Archives
Arch Virol (1990) 110:121-127
Vi rology
© by Springer-Verlag1990
Role of DNA topoisomerase I in the replication of herpes simplex virus type 2 Brief Report Y. Yamada, N. Yamamoto, K. Maeno, and Y. Nishiyama
Laboratory of Virology, Research Institute for Disease Mechanism and Control, Nagoya University School of Medicine, Showa-ku, Nagoya, Japan Accepted October 26, 1989
Summary. Camptothecin (CPT), a specific inhibitor of DNA topoisomerase I, effectively inhibited the replication of herpes simplex virus type 2 (HSV-2). CPT was found to reduce the level of immediate early viral mRNA in a dose-response manner. On the other hand, we could not find any evidence that a novel DNA topoisomerase I was induced in HSV-2-infected cells. Our observations suggest that host cell DNA topoisomerase I is involved in the replication of HSV-2.
DNA topoisomerases are enzymes which change the topological state of DNA, and are classified into two types according to their mechanism of action. DNA topoisomerase I catalyzes the breaking and the rejoining of only one strand of D N A at a time, while DNA topoisomerase II works by passing a segment of DNA through a transient double-stranded break. Although the precise roles of both enzymes are still unknown, it has been suggested that they play important roles in a variety of genetic events such as DNA replication, transcription, recombination and transposition [1]. Herpes simplex virus (HSV) is a large DNA virus and encodes a number of its own enzymes which are involved in DNA metabolism. Several investigators have shown that a novel DNA topoisomerase activity is induced in HSV-infected cells [2, 3]. We have reported that DNA topoisomerase II is involved in HSV replication, but the enzyme appeared to be of host cell origin [4]. Although the whole nucleotide sequence of HSV-1 genome has been recently revealed [5], it is still uncertain whether HSV-specific topoisomerases are encoded or not. In this study, we investigated the effect of camptothecin, a specific inhibitor
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Y. Yamada et al. !
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!
t00 o
8
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0.01 0.02 0,03 Carnptothecin (,ug/mi)
0,04
Fig. 1. Effect of CPT on the replication of HSV-2. The sensitivity of the virus was measured by plaque reduction assays on monolayers of HEF with virus adsorption for 1 h at 37°C and maintenance medium containing various concentrations of the drug. Results are expressed as a percentage of the values obtained in control cultures without any drug
Fig. 2. Effect of CPT on the synthesis of HSV2-induced proteins. Mock-infected or HSV-2-infected HEF were labeled with [3SS]methionine (10 gCi/ml) in the absence or presence of various concentration of CPT. The labeled proteins were analyzed by SDS-PAGE followed by autoradiography. Numbers on the right indicate the molecular weights of virus specific proteins
DNA topoisomerase I in the replication of herpes simplex virus type 2
123
Fig. 3. Effect of CPT on the level of HSV-2 immediate early mRNA. Cytoplasmic RNA was prepared from infected HEF at 5 h post infection and placed on a Gene Screen Plus membrane with suction at 2-fold dilutions. Dot blot hybridization was carried out as described in the text I
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100
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Arch Virol (1990) 110:121-127
Vi rology
© by Springer-Verlag1990
Role of DNA topoisomerase I in the replication of herpes simplex virus type 2 Brief Report Y. Yamada, N. Yamamoto, K. Maeno, and Y. Nishiyama
Laboratory of Virology, Research Institute for Disease Mechanism and Control, Nagoya University School of Medicine, Showa-ku, Nagoya, Japan Accepted October 26, 1989
Summary. Camptothecin (CPT), a specific inhibitor of DNA topoisomerase I, effectively inhibited the replication of herpes simplex virus type 2 (HSV-2). CPT was found to reduce the level of immediate early viral mRNA in a dose-response manner. On the other hand, we could not find any evidence that a novel DNA topoisomerase I was induced in HSV-2-infected cells. Our observations suggest that host cell DNA topoisomerase I is involved in the replication of HSV-2.
DNA topoisomerases are enzymes which change the topological state of DNA, and are classified into two types according to their mechanism of action. DNA topoisomerase I catalyzes the breaking and the rejoining of only one strand of D N A at a time, while DNA topoisomerase II works by passing a segment of DNA through a transient double-stranded break. Although the precise roles of both enzymes are still unknown, it has been suggested that they play important roles in a variety of genetic events such as DNA replication, transcription, recombination and transposition [1]. Herpes simplex virus (HSV) is a large DNA virus and encodes a number of its own enzymes which are involved in DNA metabolism. Several investigators have shown that a novel DNA topoisomerase activity is induced in HSV-infected cells [2, 3]. We have reported that DNA topoisomerase II is involved in HSV replication, but the enzyme appeared to be of host cell origin [4]. Although the whole nucleotide sequence of HSV-1 genome has been recently revealed [5], it is still uncertain whether HSV-specific topoisomerases are encoded or not. In this study, we investigated the effect of camptothecin, a specific inhibitor
t22
Y. Yamada et al. !
!
|
!
!
t00 o
8
-~so E o
n
0
0.01 0.02 0,03 Carnptothecin (,ug/mi)
0,04
Fig. 1. Effect of CPT on the replication of HSV-2. The sensitivity of the virus was measured by plaque reduction assays on monolayers of HEF with virus adsorption for 1 h at 37°C and maintenance medium containing various concentrations of the drug. Results are expressed as a percentage of the values obtained in control cultures without any drug
Fig. 2. Effect of CPT on the synthesis of HSV2-induced proteins. Mock-infected or HSV-2-infected HEF were labeled with [3SS]methionine (10 gCi/ml) in the absence or presence of various concentration of CPT. The labeled proteins were analyzed by SDS-PAGE followed by autoradiography. Numbers on the right indicate the molecular weights of virus specific proteins
DNA topoisomerase I in the replication of herpes simplex virus type 2
123
Fig. 3. Effect of CPT on the level of HSV-2 immediate early mRNA. Cytoplasmic RNA was prepared from infected HEF at 5 h post infection and placed on a Gene Screen Plus membrane with suction at 2-fold dilutions. Dot blot hybridization was carried out as described in the text I
~
I
I
I
100
._m
E 50
