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Inhibition of herpes simplex virus replication by araT
VIROLOGY
65, 294-296 (1975)
Inhibition
of Herpes GLENN
Department
of Microbiology,
Simplex
A. GENTRY University
Virus Replication
AND
of Mississippi,
JANE
F. ASWELL
School of Medicine,
Accepted January
by araT
Jackson,
Mississippi
39216
13, 1975
AraT (l-P-o-arabinofuranosylthymine) is a selective inhibitor of herpes simplex virus types 1 and 2 in hamster cell cultures in uitro and has no effect on the growth of uninfected control cells. This differential action may be related to the broader substrate range of the herpes simplex virus-specified pyrimidine deoxyribonucleoside kinase.
The recent finding that herpes simplex virus (HSV) types 1 and 2 specify a pyrimidine deoxyribonucleoside kinase able to phosphorylate deoxycytidine, l-p-~arabinofuranosylcytosine (araC), and deoxythymidine (l-3) suggested that the same enzyme, because of its relatively broad specificity, should also phosphorylate l-/?-n-arabinofuranosylthymine (araT), a natural metabolite of the sponge Cryptotethya crypta (4-6). Since araT is apparently not a substrate for mammalian host cell deoxythymidine kinase (7), a basis for a relatively specific antiviral effect might be provided. We report here that araT does indeed selectively inhibit the replication of herpes simplex virus without affecting the growth of uninfected host (hamster) cells in culture. Baby hamster kidney (BHK) cells were cult,ured, and HSV-1 strain 17, and HSV-2 strain 52 (2,3) were cultivated as described (8). The titration, scaled down and modified, was carried out in confluent BHK monolayers in 24-well cell culture trays (Model FB16-24, Linbro Chemical Co., New Haven, CT). The supernatant fluid was removed, 50 ~1 of the appropriate dilution of virus was added to each well, and the plates incubated for 2 hr with intermittent agitation at 37” in 5% CO,-95% air and 100% humidity. To each well was then added 1 ml of Eagle’s medium/tryptose phosphate broth/calf serum (ETC) (8), and the plates were returned to 294 Copyright 0 1975 by Academic Press. Inc. All rights of reproduction in any form reserved.
the incubator. Plaques were counted, with the aid of an inverted microscope, 24 hr later. In other experiments, cells were infected with HSV-1 or HSV-2 and harvested at 72 hr postinfection. Virus production was assayed by titration. Cell growth studies were also carried out in 24-well plates. Because sensitivity to inhibitors of DNA synthesis would be greatest during cell growth, the wells were seeded with relatively few cells (104/well; less than 10% surface coverage) in order to minimize the effect of cell contact and nutritional depletion on the growth rate. Replicate monolayers were trypsinized (8) at the designated times, and the completion of trypsinization was monitored with the inverted microscope. The cells were counted directly with a hemocytometer. AraT, generously provided by Dr. Jack Fox, was added to infected monolayers in ETC at the end of the virus-absorption period or to uninfected cells at the time of subculture. It is quite clear from Fig. 1 that the growth of BHK cells was totally unaffected by araT at 2 x lOA4 A4 (approximately 50 pg/ml). In dramatic contrast, however, the replication of HSV-1 and HSV-2 was completely blocked by araT at this concentration (Table 1, Fig. 2). Indeed the cells infected with HSV-1 and treated with araT were subcultured and maintained in the absence of araT for 3 days further without any signs of viral cytopathology. The corre-
SHORT
TIME
AFTER
COMMUNICATIONS
SUBCULTURE
FIG. 1. Replicate
monolayers of BHK cells were prepared and sampled at the indicated intervals as described in the text. To each well at 0 time was added 1 ml of ETC with araT (2 x 10m4 M) and 10’ cells. AraT was omitted from the control wells.
