Vol. 176, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 805-81 2
April 30, 1991
MECHANISM OF INHIBITION OF HUMAN CYTOMEGALOVIRUS REPLICATION BY OXETANOCIN G Tohru Dalkoku 1, Naohiko Yamamoto 1, Seiichi Saito 2, Masayuki Kitagawa 2, Nobuyoshi Shimada 2, and Yukihiro Nishiyama 1 1Laboratory of Virology, Research Institute for Disease Mechanism and Control, Nagoya University School of Medicine, Nagoya 466, Japan 2Research Laboratories, Pharmaceutical Group Nippon Kayaku Co., Ltd., Tokyo 115, Japan Received March 14, 1991
SUMMARY: Oxetanocin G(9-(2-deoxy-2-hydroxymethyl- ~ -D-erythro-oxetanosyl)guanine, OXT-
G) is a potent and selective agent against human cytomegalovirus (HCMV). In this study we synthesized the triphosphate form of OXT-G, OXT-GTP, and examined its effect on the activities of HCMV DNA polymerase, herpes simplex type 2 (HSV-2) DNA polymerase and human DNA polymerase a . OXT-GTP was found to inhibit all these polymerases in a competitive manner with respect to dGTP. The Km for dGTP and the Ki for OXT-GTP of HCMV DNA polymerase were 0.86 and 0.53/~ M, respectively, while the corresponding values of DNA polymerase a were 2.2 and 3.6 /~ M, respectively. HPLC analysis using [3H]OXT-G also revealed that OXT-G was converted to its triphosphate form 7- to 8-fold more efficiently in HCMV-infected cells than in uninfected cells. The results suggest that both the preferential phosphorylation of OXT-G in HCMV-infected cells and the preferential inhibition of HCMV DNA polymerase by OXT-GTP may contribute towards the selective activity of OXT-G against HCMV replication. ~ 1991 AoademioP..... Znc.
Recently, a novel nucleoside with an oxetanosyl-N-glycoside was isolated from a culture filtrate of Bacillus megaterium (1), and it has been shown that the compound and its derivatives possess antiviral activity against DNA viruses and retroviruses in vitro
(2-5). Among the related
compounds, a guanosine analog, 9-(2-deoxy-2-hydroxymethyl-¢3-D-erythro-oxetanosyI)guanine (OXT-G) showed the most potent activity against human cytomegalovirus (HCMV) (3). The activity and selectivity of OXT-G against HCMV was comparable to that of DHPG (3,6,7). The compound was also highly active in vitro (8,9). In a guinea pig model of CMV infection, OXT-G exhibited much higher efficacy than DHPG (9). Although we have shown that OXT-G inhibits the synthesis of HCMV DNA and viral late proteins in a dose response manner when the drug is added at the beginning of infection (3), the mechanism of action have remained unexplored. In this study, we investigated the effect of OXT-G0006-291X/91 $1.50 805
Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 176, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
triphosphate(OXT-GTP) against HCMV DNA polymerase and the phosphorylation of OXT-G in HCMV-infected cells. MATERIALS AND METHODS
Chemicals. OXT-G was produced from 9-(2-deoxy-2-hydroxymethyl-/3-D.erythro-oxetanosyl) adenine by chemical and biological transformation as described previously (10). The synthesis of OXT-GTP was carried out as follows(Fig.l). Briefly, OXT-G[1] was treated with 4,4'dimethoxytrityl chloride(DMTrC1) in DMF to give a selectively protected compound[2] in 25.8% yield, and a position isomer[3] was given in 16.6% yield. Acetylation of the compound[2] with triethylamine, N,N-dimethylaminopyridine and acetic anhydride gave the compound[4], which was converted into OXT-G-monoacetate[5] in aqueous acetic acid. The compound[5] was treated with phosphoryl chloride in triethyl phosphate to give the compound[6] in 71.5% yield, and OXT-Gmonophosphate[7] was given to treat the compound[6] with 0.1N NaOH at 0°C. Finally, OXTGMP[7] was activated with 4-morpholine in the presence of DCC, and then was converted into OXTGTP[9] with tributylammonium pyrophosphate in 