Topoisomerase I and II activities are required for Epstein-Barr virus replication

Transcription

1 Journal of General Virology (1993), 74, Printed in Great Britain 2263 Topoisomerase I and II activities are required for Epstein-Barr virus replication Michiko Kawanishi Department of Microbiology, Faculty of Medicine, Kyoto University, Kyoto 606, Japan The roles of topoisomerases I and II in Epstein-Barr virus (EBV) replication were investigated using Raji cells infected with EBV. The topoisomerase II inhibitor ellipticine inhibited the synthesis of EBV polypeptides at concentrations which did not affect total protein synthesis. Slot blot analysis of total cellular DNA showed that camptothecin and ellipticine inhibited replication of progeny EBV DNA in superinfected Raji cells at concentrations which did not inhibit synthesis of EBV early polypeptides prerequisite for EBV DNA replication. Analysis of the structure of EBV DNA termini demonstrated that both inhibitors affected the replicating EBV DNA. Gardella gel electrophoresis showed that both inhibitors affected the formation of the linear form of EBV DNA. However, restriction analysis of EBV DNA in superinfected Raji cells demonstrated that both inhibitors degraded neither endogenous nor exogenous EBV DNA. Cell viability was not affected by either inhibitor at the concentrations tested. These findings suggest that topoisomerase II is required for expression of the EBV genome and that both topoisomerases I and II are involved in replication of the EBV genome during the lytic phase of the life cycle. The effects of topoisomerase inhibitors on the circular form of EBV DNA during virus replication are discussed. Topoisomerases I and II are involved in gene expression and replication of eukaryotes (Maxwell & Gellert, 1985; Wang, 1985). In the case of animal viruses, topoisomerases play important roles in viral DNA replication, transcription, packaging and integration (Foglesong & Bauer, 1984; Snapka, 1986; Nishiyama et al., 1987; Benson & Huang, 1988; Champoux, 1988; Richter & Strausfeld, 1988; Ebert et al., 1990; Priel et al., 1990, 1991; Schaack et al., 1990a, b; Wong & Hsu, 1990; Yamada et al., 1990; Gu & Rhode, 1991; Priel et al., 1991 ; Wang & Rogler, 1991). Topoisomerase I changes the linking numbers of topological DNA domains in integral steps of one by introducing transient singlestrand breaks. Topoisomerase II breaks both DNA strands and resolves them, thereby changing topological linking numbers in steps of two. Recently, several specific inhibitors for mammalian DNA topoisomerases have been identified and characterized (Drlica & Franco, 1988). Camptothecin and ellipticine are specific inhibitors of mammalian topoisomerases I and II, respectively (Tewey et al., 1984; Hsiang et al., 1985). Camptothecin stablizes the covalent topoisomerase I-DNA complex, resulting in blockage of the rejoining step of the breakage-reunion reaction of topoisomerase I. Ellipticine affects the breakage-reunion reaction of topoisomerase II by stablizing the cleavable complex formed between topoisomerase II and DNA. These inhibitors are suitable for studying the functional involvement of topoisomerase activities in Epstein-Barr virus (EBV) gene expression and replication. EBV early antigen (EA) synthesis is induced in Raji cells by infection with P3HR-1 virus, followed by viral DNA replication and EBV late antigen synthesis. In this study, the effects of camptothecin and ellipticine on EBV replication were analysed using this cell system. Cells were grown in RPMI-1640 medium supplemented with 10 % fetal calf serum, penicillin (100 units/ ml) and streptomycin (250 gg/ml). P3HR-1 virus was obtained from culture fluid of a P3HR-1 substrain containing heterogeneous EBV DNA. Raji cells were infected with concentrated P3HR-1 EBV at an m.o.i, of 500. After adsorption at 37 C for 