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Effect of zinc ions on the synthesis of herpes simplex virus DNA in infected BSC-1 cells
VIROLOGY 66, 330-335 (1975)
Effect of Zinc Ions on the Synthesis DNA in Infected
of Herpes
BSC-1
Simplex
Virus
Cells
JOSEPH SHLOMAI, YAEL ASHER, YITZHAK J. GORDON, UDY OLSHEVSKY, AND YECHIEL BECKER Laboratory
for Molecular
Virology,
Hebrew
University-Hadassah
Accepted
March
Medical
School, Jerusalem,
Israel
II, 1975
Treatment of herpes simplex virus-infected cells with Z&O, (0.2 mM) inhibited the synthesis of viral DNA. A similar concentration of Z&O, had no effect on DNA synthesis in uninfected cells. In vitro analyses revealed a marked depression of the DNA-polymerase activity in nuclei obtained from zinc-treated infected cells.
Zinc ions inhibit the replication of rhinoviruses in infected cells (I) and prevent the posttranscriptional processing of the precursor peptides to rhinovirus and picornavirus structural proteins (2). Zinc ions are also inhibitory to the formation of giant cells (3) and to virus replication in herpes simplex virus (HSV)-infected cells (4). A concentration of 0.2 mM ZnSO, added at the end of the virus adsorption period irreversibly inhibited the replication of HSV in BSC-1 cells (4). Since zinc ions are required for the function of DNA- and RNA-polymerases (5-8), it was of interest to study the effect of zinc on the synthesis of HSV DNA. Two approaches were used: a) DNA molecules synthesized in HSVinfected cells in the absence or presence of zinc ions were analysed in CsCl density gradients. This technique separated viral DNA (density, 1.718 g/ml) from cellular DNA (density, 1.700 g/ml) (9), and b) the DNA-dependent DNA-polymerase activity in nuclei isolated from zinc-treated and untreated infected cells was tested under in vitro conditions that permit the assay of the viral polymerase only (9). The present study demonstrates that the synthesis of viral DNA is inhibited in the presence of 0.2 mM zinc sulfate. Zinc ions markedly affected the DNA-polymerase activity in infected nuclei, assayed in uitro.
Herpes simplex virus type 1 (HF strain) was propagated in BSC-1 cells. At 3 hr postinfection (p.i.) the infected cells were labeled with [3H]thymidine in the absence or presence of ZnSO, at concentrations of 0.1, 0.2 and 0.3 mM. The cultures were harvested at the end of the virus growth cycle (18 hr p.i.) and the DNA was isolated and centrifuged in CsCl density gradients. The viral DNA, banding at a density of 1.718 g/ml, was diluted 1:40 in TEN buffer (0.01 M Tris-HCl, pH 8.1, 0.001 M EDTA and 1 M NaCl) and centrifuged in sucrose gradients to determine its molecular size. Purified HSV-DNA was used as a marker. The viral DNA-polymerase activity in nuclei from infected untreated and zinctreated cells was assayed in vitro (9). Virus infectivity was determined by plaque assay on BSC-1 monolayers. Treatment of HSV-infected BSC-1 cells with 0.2 mM ZnSO, prevented the formation of herpesvirus particles and markedly affected the synthesis of viral DNA. In untreated cells, virions labeled with [3H]thymidine were produced (Fig. 1). This agrees with the finding that 0.2 mM ZnSO, completely and irreversibly prevented the synthesis of infectious HSV (4). Titration of HSV progeny from infected untreated cells revealed the production of 165 plaque-forming units per cell. The
330 Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
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synthesis of virus in infected cells treated with 0.2 mM ZnSO, was reduced to 0.22 plaque-forming units per cell, most likely the residual infectious virus in these cells. This result confirms the results presented in Fig. 1. The effect of 0.2 mM ZnSO, on the rate of DNA synthesis in HSV-infected BSC-1 cells is presented in Fig. 2. The infected zinc-treated and untreated cells were pulselabeled for 1 hr with [SH]thymidine at
u-3 ‘2 , I
Ii ”
FRACTION
FIG. 1. Inhibition of HSV synthesis in infected cells by 0.2 mM Z&O,. Two cultures of BSC-1 cells were infected with HSV (m.o.i., 10 PFU/cell). After incubation for 3 hr at 37” the medium was removed, the cells were washed and reincubated in fresh Dulbecco’s modified Eagle’s medium without serum. Both cultures were labeled with 2 &i/ml of [3H]thymidine (specific activity, 12.3 Ci/mmole, Nuclear Research Center, Negev). To one culture, 0.2 m&f ZnSO, was added. The cultures were further incubated at 37” for 15 hr and then harvested by scraping into reticulocyte standard buffer (RSB) without Mgz+ (0.01 M Tris-HCl, pH 7.7, 0.01 A4 KCl). The cell suspensions were sonicated, and the homogenates were layered onto sucrose gradients (12-52%, w/w) prepared in TBS buffer (0.2 M Tris-HCl, pH 8.3, 0.85% NaCl). The gradients were centrifuged in an SW 50.1 rotor of a Beckman ultracentrifuge at 40,000 rpm for 15 min at 4’ to isolate the virus particles. The gradients were collected dropwise and the trichloroacetic acid (TCA)-precipitable radioactivity was determined. 04, Untreated, infected cells; O-----O, zinctreated infected cells.
