VIROLOGY
84, 551-554
Selective
BERTOLD
(1978)
Inhibition
of Herpes Simplex Virus Type 1 DNA Polymerase by Zinc Ions
FRIDLENDER,’
Laboratory for Molecular
NOR
CHEJANOVSKY,
AND YECHIEL
Viorlogy, Hebrew University-Hadassah Accepted October 20,1977
BECKER
Medical School, Jerusalem, Israel
The DNA polymerase of herpes simplex virus (HSV) was isolated from nuclei of infected BSC-1 cells by centrifugation in sucrose gradients and by chromatography on double-stranded DNA cellulose columns. The viral enzyme was almost completely inhibited in vitro by 0.1 mM zinc acetate. The (Yand p cellular DNA polymerases were resistant to 0.3 m&f zinc acetate when tested under in vitro conditions. Inhibition of HSV renlication can be explained by the selective inhibitory effect of zinc ions on the viral DNA polymerase.
Badiochemical Centre, Amersham, England), 500 pglml of activated calf thymus DNA, 10 mM Tris * HCl, pH 8.1, and 0.5 mglml of bovine serum albumin. The assays for the cellular DNA polymerase at low salt concentration were done with 10 mikf MgCl,. The HSV DNA polymerase was isolated from homogenates of infected nuclei by chromatography on double-stranded DNA cellulose columns. This was similar to the method used by Powell and Purifoy (51. The nuclear homogenates were prepared by a modification of the technique of Alberts and Herrick (6), After preincubation for 2 min at 37”, the nuclei were treated with micrococcal endonuclease (Worthington Biochemical Co.j in 20 mM Tris * HCl, pH 7.5, 8% (w/v) sucrose, and 0.1 n&f CaCl, for 2 min at 37” to digest the chromatin. The reaction was stopped by the addition of 10 pg/ml each of ethylene glycol tetraacetic acid (EGTA) and MgC12. Phosphate buffer (1.0 M, pH 7.5) was added to a final concentration of 50 n-&f for 10 min at 4”. To this was added 10 pglml of pancreatic bovine DNase (Sigma, St. Louis, MO.) in a buffer containing 20 mM Tris. HCl, pH 8.1, 2 mM CaCl,, 10 mM MgC&, 1 mM EDTA, 1 mM 2-mercaptoethanol, 500 pg of bovine serum albumin (A grade, Sigma, St. Louis, MO.), 10% (v/ v) glycerol, and 0.6 M NaCl for 20 min at
Zinc ions were found to inhibit the replication of herpes simplex virus (HSV) in BSC-1 cells (1). Further studies (2) revealed that 0.2 mM ZnSO, markedly inhibited the synthesis of HSV DNA in isolated nuclei in vitro. It was also shown that zinc ions did not affect cell DNA synthesis in the isolated nuclei, but markedly inhibited the synthesis of viral DNA. The present study was undertaken to study the effect of zinc ions on the partially purified HSV-specific and cellular DNA polymerases. BSC-1 cells were infected with the HF strain of HSV type 1. Infected and mockinfected cells were harvested at 12 hr postinfection and fractionated to separate the cytoplasm and nuclei as previously described (3). The cellular and viral DNA polymerases were isolated from the nuclear extracts by precipitation with 70% (w/v) ammonium sulfate and centrifugation in sucrose gradients exactly as described by Hirai and Watanabe (4). The cellular and viral enzymes were assayed in the presence of low (10 mM) and high (250 n-&f) concentrations of KCl, respectively. The reaction mixtures contained 2 mM MgC&, 0.05 mM (each) dGTP, dATP, and dCTP, 0.01 n&f ‘ITP, and 3.75 &i of [3H17.“l’P(specific activity 50 Ci/mmol, The 1 Visiting scientist from the Lady Davis Fellowship Trust, Jerusalem. 551
004%6822/78/0842-0551$02.00/O
Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.
