86,577~580 (1978)

Levels of DNA-Dependent RNA Polymerases in Herpes Simplex VirusInfected BHK21 Cl3 Cells PETER A. LOWE’ M. R. C. Virology Unit, University of Glasgow, Glasgow Gil &JR, Scotland Accepted January 14, 1978 DNA-dependent RNA polymerases I, II, and III were partially purified from Herpes simplex virus-l (HSV-l)-infected and mock-infected BHK21 Cl3 cells. The enzymes were solubilized and separated by DEAE-Sephadex chromatography. RNA polymerase levels were monitored throughout the course of infection by assay with exogenous template or binding of polymerase II to [3H]amanitin. No new RNA-polymerizing activities were detected during the course of infection. Infected cell polymerase levels remained virtually constant (with respect to mock-infected cells) up to 8 hr postinfection. Chromatographic profiles of infected and mock-infected cell RNA polymerases on DEAE-Sephadex and DEAE-cellulose were closely similar, as were the responses of their polymerase II to OLamanitin.

a-Amanitin-sensitive RNA polymerase II has been strongly implicated in HSV-1 RNA synthesis, since low concentrations of a-amanitin markedly inhibit HSV-1 transcription in isolated infected nuclei (1, 2) and cells (3). Under optimum in vitro assay conditions RNA pohnerase activity in HSV-l-infected nuclei has been reported to exhibit an approximately threefold stimulation by 8 hr postinfection with 85% of the polymerizing activity due to RNA polymerase II, compared with 50% in the nuclei of uninfected cells. (Similarly, nuclei infected with adenovirus-2, a linear duplex DNA virus, show a lo-fold increase. in the levels of polymerase II and III activity with a concomitant increase in the rate of viral RNA synthesis [4].) Marked changes also occur in the location along the virus genome of HSV-1 RNA transcripts synthesized before and after the onset of viral protein synthesis (5). Prior to protein synthesis the transcripts arise from a restricted region of the genome, but the transcripts made after protein synthesis are distributed throughout the genome. The present work de’ Present address: McArdle Laboratory for Cancer Research, University of Wisconsin, 450 N. Randall Avenue, Madison, Wisconsin 53706.

scribes the partial purification, levels, and some properties of the three classes of cellular RNA polymerase during HSV- 1 infection. Partial purification of RNA polymerases I, II, III*, and 111xfrom HSV-l-infected and mock-infected cells was carried out by the method of Sugden and Keller (6) as described in Fig. 1. The enzymes were reproducibly obtained in high-activity yield from small amounts of starting material (l-2 g). Assay of early purification fractions for RNA polymerase activity in the presence and absence of cu-amanitin gives a rough determination of the relative levels of the enzymes. [However, as noted by Chambon ( 7) the activities in these crude extracts are not an accurate reflection of RNA polymerase levels.] There was no observable loss of RNA polymerase activities in the early stages of the enzyme solubilixation procedure. Furthermore, there was virtually no difference between the relative levels of activities found in HSV-l-infected and mock-infected cells during the first 8 hr of infection. The three classes of RNA polymerase activity from HSV-l-infected and mock-infected BHK cells were resolved by chromatography on DEAE-Sephadex-A25

577 0042~6622/78/0862-0577$02.00/O Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.