1;:I ,‘~~l~l~~........*~~~ ZnSOL ; 36 13
TIME
18
(HRS)
FIG. 2. Effect of 0.2 mA4 ZnSO, on the rate of DNA synthesis in HSV-infected cells. BSC-1 cells (in 30-mm plates) were infected with HSV at a m.o.i. of 10 PFU/cell. At 3 hr pi., the medium was replaced with fresh medium with or without 0.2 mM ZnSO,, and the cultures were reincubated at 37”. At different time intervals after infection (3, 6, 13 and 18 hr), the medium was replaced with 10 ml of medium per plate containing 5 rCi/ml of [sH]thymidine with or without 0.2 mM ZnSO,. After incubation for 1 hr, the cultures were harvested with 0.5% (w/v) sodium dodecyl sulfate (SDS). The TCA-precipitable radioactivity was determined. The radioactivity incorporated into the infected untreated cells during a l-hr pulse at 3 hr p.i. was regarded as 100% and was used to calculate the radioactivity incorporated during each l-hr pulse. 04, Infected, untreated; O-----O, infected zinctreated (0.2 mM ZnSO,).
different time intervals. Untreated infected cells synthesized DNA at a constant rate up to 13 hr p.i. when DNA synthesis was reduced due to termination of the virus growth cycle. The time course of HSVDNA synthesis in infected BSC-1 cells was recently reported (9). CsCl density gradient analysis of the DNA molecules synthesized in the infected cells revealed that 90-95% of the labeled DNA molecules are viral and the rest are cellular DNA molecules. Zinc ions markedly inhibited the synthesis of DNA in the infected cells throughout the virus growth cycle (Fig. 2). The effect of zinc ions on the synthesis of virus-specific DNA, banding at a density of 1.718 g/ml, was determined. DNA labeled with [3H]thymidine from infected cultures untreated or treated with zinc sulfate was analyzed by centrifugation in CsCl density gradients. Less DNA was synthesized in the presence of 0.1 mM ZnSO, (Fig. 3B) than in untreated infected cells (Fig. 3A). The analysis of five experiments revealed that 0.1 mM ZnSO, reduced HSV-DNA
-loQ.lo6
a
I (Y ‘S ;
-
;L . 0
B 0.1 mM
Zn”
t l> tv 6 0 0 6 a
boo 0 aa OtrlY
I (3
FRACTION FIG. 3. Effect of zinc ions on viral and cellular DNA synthesis in HSV-infected cells. Three BSC-1 cultures were infected with HSV and labeled with [qlthymidine as described in Fig. 1. At the end of the adsorption period one culture served as a control (A), the second culture was treated with 0.1 mM ZnSO, (B) and the third culture was treated with 0.2 mM ZnSO, (C). The cultures were incubated for an additional period of 15 hr at 37” and then harvested. The cells were scraped into TBS buffer, the suspension was adjusted to a final concentration of 1 x SSC (0.15 M NaCl, 0.015 M Na citrate), SDS (l.O%, w/v) and 0.3 mg/ml of Pronase (80,000 U/mg, free of nucleases; Calbiochem) were added. The Pronase was preincubated for 1 hr at 37”. To each cell suspension, purified HSV-DNA labeled with [‘Clthymidine was added to serve as a density marker. The suspensions were incubated at 37” for 5 hr and the lysates were then carefully transferred to polyallomer centrifuge tubes containing 8.0 ml of CsCl at a density of 1.70 g/ml. The gradients were centrifuged in a 50 Ti fixed-angle rotor of a Beckman ultracentrifuge at 37,500 rpm for 48 hr at 20”. The gradients were collected dropwise into 300~pl fractions. One-third of each fraction was removed to determine the TCA-precipitable radioactivity in the synthesized DNA (O--O) and the ‘E-labeled HSV-DNA marker (O-----O). To determine the molecular size of the HSV-DNA, 75 ~1 from every fraction with a density of 1.718 g/ml was diluted 1:40 in TEN buffer and carefully layered onto 5-20% (w/w) sucrose gradients prepared in neutral DNA buffer (12). The gradients in nitrocellulose tubes (a, b, c, respectively) were centrifuged in an SW 27 rotor of a Beckman ultracentrifuge at 25,000 rpm for 5 hr at 20”. One-milliliter fractions were collected dropwise and the TCA-precipitable radioactivity was determined. Note different scales in a, b, and c. Purified [‘Hlthymidinelabeled HSV-DNA extracted from purified virions by treatment with Sarkosyl (4%, w/w) for 2 min at 63” was used as an external marker and centrifuged in a separate sucrose gradient (not shown). 332
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synthesis by 13.2% as compared to HSVDNA synthesis in untreated infected cells which are taken to be 100%. A marked inhibition of viral DNA synthesis occurred when the infected cells were treated with 0.2 mM ZnSO, (Fig. 3C). Removal of ZnSO, (0.2 mA4) from the infected cells after treatment for 18 hr and reincubation for 4 hr in the absence of the inhibitor resulted in an increase in the amount of viral DNA synthesized in the infected cells. The DNA molecules banding at a density of 1.718 g/ml in the CsCl gradients described above were centrifuged in sucrose gradients to determine their molecular weight. A molecular weight of 100 x lo6 (Figs. 3A-C), identical to that of DNA isolated from intact virus particles, was obtained. The effect of different concentrations of ZnSO, on the synthesis of HSV and cellular DNA is presented in Fig. 4. The infected, zinc-treated cells were labeled for 15 hr (3-18 hr p.i.) with [3H]thymidine. The DNA was then extracted and centrifuged in CsCl density gradients. The amounts of radioactivity banding at a density of 1.718 g/ml (viral DNA) and 1.700 g/ml (cellular DNA) were determined. Since HSV infection inhibits the synthesis of cellular DNA, the latter constituted only about 10% of the total DNA. It is of interest that zinc ions at a concentration of 0.1 mM had only a slight inhibitory effect on the synthesis of viral DNA, while at a concentration of 0.2 mA4 the synthesis of viral DNA was markedly inhibited. Residual cellular DNA synthesis and DNA synthesis in uninfected cells were not affected by 0.2 mM ZnSO, (Fig. 4). ZnSO, at a concentration of 0.3 mM inhibited the synthesis of both viral and the residual cellular DNA (Fig. 4). These results suggest that at a concentration of 0.2 mM, ZnSO, affects the mechanism responsible for synthesis of viral DNA. To determine the mode of action of zinc ions, their effect on the viral DNA-polymerase present in the nuclei of HSV-infected cells was studied in vitro. Under the in vitro assay conditions previously reported (9), no DNA-polymerase activity was demonstrable in nuclei from uninfected cells,
150-
.