FIG. 2. Monolayers of BHK cells were infected with HSV-1 or HSV-2, as described in the text. After 2 hr for virus fixation, 1 ml of ETC containing the desired amount of araT was added to each well. Plaques were counted 24 hr later. The control well (0 araT) with HSV-1 produced 40 plaques; with HSV-2, 18. TABLE
1
EFFECTOF ARAT ON HERPESSIMPLEXVIRUS REPLICATION Virus
Input multiplicity
Virus producedb Control
araT’
HSV-1
4.5 x 10-d
5 x 105
<2 x 1O’d
HSV-2
7.7 x 10 4
2 x 106
<2 x 10’d
” In plaque-forming units per cell. 0 In plaque-forming units per ml. Infected cultures were incubated 72 hr before harvesting. ( Final concentration, 2 x lo-’ M (50 &ml). d No plaques were seen; 2 x 10’ is the limit of sensitivity of the assay.
295
sponding cells infected with HSV-2 did produce one small plaque after 48 hr, suggesting, together with the data in Fig. 2, that HSV-2 does not respond as strikingly to araT in this system as does HSV-1. If araT should be relatively labile under our experimental conditions, it might persist long enough to block viral replication (O-12 hr) but not long enough to inhibit cell growth, which was measured over a much longer time scale (O-96 hr). We therefore tested its lability by preincubating supernatants containing araT (2 x 10e4 M) in wells of growing BHK cells for 72 hr and comparing the activity against HSV-2 of these “aged” supernatants, diluted l/10 with fresh ETC, with the activity of fresh araT at the same concentration (2 x 10m5 n/f). A plaque-inhibition test similar to that described in Fig. 2 was used. The plaque reduction with the diluted, aged araT was 50%, which corresponds to an araT concentration of approximately 1.2 x 10e5 M (from Fig. 2), or 1.2 x lo-” M in the undiluted preparation. This represents a loss of 40% of activity during 72 hr incubation, but, even with this drop, a substantial antiviral effect would still be observed. The fresh araT reduced plaque formation by about 75%, which is what would be expected from Fig. 2. In a second experiment, a DNA precursor, [%]deoxycytidine (final concentration, 4 x 1O-6 M, specific activity, 31 Ci/mole, New England Nuclear, Boston, MA), was added to wells containing lo4 uninfected cells each and either araT (2 x 10m4M) in ETC or ETC alone. After 12-hr incubation at 37”, the cells were trypsinized, collected on GFA glass fiber disks, washed with 5% trichloracetic acid and 95% ethanol, dried and counted in a liquid scintillation spectrometer. Incorporation into control cells (1,054 cpm/well) was not significantly different from that into araTtreated cells (1,167 cpm/well). We conclude that the lack of effect of araT on uninfected cells cannot be explained by its lability. Although there are several studies of the inhibition by araT of HSV replication in vitro (g-12), in none of these was the
SHORT
296
COMMUNICATIONS
ability of araT to affect the normal growth of the uninfected host cells tested. On the other hand, araT has been screened as a potential antitumor agent against several mouse cell lines (13, 14). It was relatively ineffective. AraT has been tested cursorily against HSV cornea1 keratitis in rabbits, with indifferent success (11, 12). Our data lead us to suggest that a more thorough evaluation of its toxicity as well as of its antiviral effect be made in uiuo and in. vitro. If, as suggested above, it should be phosphorylated by the HSV pyrimidine deoxyribonucleoside kinase but not by normal mammalian cell enzymes, it might prove useful as a probe for detecting the HSV kinase in transformed (15, 16) and other genetically manipulated (17) cells. ACKNOWLEDGMENTS This and by Cancer Conerly
work was supported by NIH Grant No. AI-69 the Mississippi Division of the American Society. We thank J. Mize, R. Mims, S. and D. Holmes for technical assistance.
REFERENCES 1. HAY, J., PERERA,P., GENTRY, G. A., and SUBAKSHARPE, J. H., In “Strategy of the Viral Genome,” Ciba Foundation (G. E. W. Wolsten-
2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.