1 h, infected cells were cultivated in fresh medium in the presence or absence of camptothecin and ellipticine at the indicated concentrations. To investigate the effects of camptothecin and ellipticine on EBV gene expression, EBV-induced polypeptides were analysed by the immunoprecipitation method as previously described (Kawanishi et al., 1981). Cells labelled with [35S]methionine were harvested at 16 h post-infection (p.i.). More than 90% of cells were EA/viral capsid antigen (VCA)-positive by immunofluorescent staining in the absence of topoisomerase inhibitors. The immunoprecipitates obtained from in SGM

2 2264 Short communication 94K,- 67K ~- 43K ~- 30K 20K '~ (a) (b) Fig. 1. Effect of camptothecin and ellipticine on EBV-induced (a) and total (b) protein synthesis. Superinfected Raji cells were labelled with [aas]methionine in the presence or absence of camptothecin and ellipticine, and cell extracts were prepared. (a) EBV-induced polypeptides from mock-infected (lane 9) or infected Raji cells untreated (lane 1) or treated with 0.1, 0.25 and 1 gn-camptothecin (lanes 2 to 4, respectively), or with 1, 2-5 and 5 gm-ellipticine (lanes 5 to 7, respectively), or with 300 gg/ml PAA (lane 8) were precipitated with EA/VCA-positive NPC serum and analysed by 10% SDS-PAGE. (b) Total proteins from mock-infected (lane 7) or infected Raji cells untreated (lane 1), and treated with 1 and 2.5 ~tm-camptothecin (lanes 2 and 3, respectively), 2.5 and 5 ~tm-ellipticine (lanes 4 and 5, respectively), or 300 lag/ml PAA (lane 6) were analysed by 10% SDS PAGE. dividual cell extracts with serum from a nasopharyngeal carcinoma (NPC) patient (anti-ea and -VCA titres are 1:320 and 1:80, respectively) were collected with Protein A-Sepharose, and analysed by 10% SDS- PAGE followed by autoradiography. As shown in Fig. 1, numerous EBV-specific polypeptides were induced in Raji cells by infection with EBV. Treatment of infected cells with 0"1 and 0.25 gm-camptothecin only slightly inhibited the synthesis of EBV-induced polypeptides (Fig. 1 a, lanes 2 and 3, respectively). At these concentrations, total protein synthesis was also only slightly affected (data not shown). However, viral and total protein synthesis was drastically inhibited by treatment with 1 lam-camptothecin (Fig. 1 a, lane 4, and b, lane 2, respectively). As the inhibition pattern of viral protein synthesis was quite similar to that of total protein synthesis, viral and cellular protein synthesis appeared to be equally sensitive to camptothecin. Based on these results, it can not be concluded that topoisomerase I is involved in EBV gene expression. Ellipticine slightly affected only the synthesis of EBV-induced polypeptides with rather high Mr values at a concentration of 2.5 ~tu (Fig. 1 a, lane 6). At 5 pi, ellipticine drastically inhibited total viral protein synthesis (Fig. 1 a, lane 7) without affecting synthesis of cellular proteins (Fig. 1 b, lane 5). The results suggest that viral gene expression may be more highly sensitive to ellipticine than most cellular gene expression and that topoisomerase II plays a more direct role in EBV gene expression. The inhibitory effect of ellipticine on EBV gene expression was considered not to be due to side-effects, because cell viability determined by trypan blue exclusion staining was not affected by 16 h treatment at the concentrations tested (data not shown). The effects of camptothecin and ellipticine on EBV DNA replication were determined by three independent assays. First, relative amounts of EBV DNA were analysed by the slot blot technique. High M r DNA was purified from the infected cells by the method of Sambrook et al. (1989). Two gg of DNA was denatured by treating at 100 C for 5 min in 0.4 M-NaOH and 10 mm-edta and transferred to nitrocellulose filters using the Bio-Dot apparatus (Bio-Rad). Filters were hybridized