UNINFECTED
INFECTED (Residual
0
I
I 0
I 0.l ZnSOL
1 0.2 Concentration
Cellular
DNA(l.7OqCrU)
, 0.3
I
(mM)
FIG. 4. Effect of ZnSO, on the synthesis of viral and cellular DNA in HSV-infected and uninfected cells. BSC-1 cells were infected as described in Fig. 1. Infected cells were either untreated or treated with 0.1,0.2 and 0.3 mM ZnSO,. The cultures were labeled with [SH]thymidine and harvested at 18 hr p.i. by treating with 1% (w/v) SDS and 0.3 mg/ml of Pronase. The DNA was extracted twice with phenol and three times with chloroform:isoamyl alcohol (25:l). The purified DNA preparations were centrifuged in CsCl gradients as described in Fig. 3. The gradients were collected dropwise, the density and the TCA-precipitable radioactivity were determined. The total radioactivity which bands in the CsCl gradients of infected cells with a mean density of 1.718 g/ml (viral DNA, O----O) and with a mean density of 1.700 g/ml (residual cellular DNA, O-----O) was calculated. The viral DNA which was obtained from infected untreated cells (no ZnSO,) was regarded as lOO%, and the viral DNA isolated from ZnSO,-treated cells was compared to it. The residual cellular DNA synthesized in the infected untreated cells (no ZnSO,) was regarded as 100% and the residual cellular DNA in the ZnSO,-treated cells was compared to it. It should be indicated that the residual cellular DNA is about 10% of the total labeled DNA in untreated HSV-infected cells. Uninfected BSC-1 cell cultures were labeled with [Wlthymidine in the absence and the presence of 0.1 and 0.2 mM ZnSO,. The TCA-precipitable radioactivity in the untreated and treated cells was determined (x-x). The radioactive-DNA in untreated uninfected cells was regarded as 100%.
whereas viral DNA was synthesized in nuclei from infected cells. The results presented in Fig. 5A demonstrate that nuclei from HSV-infected cells linearly incorporated [3H]TMP into viral DNA during a 30-min period of incubation in vitro, as was previously shown (9). The DNA-polym-
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TIME
(min)
FIG. 5. In vitro DNA-polymerase activity in nuclei from HSV-infected cells, untreated and treated with ZnSO,. Six BSC-1 cultures were infected with HSV as described in Fig. 1. At 3 hr pi. the cultures were either left untreated (A) or were treated with 0.1 (B), 0.2 (C, E, and F) or 0.3 mM ZnSO, (D) and incubated for an additional period of 15 hr. Two cultures (E and F) were washed at 18 hr p.i. to remove the inhibitor and reincubated for an additional period of 1.5 hr, prior to harvest. To culture F, puromycin (100 pg/ml) was added to inhibit protein synthesis. The cells were harvested and disrupted in a Dounce homogenizer. The nuclei were isolated and resuspended, at a concentration of 10’ per 100 pl of 75 mM phosphate, pH 7.4, to which the following were added: 3 mM 2-mercaptoethanol, 8% (w/v) sucrose (RNase free), 6 mM MgCI,, 40 mA4 each of dATP, dCTP, dGTP (Sigma, St. Louis, MO) and 50 &i of [SH]TTP (specific activity, 15 Wmole; Radiochemical Center, Amersham, England), in a final volume of 200 ~1 of phosphate buffer. The reaction mixtures were incubated at 37Oand duplicate samples of 25 ~1 each were removed at different times to tubes containing 10% (w/v) TCA. The TCA-precipitable radioactivity was determined.
erase activity, in nuclei isolated from HSVinfected cells treated in uiuo with 0.1 mM ZnSO, during a 15hr period (Fig. 5B) was as high as in untreated infected cells (Fig. 5A). It is not yet known why, under in uiuo conditions, 0.1 mM ZnSO, slightly inhibits HSV-DNA synthesis while the DNApolymerase activity in nuclei isolated from 0.1 mM ZnSO,-treated infected cells is unaffected. Inhibition of the DNA-polymerase activity was noted in nuclei isolated from infected cells treated with 0.2 mM (Fig. 5C) or 0.3 mA4 (Fig. 5D) ZnSO,.