FIG. 1. Chromatographic separation of RNA polymerases on DEAE-Sephadex-A25 (a, b) and DEAE-cellulose (c, d). Enzymes were partially purified from mock-infected (a, c) and HSV-l-infected cells (b, d) harvested 8 hr postinfection. Assays for RNA polymerase activity (5) contained no a-amanitin (O), 0.1 pg/ml of cu-amanitin (O), or 100 ag/ml of aamanitin (A). Extracts were prepared from BHK21 Cl3 cells grown as monolayers (12) in 80-0~ bottles in Eagle’s medium supplemented with 10% (v/v) calf serum. Glasgow strain 17 syn+ HSV-1 grown as described by Bayliss et al. (13) and purified by the method of Spear and Roizman (14) was either added at a multiplicity of 20 or omitted. Cells were harvested at 0, 2,4,6, and 8 hr postinfection and simultaneously extracted in high salt followed by high-speed centrifugation to give a crude extract (Fraction A), followed by ammonium sulphate precipitation to give Fraction B (6). For DEAE-Sephadex-A25 chromatography, 340 RNA polymerase activity units of Fraction B from mock-infected and HSV-l-infected cells were simultaneously applied to two 1.1 X lo-cm columns equihbrated in 0.05 M Tris-HCl (pH 7.9), 1 n&f EDTA, 1 n&f dithiothreitol, 6 mM MgCh, 30% (v/v) glycerol, and 0.02 M ammonium sulphate. After the columns were washed in the same buffer, RNA polymerases were eluted by a linear gradient of 0.02 to 0.5 M ammonium sulphate (four column volumes) in the same buffer. Fractions, 1.2 ml, were taken and bovine serum albumin was added to give a final concentration of 1 mg/ml. Aliquots, 25 cl, were taken for assay of RNA polymerase activity. For DEAE cellulose chromatography, 130 RNA polymerase activity units of Fraction B from mock-infected and HSV-l-infected cells were simultaneously applied to two 0.7 x IO-cm columns equilibrated in the above buffer containing 0.04 M ammonium sulphate. After the columns were washed in the same buffer, RNA polymerases were eluted by a linear gradient of 0.04 to 0.4 M ammonium sulphate in the same buffer. Fractions, 0.6 ml, were taken and processed as described above. Each point constituted a genuine eukaryotic DNA-dependent

(Figs. la and lb). The polymerases eluted with increasing ammonium sulphate in the order I (cu-amanitin insensitive), II (sensitive to 0.1 pg/ml of a-amanitin), and III (sensitive to 100 pg/ml of a-amanitin). Polymerase III appeared as a double peak (111~ and 111~) similar to that found in a number of eukaryotic cell sources. The number of activity peaks, their elution position (with respect to ammonium sulphate concentration), and yield, were virtually identical in profiles obtained from HSV-linfected and mock-infected cells harvested at 2, 4, 6, and 8 hr postinfection. RNA polymerases freed of endogenous temby DEAE-Sephadex plates, separated chromatography, and assayed on exogenous templates appear to have activities proportional to actual cellular enzyme concentrations (8). Hence, by this type of analysis the levels of RNA polymerase in HSVl-infected cells remain constant relative to mock-infected cells over the first 8 hr of infection. RNA polymerases from HSV-linfected and mock-infected cells were also analyzed by DEAE-cellulose chromatography (Figs. lc and Id). The order in which the enzymes elute from DEAE-cellulose differs from that of DEAE-Sephadex-A25 (9). Increasing concentrations of ammonium sulphate co-elute RNA polymerase I and III between 0.05 and 0.1 it4 ammonium sulphate, followed by RNA polymerase II between 0.1 and 0.2 M ammonium sulphate. Again, there was close agreement between the profiles from HSV-l-infected and mock-infected cells in terms of the elution position of the two peaks and their yield. No evidence for an HSV-l-induced ribonucleoside triphosphate-incorporating activity was found. These data, however, do not exclude the possibility of loss of an RNA polymerase activity inhibited in the RNA polymerase activity, in that synthesis was uninhibited by rifampicin (added to assays at 10 ag/ml) dependent on added DNA and required the presence of all four ribonucleoside triphosphates. The level of enzyme activities was not significantly affected by endogenous ribonuclease activity during the time course of the assays as determined by the ability of semipurified enzyme fractions to digest 18s ribosomal rz2P]RNA (approximately 20,000 cpm/pg) under standard assay conditions (minus DNA and nucleoside triphosphates).








