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The effect of 5-iodo-2′-deoxyuridine-5′-triphosphate on DNA polymerase of uninfected and herpes simplex virus infected cells
Pharmacological Research Communications, Vol. 1, No. 1, 1969
3
THE EFFECT OF 5-IODO-2'-DEOXYURIDINE-5'-TRIPHOSPHATE
ON DNA POLYMERASE
OF I~qINFECTED AND HERPES SIMPLEX VIRUS INFECTED CELLS +
L. Sekely* and William H. Prusoff Department of Pharmacology, Yale University School of Medicine New Haven, Connecticut Received
1968
28 N o v e m b e r
A study has been in progress-in this laboratory to determine whether enzymes that are either viral specified or have greater activity in the virusinfected cell have increased susceptibility to inhibition by 5-iodo-2'-deoxyuridlne
(IUdR) or its mono- or triphosphate derivatives.
Thymidine kinase and
thymidylic acid kinase of mammalian cells present before and after infection with herpes simplex virus showed no differences
in susceptibility
by IUdR and 5-iodo-2'-deoxyurldine monophosphate respectively and Sekely,
]965).
to inhibition
(Prusoff, Bakhle,
This report reveals that the DNA-polymerase
present in
mammalian cells and that induced or specified by herpes simplex virus are equally susceptible to inhibition by the triphosphate of IUdR (dIUTP). The molecular basis of the antiviral activity of IUdR has been under in several laboratories (Prusoff, Bakhle, and Sekely, 1965;
investigation Prusoff,
1967).
Three general areas have been observed where this analog or
its phosphorylated derivatives exert biochemical effects:
(a) competitive
inhibition of several enz}~nes concerned with a biosynthesis of DNA-thymine; @llosteric or fee4back inhibition by 5-iodo-2'-deoxyuridine deoxycytidylate
deaminase
thymidine kinase reductase ,
i
(Diwan and Prusoff,
(Prusoff and Chang,
(Prusoff, unpublished); i,
,
,
i
triphosphate of
1968; Prusoff and Chang,
1966) and of ribonucleoslde
(c) incorporation
(b)
1968),
diphosphate
into virus-DNA with a
J
+This investigation was supported by U.S. Public llealth Service Researzh Grant CA-05262 from the National Cancer Institute. *Present address: New York I0021.
Hospital of Special Surgery, 545 East 70th Street, New York,
4
Pharmacological Research Communications, VoL 1, No. 1, 1969
subsequent effect on the expression of genetic information during either replication or transcription
(Goz and Prusoff, 1968; other pertinent references
cited in Prusoff, Bakhle and Sekely, 1965; Prusoff, 1967). African monkey kidney cells (BSC-I) were seeded and grown according to methods already described (Prusoff, Bakhle and Sekely, 1965).
Fourteen day non-
growing cultures of these cells were infected with a suspension of herpes simplex virus at a multiplicity of 20 pfu/cell.
After a period of one hour at 37 ° , the
medium was removed, the infected cells washed with phosphate (0.05 M, pH 8.0) buffered saline, and fresh medium was added.
Five hours later the medium was
decanted and the cells were suspended in 1.5 volumes phosphate buffer (0.05 M, pH 8.0) containing mercaptoethanol.
The cells were disrupted by freezing and
thawing three times, and a clear supernate was obtained by ultra-centrifugation at I00,000 x g for I hour.
DNA-polymerase activity was determined by a minor
modification of the procedure described by Mantsavinos
(1964).
Table i indicates that infection with herpes simplex virus results in a progressive increase in DNA-polymerase activity which reached a maximum 6 hours
Table I Relative DNA-polymerase activity of African green monkey kidney cells at various times after infection with herpes simplex virus
~-
- Time of ~infection . .
.
.
.
~eiative DNA-polymerase activity
I
i .0"
4
1.2
5
1.3
6
1.4
7
0.97
The composition of the basic reaction mixture is described by Mantsavinos (1964). The amount of incorporation of thymidine triphosphate-2-C 14 in the acid insoluble material in the reaction mixture that contained the enzyme from the non-infected cells was 6,100 cpm (40 per cent conversion) and this value was equated to 1.0. (Duplicate experiments)
Pharmacological Research CommunicaNons, Vol. 1, No.l, 1969 after infection.
