NaOAc, and reprecipitated with 2 volumes of ethanol. The DNA pellet was rinsed with cold 80% ethanol, dried in vacuo and suspended in T E buffer (10 mM Tris-HC1, pH 8.0, 1 mM EDTA) at about 0.5 mg/ml.

Preparation of pTK5 Plasmid-The recombinant plasmid pTK5 in Escherichia coli HBlOl was obtained from Saul Silverstein (Co- lumbia University). It consists of BamHI fragment of HSV(F) DNA containing the viral thymidine kinase gene inserted in pBR322. The bacteria were grown in 1 liter of L broth medium containing 20 pg/ml ampicillin. At a Klett value of 100, chloramphenicol was added to 100 pg/ml and the culture was further incubated overnight a t 37 "C. The cells were centrifuged a t 6,000 rpm for 10 min in a Sorvall GSA rotor, suspended in 20 ml of sucrose buffer (50 mM Tris-HCI, pH 7.4.40 mM EDTA, 25% sucrose) containing 0.858 lysozyme, and incubated 5 min a t 0 "C (33). Then 1.6 ml of 0.5 M EDTA, pH 8.0, were added and the mixture was incubated 5 min at 0 "C. Following the addition of 12.5 ml of Brij lysing buffer (1% Brij 58, 4% deoxycholate, 50 mM Tris- HCI, pH 8) with vortexing, the lysate was centrifuged for 45 min a t 50,000 rpm in a Beckman Ti-60 rotor. The supernatant liquid was decanted and to it were added 0.7 ml of 10 mg/ml ethidium bromide and approximately 19 g of CsCl to give a refractive index of 1.3995- 1.4000. The mixture was centrifuged a t 6,000 rpm for 3 min, the surface protein layer was removed with a spatula, and the liquid was centrifuged a t 31,000 rpm for 60 h a t 20 "C in a Beckman Ti-50 rotor. DNA was located with a long wave ultraviolet light and the lower band containing form I DNA was removed with a syringe from the side of the tube. This was diluted 2-fold with T E buffer, extracted once with butanol saturated with NaC1, and passed through a 5-ml Dowex Ag 50-X4 (100-200 mesh) column pre-equilibrated and eluted as described by Zimmer and Millette (34). The plasmid DNA was then dialyzed against three changes of 1.5 liters of T E buffer. Plasmid preparation was carried out under P2 conditions.

Restriction a n d Electrophoresis of DNA"pTK5 DNA for in wtro transcription was digested with 5 enzyme units of BamHI (Bethesda Research Laboratories, Rockville, MD)/1 pg of DNA for 2 h a t 37 "C in 20 mM Tris-HC1, pH 7.0, 100 mM NaCl, 7 mM MgCl,, 2 mM 2- mercaptoethanol, 100 pg/ml bovine serum albumin. The reaction was terminated by adding EDTA to twice the molar concentration of Mg", SDS to 0.296, heating a t 65 "C for 3 min, and chilling on ice. The DNA was extracted twice with phenol/chloroform/isoamyl al- cohol (49:49:2) and precipitated with 2 volumes of ethanol a t -20 "C. The DNA was pelleted by centrifugation in a Microfuge a t 9980 X g for 15 min. The DNA was dissolved in 10 mM Tris-HCI, pH 8, 1 mM EDTA, 120 mM NaOAc and precipitated with ethanol as described above. The resulting DNA pellet was washed with 80% ethanol, dried in uacuo, and dissolved in T E buffer a t about 0.5 mg/ml.

HSV DNA for Southern blot analysis was prepared by digesting 26 pg of HSV DNA with 150 enzyme units of Sal1 in 9 mM Tris-HCI, pH 7.6, 6 mM Mg(OAc)p, 0.2 mM EDTA, 150 mM NaCl, 100 pg/ml bovine serum albumin for 2.5 h at 37 "C. The reaction was terminated as described above and, after addition of Tris-HCI, pH 7.8, NaH2P0, to 30 mM, and sucrose to 10% (w/v), the sample was fractionated by electrophoresis for 12 h at 50 V through a horizontal 1% agarose gel

12782 In Vitro Transcription of HSV DNA by RNA Polymerase 11

(22 X 13 X 0.5 cm) with the recirculating buffer system of Loening (35). The gel was stained with 1 pg/ml ethidium bromide and photo- graphed under UV light.

Standard Transcription Mixture-Transcription of HSV-1 DNA was performed essentially as described above (RNA polymerase as- say) but in 25-50-pl reaction volumes containing 50 mM Tris-HCI, pH 8, 7 mM MgCla, 120 mM KCI, 0.6 mM of ATP, GTP, and UTP, 50 p~ [a-"'P]CTP (Amersham or New England Nuclear) a t 7-20 pCi/nmol, HSV DNA at 50 pg/ml, and 10 p1 of RNA polymerase 11. This gave a final glycerol concentration of 20% and 14-140 units of RNA polymerase 11/25-pl reaction mixture. In some experiments, 50 pg/ml of the recombinant plasmid pTK5 DNA cleaved with BamHI was used as a template. Reaction mixtures were incubated for 60 min at 37 "C and chilled on ice.

Purification of RNA-For size analysis, RNA was prepared by

mM NaOAc, pH 5.2,0.5% SDS, 1 mM EDTA, 25 pg/ml E. coli tRNA. bringing a 25-pl reaction mix to 0.5 ml with a solution containing 120

Nucleic acids were isolated by two extractions with phenol/chloro- form/isoamyl alcohol (4949:2), precipitation with 2 volumes of ethanol a t -70 "C for 30-60 min, and centrifugation in a microcentri- fuge for 15 min a t 4 "C and 9980 X g. The resulting nucleic acid pellet was redissolved in 0.5 ml of a solution of 0.3 M NaOAc, pH 5.2, 0.2% SDS, 1 mM EDTA and precipitated with 2 volumes of ethanol as described above. The ethanol was decanted, and the pellet was dried in uacuo and dissolved in 25 pl of 10 mM Tris-HC1, pH 7.9, 5 mM MgCL, 50 pg/ml DNase I (Worthington) (iodoacetate-treated) and incubated 5 min at 37 "C. The sample was then extracted as described above and precipitated with 2 volumes of ethanol without addition of 2 M NaOAc.

