polymerase of herpes simplex virus type 1 was partiallypurified and isolated in ... samte molecular weight as the wild-type enzyme (140,000 to 149,000) but was.
ANTIMICROBiAL AGENTS AND CHZMOTHERAPY, Jan. 1978, p. 124-127 Copyright 0 1978 American Society for Microbiology
Vol. 13, No. 1 Printed in U.S.A.
Deoxyribonucleic Acid Polymerase of Wild-Type and Phosphonoacetic Acid-Resistant Mutant of Herpes Simplex Virus BERTOLD FRIDLENDER, NOR CHEJANOVSKY, AND YECHIEL BECKER* Laboratory for Molecular Virology, The Hebrew University-Hadassah Medical School, Jerusalem, Israel Received for publication 27 July 1977
The phosphonacetic acid (PAA)-susceptible deoxyribonucleic acid (DNA) polymerase of herpes simplex virus type 1 was partially purified and isolated in sucrose gradients and on double-strand DNA cellulose columns. The DNA polymerase isolated from cells infected with the PAA-resistant mutant had the samte molecular weight as the wild-type enzyme (140,000 to 149,000) but was consistently more resistant to PAA. Phosphonoacetic acid (PAA) was shown to 5'-triphosphate, 0.01 mM thymidine triphosinhibit the replication of herpes simplex virus phate, and 3.75 ,uCi of [3H]thymidine triphos(HSV) in vitro (11, 12) and in vivo (3, 10). phate (specific activity 50 Ci/mM; The RadiFurther studies showed that PAA inhibits the ochemical Center, Amersham, England), 0.5 replication of herpesviruses by selectively bind- mM dithiothreitol, 500 Mug of activated calf ing to the virus-coded deoxyribonucleic acid thymus DNA per ml, 100 mM tris(hydroxy(DNA) polymerase (6-9, 13). Hay and Subak- methyl)aminomethane (Tris)-hydrochloride (pH Sharpe (4) and Becker et al. (2) isolated PAA- 8.1) and 500 Mug of bovine serum albumin (A resistant mutants of HSV. Although the PAA- grade, Sigma Chemical Co., St. Louis, Mo.) resistant mutants were isolated in the presence per ml. The low salt reaction mixture was of 100 ,ug of PAA per ml, it was reported (4) prepared in the absence of KCI and with 10 that the viral DNA polymerase (assayed in mM MgCl2. The HSV DNA polymerases from nuclear crude extracts) from the PAA-resistant mutants was inhibited over 50% by 2 to 5 ,ug of homogenates prepared by a modification of the PAA per ml. In our study (2), it was demon- technique of Alberts and Herrick (1) were chrostrated that the in vitro DNA synthesis in matographed on double-stranded DNA cellunuclei isolated from cells infected with a PAA- lose columns (1). After preincubation for 2 min resistant mutant of HSV type 1 was fully resist- at 37°C, the nuclei were treated with micrococant to 100 ,ug of PAA per ml. The aim of the cal endonuclease (Worthington Biochemical present study was to isolate the DNA polymer- Co.) in 20 mM Tris-hydrochloride (pH 7.5), 8% ase from cells infected with the wild-type and (wt/vol) sucrose, and 0.1 mM CaCl2 for 2 min the PAA-resistant mutants of HSV and to at 37C. The reaction was stopped by the addistudy the effect of PAA on the two enzyme tion of 10 Mg each of ethyleneglycol-bis(,8-amipreparations. noethyl ether)-N,N-tetraacetic acid per ml and BSC-1 cells were infected with the wild-type 10 mM MgCl2. Phosphate buffer (1.0 M, pH HF strain of HSV type 1 and the PAA-resistant 7.5) was added to a final concentration of 50 mutant as described previously (2). The in- mM for 10 min at 4°C. To this was added 100 fected cells were harvested 12 h after infection. MAg of pancreatic bovine deoxyribonuclease The DNA polymerases were isolated from ho- (Sigma, St. Louis, Mo.) per ml in a buffer mogenates of infected nuclei by ammonium containing 20 mM Tris-hydrochloride (pH 8.1) sulfate precipitation followed by centrifugation 2 mM CaCl2, 10 mM MgCl2, 1 mM ethylenediain sucrose gradients as described by Hirai and minetetraacetic acid (EDTA), 1 mM 2-mercapWatanabe (5). The viral DNA polymerases toethanol, 500 ug of bovine serum albumin per were assayed in reaction mixtures containing ml, 10% (vol/vol) glycerol, and 0.6 M NaCl for either high or low salt concentrations. The 20 min at 20°C. The reaction was stopped by high salt reaction mixture contained 250 mM transfer to ice. This was followed by dialysis KCI [or 100 mM (NH4)2SO41, 2 mM MgCl2, 0.05 against the column buffer (20 mM Tris-hydromM each of deoxyguanosine 5'-triphosphate, chloride (pH 8.1)-50 mM KCl-1 mM EDTA-1 deoxyadenosine 5'-triphosphate, deoxycytidine mM 2-mercaptoethanol-10% (vol/vol) glycerol124
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VOL. 13, 1978