5
Similar results were obtained by other investigators (Russell
and co-workers, 1964).
The herpes virus induced DNA polymerase activity has
been shown to be virus specified by Keir and co-workers (1966a, 1966b). The relative susceptibility to inhibition by dlUTP of DNA polymerase deri~ed from uninfected cells and from cells infected with herpes simplex virus (Rolly Strain IZ:~ and an IUdR resistant varisnt of this straln is shown in Table 2.
No differences in susceptibility to inhibition by dlUTP was
observed, the 50 percent inhibition index of DNA polymerase present in both the control and virus infected cell extracts hein E about 2.
Studies of DNA-
polymerase derived fro~ a routine neoplasm indicated that the inhibition of the utilization of dTTP by dlUTP was competitive (Bakhle and Prusoff, In press). The ahillty of DNA polymerase present in murine neoplasms to utilize either dTTP or dlUTP was indicated by the failure of the halogenated analog to inhibit the utilization of radioactive deoxyadenosine triphosphate for the biosynthesis of DNA (Bakhle and Prusoff, In press).
Table 2 Inhibition by dlUTP of the utilization of dTTF-2-C 14 by DNA-polymerase derived from either non-lnfected African green monkey kidney cells or from cells infected with herpes simplex virus of which one strain was resistant to the inhibitory effects of IUdR* Concentration of dlUTP (~M)
Noninfected
Percent Inhibition Normal herpes simplex virus
Resistant herpes simplex virus
40
51
59
54
80
75
69
71
Composition of the basic DNA-polymerase reaction mixture is described by M~=tsavlnos (1964). Concentration of dTTP is 20 ~M. (Duplicate experlment:~) Thus the present report extends to another enzyme (DNA polymerase) o u r previous findings observed with thvmidine kinase, thymidylate kinase, a n d deoxycytldylate deaminsse, that although IUdR inhibits herpes simpleK virus replication
6
Pharmacological Research Communications, Vol. 1, No. I, 1969
selectively it does not preferentially inhibit the virus induced or specified enzymes.
The ability of IUdR to inhibit replication of herpes simplex virus
at a concentration that is not inhibitory to the uninfected host cell was reported by Cramer, Wacker and Welch (1963).
This selectivity of action is
probably a result 6f the enhanced thymidine kinase activity induced or specifie by the virus (Klemperer et al., 1967), an action that results in a selective increase in the amount of phosphorylated derivatives of IUdR in the infected cell. The biological and biophysical consequences of incorporation of IUdR into DNA have been reviewed (Prusoff, Bakhle and Sekely, 1965; Prusoff, 1967), and recent studies with a bacterial virus have indicated very marked effects on the rate and amount of formation of viral specific enzb~nes (Goz and Prusoff, 1968).
It is believed that the mechanism for the antiviral activity of IUdR
will probably be elucidated from a study of the effects that result from incorporation of this analog into the DNA of the virus.
Re ference s Bakhle, Y. S. and Prusoff, W. H., Biochim. et Biophys. Acta. (In press). Cramer, J. W., Wacker, A. and Welch, A. D., Biochem. J., 87, 26P (1963). Goz, B. and Prusoff, W. H., J. Biol. Chem.,24_~_3, 4750 (1968). Diwan, A. and Prusoff, W. H., Virology, 34, 184 (1968). Keir, H. M., Hay, J., Morrison, J. M. and Subak-Sharpe, H., Nature, 210, 369
(1966a). Keir, H. M., Subak-Sharpe, H., Shedden, W. I. H., Watson, D. H., and Wildy, P., Virology, 30, 154 (1966b). Klemperer, H. G., Haynes, G. R., Sheddon, W. I. H., and Watson, D. H., Virology 31, 120 (1967). Mantsavinos, R., J. Biol. Chem., 239, 3431 (1964). Prusoff, W. H., Pharmacol. Rev., 19, 209 (1967). Prusoff, W H., Bakhle, Y. S. and Sekely, L., Ann. N. Y. Acad. Sci., 130, 135 (1965). Prusoff, W. H. and Chang, P. K., Fed Proc., 25, 755 (1966). Prusoff, W. H. and Chang, P. K., J. Biol. Chem., 243, 223 (1968). Russell, W. C., Gold, E., Kelr, H. M., Omura, H., Wilson, D. H., and Wildy, P., Virology, 22, 103 (1964). L _i
,J i
3
THE EFFECT OF 5-IODO-2'-DEOXYURIDINE-5'-TRIPHOSPHATE
ON DNA POLYMERASE
OF I~qINFECTED AND HERPES SIMPLEX VIRUS INFECTED CELLS +
L. Sekely* and William H. Prusoff Department of Pharmacology, Yale University School of Medicine New Haven, Connecticut Received
1968
28 N o v e m b e r
A study has been in progress-in this laboratory to determine whether enzymes that are either viral specified or have greater activity in the virusinfected cell have increased susceptibility to inhibition by 5-iodo-2'-deoxyuridlne
(IUdR) or its mono- or triphosphate derivatives.