RNA to be used for Southern blot analysis was prepared in an analogous manner, except that following the first precipitation step the samples were dried in uacuo, dissolved in 10 mM Tris-HC1, pH 7.9,5 mM MgCl,, 100 pg/ml DNase, and incubated for 30 min a t 37 "C. The samples were brought to 0.5 ml with 120 mM NaOAc, pH 5.2, 0.2%' SDS, 1 mM EDTA, extracted twice with phenol/chloroform/ isoamyl alcohol and precipitated with 2 volumes of ethanol. The DNase treatment was repeated and the RNA was extracted as de- scribed above, twice precipitated with ethanol from 0.3 M NaOAc redissolved in 120 mM NaOAc, pH 5.2, 0.2% SDS, 1 mM EDTA, and reprecipitated with 2 volumes of ethanol.

Glyoxalation a n d Electrophoresis of in Vitro RNA-Glyoxal (Fisher Scientific) was deionized by stirring with a mixed-bed ion exchange resin (Bio-Rad AG 501-X8D) for 4 h a t 4 "C and stored a t -70 "C (11). RNA extracted from the in uitro reaction was resus- pended in glyoxal reaction buffer (50% (v/v) dimethyl sulfoxide, 1 M glyoxal, 10 mM NaHnP04/NazHPOc pH 6.8) and incubated for 1 h a t 50 "C (36). Bromphenol blue was added to 0.01% and the samples were analyzed by electrophoresis a t 50 V for 12 h in a 1.6% agarose (Bethesda Research Laboratories) horizontal slab gel (22 X 13.3 X 0.5 cm) containing 10 mM NaHZPO4/Na2HPO4, pH 6.8, 0.1% SDS, 5% glycerol (11). The gels were dried onto Whatman 3MM paper in c'acuo and exposed to x-ray film (Kodak XRP-1) at -70 "C with an intensifying screen (Quanta 111, E. I. duPont De Nemours and Co.).

Southern Blot Hybridizations-The HSV DNA fragments were transferred from the gel to nitrocellulose membranes (BA85, Schleicher and Schuell) by the method of Southern (37). The mem- brane was cut into 5-mm strips (approximately 1 pg of DNA/strip) and these were incubated 3 h at 80 "C in Denhardt's hybridization buffer (38) containing 10 pg/ml yeast RNA. The samples to be hybridized were either ,"P-labeled in uitro RNA samples (1.25 X lo4 cpm/ng), "'P-labeled HSV DNA prepared by nick translation (39) with [a-"PIdCTP (New England Nuclear) (2.00 X IO' cpm/ng), or ""P-labeled HEp-2 cell RNA (5.00 X lo4 cpm/pg). These were heated in water for 5 min a t 80 "C and chilled on ice. An equal volume of 2X Denhardt's hybridization buffer was added, and the RNA or DNA samples were sealed with the nitrocellulose strips in Seal-N-Save plastic bags (Sears, Roebuck and Co.) in 1.5-2.0-ml total volume. Hybridization was performed with constant agitation for 40 h at 80 "C. The nitrocellulose membranes were rinsed with 3X SSC, washed once with 100 ml of 1X hybridization buffer a t 80 "C for 1 h, three times in 150 ml of 3X SSC, 0.16 SDS, 0.1% sodium pyrophos- phate at 65 "C for I h each, and two times in 150 ml of 3~ SSC a t 65 "C for 30 min each. The filters were air-dried and exposed to x-ray film (Kodak X-Omat) with an intensifying screen. Autoradiographs from two exposures were scanned with a Quick Scan R & D densitom- eter (Helena Laboratories) and the appropriate peaks were excised and weighed to estimate the relative amount of RNA hybridized to each fragment.

Assay of Enzymatic Impurities-The conversion of form I to form

contained 1.25 p g of "2P-labeled plasmid pSG 18F DNA (which I1 DNA was used as an assay for endonuclease activity. The assay

contains the EcoRI F fragment of HSV DNA inserted into pBR325) a t 2.2 X loJ cpm/pg composed of 27% form I DNA in place of HSV DNA in the standard transcription mixture, and 70 units of PolII or 30 units of PolII-H. The reaction tubes were incubated for 2 h a t 37 "C. EDTA and SDS were added to 14 mM and 0.2%, respectively, heated to 65 "C for 3 min, and analyzed by electrophoresis through a 1% agarose gel. The gel was dried in uacuo, exposed to x-ray film, and analyzed by densitometry. The presence of DNase was indicated by a decrease in the percentage of total radioactivity migrating with form I DNA.

RNase activity was assayed by measuring the degradation of bac- teriophage R17 RNA. Two pg of 'JH-labeled R17 RNA (1.28 X 10" cpm/pg) were incubated for 1 h a t 37 "C with 60 units of Pol11 under standard transcription conditions except that DNA and ribonucleo- tide triphosphates were omitted and the total volume was 50 pl. EDTA and SDS were added to 14 mM and 0.2%, respectively, and then the reaction mixtures were heated to 65 "C for 3 min, chilled, and analyzed by electrophoresis through a 1% agarose gel. The gel was sliced into 0.2-cm sections, heated to 65 "C in 0.5 ml of H20, and cooled. Each sample was then counted by liquid scintillation spec- troscopy in 10 ml of ACS (Amersham). RNase activity was measured as the decrease in percentage of total radioactivity migrating with the R17 RNA peak.

RESULTS

Partial Purification of RNA Polymerase 11-In prelimi- nary studies using gradient elution from DEAE-cellulose, we found that RNA polymerases I and I11 from HEp-2 cell extracts eluted at about 0.10 M and RNA polymerase I1 eluted at 0.20 M (NH4)2S04. As reported previously by Lowe (40), we observed no differences in the salt concentrations at which the RNA polymerases from HSV-1-infected and uninfected HEp-2 cells elute from DEAE-cellulose. However, since the enzymes obtained by gradient elution were too dilute for using in in vitro transcription studies, we chose to use a stepwise elution procedure.

A summary of the preparation of RNA polymerase I1 from uninfected (PoZII) and HSV-1-infected cells harvested at 8 h postinfection (PoZII-H) is presented in Table I. Step elution of RNA polymerases I and I11 activities from DEAE-cellulose with 160 m~ (NH&S04 and subsequent elution of RNA polymerase I1 with 500 mM (NH4)&304 resulted in a prepara- tion that is greater than 97% polymerase I1 as determined by the sensitivity of transcription to 0.2 p g / d a-amanitin as described under "RNA Polymerase Assay.'' In contrast to RNA polymerase I1 prepared by gradient elution, this prepa- ration is suffkiently concentrated for in vitro transcription without further concentration steps. The recoveries for PolII have ranged from 23 to 42% of the initial RNA polymerase activity and the specific activities have ranged from 4430 to 8920 units/mg. This represents a 40- to 80-fold purification over the crude extract. The PolII-H preparations have yielded similar values for these parameters except that the specific activities and recoveries for PoEII-H are somewhat lower than for PolII. This may be in part due to differences between the two enzymes with respect to their activity on calf thymus DNA and to their Mn2+ optima under standard assay condi- tions.