FIG. 2. Determination of RNA polymerase II levels during the course of HSV-1 infection using the technique of r3H]amanitin binding (10). Fraction A, 0.15 ml, from mock-infected, (0) and HSV-l-infected (0) cells were reacted with lo-’ pg (approximately 4 x IO4 cpm) of [O-3H]-methyl-demethyl-y-amanitin, (a generous gift from Professor P. Chambon, Strasbourg). Aliquots, 0.15 ml, gave approximately 800 cpm. Blank values, obtained by preincubation of the enzyme with unlabeled o-amanitin prior to the addition of [3H]amanitin, were approximately 15%.

early stages of the isolation procedure or an enzyme unable to function under the assay conditions chosen. RNA polymerase II binds cw-amanitin tightly, with absolute specificity in a 1:l molar complex (10). Hence, RNA polymerase II levels were measured directly by determination of the [3HJamanitin-binding ability of crude extracts of HSV-l-infected and mock-infected cells (Fig. 2). By this criterion RNA polymerase II levels remain virtually constant over the first 8 hr of infection. Because of its specificity, this type of determination is not subject to the inaccuracy of the “assay” method of estimating the level of RNA polymerase II in crude cell extracts. a-Amanitin inhibition curves for RNA polymerase II (partially purified by DEAE-Sephadex-A25 chromatography) from HSV-l-infected and mockinfected cells 8 hr postinfection were superimposable. Both were almost completely inhibited by 0.1 pg/ml of cw-amanitin and 50%inhibited by approximately 0,005pg/ml of a-amanitin (data not shown). It appears, therefore, that RNA polymerase II levels remain invariant during the course of infection of BHK cells with HSV1. Similar results have been obtained for

the RNA polymerases of KB cells infected with adenovirus-2 (II). It may be that the reported changes in RNA synthesis levels are due to an increased concentration of HSV-1 DNA in the nucleus or a preferential affinity of RNA polymerase II for the viral genome. Changes in the transcription pattern from “immediate early” to “early” may be due to HSV-l-induced modification of polymerase II or the synthesis of viral factors which interact with it. ACKNOWLEDGMENTS I wish to thank Professor J. H. Subak-Sharpe for his interest in this work and critical reading of the manuscript. Mr. Alexander Orr provided expert technical assistance. REFERENCES 1. ALWINE, J. C., STEINHART, W. L., and HIM,, C. W. Virology 00.302-307 (1974). 2. BEN-ZEEV, A., ASHER, Y., and BECKER, Y. Virology 71,302-311 (1976). 3. COSTANZO, R., CAMPADELLI-FIIJMF,, G., FOA-TOMASI, L., and CASSAI, E. J. Viral. 21, 9961001 (1977). 4. JAEHNING, J. A., WEINMANN, R., BRENDLEH, T. G., RASKAS, H. J., and ROEDER, R. G. In “RNA Polymerase” (R. Losik and M. Chamberlin, eds.), pp. 819-834. Cold Spring Harbor Labora-




tory, Cold Spring Harbor, N.Y. 5. CLEMENTS, J. B., and HAY, J. J. G’en. Viral. 36, l-12 (1977). 6. SUGDEN, B., and KELLER, W. J. Bid. Chem. 248, 3777-3788 (1973). 7, CHAMBON, P. Anna Rev. B&hem. 44, 613-633 (1975). 8. SCHWARTZ, L. B., SKLAR, V. E. F., JAEHNING, J. A., WEINMANN, R., and ROEDER, R. G. J. Bid. Chem. 249,5889-5897 (1974). 9. HOSSENLOPP, P., WELLS, D., and CHAMBON, P.

Eur. J. Biochem. 68,237-251 (1975). 10. COCHET-MEILHAC, M., and CHAMBON, P. Biochim. Bbphys. Acta 353, SO-164 (1974). 11. WEINMANN, R., JAEHNING, J. A., RASKAS, H. J., and ROEDER, R. G. J. Virol. 17.114-126 (1976). 12. MACPHERSON, I., and STOKER, M. Virology 16, 147-151 (1962). 13. BA~LISS, G. J., MARSDEN, H. S., and HAY, J. Virology 68, 124-134 (1975). 14. SPEAR, P., and ROIZMAN, B. J. Viral. 9, 143-159 (1972).

Levels of DNA-dependent RNA polymerases in herpes simplex virus-infected BHK21 C13 cells.

VIROLOGY 86,577~580 (1978) Levels of DNA-Dependent RNA Polymerases in Herpes Simplex VirusInfected BHK21 Cl3 Cells PETER A. LOWE’ M. R. C. Virology...
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