100 ,ug of bovine serum albumin per ml) overnight with at least two changes of buffer. The preparations were placed on the columns and stepwise elution was performed at a rate of 6 ml/h with 10 ml each of 0.15 M, 0.3 M, 0.6 M, 1.0 M, and 2.0 M KCl. Samples of 1 ml were collected, and 25-Al portions were removed from each tube and incubated in a high salt reaction mixture for DNA synthesis. Centrifugation of ammonium sulfate-precipitated nuclear homogenates in sucrose gradients led to isolation of the viral DNA polymerase from cells infected with both the wildtype (Fig. 1A) and the PAA-resistant mutants (Fig. 1B) of HSV. The enzyme was able to synthesize DNA on activated calf thymus DNA in vitro in the presence of the four deoxyribonucleoside trisphosphates and 250 mM KCl. In a reaction mixture containing 10 mM KCl the viral DNA polymerase was inactive. In this respect, the enzyme had the properties reported
125
by Weissbach et. al (14) for the HSV DNA polymerase. The wild-type DNA polymerase activity was completely inhibited by the addition of 5 p.g of PAA per ml to the reaction mixture. The viral DNA polymerase from cells infected with the PAA-resistant mutant was more resistant to PAA although only about 30% of the DNA polymerase activity remained in the presence of PAA (Fig. 1B). The virus DNA polymerase had a mean molecular weight of 140,000 to 149,000 in relation to the markers included in the sucrose gradients (Fig. 1). This differs from Weissbach et al. (14) who found that the HSV DNA polymerase has a molecular weight of 180,000. The difference in molecular weight may be due to differences in technique. To clarify the differences between the PAAsensitive and -resistant DNA polymerases, samples of the enzyme preparations isolated in sucrose gradients were incubated with increasing concentrations of PAA (Fig. 1C). The activ-
200 100
A
50
IgG 12-, 0
-I>
,l ,,0 i
DIN5e
100
so PAA
P9/mI
\c-AmnyLase 0.
80 80
~~~~~~~DNase
20
LOJ 100
F R A C T IO N FIG. 1. Sucrose gradient analysis of DNA polymerases from wild-type and PAA-resistant HSV. Nuclear extracts from wild-type virus (A) and the PAA-resistant mutant (B) were layered onto 10 to 20% (wtlvol) sucrose gradients and centrifuged at 35,000 rpm for 15 h at 4°C in the SW 50.5 rotor of the Beckman ultracentrifuge. Immunoglobulin G, aeamylase, and DNase were centrifuged in separate gradients as markers and identified by ultraviolet absorption at 280 nm. The tubes were fractionated, and portions from each sample were assayed for DNA polymerase activity in vitro in the presence of 250 mM KCI (O), 100 mM (NH4) 204 (0), 10 mM KCl (A), and 250 mM KCl plus 5 pg of PAA (A) per ml. To determine the PAA sensitivity of the wild-type and PAA-resistant enzymes (C), samples from the peak region in A (0) and B (O), respectively, were tested against increasing concentrations of PAA.
126
NOTES
ANTIMICROB. AGENTs CHEMOTHER.
ity of the wild-type DNA polymerase was inhibited 95% by 2.5 yg of PAA per ml, whereas the PAA-resistant DNA polymerase was inhibited only 50% by the same drug concentration. About 30% of the DNA polymerase activity of the mutant strain was resistant to 50 Mug of PAA per ml. Chromatography on double-stranded DNA cellulose columns of nuclear homogenates from cells infected with the PAA-resistant mutant showed that most of the DNA polymerase activity eluted with 0.3 M KCI and was resistant to 0.25 Mug of PAA per ml when added to the reaction mixture (Fig. 2A). A similar elution pattern was obtained with the wild-type enzyme, which was sensitive to 0.25 Mug of PAA per ml (not shown). The viral DNA polymerases from cells infected with the wild-type and PAA-resistant virus, which eluted from doublestranded DNA cellulose columns with 0.3 M
KCO, were incubated in vitro with increasing concentrations of PAA (Fig. 2B). The enzyme from cells infected with the mutant virus was consistently more resistant to PAA than the wild-type DNA polymerase. The results of the present study reveal that the HSV DNA polymerase obtained from cells infected with the PAA-resistant mutant is only partially resistant to PAA at concentrations higher than 2.5 ,g/ml when assayed in vitro, even though the PAA-resistant mutant was selected in the presence of 100 ,ug/ml. The partial resistance of the enzyme in vitro, together with our previous observation (2) that in isolated nuclei the mutant viral DNA polymerase if fully resistant to 100 ug of PAA per ml, lead us to conclude that the nuclear membrane prevents penetration of the drug into the nucleus.