TO determine the level of nuclease contamination in these polymerase preparations, we incubated each enzyme with "P- labeled form I plasmid pSG18F for 2 h at 37 "C. Agarose gel electrophoresis of the reaction mixtures with and without added polymerase showed that the percentage of radioactivity migrating with form I DNA was 27% with no added polyrn- erase and 26% following incubation with either Pol11 or PolII- H. To assay for RNase activity, we incubated 'H-labeled bacteriophage R17 RNA with and without added Pol11 for 1 h at 37 "C. The assay mixtures were analyzed by electropho-

In Vitro Transcription of HSV DNA by R N A Polymerase II 12783

TABLE I Summary of RNA polymerase preparation from uninfected and HSV-1-infected HEp-2 cells

RNA polymerase was solubilized and purified from 30 roller bottles of uninfected or HSV-I-infected HEp-2 cells as described under “Experimental Procedures.”

Protein Activity Specific activity Purification step

Recovery

[Jninfected Infected Uninfected Infected IJninfected Infected IJninfected Infected

mg 10 X umts unlts/rng c:,

Cell sonicate 624 602 6.2“ 5.8“ 99 96 100 100 200,000 X g supernatant 366 397 5.8 2.9 159 73 94 50 Dialysate 355 285 8.0 7.7 225 270 129 133 20,000 X g supernatant 274 169 13.9 12.3 507 728 224 212 DEAE-cellulose fractions

Polymerases I and 111 14.4 8.9 232 153 Polymerase I1 2.9 2.1 1.95 0.86 6720 4100 31 15

“Approximately 30% of the polymerase activity in the cell sonicate is due to RNA polymerase I1 as determined by the sensitivity to u-amanitin ( 1 pg/ml).

resis to determine the percentage of label migrating with the intact R17 RNA peak. For incubations in the absence and presence of PoZII, this value was found to be 65 and 6656, respectively. From these results, we conclude that nuclease contamination of the polymerase preparations is negligible.

Optimal Conditions for Transcription of HSV-1 DNA-To optimize in vitro transcription of HSV DNA, a number of parameters of the reaction were investigated. First, we ana- lyzed the effect of varying the HSV DNA concentration. As shown in Fig. 1, the polymerase reaction is highly dependent on added DNA and reaches saturation at 25 pg/ml DNA. To ensure that the DNA concentration was saturating, all further experiments to be described were performed at 50 pg/ml.

The effect of divalent cations on the transcription of HSV DNA by PolII and PolII-H is shown in Fig. 2. RNA synthesis in the presence of MgC12 reaches a maximum level from 7 to 10 mM with both enzymes. However, the optimum MnClz concentrations for PolII and PoZII-H activities differ slightly, occurring at 2 and 3 m, respectively.

The influence of KC1 concentration on the transcription of HSV DNA by the two enzymes is shown in Fig. 3. The addition of KC1 stimulates transcription of HSV DNA with an optimum being reached at 80 and 120 m~ for PolII-H and PoZII, respectively. Concentrations above 200 m are inhibi- tory for both enzymes. These data establish the optimal conditions for transcription of HSV-1 DNA and suggest that Pol11 and PoZII-H may differ in their ionic strength and Mn2’ optima. However, these differences are so subtle that we must conclude that Pol11 and PoZII-H do not differ significantly in their response to KC1, MnClZ, and MgClZ.

In Vitro Transcription Properties of PoZII and PoZII-H- As shown in Table 11, incorporation of [3H]UMP by PolII and PoZII-H is dependent upon added DNA and ribonucleotide triphosphate. The reaction is about 90% inhibited by actino- mycin D (100 pg/ml) and 99% inhibited by low concentrations of a-amanitin (1 pg/ml). The products of both enzymes are 100% sensitive to alkaline hydrolysis, but less than 8% sensitive to DNase treatment. These results indicate that the incorpo- ration of [3H]UMP by these enzymes represents RNA polym- erase 11-directed RNA synthesis.

The relative activities of Pol11 and PoZII-H with various DNA templates are compared in Table 111. Under standard transcription conditions, the PoZII-H-to-Pol11 activity ratio on HSV DNA or adenovirus 2 EcoRI-C fragment (containing early region IV) is 1.0. This ratio is somewhat lower with calf thymus or salmon sperm DNA (0.91 and 0.69, respectively). However, PoZII-H shows a preference for DNA containing the early (p) promoter for the viral thymidine kinase gene. The PolII-H-to-Pol11 activity ratio on plasmid pTK5 and its 1.6-kilobase pair SmaI fragment is 1.48 and 3.41, respectively. This indicates that PolII and PoZII-H differ in the efficiency

“” 6 t

I 1

I 1

0 20 40 60 HSV DNA (pg/ml)

FIG. 1. Effect of increasing concentrations of HSV-1 DNA on RNA synthesis by RNA polymerase 11. RNA synthesis by RNA polymerase I1 was measured in the presence of various concentrations of HSV DNA. Reaction mixtures (50 pl) contained 50 m~ Tris-HCI, pH 8.0, 2 mM MnC12, 100 m~ (NH4)2S04, 0.6 m~ each of ATP, GTP, and CTP, 50 PM [3H]UTP (234 cpm/pmol), 15% (v/v) glycerol, 35 units of PolII (5 pl), and HSV DNA at the concentrations indicated. Incubation was carried out at 37 “C for 30 min, the reactions were terminated by chilling on ice, and trichloroacetic acid precipitation was performed as described under “Experimental Procedures.”

FIG. 2. Effect of divalent cation concentration on the tran- scription of HSV DNA by PoZII or PolII-H. RNA synthesis by PolII (A, A) or PolII-H (0, 0) was measured in the presence of various concentrations of MnC12 (open symbols) or MgC12 (closed symbols). Reaction mixtures (25 pl) contained 50 m~ Tris-HC1, pH 8.0, 120 m~ KCI, 0.6 m~ each of ATP, GTP, and UTP, 50 p~ (a-

of PolII-H (5 pl each), and various concentrations of MnC12 or MgC12 as indicated. Incubation was carried out for 60 min at 37 “C, the reactions were terminated by chilling the reaction tubes on ice and adding 0.4 ml of H20, 0.1 ml of 50 m~ ATP, pH 7.0, and 0.5 ml of ice- cold 10% trichloroacetic acid. After holding the tubes on ice for 5 min, the precipitates were collected onto glass fiber filters by suction filtration and washed as described under “Experimental Procedures.”