15. 16. 17.
holme and M. O‘Connor, eds.), pp. 355-376. Churchill Livingstone, London, 1970. JAMIESON, A. T., and SUBAK-SHARPE, J. H., J. Gen. Viral. 24,481-492 (1974). JAMIESON,A. T., GENTRY, G. A., and SUBAKSHARPE,J. H., J. Gen. Viral. 24,465-480 (1974). BERGMANN,W., and FEENY,R. J., J. Amer. Chem. Sot. 72, 2809-2810 (1950). BERGMANN,W., and FEENY, R. J., J. Org. Chem. 16, 981-987 (1951). COHEN,S. S., Prog. Nucleic Acid Res. Mol. Biol. 5, 2-88 (1966). KIT, S., DE TORRES, R. A., and DUBBS, D. R., Cancer Res. 26, 1859-1866 (1966). RUSSELL,W. C., Nature (London) 195,1028-1029 (1962). RENIS, H. E., and BUTHALA,D. A., Ann. N.Y. Acad. Sci. 130, 345-354 (1965). DE RUDDER,J., and DE GARILHE,M. P., Antimicrab. Ag. Chemother. 1965, 578-584 (1965). RUDDER,J., and DE GARILHE,M. P., Int. Congr. Chemother. Proc. 2, 29-35 (1967). UNDERWOOD,G. E., WISNER, C. A., and WEED, S. D. Arch. OphthaEmoL 72, 505-512 (1964). CHU, M. Y., and FISCHER,G. A., Biochem. Pharmacol. 11, 423-430 (1962). DE GARILHE, M. P., and DE RUDDER, J., Progr. Antimicrob. Anticancer Chemother. 2, 180-184, (1970). DUFF,R., and RAPP, F., J. Viral. 8,469-477 (1971). DUFF, R., and RAPP, F., J. Viral. 12. 209-217 (1973). MUNYON, W., KRAISELBURD,E., DAVIS, D., and MANN, J., J. Viral. 7, 813-820 (1971). DE
65, 294-296 (1975)
Inhibition
of Herpes GLENN
Department
of Microbiology,
Simplex
A. GENTRY University
Virus Replication
AND
of Mississippi,
JANE
F. ASWELL
School of Medicine,
Accepted January
by araT
Jackson,
Mississippi
39216
13, 1975
AraT (l-P-o-arabinofuranosylthymine) is a selective inhibitor of herpes simplex virus types 1 and 2 in hamster cell cultures in uitro and has no effect on the growth of uninfected control cells. This differential action may be related to the broader substrate range of the herpes simplex virus-specified pyrimidine deoxyribonucleoside kinase.
The recent finding that herpes simplex virus (HSV) types 1 and 2 specify a pyrimidine deoxyribonucleoside kinase able to phosphorylate deoxycytidine, l-p-~arabinofuranosylcytosine (araC), and deoxythymidine (l-3) suggested that the same enzyme, because of its relatively broad specificity, should also phosphorylate l-/?-n-arabinofuranosylthymine (araT), a natural metabolite of the sponge Cryptotethya crypta (4-6). Since araT is apparently not a substrate for mammalian host cell deoxythymidine kinase (7), a basis for a relatively specific antiviral effect might be provided. We report here that araT does indeed selectively inhibit the replication of herpes simplex virus without affecting the growth of uninfected host (hamster) cells in culture. Baby hamster kidney (BHK) cells were cult,ured, and HSV-1 strain 17, and HSV-2 strain 52 (2,3) were cultivated as described (8). The titration, scaled down and modified, was carried out in confluent BHK monolayers in 24-well cell culture trays (Model FB16-24, Linbro Chemical Co., New Haven, CT). The supernatant fluid was removed, 50 ~1 of the appropriate dilution of virus was added to each well, and the plates incubated for 2 hr with intermittent agitation at 37” in 5% CO,-95% air and 100% humidity. To each well was then added 1 ml of Eagle’s medium/tryptose phosphate broth/calf serum (ETC) (8), and the plates were returned to 294 Copyright 0 1975 by Academic Press. Inc. All rights of reproduction in any form reserved.