Thymidine kinase and
thymidylic acid kinase of mammalian cells present before and after infection with herpes simplex virus showed no differences
in susceptibility
by IUdR and 5-iodo-2'-deoxyurldine monophosphate respectively and Sekely,
]965).
to inhibition
(Prusoff, Bakhle,
This report reveals that the DNA-polymerase
present in
mammalian cells and that induced or specified by herpes simplex virus are equally susceptible to inhibition by the triphosphate of IUdR (dIUTP). The molecular basis of the antiviral activity of IUdR has been under in several laboratories (Prusoff, Bakhle, and Sekely, 1965;
investigation Prusoff,
1967).
Three general areas have been observed where this analog or
its phosphorylated derivatives exert biochemical effects:
(a) competitive
inhibition of several enz}~nes concerned with a biosynthesis of DNA-thymine; @llosteric or fee4back inhibition by 5-iodo-2'-deoxyuridine deoxycytidylate
deaminase
thymidine kinase reductase ,
i
(Diwan and Prusoff,
(Prusoff and Chang,
(Prusoff, unpublished); i,
,
,
i
triphosphate of
1968; Prusoff and Chang,
1966) and of ribonucleoslde
(c) incorporation
(b)
1968),
diphosphate
into virus-DNA with a
J
+This investigation was supported by U.S. Public llealth Service Researzh Grant CA-05262 from the National Cancer Institute. *Present address: New York I0021.
Hospital of Special Surgery, 545 East 70th Street, New York,
4
Pharmacological Research Communications, VoL 1, No. 1, 1969
subsequent effect on the expression of genetic information during either replication or transcription
(Goz and Prusoff, 1968; other pertinent references
cited in Prusoff, Bakhle and Sekely, 1965; Prusoff, 1967). African monkey kidney cells (BSC-I) were seeded and grown according to methods already described (Prusoff, Bakhle and Sekely, 1965).
Fourteen day non-
growing cultures of these cells were infected with a suspension of herpes simplex virus at a multiplicity of 20 pfu/cell.
After a period of one hour at 37 ° , the
medium was removed, the infected cells washed with phosphate (0.05 M, pH 8.0) buffered saline, and fresh medium was added.
Five hours later the medium was
decanted and the cells were suspended in 1.5 volumes phosphate buffer (0.05 M, pH 8.0) containing mercaptoethanol.
The cells were disrupted by freezing and
thawing three times, and a clear supernate was obtained by ultra-centrifugation at I00,000 x g for I hour.
DNA-polymerase activity was determined by a minor
modification of the procedure described by Mantsavinos
(1964).
Table i indicates that infection with herpes simplex virus results in a progressive increase in DNA-polymerase activity which reached a maximum 6 hours
Table I Relative DNA-polymerase activity of African green monkey kidney cells at various times after infection with herpes simplex virus
~-
- Time of ~infection . .
.
.
.
~eiative DNA-polymerase activity
I
i .0"
4
1.2
5
1.3
6
1.4
7
0.97
The composition of the basic reaction mixture is described by Mantsavinos (1964). The amount of incorporation of thymidine triphosphate-2-C 14 in the acid insoluble material in the reaction mixture that contained the enzyme from the non-infected cells was 6,100 cpm (40 per cent conversion) and this value was equated to 1.0. (Duplicate experiments)
Pharmacological Research CommunicaNons, Vol. 1, No.l, 1969 after infection.
5
Similar results were obtained by other investigators (Russell
and co-workers, 1964).