32 P)CTP (758 cpm/pmol), 10% glycerol, 35 units of PolII or 14 units

12784 In Vitro Transcription of HSVDNA by RNA Polymerase II

FIG. 3. Effect of KC1 concentrations on the transcription of HSV DNA by Pol11 or PoZII-H. Reactions were performed as described in Fig. 2 except that each tube contained 7 m~ MgCL and the indicated concentration of KCl. Each tube contained 35 units of PolII (A) or 14 units of PolII-H (0).

TABLE I1 Characteristics of transcription of HSV DNA by PoMI and PolII-H

Fourteen 25-pl reaction mixtures were set up containing 50 pg/ml HSV DNA and either 35 units of PolII or 14 units of PolII-H as described in Fig. 2 except that all tubes contained 7 mM MgC12, and 50 p~ [.’H]UTP (178 cpm/pmol) was substituted for [“*P]CTP with the other NTPs at 0.6 mM. All reactions were complete except for the omissions or additions indicated. Reactions 4 and 5 received n-aman- itin or actinomycin D, respectively, at the beginning of the incubation. All reactions were incubated for 1 h at 37 “C. Reaction 6 was then treated with 100 pg/ml DNase (Rnase-free) for 30 min at 37 “C and reaction 7 was hydrolyzed in 1 N KOH at 70 “C for 1 h and then neutralized by addition of 2.2 pl of concentrated HCI. Acid-precipi- table radioactivity in each reaction was determined as described under “Experimental Procedures.”

Reaction incorporated incorporated PolII UMP PolII-H UMP

pmol 1. Complete 1.84 2. -DNA 0.02 3. -ATP, GTP, CTP 0.20 4. +1 pg/ml a-amanitin 0.01 5. +lo0 pg/ml actinomycin D 0.15 6. +DNase 1.70 7. +KOH 0

‘5 pmol T 100.0 1.36 100.0

1.3 0.03 2.1 10.8 0.11 7.9 0.5 0 0 7.9 0.21 15.4

92.4 1.69 124.0 0 0 0

TABLE I11 Comparison of PolII and PolII-H activity on various templates Transcription was carried out as described in Table I1 except that

each reaction mixture contained 50 pg/ml of the DNA indicated and 98 units of PolII or 30 units of PolII-H in a total reaction volume of

pmol Calf thymus 9.09 8.28 0.91 Salmon sperm 9.56 6.61 0.69 HSV 2.54 2.62 1.03 pTK5 11.2 16.0 1.48 1.6-kilobase pair SmaI frag- 0.73 2.49 3.41

Adenovirus 2 EcoRI-C frag- 5.41 5.48 1.01 ment from pTK5

ment

with which they can transcribe various DNA templates. Kinetics and Initiation of HSV-1 DNA Transcription by

PoZII-The kinetics of transcription of HSV-1 DNA by Pol11 in the presence of optimal concentrations of either Mn2+ or Mg2+ is presented in Fig. 4. In the presence of either divalent

Mlnutes

FIG. 4. Time course of HSV DNA transcription at optimal MgClz or MnCL concentration. Four 150-pl transcription mixtures were prepared as described in Fig. 2 except that 50 mM [3H]UTP (183 cpm/pmol) was used in place of [a-32P]CTP and two reaction mixtures contained 2 m~ MnCL (as indicated) in place of 7 mM MgC12. Each tube contained 90 units of PolII and at the times indicated, 25 pl were removed and diluted into 0.5 ml of 50 m~ sodium pyrophosphate (0 “C) and assayed by trichloroacetic acid precipitation as described under “Experimental Procedures.” Transcription was performed in the presence of (0) MgC12, (0) MgCL plus 0.5 pg/ml a-amanitin added at 30 min, (.) MnC12, (0) MnC12 plus 0.5 pg/ml a-amanitin added at 30 min. The arrow indicates the time of addition of inhibitor.

cation, the reaction proceeds nearly linearly for 60 to 90 min. However, RNA synthesis in the presence of Mg2+ exhibits an initial lag phase and proceeds with a somewhat slower rate relative to that in the presence of Mn2+. Addition of a-aman- itin (1 pg/ml) 30 min after initiation of RNA synthesis com- pletely inhibits further incorporation of [3H]UTP and results in plateau kinetics. This indicates that the transcription we are observing with both divalent cations is strictly due to RNA polymerase 11.

The continued synthesis of RNA for extended periods of time suggested that RNA polymerase I1 re-initiates RNA chains in vitro. To determine if this was the case, we used several inhibitors of initiation of transcription. The polyanion heparin binds to and inactivates free RNA polymerase but does not affect enzyme actively engaged in RNA synthesis on a double-stranded DNA template (41,42). Similarly, high salt concentrations, for example, 0.4 M (NH4)2S04, prevent the binding of free RNA polymerase to DNA, but allow the elongation of RNA chains initiated a t low salt concentration (43). a-Amanitin inhibits phosphodiester bond formation by RNA polymerase I1 and thus inhibits both the initiation and elongation of RNA chains by this enzyme (44). Table IV shows the effect of adding these inhibitors 30 min after the initiation of RNA synthesis by Pol11 on HSV DNA and allowing transcription to continue for an additional 60 min. Addition of a-amanitin (0.5 pg/ml) a t 30 min completely abolishes further RNA synthesis. RNA synthesis is inhibited by heparin (15 pg/ml) or (NH4)2S04 (0.4 M) to 61 and 76% of the control, respectively. Furthermore, we have found that the continued synthesis of RNA is accompanied by the con- tinued incorporation of [-p3’P]ATP and, as with [‘HIUTP incorporation, it is sensitive to low concentrations of a-aman- itin. These data indicate that the continued incorporation of nucleotides is mainly the result of continued initiation of RNA chains by RNA polymerase I1 in vitro.

Size Analysis of RNA Synthesized from HSV DNA by PolII and PolII-H-To determine the sue of RNA synthe- sized in vitro from HSV DNA by Pol11 and PoZII-H, RNA was isolated as described under “Experimental Procedures,”

In Vitro Transcription of HSV DNA by RNA Polymerase I I 12785

TABLE IV Effect of inhibitors on initiation of transcription by PoMI on Hsv

DNA Four 100-pl reaction mixtures were set up containing 50 pg/ml HSV

DNA, 70 units of PoLII, and 120 mM ( N H s ) 8 0 4 as described under "Experimental Procedures." The reactions were incubated 30 min at 37 "C, inhibitors were added as indicated, and 25-pl aliquots were removed for trichloroacetic acid precipitation. The reaction mixtures were further incubated 60 min and another 25-pl alqiuot was removed for trichloroacetic acid precipitation. Values expressed are the differ- ence between the 30- and 90-min time points.