552
SHORT
COMMUNICATIONS
20”. This was to remove completely all cellular and viral DNA. The reaction was stopped by transfer into ice. This was followed by dialysis against the column buffer (20 n&f Tris * HCI, pH 8.1, 50 mM KCI, 1 mM EDTA, 1 mM 2-mercaptoethanol, JO% (V/V) glycerol, and 10 pg/ml of bovine serum albumin) 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/hr with 10 ml each of 0.15, 0.3, 0.6, 1.0, and 2.0 M KCl. One-milliliter samples were collected and 25-4 aliquots were removed from each tube and incubated in high-salt reaction mixture for DNA synthesis. Mock-infected cells were similarly treated. The viral DNA polymerase was
incubated with different concentrations of zinc acetate to determine the effect on the ability of the enzyme to synthesize DNA in vitro. Centrifugation of nuclear homogenates from infected cells in sucrose gradients resulted in the isolation of the viral DNA polymerase (Fig. 1A) that was active when incubated with 250 m&f KCI. Incubation in the presence of 0.1 n&f zinc acetate resulted in almost complete inhibition of the viral DNA polymerase activity. In numerous experiments the (Y and /? cellular DNA polymerases could be detected in both infected and uninfected cells when incubated in the presence of a low salt concentration (10 mM KCl). In uninfected cells, both cellular DNA polymerases
B
J 0
10
20
30
40
50
60
FRACTION
FIG. 1. Sucrose gradient analysis of viral and cellular DNA polymerases. Nuclear extracts from infected (A) and mock-infected (B) BSC-1 cells were layered onto lo-208 (w/v) sucrose gradients and centrifuged for 15 hr at 36,000 rpm at 4” in the SW 50.1 rotor of the Beckman ultracentrifuge. The tubes were fractionated and aliquots from each sample were assayed for DNA polymerase activity in uitro. Symbols: (A) in the A); in the presence of 250 mM KC1 without (0-O) or with (O- - - - -0) 0.1 presence of 10 mM KC1 (AmM zinc acetate. (B) In the presence of 10 mM KC1 without (0-O) or with (A-A) 0.3 mil4 zinc acetate, or with 5 pg/ml of PAA (O- - - - -0).
SHORT
COMMUNICATIONS
showed resistance to the effect of 0.3 mM zinc acetate (Fig. 1B). Thus the a! and p cellular DNA polymerases are resistant to zinc ions, while the HSV DNA polymerase is highly sensitive. The positions of the (Y- and P-DNA polymerases in the sucrose gradients were determined by sensitivity to phosphonoacetic acid (PAA) and by comparison with other gradients that contained the a-DNA polymerase in larger amounts. Inclusion of 5 pg/ml of PAA in the reaction mixture distinguished between the cellular P-DNA polymerase which is resistant and the (YDNA polymerase which is partially sensitive to PAA (Fig. 1B).
553
Chromatography on double-stranded DNA cellulose columns revealed that most of the HSV DNA polymerase was eluted from the column by 0.3 M KC1 (Fig. 2A). Such a DNA polymerase was not found in mock-infected cells (Fig. 2B). Some viral DNA polymerase (about 10% of the main peak) eluted with 0.6 M KC1 (Fig. 2A). This is an enzyme with the same properties as that eluted by 0.3 M KCI. A concentration of 0.1 n-144zinc acetate caused 70% inhibition of the activity of the viral DNA polymerase (Fig. 20, whereas 5 mM zinc acetate almost completely inhibited the viral enzyme. This study extends the previous reports
x
E *oa . ”
FRACTION FIG. 2. Chromatography on double-stranded DNA cellulose columns. Nuclear extracts from infected (A) and mock-infected (B) BSC-1 cells, prepared as described in the text, were eluted with 0.15, 0.3, 0.6, 1.0, and 2.0 M KCl. Fractions from the column were assayed in uitro for DNA polymerase activity with 250 mM KC1 in the absence (0-O) or presence (O- - - - -0) of 0.25 pg/ml of PAA to identify the viral enzyme which is sensitive to PAA (8). The viral enzyme that eluted with 0.3 M KC1 was tested with increasing concentrations of zinc acetate (C).