The herpes virus induced DNA polymerase activity has
been shown to be virus specified by Keir and co-workers (1966a, 1966b). The relative susceptibility to inhibition by dlUTP of DNA polymerase deri~ed from uninfected cells and from cells infected with herpes simplex virus (Rolly Strain IZ:~ and an IUdR resistant varisnt of this straln is shown in Table 2.
No differences in susceptibility to inhibition by dlUTP was
observed, the 50 percent inhibition index of DNA polymerase present in both the control and virus infected cell extracts hein E about 2.
Studies of DNA-
polymerase derived fro~ a routine neoplasm indicated that the inhibition of the utilization of dTTP by dlUTP was competitive (Bakhle and Prusoff, In press). The ahillty of DNA polymerase present in murine neoplasms to utilize either dTTP or dlUTP was indicated by the failure of the halogenated analog to inhibit the utilization of radioactive deoxyadenosine triphosphate for the biosynthesis of DNA (Bakhle and Prusoff, In press).
Table 2 Inhibition by dlUTP of the utilization of dTTF-2-C 14 by DNA-polymerase derived from either non-lnfected African green monkey kidney cells or from cells infected with herpes simplex virus of which one strain was resistant to the inhibitory effects of IUdR* Concentration of dlUTP (~M)
Noninfected
Percent Inhibition Normal herpes simplex virus
Resistant herpes simplex virus
40
51
59
54
80
75
69
71
Composition of the basic DNA-polymerase reaction mixture is described by M~=tsavlnos (1964). Concentration of dTTP is 20 ~M. (Duplicate experlment:~) Thus the present report extends to another enzyme (DNA polymerase) o u r previous findings observed with thvmidine kinase, thymidylate kinase, a n d deoxycytldylate deaminsse, that although IUdR inhibits herpes simpleK virus replication
6
Pharmacological Research Communications, Vol. 1, No. I, 1969
selectively it does not preferentially inhibit the virus induced or specified enzymes.
The ability of IUdR to inhibit replication of herpes simplex virus
at a concentration that is not inhibitory to the uninfected host cell was reported by Cramer, Wacker and Welch (1963).
This selectivity of action is
probably a result 6f the enhanced thymidine kinase activity induced or specifie by the virus (Klemperer et al., 1967), an action that results in a selective increase in the amount of phosphorylated derivatives of IUdR in the infected cell. The biological and biophysical consequences of incorporation of IUdR into DNA have been reviewed (Prusoff, Bakhle and Sekely, 1965; Prusoff, 1967), and recent studies with a bacterial virus have indicated very marked effects on the rate and amount of formation of viral specific enzb~nes (Goz and Prusoff, 1968).
It is believed that the mechanism for the antiviral activity of IUdR
will probably be elucidated from a study of the effects that result from incorporation of this analog into the DNA of the virus.
Re ference s Bakhle, Y. S. and Prusoff, W. H., Biochim. et Biophys. Acta. (In press). Cramer, J. W., Wacker, A. and Welch, A. D., Biochem. J., 87, 26P (1963). Goz, B. and Prusoff, W. H., J. Biol. Chem.,24_~_3, 4750 (1968). Diwan, A. and Prusoff, W. H., Virology, 34, 184 (1968). Keir, H. M., Hay, J., Morrison, J. M. and Subak-Sharpe, H., Nature, 210, 369
(1966a). Keir, H. M., Subak-Sharpe, H., Shedden, W. I. H., Watson, D. H., and Wildy, P., Virology, 30, 154 (1966b). Klemperer, H. G., Haynes, G. R., Sheddon, W. I. H., and Watson, D. H., Virology 31, 120 (1967). Mantsavinos, R., J. Biol. Chem., 239, 3431 (1964). Prusoff, W. H., Pharmacol. Rev., 19, 209 (1967). Prusoff, W H., Bakhle, Y. S. and Sekely, L., Ann. N. Y. Acad. Sci., 130, 135 (1965). Prusoff, W. H. and Chang, P. K., Fed Proc., 25, 755 (1966). Prusoff, W. H. and Chang, P. K., J. Biol. Chem., 243, 223 (1968). Russell, W. C., Gold, E., Kelr, H. M., Omura, H., Wilson, D. H., and Wildy, P., Virology, 22, 103 (1964). L _i
,J i