Inhibitor IJMP incorpo- Inhibition rated pmol Q

None 4.53 0 Heparin (15 pg/ml) 1.78 60.7

a-Amanitin (0.5 pg/ml) 0 100 (NH4)aSO'l 1.09 75.9

denatured with glyoxal, and analyzed by electrophoresis through an agarose gel. A densitometer scan of the autoradi- ograph of the gel and the position of rRNA size standards are shown in Fig. 5. These results demonstrate that both Pol11 and PoZII-H synthesize high molecular weight RNA ranging in size from about 600 to greater than 5000 nucleotides. When synthesis was carried out in the presence of a-amanitin, no RNA was detected, indicating that these are bona fide RNA polymerase I1 products. From these data, we have calculated weight average chain lengths of 4000 and 3500 nucleotides for PolII and PoZII-H products, respectively. These values are strikingly close to the average UV target sizes of the imme- diate early (4070 nucleotides) and early (3480 nucleotides) transcription units as calculated from the data of Millette and Klaiber (29).

Hybridization Analysis of RNA Synthesized on HSV DNA by PolII and PolII-H-To determine if PolII and PolII-H show transcriptional selectivity on HSV DNA, RNA products generated in vitro by the two enzymes were hybridized to SalI restriction fragments of HSV DNA immobilized on ni- trocellulose filters. RNA synthesized in the presence of a- amanitin, 32P-labeled, nick-translated HSV DNA, and 32P- labeled HEp-2 cell RNA served as controls. Fig. 6 shows the hybridization patterns and the corresponding densitometer tracings for the individual hybridizations. PolII-H (Fig. 6B) transcribes all regions of the HSV DNA and the hybridization pattern is similar to that produced by 32P-labeled HSV DNA (Fig. 6A). However, there appears to be some preferential transcription of RNA homologous to SalI fragments r, s, t, and u (Table V). This is in general agreement with the mapping data of Clements et al. (20) and Jones and Roizman (19) showing that RNA present at early and late times during HSV infection hybridized to all regions of the HSV genome but with more abundant transcripts mapping approximately at coordinates 0.1-0.3, 0.6-0.73, and 0.83-0.9. These same regions contain the SalI fragments r, s, and u of HSV DNA.

Similarly, when the transcription of HSV DNA is carried out with PolII, the RNA produced hybridizes to all regions of the HSV genome (Fig. 6C). However, certain regions, corre- sponding to SalI fragments a, j,, j,, and k are transcribed preferentially. Quantitation of the level of hybridization within these regions relative to the HSV DNA control reveals a 2- fold greater transcription from these regions (Table v). Fur- thermore, we have consistently observed a small amount of preferential transcription from the region of Sal1 fragment t. RNA polymerase I1 concentrations in the range of 35-140 units have yielded similar results. The regions giving enhanced transcription map in the internal (fragment a) and terminal (fragments j and k) repeat units and adjacent regions (frag- ment t). Several investigators (45, 46) have shown that these

regions contain the immediate early genes. These results are in general agreement with the results of hybridization studies of Clements et aZ. (20) and Jones and Roizman (19) which showed that RNA present in infected cells in the absence of

FIG. 5. Electrophoretic analysis of RNA transcribed in vitro from HSV-1 DNA by PolII and PolII-H. Transcription mixtures were prepared as described in Fig. 2 except that each contained 7 mM MgC12,50 ~ L M [CX-~~P]CTP (10 pCi/nmol), and 20 p1 of Pol11 (140 units) or PolII-H (56 units) in a total volume of 50 pl. RNA products were extracted, denatured, and electrophoresed through a 1.6% agarose gel. Densitometer tracings are shown of an autoradiogram from the indi- vidual gel tracks containing RNA transcribed in vitro by PolII ( A ) and PolII-H (B) . The position of 28 S, 23 S, 18 S, and 16 S ribosomal RNAs in the gel are indicated by arrows. Weight average chain lengths were calculated from the electrophoretic mobility relative to the E. coli ribosomal RNAs using values obtained by DNA sequence analysis (50,51) of 2904 nucleotides and 1541 nucleotides for 23 S and 16 S RNAs, respectively.

Sal I de

',g/ , c b b,

FIG. 6. Hybridization analysis of RNA transcribed in vitro from HSV DNA by PolII and PolII-H. RNA was transcribed in vitro as described in Fig. 5. RNA products were extracted and hybrid- ized to SalI restriction fragments of HSV-1 DNA immobilized on nitrocellulose strips. The densitometer tracing of the resulting auto- radiogram shows the hybridization patterns for (A) 32P-labeled, nick- translated HSV-1 DNA, (B) [32P]RNA transcribed by PolII-H, and (0 [32P]RNA transcribed by PolII; the input radioactivities were 58,400, 38,000, and 33,200 cpm, respectively. The position of the SalI restriction fragments are indicated by a-z. The SalI restriction map for HSV-1 DNA taken from Locker and Frenkel (54) is shown in D. The HSV genome consists of two unique regions, unique long (UL) and unique short (US), each flanked by terminal or internal repetitive sequences (TRL/IRL; TRs/IRs), which are covalently joined at IRL- IRs. Fragment a is the combination of fragmentsj and k derived from the IRL-IRs junction.

12786 In Vitro Transcription of HSV DNA by RNA Polymerase I1

TABLE V Quantitation of hybridization to Sall restriction fragments of

RNA polymerase products from HSV DNA transcription and nick- translated ”’P-HSV DNA were hybridized to blots of SaA-restricted HSV-1 DNA as described under “Experimental Procedures.” Values shown are the percentage of total radioactivity hybridized and are given as the mean f S.D. for two autoradiograms of two separate hybridizations. The SUA restriction map of HSV-1 DNA is shown in Fie. 6D.