with 3~P-labelled EBV BamHl W fragments. Scans of the autoradiogram were obtained by using a laser densitometer (LKB 2222 UltroScan XL). As shown in Fig. 2, there was an approximately 100-fold increase in overall amounts of EBV DNA at 24 h p.i., when compared with those at 4 h p.i. Treatment of infected cells with camptothecin and ellipticine inhibited the amplification of EBV DNA in a dose-dependent manner. Camptothecin (0.1 gu) and ellipticine (2.5 lam) reduced amounts of amplified viral DNA to the same level as those in cells treated with phosphonoacetic acid (PAA) (300 ~tg/ml), a specific inhibitor of EBV-induced DNA polymerase. At these concentrations, camptothecin inhibited neither viral protein synthesis (Fig. 1 a, lane 2) nor total protein synthesis (data not shown), and ellipticine only slightly affected viral protein synthesis (Fig. 1 a, lane 6) without affecting total protein synthesis (Fig. 1 b, lane 4). Twenty-four hours treatment with both inhibitors did not affect cell viability at the concentrations tested (data not shown). These results indicate that the inhibitory effects of camptothecin and ellipticine on EBV DNA replication are neither due to the suppression of viral protein synthesis required for viral DNA replication nor due to their side-effects. To examine more directly the effects of topoisomerase inhibitors on replicating EBV DNA, the structure of the EBV genome termini was analysed. EBV DNA has homologous direct repeats of approximately 500 bp at each terminus. The structure of the EBV termini contributes evidence for viral replication, as indicated by

3 Short communication 2265 (a) kb 21~ A I 12~" 7~,~ (b) ~ N z Fig. 2. Slot blot analysis of camptothecin and ellipticine effects on viral DNA synthesis. Various concentrations of camptothecin (Camp.) and ellipticine (Ellip.), or 300 lag/ml PAA were added to superinfected Raft cells at the time of infection. After incubation for 24 h, high M~ DNA was purified, and hybridized with EBV BamHI W fragments labelled with [32P]dCTP by nick translation, and exposed to RP X-Omat film (Kodak) with an intensifying screen. Densitometric scans of the autoradiograms were performed. kb :< the appearance of ladder arrays of terminal fragments representing fused and linear forms of EBV DNA (Raab- Traub & Flynn, 1986; Katz et al., 1989). Total cellular DNA was digested with BamHI, electrophoresed in a 0.6 % agarose gel and transferred to nitrocellulose filters. EBV-specific DNA was probed with the a2p-labelled 1.9 kb XhoI fragment located near the right terminus (Raab-Traub & Flynn, 1986). As shown in Fig. 3(a), a 21 kb fragment represented the fused terminus of the episomal form of endogenous EBV DNA in Raji cells (lane 1), and 7 kb and smaller ladder DNA fragments represented the right termini of linear form EBV DNA from exogenous P3HR-1 virus (lane 2). Multiple fused terminal fragments ranging from 9 to 12 kb appeared at 12 h p.i. (lane 4) and increased in density with time after infection (lanes 5 and 7). DNA fragments representing linear termini were also increased in density with time after infection. These fragments were greatly reduced by treatment with PAA (lane 6). These results indicated that fused terminal fragments and increased DNA fragments representing linear termini were replication-dependent Fig. 3. Southern blot of right-sided EBV DNA termini in superinfected Raji cells. Ten lag of total cellular DNA was digested with BamHI and probed with a ~2p-labelled 1.9 kb XhoI fragment. (a) Latent and replicating forms of EBV DNA. DNA was extracted from superinfected Raji cells at 0, 8, 12, 24 and 48 h p.i. (lanes 2 to 5 and 7, respectively) or superinfected Raji cells in the presence of PAA (300 lag/ml) for 24 h (lane 6). DNA from mock-infected Raji cells and P3HR-1 cells was analysed (lanes 1 and 8, respectively). (b) Effects of topoisomerase inhibitors on latent and replicating forms of EBV DNA. At 24 h p.i., DNA was purified from infected Raji cells untreated (iane i), or treated with 0.25 lam-camptothecin, 2-5 lam-ellipticine or 300 lag/ml PAA (lanes 2 to 4, respectively). Lane 5 shows the restriction pattern of DNA from mock-infected Raji cells. forms. As shown in Fig. 3(b), after treatment with 0.25 gm-camptothecin and 2.5 gm-ellipticine, the amounts of increased ladder fragments representing fused and linear termini were reduced to the same level as those in