the incubator. Plaques were counted, with the aid of an inverted microscope, 24 hr later. In other experiments, cells were infected with HSV-1 or HSV-2 and harvested at 72 hr postinfection. Virus production was assayed by titration. Cell growth studies were also carried out in 24-well plates. Because sensitivity to inhibitors of DNA synthesis would be greatest during cell growth, the wells were seeded with relatively few cells (104/well; less than 10% surface coverage) in order to minimize the effect of cell contact and nutritional depletion on the growth rate. Replicate monolayers were trypsinized (8) at the designated times, and the completion of trypsinization was monitored with the inverted microscope. The cells were counted directly with a hemocytometer. AraT, generously provided by Dr. Jack Fox, was added to infected monolayers in ETC at the end of the virus-absorption period or to uninfected cells at the time of subculture. It is quite clear from Fig. 1 that the growth of BHK cells was totally unaffected by araT at 2 x lOA4 A4 (approximately 50 pg/ml). In dramatic contrast, however, the replication of HSV-1 and HSV-2 was completely blocked by araT at this concentration (Table 1, Fig. 2). Indeed the cells infected with HSV-1 and treated with araT were subcultured and maintained in the absence of araT for 3 days further without any signs of viral cytopathology. The corre-
SHORT
TIME
AFTER
COMMUNICATIONS
SUBCULTURE
FIG. 1. Replicate
monolayers of BHK cells were prepared and sampled at the indicated intervals as described in the text. To each well at 0 time was added 1 ml of ETC with araT (2 x 10m4 M) and 10’ cells. AraT was omitted from the control wells.
FIG. 2. Monolayers of BHK cells were infected with HSV-1 or HSV-2, as described in the text. After 2 hr for virus fixation, 1 ml of ETC containing the desired amount of araT was added to each well. Plaques were counted 24 hr later. The control well (0 araT) with HSV-1 produced 40 plaques; with HSV-2, 18. TABLE
1
EFFECTOF ARAT ON HERPESSIMPLEXVIRUS REPLICATION Virus
Input multiplicity
Virus producedb Control
araT’
HSV-1
4.5 x 10-d
5 x 105
<2 x 1O’d
HSV-2
7.7 x 10 4
2 x 106
<2 x 10’d
” In plaque-forming units per cell. 0 In plaque-forming units per ml. Infected cultures were incubated 72 hr before harvesting. ( Final concentration, 2 x lo-’ M (50 &ml). d No plaques were seen; 2 x 10’ is the limit of sensitivity of the assay.
295
sponding cells infected with HSV-2 did produce one small plaque after 48 hr, suggesting, together with the data in Fig. 2, that HSV-2 does not respond as strikingly to araT in this system as does HSV-1. If araT should be relatively labile under our experimental conditions, it might persist long enough to block viral replication (O-12 hr) but not long enough to inhibit cell growth, which was measured over a much longer time scale (O-96 hr). We therefore tested its lability by preincubating supernatants containing araT (2 x 10e4 M) in wells of growing BHK cells for 72 hr and comparing the activity against HSV-2 of these “aged” supernatants, diluted l/10 with fresh ETC, with the activity of fresh araT at the same concentration (2 x 10m5 n/f). A plaque-inhibition test similar to that described in Fig. 2 was used. The plaque reduction with the diluted, aged araT was 50%, which corresponds to an araT concentration of approximately 1.2 x 10e5 M (from Fig. 2), or 1.2 x lo-” M in the undiluted preparation. This represents a loss of 40% of activity