HSV-1 DNA

”

Son Total radioactivity hybridized Ratio of radioactivity hybridized

a 8.9 f 1.2 b.c 13.2 f 1.0 c,e 14.0 f 2.4 f.p:h 15.3 f 0.1

l.m,n,o 12.0 f 0.4 J2,I.Jl.k 14.6 f 2.1

PSI 6.4 f 0.3 r s 7.7 f 0.9 t,u 4.7 f 0.9 v,w 1.4 f 0.8 X 0.4 f 0.1 v.z.a 1.4 f 0.7

18.7 f 1.0 7.6 f 0.6 9.4 f 0.6 1.27 f 2.1 9.7 f 1.1 15.1 f 1.4

11.9 f 2.0 16.1 f 1.0 28.4 f 1.4 14.3 f 0.3

7.3 f 0.5 14.5 f 0.7 3.0 f 0.7 6.2 f 1.5 4.3 f 0.3 5.3 f 0.7 4.1 f 0.2 3.3 f 0.2 1.6 f 0.2 2.5 f 0.9 0.5 f 0.2 0.4 f 0.0 1.1 f 0.5 2.0 f 0.7

1.17 1.04 0.93 0.95 1.02 0.83 1.03 1.50 1.50 0.56 1 .oo 0.70

2.46 0.77 0.64 0.74 1.99 0.50 0.48 0.81 1.24 0.64 1.25 0.55

~ ~~

protein synthesis hybridizes to the internal and terminal re- peats and adjacent regions. From these results, we conclude that PolII, in contrast to PolII-H, transcribes with greater preference the immediate early regions of the HSV genome.

Transcription of the Cloned HSV DNA Bam Fragment Containing the Viral Thymidine Kinase Gene-To obtain more definitive evidence for differences in the transcriptional specificity of the polymerase preparations, we used the HSV- 1 BamHI-Q fragment containing the viral thymidine kinase gene cloned in pBR322 (pTK5) as a template for in vitro transcription. A large part of this fragment has been sequenced (47, 48) and a considerable amount of data is available con- cerning the regulation of thymidine kinase gene expression as an early (p ) transcription unit in vivo (24-26, 28). Plasmid pTK5 was cleaved with BamHI and transcribed in vitro with PolII and PolII-H. The RNA products were sized after de- naturation with glyoxal by electrophoresis in a 1.6% agarose gel (Fig. 7). With this template, PolII generates two major transcripts of 2.12 f 0.03 and 1.53 * 0.02 kb (Fig. 7, lanes 2- 4). In addition, several minor species of 1.19 f 0.05, 1.03 f 0.05, and 0.63 f 0.02 kb are occasionally observed. RNA polymerase I1 prepared from mock-infected cells produced a pattern of transcripts identical with those produced by PolII. When low concentrations of a-amanitin (1 pg/ml) were added, production of these RNAs was not completely inhibited (lane 5) .

Aside from the common end-to-end transcripts of BamHI- Q and pBR322, transcription of pTK5 by PolII-H resulted in the production of a completely different set of major tran- scripts having sues of 2.84 f 0.03, 1.62 & 0.02, and 1.47 f 0.02 kb (Fig. 7, lanes 6-8). The 2.84-kb transcript has the appro- priate size for a run-off transcript from the TK gene promoter. It should be noted that the minor species occasionally seen with PolII are present in RNA generated by PolII-H (Fig. 7, lanes 6-8). When transcription was performed in the presence of a-amanitin, the production of all transcripts was fully inhibited (lane 9).

As shown in Fig. 7, the pattern of RNAs produced by both preparations is independent of the concentration of PolII and PolII-H in the range of 20-60 units/50-pl reaction. In fact, the patterns remain unchanged with 140 units of polymerase (data

1 2 3 4 5 6 7 8 9 1 0

-1.19 - 1.03

-0.63

from the BmHI-Q fragment of HSV-1 DNA by Pol11 and PolII- FIG. 7. Electrophoretic analysis of RNA transcribed in uitm

H. RNA was synthesized as described in Fig. 5 except that the template was the recombinant plasmid pTK5 (consisting of the BamHI-Q fragment of HSV-1 DNA inserted into the Barn site in pBR322) cleaved with BamHI and the concentration of PolII or PolII-H was varied as indicated. RNA products were extracted, denatured, and electrophoresed as described under “Experimental Procedures.’’ The gel tracks contain RNA synthesized with: lane 2, 20 units of PolII; Lane 3,40 units of PolII; lane 4, 60 units of PolII; lane 5, 60 units of PolII in the presence of 1 p g / d a-amanitin; lane 6,20 units of PolII-H; lune 7.40 units of PolII-H; lane 8,60 units of PolII-H; lane 9, 60 units of PoZII-H in the presence of 1 pg/d a- manitin. Each lane contained 25% of the total reaction volume. Lanes I and IO contain labeled marker HEp-2 and E. coli ribosomal RNAs, respectively. The position of end-to-end transcripts of pBR322 and BamHI-Q DNA are indicated by dots.

not shown). Although no transcripts are produced in the absence of DNA, varying the DNA concentration from 25 to 100 p g / d produced no changes in the transcript patterns or relative band intensities (data not shown). These results dem- onstrate that the infected cell RNA polymerase I1 (PolII-H), but not the uninfected (PolII) or mock-infected cell enzyme, can transcribe the DNA fragment containing the early (p) TK gene with high efficiency in vitro.

DISCUSSION

In this report, we have described an in vitro transcription system, consisting of partially purified RNA polymerase I1 from HSV-1-infected (PolII-H) and uninfected (PolII) HEp- 2 cells. We have shown that this transcription system has a number of properties making it useful for studying transcrip- tional regulation in vitro. First, this preparation provides continuous initiation and synthesis of RNA chains for at least 90 min. Second, being essentially free from other cellular polymerases and nucleases, it alleviates possible complications caused by the presence of these components during in vitro transcription. Finally, our studies on the transcription of both whole HSV DNA and cloned fragments have indicated that this system preserves several features of in vivo regulation.

Partial Purification of RNA Polymerase ZZ-The prepa- ration of RNA polymerase I1 described here takes advantage of the fact that RNA polymerase I1 elutes at a higher salt concentration from DEAE-cellulose than do RNA polymer- ases I and 111. By using appropriate stepwise elution proce- dures, one can obtain polymerase preparations that are greater

In Vitro Transcription of HSV DNA by RNA Polymerase II 12787

than 97% polymerase I1 as determined by the sensitivity of transcription to low concentrations of a-amanitin. Since the bulk of cellular protein (greater than 90%) is eluted from DEAE-cellulose prior to DNA polymerase 11, the enzyme is purified 40- to 80-fold over the crude extract with specific activities as high as 8900 units/mg. Furthermore, these prep- arations are essentially free of contaminating DNase and RNase activities which might interfere with in vitro transcrip- tion studies.

Kinetics and Initiation of Transcription by RNA Polym- erase 11-Under conditions optimized for transcription of HSV DNA, RNA synthesis is nearly linear for 60 min and continues for at least 90 min. We have shown that the contin- uous transcription is mainly due to the re-initiation of RNA chains by RNA polymerase I1 since it is 60-80% inhibited by treatments (heparin or high ionic strength) known to block free but not “engaged” RNA polymerase and it is greater than 97% inhibited by low concentrations of a-amanitin. This is supported by the preliminary observation that transcription is accompanied by the continued incorporation of [y-’”P]ATP and this is similarly sensitive to a-amanitin.