20.0% yield. Radioactive OXT-G was prepared as 9-(2-deoxy-2-hydroxymethyl- fl -D-erythro-[4-3H]-oxetanosyl)guanine. Detailed procedures of the synthesis of these compounds will be published elsewhere. Deoxynucleoside triphosphates were purchased from Sigma Chemical Co., St.Louis,Mo. [8-3H]dGTP(specific activity, 16Ci/mmol) and [methyl-3H]dTFP(specific activity, 67Ci/mmol) were obtained from Amersham Laboratories, United Kingdom. Activated calf thymus DNA was purchased from Anglian Biotechnology Ltd., Essex, United Kingdom. Cell and viruses. Human embryonic fibroblasts(HEF) were prepared as described previously (11) and grown in Eagles' minimal essential medium(MEM) supplemented with 10% fetal calf serum, and used in passage 5 to 20. HCMV strain AD 169 was used throughout this study. Herpes simplex type 2(HSV-2) strain 186 was also used to prepare the partially purified HSV-2 DNA polymerase. Purification of DNA polymerases. HCMV and HSV-2 DNA polymerases were partially purified from HCMV-infected HEF and HSV-2-infected Vero cells, respectively. Human DNA polymerase a was purified from HCMV-infected HEF, and the method of purification of these polymerases has been described elsewhere (11,12). The specific activities of HCMV and HSV-2 DNA polymerases and human DNA polymerase a were 3.6 × l0 s, 4.8 × 10 6, and 1.4 × l0 s units/mg protein, respectively. Assay of DNA polymerases. The standard mixture for the assay of HCMV and HSV-2 DNA polymerases contained 50mM Tris-HCi(pH7.8), 8mM MgC12, lmM dithiothreitol(DTT), 200mM KC1, 64/~ M dATE dTTP, dCTP and 32/e M [3H]dGTP(1.5/z Ci/nmol), 2.5 ¢z g of activated calf thymus DNA and enzyme in 25 ft 1. Identical conditions were used as the standard assay of human DNA polymerase a , except that 200mM KC1 was omitted. Incubation was carried out at 37°C for 15min before acid-insoluble radioactivity was measured. One unit of DNA polymerase activity was defined as the amount of enzyme that catalyzed the incorporation of lpmol of deoxynucleoside monophosphates into DNA in an hour. Measurement of drug anabolites. Monolayers of HEF in 100ram plastic dishes were infected with HCMV at a multiplicity of approximately 3PFU/cell. At 48h postinfection, [3H]OXT-G was added to HCMV-infected or mock-infected cultures and the cultures were incubated for 12h at 37°C. After incubation, the medium was removed, and the cells were washed twice with cold phosphate-buffered saline(PBS), scraped by rubber policemen and pelleted by centrifugation at 200 × g for 5min. Preparation of cell extracts for nucleotide analysis was carried out as described by Tanaka etal (13). Briefly, the cells were suspended in PBS and cold trichloroacetic acid was added to the suspension to give a final concentration of 0.3M. After mixing well, the mixture was kept for 30rain at 4°C and centrifuged. The acidic supernatant was added to 1.1 volume of cold Freon containing 0.5M tri-noctylamine, and centrifuged. The aqueous upper layer was separated and then analysed with a Shimadzu LC-6A high-pressure liquid chromatography(HPLC), fitted with a JASCO Biofine IEC806
Vol. 176, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
DEAE column(7.5 × 75mm). Elution was with a linear gradient of NaCI, 0-0.5M in 100mM sodium acetate, pH4.5 with a flow rate of lml/min. The UV adsorption was monitored at 254nm. Fractions of lml were collected and mixed with 10ml of aqueous counting scintillant ACS II(Amersham) for determination of radioactivity. RESULTS Inhibition of HCMV DNA polymerase by OXT-GTP. Since it has been shown that OXT-G inhibit HCMV replication by impairing viral DNA synthesis (3), we synthesized OXT-GTP (Fig. 1) and studied its inhibitory effect against HCMV DNA polymerase. HCMV DNA polymerase was inhibited by OXT-GTP in a dose response manner, while neither OXT-G