To determine whether zinc ions prevent the synthesis of the virus-induced DNA polymerase or alternatively affect the activity of this enzyme, the kinetics of [$H]TMP incorporation into DNA were determined after removal of the zinc ions and inhibition of protein synthesis. Two cultures of infected BSC-1 cells, treated with 0.2 mM ZnSO, during a 15 hr period, were washed to remove the zinc ions and reincubated for an additional period of 1.5 hr at 37” in fresh medium. To prevent further protein synthesis, one of the cultures received puromycin (100 pg/ml) with the fresh medium. The nuclei were isolated at the end of a 1.5-hr incubation period in the absence of ZnSO,, and the DNApolymerase activity was determined. Removal of the zinc ions from the infected cells partially reactivated the DNApolymerase activity (Fig. 5E). In the absence of protein synthesis, the DNA polymerase was reactivated to an even greater extent (Fig. 5F). These results might suggest that the viral DNA polymerase is synthesized in HSV-infected cells treated with 0.2 mM ZnSO,, but the enzymatic activity is reduced in the presence of the zinc ions. It is of interest that, although removal of the zinc ions partially restores the activity of the viral DNA polymerase, no infectious virus progeny could be obtained from such cells even after an additional incubation period of 18 hr (4). The present study demonstrates that ZnSO, at a concentration of 0.2 mM prevents the synthesis of HSV-DNA by a mechanism which involves inhibition of the viral DNA-polymerase activity. In contrast, 0.2 mM ZnSO, had no effect on cellular DNA synthesis in uninfected cells. The mechanism by which zinc ions inhibit the viral DNA polymerase is not yet known. It is possible that zinc ions are required for the activity of the virusinduced DNA polymerase, but an excess of zinc ions inhibits this enzymatic activity. Alternatively, zinc ions may affect the cleavage of a peptide precursor to the viral DNA polymerase, thus leading to the inhibition of the viral enzyme; upon removal of zinc ions, such a precursor peptide may be processed causing enzymatic activity to
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appear. The ability of zinc ions to prevent the synthesis of HSV-DNA results in the inhibition of both virus replication and the formation of infectious virus progeny (4). Another phenomenon of interest is the effect of 0.1 mM Z&O, on the synthesis of herpesvirus progeny. It was shown that 0.1 mM ZnSO, only slightly inhibits the synthesis of intact viral DNA molecules. However, under these conditions treated cells synthesize only 5% of the infectious virus progeny (4). These observations suggest that either the HSV-DNA molecules synthesized in the presence of 0.1 mA4 ZnSO, are not functional or that ZnSO, inhibits other molecular processes associated with the assembly of the virus particles. Further studies are needed to determine the effect of ZnSO, on the synthesis of HSV-specified messenger RNA and proteins. Since ZnSO, (0.5%, w/v) has been reported to be effective in the treatment of herpetic keratitis in man (13), studies on the mechanism of its antiviral action may help in the evaluation of zinc as an antiviral agent. ACKNOWLEDGMENTS This study was supported by Contract No. NO1 CP 33310 from the Special Virus Cancer Program, National Cancer Institute, National Institutes of Health, Bethesda Md, U.S.A. Dr. Y. J. Gordon was on leave from the Department of Ophthalmology, Hadassah Hospital, Jerusalem.
The interest of Prof. H. Zauberman, head of the Department of Ophthalmology, is greatly acknowledged. REFERENCES 1. KORANT, B. D., KANER, J. C., and BUTTERWORTH, B. E., Nature (London) 248.588-590 (1974). 2. BUTTERWORTH,B. E., and KORANT, B. D., J. Viral. 14, 282-291 (1974). 3. FALKE, D., and KAHL, G. F. 2. Med. Microbial. Immunol. 153, 175-189 (1967). 4. GORDON, Y. J., ASHER, Y., and EECKER, Y., Antimicrob. Ag. Chemother., In press. 1975. 5. SLATER, J. P., MILDVAN, A. S., and LOEB, L. A., Biochem. Biophys. Res. Commun. 44, 37-43 (1968). 6. SCRUTFON, M. C., Wu, C. W., and GOLLYMIWAIT, D. A., Proc. Nat. Acad. Sci. USA 68, 2497-2501 (1971). 7. VALLEE, B. L., and WACKER, W. E. C., In The Proteins (H. Neurath, ed.), 2nd Ed, Vol. 5, Academic Press, New York, 1970. 8. AULD, D. S., KAWAGUCHI, H., LIVINGSTON, D. M., and VALLEE, B. L., Proc. Nat. Acad. Sci. USA 71, 2091-2095 (1974). 9. BECKER, Y., and ASHER, Y., Virology, 63, 209-220 (1975). 10. GORDIN, M., OLSHEVSKY, U., ROSENKRANZ, H. S., and BECKER, Y., Virology 55, 280-284 (1973). 11. BECKER, Y., DYM, H., and SAROV, I., Virology 36, 185-192 (1968). 12. FRENKEL, N., and ROIZMAN, B., J. Viral. 10, 565-572 (1972). 13. DE ROETH, A., Amer. J. Ophthalmol. 56, 729-731 (1963).