554
SHORT COMMUNICATIONS
on the effect of zinc ions on HSV replication (1, 2). It is shown that zinc ions interfere with the activity of the viral DNA polymerases under in vitro conditions, while only a slight effect was noted on the cellular (Y- and P-DNA polymerases. These findings provide an explanation for the selective inhibitory effect of zinc ions on HSV DNA replication in infected cells and may account for the finding of de Roeth (7) that zinc sulfate (0.5%, w/v) is effective in the treatment of herpes keratitis in man. ACKNOWLEDGMENTS This research was supported by Contract No. NOl-CP-3-3310 within the Virus Cancer Program of the National Cancer Institute, Bethesda, Maryland. The excellent technical assistance of Yaffa Cohen is acknowledged.
REFERENCES 1. GORDON, Y. J., ASHER, Y., and BECKER, Y., Antimicrob. Agents Chemother. 8, 377-380 (1975). 2. SHLOMAI, J., ASHER, Y., GORDON, Y. J., OLSHEVSKY, U., and BECKER, Y., Virology 66, 330-335 (1975). 3. BEN-ZEEV, A., ASHER, Y., and BECKER, Y., Virology 71,302-311 (1976). 4. HIRAI, K., and WATANABE, Y., Biochim. Biophys. Actu 447,328-339 (1976). 5. POWELL, K. L., and PURIFOY, D. J. M., Zntervirology 7,225-239 (1976). 6. ALBERTS, B., and HERRICK, G., In “Methods in Enzymology. Nucleic Acids. Part D” (L. Grossman and K. Moldave, eds.), Vol. 21, pp. 198-217. Academic Press, New York, 1971. 7. DE ROETH, A., Amer. J. Ophthamol. 56.729-731 (1963). 8. F’RIDLENDER, B., CHEJANOVSKY, N., and BECKER, Y., Antimicrob. Agents Chemother. 13, (1978).
84, 551-554
Selective
BERTOLD
(1978)
Inhibition
of Herpes Simplex Virus Type 1 DNA Polymerase by Zinc Ions
FRIDLENDER,’
Laboratory for Molecular
NOR
CHEJANOVSKY,
AND YECHIEL
Viorlogy, Hebrew University-Hadassah Accepted October 20,1977
BECKER
Medical School, Jerusalem, Israel
The DNA polymerase of herpes simplex virus (HSV) was isolated from nuclei of infected BSC-1 cells by centrifugation in sucrose gradients and by chromatography on double-stranded DNA cellulose columns. The viral enzyme was almost completely inhibited in vitro by 0.1 mM zinc acetate. The (Yand p cellular DNA polymerases were resistant to 0.3 m&f zinc acetate when tested under in vitro conditions. Inhibition of HSV renlication can be explained by the selective inhibitory effect of zinc ions on the viral DNA polymerase.