nor OXT-GMP showed such an inhibitory effect for the DNA polymerase(data not shown). The mode of the inhibitory action of OXT-GTP was analysed using Lineweaver-Burk plots (Fig.2), and the compound was found to inhibit HCMV DNA polymerase competitively with respect to dGTP. The apparent Km value for dGTP was 0.86/x M for HCMV DNA polymerase under the assay condition we used. The Ki value, determined by replotting of the apparent Km value versus the concentrations of OXT-GTP, was 0.53 klM. Similar experiments with human DNA polymerase a (Fig.3) and HSV-2 DNA polymerase (data not shown) showed that the inhibition was competitive with dGTP for these polymerases, and the kinetic parameters were then calculated (Table 1). The Ki for OXT-GTP of HSV-2 DNA polymerase was similar to that of HCMV DNA polymerase, but the corresponding value of DNA O
~ 10 ft Ci/ml) for more than 12h, only undetectable levels of the radioactivity was associated with the acidinsoluble fractions of HCMV-infected cells. At present, however, we can not rule out the possibility that OXT-GMP is incorporated into DNA and acts as a DNA chain terminator. The HPLC analysis demonstrates that OXT-G was indeed phosphorylated to the triphosphate form in HCMV-infected cells. Although mock-infected cells were also capable of producing OXT-G, the level of the triphosphate was 7- to 8-fold lower than that in HCMV-infected cells. It is well known that infection of cells with HCMV results in the induction of host cell nucleoside kinases such as thymidine and deoxyguanosine kinases (14,15). On the other hand, Biron et al. (16) have pointed out the possibility that HCMV encodes a nucleoside kinase. We think that the 7- to 8-fold difference in the OXT-GTP level between the HCMV-infected and uninfected cells may be explained by the induction of host cell nucleoside and nucleotide kinases. The identification of enzymes responsible for the phosphorylation of OXT-G remains to be determined.
REFERENCES 1. 2. 3. 4. 5.
Shimada, N., Hasegawa, S., Saito, S., Nishikiori, T., Fujii, A., and Takita, T. (1987) J. Antibiot. 4 0, 1788-1790. Hoshino, H., Shimizu, N., Shimada, N., Takita, T., and Takeuchi, T. (1987) J. Antibiot. 40, 1077-1078. Nishiyama, Y., Yamamoto, N., Takahashi, K., and Shimada, N. (1988) Antimicrob. Agents Chemother. 32, 1053-1056. Ueda, K., Tsurimoto, T., Nagahata, T., Chisaki, O., and Matsubara, K. (1990) Virology 169, 213-233. Seki, J., Shimada, N., Takahashi, K., Takita, T., Takeuchi, T., and Hoshino, H. (1989) Antimicrob. Agents Chemther. 3 3, 773-775. 811
Vol. 176, No. 2, 1991
6. 7. 8. 9. 10. 11. 12. 13. I4. 15. 16.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Cheng, Y.-H., Huang, E.-S., Lin, J.-C., Mar, E.-C., Pagano, T.S., Dutschman, G.E., and Grill, S P. (1983) Proc. Natl. Acad. Sci. USA 80, 2767-2770. Mar, E.-C., Cheng, Y.-C., and Huang, E.-S., (1983) Antimicrob. Agents Chemother. 2 4, 518-521. Nishiyama, Y., Yamamoto, N., Yamada, Y., Fujioka, H., Shimada, N., and Takahashi, K. (1989) J. Antibiot. 42, 1308-13t 1. Yamamoto, N., Yamada, Y., Daikoku, T., Nishiyama, Y., Tsutsui, Y., Shimada, N., and Takahashi, K. (1990) J. Antibiot. in press. Shimada, N., Hasegawa, S., Harada, T., Tomisawa, T., Fujii, A., and Takita, T. (1986) J. Antibiot. 3 9, 1623-1625. Nishiyama, Y., Maeno, K., and Yoshida, S. (1983) Virology 124, 221-231. Nishiyama, Y., Suzuki, S., Yamauchi, M., Maeno, K., and Yoshida, S. (1984) Virology 135, 87-96. Tanaka, K., Yoshioka, A., Tanaka, S., and Wataya, Y. (1984) Anal. Biochem. 139, 35-41. Zavada, V., Erban, Rezacova, D., and Vonka, V. (1976) Arch. Virol. 5 2, 333-339. Estes, J., and Huang, E.-S. (1977) J. Virol. 23, 13-21. Biron, K.K., Fyfe, J.A., Stanat, S.C., Leslie, L.K., Sorrell, J.B., Lambe, C.U., and Coen, D.M. (1986) Proc. Natl. Acad. Sci. USA 83, 8769-8773.