2 0 Icx 0. 0 cr u 0
z x a.
I I
PAA. ( pg/nL )
FIG. 2. Chromatography of PAA-resistant DNA polymerase on double-stranded DNA cellulose columns. Elution with 0.15, 0.3, 0.6, 1.0, and 2.0 M KCI was performed as described in the text (A). Portions from each fraction were assayed for DNA polymerase activity in the presence of 250 mM KCI (a) and with the addition of 0.25 pg of PAA (0). The enzyme that eluted with 0.3 M KCl was tested with increasing concentrations of PAA (B). Both the wild type (0) and PAA resistant (a) enzymes were tested.
VOL. 13, 1978 This research was supported by Public Health Service contract no. NO1-CP-3-3310 within the Virus Cancer Program of the National Cancer Institute. We thank Julia Hadar for assistance in preparing the manuscript and Yaffa Cohen for excellent technical assistance. B. F. is a visiting scientist of the Lady Davis Fellowship Trust, Jerusalem, Israel. LITERATURE CITED 1. Alberts, B., and G. Herrick. 1971. DNA-cellulose chromatography. Methods Enzymol. 21:198-217. 2. Becker, Y., Y. Asher, Y. Cohen, G. Weinberg-Zahlering, and J. Shlomai. 1977. Phosphonoacetic acid-resistant mutants of herpes simplex virus: effect of phosphonoacetic acid on virus replication and in vitro DNA synthesis in isolated nuclei. Antimicrob. Agents Chemother. 11:919-922. 3. Gerstein, D. D., C. R. Dawson, and J. 0. Oh. 1975. Phosphonoacetic acid in the treatment of experimental herpes simplex keratitis. Antimicrob. Agents Chemother. 7:285-288. 4. Hay, J., and J. Subak-Sharpe. 1976. Mutants of herpes simplex virus type 1 and 2 that are resistant to phosphonoacetic acid induce altered DNA polymerase activities in infected cells. J. Gen. Virol. 31:145-148. 5. Hirai, K., and Y. Watanabe. 1976. Induction of a-type DNA polymerases in human cytomegalovirus-infected WI-38 cells. Biochim. Biophys. Acta 447:328339. 6. Huang, H. S. 1975. Human cytomegalovirus IV. Specific inhibition of virus-induced DNA polymerase activity and viral DNA replication by phosphonoace-
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tic acid. J. Virol. 16:1560-1565. 7. Leinbach, S. S., J. M. Rer.o, L. F. Lee, A. F. Isball, and J. A. Boezi. 1976. Mechanism of phosphonoacetate inhibition of herpesvirus-induced DNA polymerase. Biochemistry 15:426-430. 8. Mao, J. C. H., and E. E. Robishaw. 1975. Mode of inhibition of herpes simplex virus DNA polymerase by phosphonoacetate. Biochemistry 14:5475-5479. 9. Mao, J. C. H., E. E. Robishaw, and L. R. Overby. 1975. Inhibition of DNA polymerase from herpes simplex virus-infected WI-38 by phosphonoacetic acid. J. Virol. 15:1281-1285. 10. Meyer, R. F., E. D. Vanell, and H. E. Kaufman. 1976. Phosphonoacetic acid in the treatment of experimental ocular herpes simplex infections. Antimicrob. Agents Chemother. 9:308-311. 11. Overby, L. B., E. E. Rubishaw, A. Schleicher, N. L. Rueter, N. L. Shipkowitz, and J. C. H. Mao. 1974. Inhibition of herpes simplex virus replication by phosphonoacetic acid. Antimicrob. Agents Chemother. 6:360-365. 12. Shipkowitz, N. L., R. R. Bower, R. N. Appell, C. W. Nordeen, L. R. Overby, W. R. Roderick, J. B. Schleicher, and A. A. Von Esch. 1973. Suppression of herpes simplex virus infection by phosphonoacetic acid. Appl. Microbiol. 26:264-267. 13. Summers, W. C., and G. Klein. 1976. Inhibition of EBV DNA synthesis and late gene expression by phosphonoacetic acid. J. Virol. 18:151-155. 14. Weissbach, A., S. L. Hong, J. Aucker, and R. Muller. 1973. Characterization of herpes simplex virus-induced deoxyribonucleic acid polymerase. J. Biol. Chem. 248:6270-6277.