Effect of Zinc Ions on the Synthesis DNA in Infected
of Herpes
BSC-1
Simplex
Virus
Cells
JOSEPH SHLOMAI, YAEL ASHER, YITZHAK J. GORDON, UDY OLSHEVSKY, AND YECHIEL BECKER Laboratory
for Molecular
Virology,
Hebrew
University-Hadassah
Accepted
March
Medical
School, Jerusalem,
Israel
II, 1975
Treatment of herpes simplex virus-infected cells with Z&O, (0.2 mM) inhibited the synthesis of viral DNA. A similar concentration of Z&O, had no effect on DNA synthesis in uninfected cells. In vitro analyses revealed a marked depression of the DNA-polymerase activity in nuclei obtained from zinc-treated infected cells.
Zinc ions inhibit the replication of rhinoviruses in infected cells (I) and prevent the posttranscriptional processing of the precursor peptides to rhinovirus and picornavirus structural proteins (2). Zinc ions are also inhibitory to the formation of giant cells (3) and to virus replication in herpes simplex virus (HSV)-infected cells (4). A concentration of 0.2 mM ZnSO, added at the end of the virus adsorption period irreversibly inhibited the replication of HSV in BSC-1 cells (4). Since zinc ions are required for the function of DNA- and RNA-polymerases (5-8), it was of interest to study the effect of zinc on the synthesis of HSV DNA. Two approaches were used: a) DNA molecules synthesized in HSVinfected cells in the absence or presence of zinc ions were analysed in CsCl density gradients. This technique separated viral DNA (density, 1.718 g/ml) from cellular DNA (density, 1.700 g/ml) (9), and b) the DNA-dependent DNA-polymerase activity in nuclei isolated from zinc-treated and untreated infected cells was tested under in vitro conditions that permit the assay of the viral polymerase only (9). The present study demonstrates that the synthesis of viral DNA is inhibited in the presence of 0.2 mM zinc sulfate. Zinc ions markedly affected the DNA-polymerase activity in infected nuclei, assayed in uitro.
Herpes simplex virus type 1 (HF strain) was propagated in BSC-1 cells. At 3 hr postinfection (p.i.) the infected cells were labeled with [3H]thymidine in the absence or presence of ZnSO, at concentrations of 0.1, 0.2 and 0.3 mM. The cultures were harvested at the end of the virus growth cycle (18 hr p.i.) and the DNA was isolated and centrifuged in CsCl density gradients. The viral DNA, banding at a density of 1.718 g/ml, was diluted 1:40 in TEN buffer (0.01 M Tris-HCl, pH 8.1, 0.001 M EDTA and 1 M NaCl) and centrifuged in sucrose gradients to determine its molecular size. Purified HSV-DNA was used as a marker. The viral DNA-polymerase activity in nuclei from infected untreated and zinctreated cells was assayed in vitro (9). Virus infectivity was determined by plaque assay on BSC-1 monolayers. Treatment of HSV-infected BSC-1 cells with 0.2 mM ZnSO, prevented the formation of herpesvirus particles and markedly affected the synthesis of viral DNA. In untreated cells, virions labeled with [3H]thymidine were produced (Fig. 1). This agrees with the finding that 0.2 mM ZnSO, completely and irreversibly prevented the synthesis of infectious HSV (4). Titration of HSV progeny from infected untreated cells revealed the production of 165 plaque-forming units per cell. The
330 Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
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synthesis of virus in infected cells treated with 0.2 mM ZnSO, was reduced to 0.22 plaque-forming units per cell, most likely the residual infectious virus in these cells. This result confirms the results presented in Fig. 1. The effect of 0.2 mM ZnSO, on the rate of DNA synthesis in HSV-infected BSC-1 cells is presented in Fig. 2. The infected zinc-treated and untreated cells were pulselabeled for 1 hr with [SH]thymidine at
u-3 ‘2 , I
Ii ”
FRACTION
FIG. 1. Inhibition of HSV synthesis in infected cells by 0.2 mM Z&O,. Two cultures of BSC-1 cells were infected with HSV (m.o.i., 10 PFU/cell). After incubation for 3 hr at 37” the medium was removed, the cells were washed and reincubated in fresh Dulbecco’s modified Eagle’s medium without serum. Both cultures were labeled with 2 &i/ml of [3H]thymidine (specific activity, 12.3 Ci/mmole, Nuclear Research Center, Negev). To one culture, 0.2 m&f ZnSO, was added. The cultures were further incubated at 37” for 15 hr and then harvested by scraping into reticulocyte standard buffer (RSB) without Mgz+ (0.01 M Tris-HCl, pH 7.7, 0.01 A4 KCl). The cell suspensions were sonicated, and the homogenates were layered onto sucrose gradients (12-52%, w/w) prepared in TBS buffer (0.2 M Tris-HCl, pH 8.3, 0.85% NaCl). The gradients were centrifuged in an SW 50.1 rotor of a Beckman ultracentrifuge at 40,000 rpm for 15 min at 4’ to isolate the virus particles. The gradients were collected dropwise and the trichloroacetic acid (TCA)-precipitable radioactivity was determined. 04, Untreated, infected cells; O-----O, zinctreated infected cells.