Badiochemical Centre, Amersham, England), 500 pglml of activated calf thymus DNA, 10 mM Tris * HCl, pH 8.1, and 0.5 mglml of bovine serum albumin. The assays for the cellular DNA polymerase at low salt concentration were done with 10 mikf MgCl,. The HSV DNA polymerase was isolated from homogenates of infected nuclei by chromatography on double-stranded DNA cellulose columns. This was similar to the method used by Powell and Purifoy (51. The nuclear homogenates were prepared by a modification of the technique of Alberts and Herrick (6), After preincubation for 2 min at 37”, the nuclei were treated with micrococcal endonuclease (Worthington Biochemical Co.j in 20 mM Tris * HCl, pH 7.5, 8% (w/v) sucrose, and 0.1 n&f CaCl, for 2 min at 37” to digest the chromatin. The reaction was stopped by the addition of 10 pg/ml each of ethylene glycol tetraacetic acid (EGTA) and MgC12. Phosphate buffer (1.0 M, pH 7.5) was added to a final concentration of 50 n-&f for 10 min at 4”. To this was added 10 pglml of pancreatic bovine DNase (Sigma, St. Louis, MO.) in a buffer containing 20 mM Tris. HCl, pH 8.1, 2 mM CaCl,, 10 mM MgC&, 1 mM EDTA, 1 mM 2-mercaptoethanol, 500 pg of bovine serum albumin (A grade, Sigma, St. Louis, MO.), 10% (v/ v) glycerol, and 0.6 M NaCl for 20 min at
Zinc ions were found to inhibit the replication of herpes simplex virus (HSV) in BSC-1 cells (1). Further studies (2) revealed that 0.2 mM ZnSO, markedly inhibited the synthesis of HSV DNA in isolated nuclei in vitro. It was also shown that zinc ions did not affect cell DNA synthesis in the isolated nuclei, but markedly inhibited the synthesis of viral DNA. The present study was undertaken to study the effect of zinc ions on the partially purified HSV-specific and cellular DNA polymerases. BSC-1 cells were infected with the HF strain of HSV type 1. Infected and mockinfected cells were harvested at 12 hr postinfection and fractionated to separate the cytoplasm and nuclei as previously described (3). The cellular and viral DNA polymerases were isolated from the nuclear extracts by precipitation with 70% (w/v) ammonium sulfate and centrifugation in sucrose gradients exactly as described by Hirai and Watanabe (4). The cellular and viral enzymes were assayed in the presence of low (10 mM) and high (250 n-&f) concentrations of KCl, respectively. The reaction mixtures contained 2 mM MgC&, 0.05 mM (each) dGTP, dATP, and dCTP, 0.01 n&f ‘ITP, and 3.75 &i of [3H17.“l’P(specific activity 50 Ci/mmol, The 1 Visiting scientist from the Lady Davis Fellowship Trust, Jerusalem. 551
004%6822/78/0842-0551$02.00/O
Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.
552
SHORT
COMMUNICATIONS
20”. This was to remove completely all cellular and viral DNA. The reaction was stopped by transfer into ice. This was followed by dialysis against the column buffer (20 n&f Tris * HCI, pH 8.1, 50 mM KCI, 1 mM EDTA, 1 mM 2-mercaptoethanol, JO% (V/V) glycerol, and 10 pg/ml of bovine serum albumin) 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/hr with 10 ml each of 0.15, 0.3, 0.6, 1.0, and 2.0 M KCl. One-milliliter samples were collected and 25-4 aliquots were removed from each tube and incubated in high-salt reaction mixture for DNA synthesis. Mock-infected cells were similarly treated. The viral DNA polymerase was
incubated with different concentrations of zinc acetate to determine the effect on the ability of the enzyme to synthesize DNA in vitro. Centrifugation of nuclear homogenates from infected cells in sucrose gradients resulted in the isolation of the viral DNA polymerase (Fig. 1A) that was active when incubated with 250 m&f KCI. Incubation in the presence of 0.1 n&f zinc acetate resulted in almost complete inhibition of the viral DNA polymerase activity. In numerous experiments the (Y and /? cellular DNA polymerases could be detected in both infected and uninfected cells when incubated in the presence of a low salt concentration (10 mM KCl). In uninfected cells, both cellular DNA polymerases
B
J 0
10
20
30
40
50
60
FRACTION
FIG. 1. Sucrose gradient analysis of viral and cellular DNA polymerases. Nuclear extracts from infected (A) and mock-infected (B) BSC-1 cells were layered onto lo-208 (w/v) sucrose gradients and centrifuged for 15 hr at 36,000 rpm at 4” in the SW 50.1 rotor of the Beckman ultracentrifuge. The tubes were fractionated and aliquots from each sample were assayed for DNA polymerase activity in uitro. Symbols: (A) in the A); in the presence of 250 mM KC1 without (0-O) or with (O- - - - -0) 0.1 presence of 10 mM KC1 (AmM zinc acetate. (B) In the presence of 10 mM KC1 without (0-O) or with (A-A) 0.3 mil4 zinc acetate, or with 5 pg/ml of PAA (O- - - - -0).