Properties of Pol I I and PolII-H-Previous studies have shown that the salt concentrations at which RNA polymerases from HSV-1-infected cells elute from DEAE-cellulose or DEAE-Sephadex are identical with that of the enzymes from uninfected cells (40, 52, 53). In addition, our present findings show that RNA polymerase I1 from HSV-1-infected HEp-2 cells (PolII-H) is, in several aspects, indistinguishable from its counterpart from uninfected cells in its transcription of HSV DNA in vitro. These include ionic strength optima, divalent cation preference, and sensitivity to inhibitors (a- amanitin and actinomycin D). These observations are con- sistent with those published previously (52, 53). However, as discussed below, the two enzyme preparations differ signifi- cantly in their in vitro transcription properties.

Template Preferences-Using concentrations of PollI-H and PolII that provide an activity ratio of unity with HSV-1 DNA, several differences in template preference were ob- served. For example, PolII shows slightly greater activity than PolII-H on calf thymus and salmon sperm DNA. This result also explains at least in part the apparent lower specific activities and recoveries we have observed in the preparation of RNA polymerase I1 from HSV-infected cells relative to uninfected controls, since calf thymus DNA is routinely used to monitor purification. A more interesting observation is that PolII-H is 3.4-fold more active than PolII with the HSV DNA fragment containing the early (p) thymidine kinase promoter. These results indicate that the two enzyme preparations pos- sess different template specificities.

Evidence for Transcriptlonal Selectivity on Whole HSV-1 DNA-We have shown that these polymerase preparations exhibit some of the characteristics of HSV transcription and regulation that are observed in vivo. Analysis of transcripts produced from whole HSV-1 DNA demonstrated that both enzyme preparations generate high molecular weight RNA products. Moreover, the weight average size of the Pol11 and PolII-H products (4.00 and 3.50 kb, respectively) was found to be very close to the average size of the immediate early ( a ) and early (p) viral transcription units, respectively, calcu- lated from our previous UV mapping data (29).

Southern blot hybridization analysis of the in vitro products transcribed from HSV DNA by these two enzymes provided evidence of differential transcriptional specificity. The results of these studies showed that transcripts produced by the uninfected cell enzyme (PoZIl) hybridized preferentially to fragments derived from the repeat regions of the genome. These sequences code in part for the immediate early gene

products (45, 46). The products of the enzyme from HSV- infected cells (PolII-H), on the other hand, hybridized to all regions of the genome with some slight preference for certain portions in the unique long regions. This is the pattern pre- dicted from the data on in vivo early (p ) and late ( y ) gene expression.

The preferred transcription of the immediate early se- quences by PoZII is not as great as one would predict from the in vivo transcription data of Clements et al. (20). There are several likely explanations for this. First, this could be the result of a requirement for one or more non-a-gene virion products as has recently been proposed by Roizman et aZ. (49). Such products would not be present in the uninfected cell RNA polymerase I1 preparation. Second, factors that might be required for transcriptional selectivity or termination might have been removed from the enzyme during purifica- tion. Third, random nicks in the HSV-1 DNA preparations might tend to randomize transcription. On the other hand, the hybridization pattern of our in vitro PolII products agrees more closely with the hybridization data of Jones and Roiz- man (19) which showed that immediate early ( a ) nuclear RNA hybridized to all fragments of HSV DNA analyzed, but with some preferential hybridization to sequences including the repeat regions.

Selectivity of Transcription from the Cloned HSV Thymi- dine Kinase Gene-In transcription studies using the cloned HSV-1 DNA fragment containing the viral early (p) thymidine kinase gene (pTK5), we showed that the sizes of the major transcripts generated by PolII-H are completely different from those produced by PoZII. Most importantly, only PolII- H produced a transcript (2.84 kb) of the size expected for a run-off RNA from the thymidine kinase gene promoter. In studies to be published elsewhere, we have mapped the var- ious PolII and PolII-H transcripts and have confirmed that PolII-H, but not PolII, initiates transcription from the thy- midine kinase gene promoter.’

We have shown that RNA polymerase I1 prepared from mock-infected cells behaves identically to PolII in transcrib- ing the BamHI-Q fragment as well as several other cloned HSV-1 DNA fragments. We have further demonstrated that the differences in transcriptional patterns produced by PolII and PoZII-H are independent of both enzyme and DNA con- centrations. These results indicate that the transcriptional differences we have observed between the uninfected and infected cell RNA polymerases are indeed the result of HSV- 1 infection.

We were surprised to find that the PolII transcripts were partially resistant to a-amanitin in view of the complete sensitivity of the transcription of whole HSV DNA to this inhibitor and the very low level of contamination by RNA polymerases I and 111. This activity is currently under inves- tigation. Nevertheless, by using this criterion, it is clear that the transcripts produced by PolII-H are bona fide RNA polymerase I1 products.

Comparison to Other in Vitro Transcription Systems-As with other eukaryotic in vitro transcription systems (1 ,2 ) , the RNA polymerase I1 preparations described in this communi- cation contain negligible nuclease activity and exhibit ribo- nucleotide incorporation into RNA that is completely depend- ent on added DNA. Although a direct comparison of the specific transcript yields between our RNA polymerase I1 preparations and the syst,ems of Weil et al. (1) and Manley et al. ( 2 ) is inappropriate because of possible differences in promoter strength of the templates used, we have calculated that 60 units of PolII-H produces approximately 3 X lo-,’

’ T. W. Beck and R. L. Millette, manuscript in preparation.

12788 In Vitro Transcription of HSV DNA by RNA Polymerase I1

pmol of the 2.84-kb transcript. This value is in the range of transcript yields obtained from the adenovirus 2 major late promoter as calculated by Manley et al. (2). However, because of the additional purification steps we have used, the concen- tration of protein present in our reactions (-300 pg/ml) is a t least 10-fold less than that used in other systems. This feature should facilitate the isolation and characterization of tran- scriptional control factors present in polymerase preparations.

At present, the mechanism of transcriptional control in HSV-1-infected cells is unknown. However, the findings pre- sented here demonstrate that viral infection somehow alters the transcriptional specificity of RNA polymerase I1 and provide an approach to studying the mechanism of HSV transcriptional regulation in vitro.

Acknowledgments-We are grateful to Dr. Joseph C. Bagshaw for kind instruction in the preparation of RNA polymerase I1 and for many helpful discussions, to Rosemarie Klaiber-Franco for technical assistance with cells and virus and in the preparation of figures, and to Ann Sesko, Kris Engstrom, and John Cleveland for technical assistance.