1;:I ,‘~~l~l~~........*~~~ ZnSOL ; 36 13
TIME
18
(HRS)
FIG. 2. Effect of 0.2 mA4 ZnSO, on the rate of DNA synthesis in HSV-infected cells. BSC-1 cells (in 30-mm plates) were infected with HSV at a m.o.i. of 10 PFU/cell. At 3 hr pi., the medium was replaced with fresh medium with or without 0.2 mM ZnSO,, and the cultures were reincubated at 37”. At different time intervals after infection (3, 6, 13 and 18 hr), the medium was replaced with 10 ml of medium per plate containing 5 rCi/ml of [sH]thymidine with or without 0.2 mM ZnSO,. After incubation for 1 hr, the cultures were harvested with 0.5% (w/v) sodium dodecyl sulfate (SDS). The TCA-precipitable radioactivity was determined. The radioactivity incorporated into the infected untreated cells during a l-hr pulse at 3 hr p.i. was regarded as 100% and was used to calculate the radioactivity incorporated during each l-hr pulse. 04, Infected, untreated; O-----O, infected zinctreated (0.2 mM ZnSO,).
different time intervals. Untreated infected cells synthesized DNA at a constant rate up to 13 hr p.i. when DNA synthesis was reduced due to termination of the virus growth cycle. The time course of HSVDNA synthesis in infected BSC-1 cells was recently reported (9). CsCl density gradient analysis of the DNA molecules synthesized in the infected cells revealed that 90-95% of the labeled DNA molecules are viral and the rest are cellular DNA molecules. Zinc ions markedly inhibited the synthesis of DNA in the infected cells throughout the virus growth cycle (Fig. 2). The effect of zinc ions on the synthesis of virus-specific DNA, banding at a density of 1.718 g/ml, was determined. DNA labeled with [3H]thymidine from infected cultures untreated or treated with zinc sulfate was analyzed by centrifugation in CsCl density gradients. Less DNA was synthesized in the presence of 0.1 mM ZnSO, (Fig. 3B) than in untreated infected cells (Fig. 3A). The analysis of five experiments revealed that 0.1 mM ZnSO, reduced HSV-DNA
-loQ.lo6
a
I (Y ‘S ;
-
;L . 0
B 0.1 mM
Zn”
t l> tv 6 0 0 6 a
boo 0 aa OtrlY
I (3
FRACTION FIG. 3. Effect of zinc ions on viral and cellular DNA synthesis in HSV-infected cells. Three BSC-1 cultures were infected with HSV and labeled with [qlthymidine as described in Fig. 1. At the end of the adsorption period one culture served as a control (A), the second culture was treated with 0.1 mM ZnSO, (B) and the third culture was treated with 0.2 mM ZnSO, (C). The cultures were incubated for an additional period of 15 hr at 37” and then harvested. The cells were scraped into TBS buffer, the suspension was adjusted to a final concentration of 1 x SSC (0.15 M NaCl, 0.015 M Na citrate), SDS (l.O%, w/v) and 0.3 mg/ml of Pronase (80,000 U/mg, free of nucleases; Calbiochem) were added. The Pronase was preincubated for 1 hr at 37”. To each cell suspension, purified HSV-DNA labeled with [‘Clthymidine was added to serve as a density marker. The suspensions were incubated at 37” for 5 hr and the lysates were then carefully transferred to polyallomer centrifuge tubes containing 8.0 ml of CsCl at a density of 1.70 g/ml. The gradients were centrifuged in a 50 Ti fixed-angle rotor of a Beckman ultracentrifuge at 37,500 rpm for 48 hr at 20”. The gradients were collected dropwise into 300~pl fractions. One-third of each fraction was removed to determine the TCA-precipitable radioactivity in the synthesized DNA (O--O) and the ‘E-labeled HSV-DNA marker (O-----O). To determine the molecular size of the HSV-DNA, 75 ~1 from every fraction with a density of 1.718 g/ml was diluted 1:40 in TEN buffer and carefully layered onto 5-20% (w/w) sucrose gradients prepared in neutral DNA buffer (12). The gradients in nitrocellulose tubes (a, b, c, respectively) were centrifuged in an SW 27 rotor of a Beckman ultracentrifuge at 25,000 rpm for 5 hr at 20”. One-milliliter fractions were collected dropwise and the TCA-precipitable radioactivity was determined. Note different scales in a, b, and c. Purified [‘Hlthymidinelabeled HSV-DNA extracted from purified virions by treatment with Sarkosyl (4%, w/w) for 2 min at 63” was used as an external marker and centrifuged in a separate sucrose gradient (not shown). 332
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synthesis by 13.2% as compared to HSVDNA synthesis in untreated infected cells which are taken to be 100%. A marked inhibition of viral DNA synthesis occurred when the infected cells were treated with 0.2 mM ZnSO, (Fig. 3C). Removal of ZnSO, (0.2 mA4) from the infected cells after treatment for 18 hr and reincubation for 4 hr in the absence of the inhibitor resulted in an increase in the amount of viral DNA synthesized in the infected cells. The DNA molecules banding at a density of 1.718 g/ml in the CsCl gradients described above were centrifuged in sucrose gradients to determine their molecular weight. A molecular weight of 100 x lo6 (Figs. 3A-C), identical to that of DNA isolated from intact virus particles, was obtained. The effect of different concentrations of ZnSO, on the synthesis of HSV and cellular DNA is presented in Fig. 4. The infected, zinc-treated cells were labeled for 15 hr (3-18 hr p.i.) with [3H]thymidine. The DNA was then extracted and centrifuged in CsCl density gradients. The amounts of radioactivity banding at a density of 1.718 g/ml (viral DNA) and 1.700 g/ml (cellular DNA) were determined. Since HSV infection inhibits the synthesis of cellular DNA, the latter constituted only about 10% of the total DNA. It is of interest that zinc ions at a concentration of 0.1 mM had only a slight inhibitory effect on the synthesis of viral DNA, while at a concentration of 0.2 mA4 the synthesis of viral DNA was markedly inhibited. Residual cellular DNA synthesis and DNA synthesis in uninfected cells were not affected by 0.2 mM ZnSO, (Fig. 4). ZnSO, at a concentration of 0.3 mM inhibited the synthesis of both viral and the residual cellular DNA (Fig. 4). These results suggest that at a concentration of 0.2 mM, ZnSO, affects the mechanism responsible for synthesis of viral DNA. To determine the mode of action of zinc ions, their effect on the viral DNA-polymerase present in the nuclei of HSV-infected cells was studied in vitro. Under the in vitro assay conditions previously reported (9), no DNA-polymerase activity was demonstrable in nuclei from uninfected cells,
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.