SHORT
COMMUNICATIONS
showed resistance to the effect of 0.3 mM zinc acetate (Fig. 1B). Thus the a! and p cellular DNA polymerases are resistant to zinc ions, while the HSV DNA polymerase is highly sensitive. The positions of the (Y- and P-DNA polymerases in the sucrose gradients were determined by sensitivity to phosphonoacetic acid (PAA) and by comparison with other gradients that contained the a-DNA polymerase in larger amounts. Inclusion of 5 pg/ml of PAA in the reaction mixture distinguished between the cellular P-DNA polymerase which is resistant and the (YDNA polymerase which is partially sensitive to PAA (Fig. 1B).
553
Chromatography on double-stranded DNA cellulose columns revealed that most of the HSV DNA polymerase was eluted from the column by 0.3 M KC1 (Fig. 2A). Such a DNA polymerase was not found in mock-infected cells (Fig. 2B). Some viral DNA polymerase (about 10% of the main peak) eluted with 0.6 M KC1 (Fig. 2A). This is an enzyme with the same properties as that eluted by 0.3 M KCI. A concentration of 0.1 n-144zinc acetate caused 70% inhibition of the activity of the viral DNA polymerase (Fig. 20, whereas 5 mM zinc acetate almost completely inhibited the viral enzyme. This study extends the previous reports
x
E *oa . ”
FRACTION FIG. 2. Chromatography on double-stranded DNA cellulose columns. Nuclear extracts from infected (A) and mock-infected (B) BSC-1 cells, prepared as described in the text, were eluted with 0.15, 0.3, 0.6, 1.0, and 2.0 M KCl. Fractions from the column were assayed in uitro for DNA polymerase activity with 250 mM KC1 in the absence (0-O) or presence (O- - - - -0) of 0.25 pg/ml of PAA to identify the viral enzyme which is sensitive to PAA (8). The viral enzyme that eluted with 0.3 M KC1 was tested with increasing concentrations of zinc acetate (C).
554
SHORT COMMUNICATIONS
on the effect of zinc ions on HSV replication (1, 2). It is shown that zinc ions interfere with the activity of the viral DNA polymerases under in vitro conditions, while only a slight effect was noted on the cellular (Y- and P-DNA polymerases. These findings provide an explanation for the selective inhibitory effect of zinc ions on HSV DNA replication in infected cells and may account for the finding of de Roeth (7) that zinc sulfate (0.5%, w/v) is effective in the treatment of herpes keratitis in man. ACKNOWLEDGMENTS This research was supported by Contract No. NOl-CP-3-3310 within the Virus Cancer Program of the National Cancer Institute, Bethesda, Maryland. The excellent technical assistance of Yaffa Cohen is acknowledged.
REFERENCES 1. GORDON, Y. J., ASHER, Y., and BECKER, Y., Antimicrob. Agents Chemother. 8, 377-380 (1975). 2. SHLOMAI, J., ASHER, Y., GORDON, Y. J., OLSHEVSKY, U., and BECKER, Y., Virology 66, 330-335 (1975). 3. BEN-ZEEV, A., ASHER, Y., and BECKER, Y., Virology 71,302-311 (1976). 4. HIRAI, K., and WATANABE, Y., Biochim. Biophys. Actu 447,328-339 (1976). 5. POWELL, K. L., and PURIFOY, D. J. M., Zntervirology 7,225-239 (1976). 6. ALBERTS, B., and HERRICK, G., In “Methods in Enzymology. Nucleic Acids. Part D” (L. Grossman and K. Moldave, eds.), Vol. 21, pp. 198-217. Academic Press, New York, 1971. 7. DE ROETH, A., Amer. J. Ophthamol. 56.729-731 (1963). 8. F’RIDLENDER, B., CHEJANOVSKY, N., and BECKER, Y., Antimicrob. Agents Chemother. 13, (1978).