4 2266 Short communication kb 11.51~ 8-6~ Circular EBV DNAI~ 4.71~ 3.4~ 2'31~ 2.0~, ~ Linear EBV DNA~" Fig. 4. Gardella gel analysis of circular and linear EBV DNA in superinfected Raji cells. At 24hp.i., infected Raji cells (0.5 x 10 ~) untreated (lane 1) or treated with 0.1, 0.25 and 1.0 gm-camptothecin (lanes 2 to 4, respectively), 1.0, 2.5 and 5 gm-ellipticine (lanes 5 to 7, respectively), or 300 gg/ml PAA (lane 8) were placed in each well of an agarose gel. DNA was transferred onto nitrocellulose filters and probed with the 32P-labelled EBV BamHI W fragment. Lanes 9 and 10 show Gardella gel patterns of mock-infected and infected Raji cells at 4 h p.i., respectively. cells treated with PAA (300 gg/ml). In contrast, the 21 kb fragment representing fused termini of endogenous circular form EBV DNA was not amplified by infection with P3HR-1 virus and was not affected by treatment with either inhibitor. These results suggest that camptothecin and ellipticine affect replicating EBV DNA but not the amount of endogenous EBV DNA. The effect of topoisomerase inhibitors on the formation of linear and circular forms of EBV DNA was analysed by Gardella gel electrophoresis (Gardella et al., 1984). The Gardella gel technique is suitable for resolution of linear and covalently closed circular DNA. As shown in Fig. 4, the infection of Raji cells with P3HR- 1 virus induced synthesis of numerous linear viral DNAs in Raji cells, but did not affect the amounts of circular DNA (lanes 1 and 9). PAA blocked the amplification of linear viral DNA (lane 8), showing this amplification was dependent on EBV-associated DNA polymerase. Treatment of superinfected Raji cells with 0.25 I.tMcamptothecin (lane 3) or 2.5 gm-ellipticine (lane 6) reduced amounts of amplified linear viral DNA to the same level as those at 4 h p.i. (lane 10), suggesting that camptothecin and ellipticine inhibit the formation of Fig. 5. Southern blot of EBV DNA in superinfected Raji cells. High M,. DNA was purified from superinfected Raji cells untreated (lane 1) or treated with 2.5 gm-camptothecin (lane 2), 5 gm-ellipticine (lane 3), and 300 gg/ml PAA (lane 4), mock-infected Raji cells (lane 5), and P3HR-1 cells (lane 6). Two gg of DNA was digested with BamHI and transferred onto nitrocellulose filters, EBV-specific DNA was detected by hybridization with the a~p-labelled EBV BamHI W fragment. linear EBV DNA by EBV DNA polymerase. The amount of circular DNA was also slightly reduced by treatment with each inhibitor. However, neither of the inhibitors affected the amount of the 21 kb fragment representing fused termini of endogenous EBV DNA (Fig. 3). Furthermore, endogenous EBV DNA has been demonstrated to be used as a template for EBV DNA replication by viral DNA polymerase (data not shown). Therefore, these inhibitors might stabilize the complex with topoisomerase I or II and the circular form of EBV DNA during the lytic cycle in Raji cells, resulting in conformational change. The amount of the circular form of endogenous EBV DNA was also reduced at 4 h (lane 10) or 8 h (data not shown) p.i. Hence a conformational change of circular EBV DNA might occur early in infection. To determine whether camptothecin and ellipticine block the process of replication of EBV DNA or nonspecifically degrade viral DNA, the BamHI restriction