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INFECTED (Residual
0
I
I 0
I 0.l ZnSOL
1 0.2 Concentration
Cellular
DNA(l.7OqCrU)
, 0.3
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FIG. 4. Effect of ZnSO, on the synthesis of viral and cellular DNA in HSV-infected and uninfected cells. BSC-1 cells were infected as described in Fig. 1. Infected cells were either untreated or treated with 0.1,0.2 and 0.3 mM ZnSO,. The cultures were labeled with [SH]thymidine and harvested at 18 hr p.i. by treating with 1% (w/v) SDS and 0.3 mg/ml of Pronase. The DNA was extracted twice with phenol and three times with chloroform:isoamyl alcohol (25:l). The purified DNA preparations were centrifuged in CsCl gradients as described in Fig. 3. The gradients were collected dropwise, the density and the TCA-precipitable radioactivity were determined. The total radioactivity which bands in the CsCl gradients of infected cells with a mean density of 1.718 g/ml (viral DNA, O----O) and with a mean density of 1.700 g/ml (residual cellular DNA, O-----O) was calculated. The viral DNA which was obtained from infected untreated cells (no ZnSO,) was regarded as lOO%, and the viral DNA isolated from ZnSO,-treated cells was compared to it. The residual cellular DNA synthesized in the infected untreated cells (no ZnSO,) was regarded as 100% and the residual cellular DNA in the ZnSO,-treated cells was compared to it. It should be indicated that the residual cellular DNA is about 10% of the total labeled DNA in untreated HSV-infected cells. Uninfected BSC-1 cell cultures were labeled with [Wlthymidine in the absence and the presence of 0.1 and 0.2 mM ZnSO,. The TCA-precipitable radioactivity in the untreated and treated cells was determined (x-x). The radioactive-DNA in untreated uninfected cells was regarded as 100%.
whereas viral DNA was synthesized in nuclei from infected cells. The results presented in Fig. 5A demonstrate that nuclei from HSV-infected cells linearly incorporated [3H]TMP into viral DNA during a 30-min period of incubation in vitro, as was previously shown (9). The DNA-polym-
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FIG. 5. In vitro DNA-polymerase activity in nuclei from HSV-infected cells, untreated and treated with ZnSO,. Six BSC-1 cultures were infected with HSV as described in Fig. 1. At 3 hr pi. the cultures were either left untreated (A) or were treated with 0.1 (B), 0.2 (C, E, and F) or 0.3 mM ZnSO, (D) and incubated for an additional period of 15 hr. Two cultures (E and F) were washed at 18 hr p.i. to remove the inhibitor and reincubated for an additional period of 1.5 hr, prior to harvest. To culture F, puromycin (100 pg/ml) was added to inhibit protein synthesis. The cells were harvested and disrupted in a Dounce homogenizer. The nuclei were isolated and resuspended, at a concentration of 10’ per 100 pl of 75 mM phosphate, pH 7.4, to which the following were added: 3 mM 2-mercaptoethanol, 8% (w/v) sucrose (RNase free), 6 mM MgCI,, 40 mA4 each of dATP, dCTP, dGTP (Sigma, St. Louis, MO) and 50 &i of [SH]TTP (specific activity, 15 Wmole; Radiochemical Center, Amersham, England), in a final volume of 200 ~1 of phosphate buffer. The reaction mixtures were incubated at 37Oand duplicate samples of 25 ~1 each were removed at different times to tubes containing 10% (w/v) TCA. The TCA-precipitable radioactivity was determined.
erase activity, in nuclei isolated from HSVinfected cells treated in uiuo with 0.1 mM ZnSO, during a 15hr period (Fig. 5B) was as high as in untreated infected cells (Fig. 5A). It is not yet known why, under in uiuo conditions, 0.1 mM ZnSO, slightly inhibits HSV-DNA synthesis while the DNApolymerase activity in nuclei isolated from 0.1 mM ZnSO,-treated infected cells is unaffected. Inhibition of the DNA-polymerase activity was noted in nuclei isolated from infected cells treated with 0.2 mM (Fig. 5C) or 0.3 mA4 (Fig. 5D) ZnSO,.