812
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 805-81 2
April 30, 1991
MECHANISM OF INHIBITION OF HUMAN CYTOMEGALOVIRUS REPLICATION BY OXETANOCIN G Tohru Dalkoku 1, Naohiko Yamamoto 1, Seiichi Saito 2, Masayuki Kitagawa 2, Nobuyoshi Shimada 2, and Yukihiro Nishiyama 1 1Laboratory of Virology, Research Institute for Disease Mechanism and Control, Nagoya University School of Medicine, Nagoya 466, Japan 2Research Laboratories, Pharmaceutical Group Nippon Kayaku Co., Ltd., Tokyo 115, Japan Received March 14, 1991
SUMMARY: Oxetanocin G(9-(2-deoxy-2-hydroxymethyl- ~ -D-erythro-oxetanosyl)guanine, OXT-
G) is a potent and selective agent against human cytomegalovirus (HCMV). In this study we synthesized the triphosphate form of OXT-G, OXT-GTP, and examined its effect on the activities of HCMV DNA polymerase, herpes simplex type 2 (HSV-2) DNA polymerase and human DNA polymerase a . OXT-GTP was found to inhibit all these polymerases in a competitive manner with respect to dGTP. The Km for dGTP and the Ki for OXT-GTP of HCMV DNA polymerase were 0.86 and 0.53/~ M, respectively, while the corresponding values of DNA polymerase a were 2.2 and 3.6 /~ M, respectively. HPLC analysis using [3H]OXT-G also revealed that OXT-G was converted to its triphosphate form 7- to 8-fold more efficiently in HCMV-infected cells than in uninfected cells. The results suggest that both the preferential phosphorylation of OXT-G in HCMV-infected cells and the preferential inhibition of HCMV DNA polymerase by OXT-GTP may contribute towards the selective activity of OXT-G against HCMV replication. ~ 1991 AoademioP..... Znc.
Recently, a novel nucleoside with an oxetanosyl-N-glycoside was isolated from a culture filtrate of Bacillus megaterium (1), and it has been shown that the compound and its derivatives possess antiviral activity against DNA viruses and retroviruses in vitro
(2-5). Among the related
compounds, a guanosine analog, 9-(2-deoxy-2-hydroxymethyl-¢3-D-erythro-oxetanosyI)guanine (OXT-G) showed the most potent activity against human cytomegalovirus (HCMV) (3). The activity and selectivity of OXT-G against HCMV was comparable to that of DHPG (3,6,7). The compound was also highly active in vitro (8,9). In a guinea pig model of CMV infection, OXT-G exhibited much higher efficacy than DHPG (9). Although we have shown that OXT-G inhibits the synthesis of HCMV DNA and viral late proteins in a dose response manner when the drug is added at the beginning of infection (3), the mechanism of action have remained unexplored. In this study, we investigated the effect of OXT-G0006-291X/91 $1.50 805
Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 176, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
triphosphate(OXT-GTP) against HCMV DNA polymerase and the phosphorylation of OXT-G in HCMV-infected cells. MATERIALS AND METHODS
Chemicals. OXT-G was produced from 9-(2-deoxy-2-hydroxymethyl-/3-D.erythro-oxetanosyl) adenine by chemical and biological transformation as described previously (10). The synthesis of OXT-GTP was carried out as follows(Fig.l). Briefly, OXT-G[1] was treated with 4,4'dimethoxytrityl chloride(DMTrC1) in DMF to give a selectively protected compound[2] in 25.8% yield, and a position isomer[3] was given in 16.6% yield. Acetylation of the compound[2] with triethylamine, N,N-dimethylaminopyridine and acetic anhydride gave the compound[4], which was converted into OXT-G-monoacetate[5] in aqueous acetic acid. The compound[5] was treated with phosphoryl chloride in triethyl phosphate to give the compound[6] in 71.5% yield, and OXT-Gmonophosphate[7] was given to treat the compound[6] with 0.1N NaOH at 0°C. Finally, OXTGMP[7] was activated with 4-morpholine in the presence of DCC, and then was converted into OXTGTP[9] with tributylammonium pyrophosphate in 