patterns of EBV DNA from infected Raji cells were analysed. EBV-specific DNA was probed with the 32p_ labelled BamHI W fragment. As shown in Fig. 5, the 8.6 kb, 4.7 kb and 2-3 kb fragments were detected in P3HR-1 virus- (lane 6) but not in mock-infected Raji cells (lane 5). The results indicate that these fragments are derived from exogenous P3HR-1 virus. Since the 2.0 kb fragment was not detected in P3HR-1 virus DNA, it was considered to be specific for endogenous EBV DNA in Raji cells. The density of the bands of the 8"6 kb, 4.7 kb and 2-3 kb fragments was increased in infected Raji cells

5 Short communication 226'7 when compared with that of PAA-treated cells (lanes 1 and 4) and was reduced by the treatment of 2"5 gmcamptothecin (lane 2) or 5 gm-etlipticine (lane 3). Therefore, P3HR-1 virus DNA was amplified in infected Raji cells and the amplification of progeny virus DNA was inhibited by both inhibitors. In addition, the BamHI digestion patterns of EBV DNA derived from infected Raji cells treated with camptothecin or ellipticine were similar to those from the cells treated with PAA. These observations indicate that topoisomerase inhibitors themselves degrade neither exogenous nor endogenous viral DNA but inhibit the process of the replication of EBV DNA. Three independent assays of EBV DNA replication and restriction analysis of BamHI W fragments of DNA from superinfected Raji cells showed that camptothecin and ellipticine suppressed replicating EBV DNA without degradation of exogenous and endogenous viral DNA. These results suggest that both topoisomerases I and II play a role in the replication of EBV DNA by virusassociated DNA polymerase. The role of topoisomerases in DNA replication has been studied in a number of organisms (Maxwell & Geltert, 1985; Wang, 1985). In the case of viruses, Snapka (1986) has reported that topoisomerase II inhibitors including ellipticine block decatenation of newly replicated simian virus 40 daughter chromosomes and lead to an accumulation of catenated dimers containing single-strand DNA breaks and that camptothecin rapidly breaks the replication fork in growing Cairns structures. Recent reports have shown that the cellular topoisomerase II can induce double-strand breaks at specific locations in herpes simplex virus type 1 DNA and is involved in aspects of viral replication at late times in the infection cycle (Ebert et al, 1990). The same mechanisms would occur in EBV DNA replication. The present study has indicated that mammalian topoisomerases are involved in various steps during the lytic phase of the EBV life cycle. However, it remains to be clarified whether the topoisomerases I and II involved in EBV replication are virus-encoded or cellular in origin. As the topoisomerase II associated with EBV gene expression was shown to be more sensitive to ellipticine than that involved in cellular gene expression, these enzymes might be different in origin. Although topoisomerase II has been shown to be associated with DNA replication of various kinds of herpesviruses (Nishiyama et al., 1987; Benson & Huang, 1988; Ebert et al., 1990) including EBV, this is the first study that demonstrates involvement of topoisomerase I in viral DNA replication among the herpesvirus group. I am grateful to Professor Yoshifumi Takeda for his encouragement throughout this work. References BENSON, J. D. & HUANG, E. S. (1988). Two specific topoisomerase II inhibitors prevent replication of human cytomegalovirus DNA: an implied role in replication of the viral genome. Journal of Virology 62, CHAMPOUX, J. J. (1988). Topoisomerase I is preferentially associated with isolated replicating simian virus 40 molecules after treatment of infected cells with camptothecin. Journal of Virology 28, DRLICA, K. & FRANCO, R.J. (1988). Inhibitors of DNA topoisomerases. Biochemist O, 27, EBERT, S. N., SHTROM, S. S. & MULLER, M. T. (1990). Topoisomerase II cleavage of herpes simplex virus type 1 DNA in vivo is replication dependent. Journal of Virology 64, FOGLESONG, P. D. & BAUER, W. R. (1984). Effect of ATP and inhibitory factors on the activity of vaccinia virus type I topoisomerase. Journal of Virology 49, 1-8. GARDELLA, T., MEDWCZKY, P., SAIRENNJI, T. & MULDER, C. (1984). Detection of circular and linear herpesvirus DNA molecules in mammalian cells by gel electrophoresis. Journal of Virology 50, Gu, M.