1.

2.

3. 4.

5. 6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

17.

REFERENCES

Weil, P. A., Luse, D. S., Segall, J., and Roeder, R. G. (1979) Cell

Manley, J. L., Fire, A., Cano, A., Sharp, P. A., and Gefter, M. L.

Lee, L). C., and Roeder, R. G. (1981) Mol. Cell. Biol. 1,635-651 Weingartner, B., and Keller, W. (1981) Proc. Natl. Acad. Sci. U.

Luse, D. S., and Roeder, R. G. (1980) Cell 20,691-699 Proudfoot, N. J., Shandler, M., Manley, J., Gefter, M., and Man-

iatas, T. (1980) Science (Wash. D. C.) 209, 1329-1336 Luse, D. S., Haynes, I. R., Van Leeuwen, D., Schan, E. A., Cleary,

M. L., Shapiro, S. G., Lingrel, J. B., and Roeder, R. G. Nucleic Acids Res. 9,4339-4354

Wasylyk, B., Kedinger, C., Corden, J., Brison, O., and Chambon, P. (1980) Nature (Lond.) 285,367-373

Tsai, S.. Tsai, M., and O’Malley, B. (1981) Proc. Natl. Acad. Sci. U. S. A . 78, 2174-2178

Myers, R. M., Rio, D. C., Robbins, A. K., and Tjian, R. (1981) Cell 25, 373-384

Rio, D., Robbins, A,, Myers, R., and Tjian, R. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 5706-5710

Gidoni, D., Kahana, C., Canaani, D., and Groner, Y. (1981) Proc. Natl. Acad. Sci. U. S. A . 78, 2174-2178

Handas, H., Kaufman, R. J., Manley, J., Gefter, M., and Sharp, P. A. (1981) J. Biol. Chern. 256,478-482

Hu, S., and ManIey, J. (1981) Proc. Natl. Acad. Sci. U. S. A. 78,

Mathis, D. J., and Chambon, P. (1981) Nature (Lond.) 290, 310-

Alwine, J. C., Steinhart, W . L., and Hill, C. W. (1974) Virology

Constanzo, F., Campadelli-Fiume, G., Foa-Tomasi, L., and Cassai,

18,469-484

(1980) Proc. Natl. Acad. Sci. U. S. A. 77,3855-3859

S. A. 78,4092-4096

820-824

315

60, 302-307

E. (1977) J. Virol. 21, 996-1001 18. Jones, P. C., Hayward, G. S., and Roizman, B. (1977) J. Virol. 21,

19. Jones, P. C., and Roizman, B. (1979) J. Virol. 31, 299-314 20. Clements, J. B., Watson, R. J., and Wilkie, N. M. (1977) Cell 12,

21. Frenkel, N., and Hoizman, B. (1972) Proc. Natl. Acad. SCL. U. S.

22. Swanstrom, R. I., and Wagner, E. K. (1974) Virology 60,522-533 23. Kozak, M., and Roizman, B. (1974) Proc. Natl. Acad. Sci. U. S.

24. Leung, W.-C. (1978) J. Virol. 27, 269-274 25. Preston, C. M. (1979) J. Virol. 29, 275-284 26. Preston, C. M. (1979) J. Virol. 32, 357-369 27. Watson, H. J., and Clements, B. J. (1980) Nature (Lond.) 285,

28. Post, L. E., Mackem, S., and Hoizman, B. (1981) Cell 24, 555-565 29. Millette, R. L., and Klaiber, R. (1980) J. Virol. 34, 604-614 30. Schwartz, L. B., Sklar, V. E. F., Jaehning, J. A., Weinmann, R.,

and Roeder, R. G. (1974) J. Biol. Chern. 249, 5889-5897 31. Bagshaw, J. C. (1976) Nucleic Acids Res. 3, 1449-1461 32. Talky-Brown, S., and Millette, R. L. (1979) J . Virol. 31, 733-740 33. Clewell, D., and Helenski, D. (1970) Biochemistry 9, 4428-4440 34. Zimmer, S. G., and Millette, R. L. (1975) Biochemistry 14, 290-

35. Loening, U. E. (1969) Biochern. J. 113, 131-138 36. McMaster, G. K., and Carmichael, G. C. (1977) Proc. Natl. Acad.

37. Southern, E. M. (1975) J . Mol. Biol. 98, 503-517 38. Denhardt, D. T. (1966) Biochem. Biophys. Res. Cornmun. 23,

39. Rigby, P. W . J., Dieckmann, M., Rhodes, C., and Berg, P. (1977)

40. Lowe, P. A. (1978) Virology 86, 577-580 41. Cox, R. F. (1973) Eur. J. Biochem. 39,49-61 42. Lescure, B., Chestier, A,, and Yaniv, M. (1978) J. Mol. Biol. 124,

43. Cedar, H. (1975) J. Mol. Biol. 95, 257-269 44. Cochet-Meilhac, M., and Chambon, P. (1974) Biochim. Biophys.

45. Watson, R. J., Preston, C. M., and Clements, J. B. (1979) J. Virol.

46. Anderson, K. P., Costa, R. H., Holland, L. E., and Wagner, E. K.

47. McKnight, S. L. (1980) Nucleic Acids Res. 8, 5949-5964 48. Wagner, M. J., Sharp, J. A., and Summers, W . C. (1981) Proc.

49. Roizman, B., Batterson, W., and Knipe, D. M. (1981) Perspect.

50. Brosium, J., Dull, T. J., and Noller, H. F. (1980) Proc. Natl. Acad.

51. Brosium, J., Palmer, M. L., Kennedy, P. J., and Noller, H. F.

52. Sasaki, Y., Sasaki, R., Cohen, G. H., and Pizer, L. I. (1974)

53. Muller, W. E. G.. Zahn, R. K., and Falke, D. (1978) Virology 84,

54. Locker, A., and Frenkel, N. (1980) J. Virol. 32,429-441

268-276

275-285

A. 69,2654-2658

A. 71,4322-4326

329-330

299

Sci. U. S. A. 74,4835-4838

64 1-646

J. Mol. Biol. 113,237-251

73-85

Acta 353, 160-184

31, 42-52

(1980) J. Virol. 34,9-27

Natl. Acad. Sci. U. S. A . 78, 1441-1445

Virol. 11, 77-92

Sci. U. S. A. 77, 201-204

(1978) Proc. Natl. Acad. Sci. U. S. A. 75,4801-4805

Zntervirology 3, 147-161

320-330