20.0% yield. Radioactive OXT-G was prepared as 9-(2-deoxy-2-hydroxymethyl- fl -D-erythro-[4-3H]-oxetanosyl)guanine. Detailed procedures of the synthesis of these compounds will be published elsewhere. Deoxynucleoside triphosphates were purchased from Sigma Chemical Co., St.Louis,Mo. [8-3H]dGTP(specific activity, 16Ci/mmol) and [methyl-3H]dTFP(specific activity, 67Ci/mmol) were obtained from Amersham Laboratories, United Kingdom. Activated calf thymus DNA was purchased from Anglian Biotechnology Ltd., Essex, United Kingdom. Cell and viruses. Human embryonic fibroblasts(HEF) were prepared as described previously (11) and grown in Eagles' minimal essential medium(MEM) supplemented with 10% fetal calf serum, and used in passage 5 to 20. HCMV strain AD 169 was used throughout this study. Herpes simplex type 2(HSV-2) strain 186 was also used to prepare the partially purified HSV-2 DNA polymerase. Purification of DNA polymerases. HCMV and HSV-2 DNA polymerases were partially purified from HCMV-infected HEF and HSV-2-infected Vero cells, respectively. Human DNA polymerase a was purified from HCMV-infected HEF, and the method of purification of these polymerases has been described elsewhere (11,12). The specific activities of HCMV and HSV-2 DNA polymerases and human DNA polymerase a were 3.6 × l0 s, 4.8 × 10 6, and 1.4 × l0 s units/mg protein, respectively. Assay of DNA polymerases. The standard mixture for the assay of HCMV and HSV-2 DNA polymerases contained 50mM Tris-HCi(pH7.8), 8mM MgC12, lmM dithiothreitol(DTT), 200mM KC1, 64/~ M dATE dTTP, dCTP and 32/e M [3H]dGTP(1.5/z Ci/nmol), 2.5 ¢z g of activated calf thymus DNA and enzyme in 25 ft 1. Identical conditions were used as the standard assay of human DNA polymerase a , except that 200mM KC1 was omitted. Incubation was carried out at 37°C for 15min before acid-insoluble radioactivity was measured. One unit of DNA polymerase activity was defined as the amount of enzyme that catalyzed the incorporation of lpmol of deoxynucleoside monophosphates into DNA in an hour. Measurement of drug anabolites. Monolayers of HEF in 100ram plastic dishes were infected with HCMV at a multiplicity of approximately 3PFU/cell. At 48h postinfection, [3H]OXT-G was added to HCMV-infected or mock-infected cultures and the cultures were incubated for 12h at 37°C. After incubation, the medium was removed, and the cells were washed twice with cold phosphate-buffered saline(PBS), scraped by rubber policemen and pelleted by centrifugation at 200 × g for 5min. Preparation of cell extracts for nucleotide analysis was carried out as described by Tanaka etal (13). Briefly, the cells were suspended in PBS and cold trichloroacetic acid was added to the suspension to give a final concentration of 0.3M. After mixing well, the mixture was kept for 30rain at 4°C and centrifuged. The acidic supernatant was added to 1.1 volume of cold Freon containing 0.5M tri-noctylamine, and centrifuged. The aqueous upper layer was separated and then analysed with a Shimadzu LC-6A high-pressure liquid chromatography(HPLC), fitted with a JASCO Biofine IEC806
Vol. 176, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
DEAE column(7.5 × 75mm). Elution was with a linear gradient of NaCI, 0-0.5M in 100mM sodium acetate, pH4.5 with a flow rate of lml/min. The UV adsorption was monitored at 254nm. Fractions of lml were collected and mixed with 10ml of aqueous counting scintillant ACS II(Amersham) for determination of radioactivity. RESULTS Inhibition of HCMV DNA polymerase by OXT-GTP. Since it has been shown that OXT-G inhibit HCMV replication by impairing viral DNA synthesis (3), we synthesized OXT-GTP (Fig. 1) and studied its inhibitory effect against HCMV DNA polymerase. HCMV DNA polymerase was inhibited by OXT-GTP in a dose response manner, while neither OXT-G nor