-L. & RHODE, S.L. (1991). Autonomous parvovirus DNA replication requires topoisomerase I and its activity is increased during infection. Journal of Virology 65, HSIANG, Y.-H., HERTZBERG, R., HECHT, S. & LIU, L.F. (1985). Camptothecin induces protein-linked DNA breaks via mammalian DNA topoisomerase I. Journal of Biological Chemistry 260, KATZ, B., RAAB-TRAUB, N. & MILLER, G. (1989). Latent and replicating forms of Epstein-Barr virus DNA in lymphoma and lymphoproliferative diseases. Journal of Infectious Diseases 160, KAWANISHI, M., SUGAWARA, K. & ITO, Y. (1981). Epstein-Barr virusinduced polypeptides: a comparative study with superinfected Raft, IUdR-treated, and n-butyrate-treated P3HR-1 cells. Virology 109, 7~81. MAXWELL, A. & GELLERT, M. (1985). Mechanistic aspects of DNA topoisomerases. Advances in Protein Chemistry 38, NISHIYAMA, Y., FUJ1OKA, H., TSURUMI, T., YAMAMOTO, N., MAENO, K., YOSHIDA, S. & SmMOKATA, K. (1987). Effects of the epipodophyllotoxin VP on herpes simplex virus type 2 replication. Journal of General Virology 68, PRIEL, E., SHOWALTER, S. D., ROBERTS, M., OROSZLAN, S., SEGAL, S., ABotm, M. & BLAIR, D.G. (1990). Topoisomerase I activity associated with human immunodeficiency virus (HIV) particles and equine infectious anemia virus core. EMBO Journal 9, PRIEL, E., SHOWALTER, S. D., ROBERTS, M., OROSZLAN, S. & BLAIR, D. G. (1991). The topoisomerase I inhibitor, camptothecin, inhibits equine infectious anemia virus replication in chronically infected CF2Th cells. Journal of Virology 65, RAAB-TRAUB, N. & FLYNN, K. (1986). The structure of the termini of the Epstein-Barr virus as a marker of clonal cellular proliferation. Cell 22, RICHTER, A. &; STRAUSFELD, U. (1988). Effects of VM26, a specific inhibitor of type II DNA topoisomerase, on SV40 chromatin replication in vitro. Nucleic Acids Research 16, SAMBROOK, J., FRITSCH, E.E. ~,~ MANIATIS, T. (1989). Molecular Cloning: A Laboratory Manual, 2nd edn. New York: Cold Spring Harbor Laboratory. SCHAACK, J., SCHEDL, P. & SHENK, T. (1990a). Topoisomerase I and II cleavage of adenovirus DNA in vivo: both topoisomerase activities appear to be required for adenovirus DNA replication. Journal of Virology 64, SCHAAK, J., SCHEDL, P. & SHI~NK, T. (1990b). Transcription of adenovirus and HeLa genes in the presence of drugs that inhibit topoisomerase I and II function. Nucleic Acids Research 18, 149%1508. SNAPKA, R.M. (1986). Topoisomerase inhibitors can selectively interfere with different stages of simian virus 40 DNA replication. Molecular and Cellular Biology 6, TEWEY, K.M., CHEN, G.L., NELSON, E.M. & LIU, L.F. (1984). Intercalative antitumor drugs interfere with the breakage-reunion

6 2268 Short communication reaction of mammalian DNA topoisomerase II. Journal of Biological Chemistry 259, WANG, H.-P. & ROGLER, C.E. (1991). Topoisomerase I-mediated integration of hepadnavirus DNA in vitro. Journal of Virology 65, WANG, J.C. (1985). DNA topoisomerases. Annual Review of Biochemistry 54, WONG, M.-L. & Hsu, M.-T. (1990). Involvement of topoisomerases in replication, transcription, and packaging of the linear adenovirus genome. Journal of Virology 64, YAMADA, Y., YAMAMOTO, N., MAENO, K. & NISHIYAMA, Y. (1990). Role of DNA topoisomerase I in the replication of herpes simplex virus type 2. Archives of Virology 110, (Received 11 December 1992; Accepted 8 June 1993)