during 72 hr incubation, but, even with this drop, a substantial antiviral effect would still be observed. The fresh araT reduced plaque formation by about 75%, which is what would be expected from Fig. 2. In a second experiment, a DNA precursor, [%]deoxycytidine (final concentration, 4 x 1O-6 M, specific activity, 31 Ci/mole, New England Nuclear, Boston, MA), was added to wells containing lo4 uninfected cells each and either araT (2 x 10m4M) in ETC or ETC alone. After 12-hr incubation at 37”, the cells were trypsinized, collected on GFA glass fiber disks, washed with 5% trichloracetic acid and 95% ethanol, dried and counted in a liquid scintillation spectrometer. Incorporation into control cells (1,054 cpm/well) was not significantly different from that into araTtreated cells (1,167 cpm/well). We conclude that the lack of effect of araT on uninfected cells cannot be explained by its lability. Although there are several studies of the inhibition by araT of HSV replication in vitro (g-12), in none of these was the
SHORT
296
COMMUNICATIONS
ability of araT to affect the normal growth of the uninfected host cells tested. On the other hand, araT has been screened as a potential antitumor agent against several mouse cell lines (13, 14). It was relatively ineffective. AraT has been tested cursorily against HSV cornea1 keratitis in rabbits, with indifferent success (11, 12). Our data lead us to suggest that a more thorough evaluation of its toxicity as well as of its antiviral effect be made in uiuo and in. vitro. If, as suggested above, it should be phosphorylated by the HSV pyrimidine deoxyribonucleoside kinase but not by normal mammalian cell enzymes, it might prove useful as a probe for detecting the HSV kinase in transformed (15, 16) and other genetically manipulated (17) cells. ACKNOWLEDGMENTS This and by Cancer Conerly
work was supported by NIH Grant No. AI-69 the Mississippi Division of the American Society. We thank J. Mize, R. Mims, S. and D. Holmes for technical assistance.
REFERENCES 1. HAY, J., PERERA,P., GENTRY, G. A., and SUBAKSHARPE, J. H., In “Strategy of the Viral Genome,” Ciba Foundation (G. E. W. Wolsten-
2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.
15. 16. 17.
holme and M. O‘Connor, eds.), pp. 355-376. Churchill Livingstone, London, 1970. JAMIESON, A. T., and SUBAK-SHARPE, J. H., J. Gen. Viral. 24,481-492 (1974). JAMIESON,A. T., GENTRY, G. A., and SUBAKSHARPE,J. H., J. Gen. Viral. 24,465-480 (1974). BERGMANN,W., and FEENY,R. J., J. Amer. Chem. Sot. 72, 2809-2810 (1950). BERGMANN,W., and FEENY, R. J., J. Org. Chem. 16, 981-987 (1951). COHEN,S. S., Prog. Nucleic Acid Res. Mol. Biol. 5, 2-88 (1966). KIT, S., DE TORRES, R. A., and DUBBS, D. R., Cancer Res. 26, 1859-1866 (1966). RUSSELL,W. C., Nature (London) 195,1028-1029 (1962). RENIS, H. E., and BUTHALA,D. A., Ann. N.Y. Acad. Sci. 130, 345-354 (1965). DE RUDDER,J., and DE GARILHE,M. P., Antimicrab. Ag. Chemother. 1965, 578-584 (1965). RUDDER,J., and DE GARILHE,M. P., Int. Congr. Chemother. Proc. 2, 29-35 (1967). UNDERWOOD,G. E., WISNER, C. A., and WEED, S. D. Arch. OphthaEmoL 72, 505-512 (1964). CHU, M. Y., and FISCHER,G. A., Biochem. Pharmacol. 11, 423-430 (1962). DE GARILHE, M. P., and DE RUDDER, J., Progr. Antimicrob. Anticancer Chemother. 2, 180-184, (1970). DUFF,R., and RAPP, F., J. Viral. 8,469-477 (1971). DUFF, R., and RAPP, F., J. Viral. 12. 209-217 (1973). MUNYON, W., KRAISELBURD,E., DAVIS, D., and MANN, J., J. Viral. 7, 813-820 (1971). DE