OXT-GMP showed such an inhibitory effect for the DNA polymerase(data not shown). The mode of the inhibitory action of OXT-GTP was analysed using Lineweaver-Burk plots (Fig.2), and the compound was found to inhibit HCMV DNA polymerase competitively with respect to dGTP. The apparent Km value for dGTP was 0.86/x M for HCMV DNA polymerase under the assay condition we used. The Ki value, determined by replotting of the apparent Km value versus the concentrations of OXT-GTP, was 0.53 klM. Similar experiments with human DNA polymerase a (Fig.3) and HSV-2 DNA polymerase (data not shown) showed that the inhibition was competitive with dGTP for these polymerases, and the kinetic parameters were then calculated (Table 1). The Ki for OXT-GTP of HSV-2 DNA polymerase was similar to that of HCMV DNA polymerase, but the corresponding value of DNA O
~ 10 ft Ci/ml) for more than 12h, only undetectable levels of the radioactivity was associated with the acidinsoluble fractions of HCMV-infected cells. At present, however, we can not rule out the possibility that OXT-GMP is incorporated into DNA and acts as a DNA chain terminator. The HPLC analysis demonstrates that OXT-G was indeed phosphorylated to the triphosphate form in HCMV-infected cells. Although mock-infected cells were also capable of producing OXT-G, the level of the triphosphate was 7- to 8-fold lower than that in HCMV-infected cells. It is well known that infection of cells with HCMV results in the induction of host cell nucleoside kinases such as thymidine and deoxyguanosine kinases (14,15). On the other hand, Biron et al. (16) have pointed out the possibility that HCMV encodes a nucleoside kinase. We think that the 7- to 8-fold difference in the OXT-GTP level between the HCMV-infected and uninfected cells may be explained by the induction of host cell nucleoside and nucleotide kinases. The identification of enzymes responsible for the phosphorylation of OXT-G remains to be determined.
REFERENCES 1. 2. 3. 4. 5.
Shimada, N., Hasegawa, S., Saito, S., Nishikiori, T., Fujii, A., and Takita, T. (1987) J. Antibiot. 4 0, 1788-1790. Hoshino, H., Shimizu, N., Shimada, N., Takita, T., and Takeuchi, T. (1987) J. Antibiot. 40, 1077-1078. Nishiyama, Y., Yamamoto, N., Takahashi, K., and Shimada, N. (1988) Antimicrob. Agents Chemother. 32, 1053-1056. Ueda, K., Tsurimoto, T., Nagahata, T., Chisaki, O., and Matsubara, K. (1990) Virology 169, 213-233. Seki, J., Shimada, N., Takahashi, K., Takita, T., Takeuchi, T., and Hoshino, H. (1989) Antimicrob. Agents Chemther. 3 3, 773-775. 811
Vol. 176, No. 2, 1991
6. 7. 8. 9. 10. 11. 12. 13. I4. 15. 16.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Cheng, Y.-H., Huang, E.-S., Lin, J.-C., Mar, E.-C., Pagano, T.S., Dutschman, G.E., and Grill, S P. (1983) Proc. Natl. Acad. Sci. USA 80, 2767-2770. Mar, E.-C., Cheng, Y.-C., and Huang, E.-S., (1983) Antimicrob. Agents Chemother. 2 4, 518-521. Nishiyama, Y., Yamamoto, N., Yamada, Y., Fujioka, H., Shimada, N., and Takahashi, K. (1989) J. Antibiot. 42, 1308-13t 1. Yamamoto, N., Yamada, Y., Daikoku, T., Nishiyama, Y., Tsutsui, Y., Shimada, N., and Takahashi, K. (1990) J. Antibiot. in press. Shimada, N., Hasegawa, S., Harada, T., Tomisawa, T., Fujii, A., and Takita, T. (1986) J. Antibiot. 3 9, 1623-1625. Nishiyama, Y., Maeno, K., and Yoshida, S. (1983) Virology 124, 221-231. Nishiyama, Y., Suzuki, S., Yamauchi, M., Maeno, K., and Yoshida, S. (1984) Virology 135, 87-96. Tanaka, K., Yoshioka, A., Tanaka, S., and Wataya, Y. (1984) Anal. Biochem. 139, 35-41. Zavada, V., Erban, Rezacova, D., and Vonka, V. (1976) Arch. Virol. 5 2, 333-339. Estes, J., and Huang, E.-S. (1977) J. Virol. 23, 13-21. Biron, K.K., Fyfe, J.A., Stanat, S.C., Leslie, L.K., Sorrell, J.B., Lambe, C.U., and Coen, D.M. (1986) Proc. Natl. Acad. Sci. USA 83, 8769-8773.
812