To determine whether zinc ions prevent the synthesis of the virus-induced DNA polymerase or alternatively affect the activity of this enzyme, the kinetics of [$H]TMP incorporation into DNA were determined after removal of the zinc ions and inhibition of protein synthesis. Two cultures of infected BSC-1 cells, treated with 0.2 mM ZnSO, during a 15 hr period, were washed to remove the zinc ions and reincubated for an additional period of 1.5 hr at 37” in fresh medium. To prevent further protein synthesis, one of the cultures received puromycin (100 pg/ml) with the fresh medium. The nuclei were isolated at the end of a 1.5-hr incubation period in the absence of ZnSO,, and the DNApolymerase activity was determined. Removal of the zinc ions from the infected cells partially reactivated the DNApolymerase activity (Fig. 5E). In the absence of protein synthesis, the DNA polymerase was reactivated to an even greater extent (Fig. 5F). These results might suggest that the viral DNA polymerase is synthesized in HSV-infected cells treated with 0.2 mM ZnSO,, but the enzymatic activity is reduced in the presence of the zinc ions. It is of interest that, although removal of the zinc ions partially restores the activity of the viral DNA polymerase, no infectious virus progeny could be obtained from such cells even after an additional incubation period of 18 hr (4). The present study demonstrates that ZnSO, at a concentration of 0.2 mM prevents the synthesis of HSV-DNA by a mechanism which involves inhibition of the viral DNA-polymerase activity. In contrast, 0.2 mM ZnSO, had no effect on cellular DNA synthesis in uninfected cells. The mechanism by which zinc ions inhibit the viral DNA polymerase is not yet known. It is possible that zinc ions are required for the activity of the virusinduced DNA polymerase, but an excess of zinc ions inhibits this enzymatic activity. Alternatively, zinc ions may affect the cleavage of a peptide precursor to the viral DNA polymerase, thus leading to the inhibition of the viral enzyme; upon removal of zinc ions, such a precursor peptide may be processed causing enzymatic activity to
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appear. The ability of zinc ions to prevent the synthesis of HSV-DNA results in the inhibition of both virus replication and the formation of infectious virus progeny (4). Another phenomenon of interest is the effect of 0.1 mM Z&O, on the synthesis of herpesvirus progeny. It was shown that 0.1 mM ZnSO, only slightly inhibits the synthesis of intact viral DNA molecules. However, under these conditions treated cells synthesize only 5% of the infectious virus progeny (4). These observations suggest that either the HSV-DNA molecules synthesized in the presence of 0.1 mA4 ZnSO, are not functional or that ZnSO, inhibits other molecular processes associated with the assembly of the virus particles. Further studies are needed to determine the effect of ZnSO, on the synthesis of HSV-specified messenger RNA and proteins. Since ZnSO, (0.5%, w/v) has been reported to be effective in the treatment of herpetic keratitis in man (13), studies on the mechanism of its antiviral action may help in the evaluation of zinc as an antiviral agent. ACKNOWLEDGMENTS This study was supported by Contract No. NO1 CP 33310 from the Special Virus Cancer Program, National Cancer Institute, National Institutes of Health, Bethesda Md, U.S.A. Dr. Y. J. Gordon was on leave from the Department of Ophthalmology, Hadassah Hospital, Jerusalem.
The interest of Prof. H. Zauberman, head of the Department of Ophthalmology, is greatly acknowledged. REFERENCES 1. KORANT, B. D., KANER, J. C., and BUTTERWORTH, B. E., Nature (London) 248.588-590 (1974). 2. BUTTERWORTH,B. E., and KORANT, B. D., J. Viral. 14, 282-291 (1974). 3. FALKE, D., and KAHL, G. F. 2. Med. Microbial. Immunol. 153, 175-189 (1967). 4. GORDON, Y. J., ASHER, Y., and EECKER, Y., Antimicrob. Ag. Chemother., In press. 1975. 5. SLATER, J. P., MILDVAN, A. S., and LOEB, L. A., Biochem. Biophys. Res. Commun. 44, 37-43 (1968). 6. SCRUTFON, M. C., Wu, C. W., and GOLLYMIWAIT, D. A., Proc. Nat. Acad. Sci. USA 68, 2497-2501 (1971). 7. VALLEE, B. L., and WACKER, W. E. C., In The Proteins (H. Neurath, ed.), 2nd Ed, Vol. 5, Academic Press, New York, 1970. 8. AULD, D. S., KAWAGUCHI, H., LIVINGSTON, D. M., and VALLEE, B. L., Proc. Nat. Acad. Sci. USA 71, 2091-2095 (1974). 9. BECKER, Y., and ASHER, Y., Virology, 63, 209-220 (1975). 10. GORDIN, M., OLSHEVSKY, U., ROSENKRANZ, H. S., and BECKER, Y., Virology 55, 280-284 (1973). 11. BECKER, Y., DYM, H., and SAROV, I., Virology 36, 185-192 (1968). 12. FRENKEL, N., and ROIZMAN, B., J. Viral. 10, 565-572 (1972). 13. DE ROETH, A., Amer. J. Ophthalmol. 56, 729-731 (1963).