We show that the anti-human immunodeficiency virus agent, 3'-azido-3'-deoxythymidine (AZT), which suppresses infectivity and cytopathic effects of human immunodeficiency virus, also effectively inhibits Epstein-Barr virus (EBV) DNA replication. However, AZT has no effect on four other human herpesviruses: cytomegalovirus, varicella-zoster virus, and herpes simplex virus types 1 and 2. The combination of acyclovir and AZT, while it is not synergistic, has an additive effect against EBV replication. AZT may prove to be a useful drug for treatment of coinfections with human immunodeficiency virus and EBV

Biron, K

Lin, J C

Pagano, J S

Smith, M C

Zhang, Z X

PubMed

We show that the anti-human immunodeficiency virus agent, 3'-azido-3'-deoxythymidine (AZT), which suppresses infectivity and cytopathic effects of human immunodeficiency virus, also effectively inhibits Epstein-Barr virus (EBV) DNA replication. However, AZT has no effect on four other human herpesviruses: cytomegalovirus, varicella-zoster virus, and herpes simplex virus types 1 and 2. The combination of acyclovir and AZT, while it is not synergistic, has an additive effect against EBV replication. AZT may prove to be a useful drug for treatment of coinfections with human immunodeficiency virus and EBV.</jats:p

J C Lin

Z X Zhang

M C Smith

K Biron

J S Pagano

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Antimicrobial Agents and Chemotherapy

Anti-human immunodeficiency virus agent 3'-azido-3'-deoxythymidine inhibits replication of Epstein-Barr virus

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ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Feb. 1988, p. 265-267 Vol. 32, No. 20066-4804/88/020265-03$02.00/0Copyright C 1988, American Society for MicrobiologyAnti-Human Immunodeficiency Virus Agent 3'-Azido-3'-Deoxythymidine Inhibits Replication of Epstein-Barr VirusJUNG-CHUNG LIN,i2* ZHEN-XI ZHANG,1 M. CAROLYN SMITH,' KAREN BIRON,3AND JOSEPH S. PAGANO' 4'5Lineberger Cancer Research Center' and Departments of Biochemistry and Nutrition,2 Medicine,4 and Microbiology,5School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27514, and The BurroughsWellcome Company, Research Triangle Park, North Carolina 277093Received 21 August 1987/Accepted 9 November 1987We show that the anti-human immunodeficiency virus agent, 3'-azido-3'-deoxythymidine (AZT), whichsuppresses infectivity and cytopathic effects of human immunodeficiency virus, also effectively inhibitsEpstein-Barr virus (EBV) DNA replication. However, AZT has no effect on four other human herpesviruses:cytomegalovirus, varicella-zoster virus, and herpes simplex virus types 1 and 2. The combination of acyclovirand AZT, while it is not synergistic, has an additive effect against EBV replication. AZT may prove to be auseful drug for treatment of coinfections with human immunodeficiency virus and EBV.In recent years, we have shown that several nucleosideanalogs selectively inhibit the replication of Epstein-Barrvirus (EBV) (14-17). One drug has been tested in trials inpatients with infectious mononucleosis (6, 21; C. M. Van derHorst, J. Joncas, G. Ahronheim, G. Stein, M. Gurwith, G.Fleisher, J. L. Sullivan, J. Sixbey, C. Sumaya, R. Schooley,S. Roland Sweezy, and J. S. Pagano, Program Abstr. 26thIntersci. Conf. Antimicrob. Agents Chemother., abstr. no.318, 1986). In searching for other antiherpetic agents, wediscovered that 3'-azido-3'-deoxythymidine (AZT), which isactive against human immunodeficiency virus (HIV) both invitro (20) and in vivo (26), strongly inhibits EBV replication.Inhibition was surprisingly selective in that the drug showedno detectable effect on replication of the other humanherpesviruses, cytomegalovirus (HCMV), varicella-zostervirus (VZV), and herpes simplex virus types 1 and 2 (HSV-1and HSV-2).To test the effect of AZT on EBV DNA replication, weused P3HR-1 (LS) cells, a high-virus-producer cell line de-rived from P3HR-1 cells by gradual adaptation to growth inlow-serum medium (1.5% newborn calf serum) (12). Underthese conditions, approximately 20 to 50% of the cells arespontaneously activated to produce virus and large numbersof linear EBV DNA genomes (unpublished data). Exponen-tially growing P3HR-1 (LS) cells were treated for 14 dayswith various concentrations of AZT in RPMI 1640 mediumcontaining 1.5% serum. The cells were harvested, and EBVgenome copy numbers were determined by complementaryRNA-DNA hybridization with an EBV-specific cRNA probe.Figure 1 shows the dose-dependent inhibition of EBVgenome replication by AZT. EBV genome copy numbersdecreased with increasing drug concentrations. The virus50% effective dose (ED50) and ED90 were determined fromthe semilogarithmic plot of drug concentrations against viralgenome copies per cell, assuming the residual genome level(30 copies per cell) achieved by an effective drug concentra-tion (100 ,uM) as zero and the viral genome level in thedrug-free control as 100. We have shown previously that theresidual EBV copy number of -30 per cell is due to episomalforms which are insensitive to antiviral drugs (16). The ED50and ED90 thus obtained were 3 and 30 ,uM, respectively. For* Corresponding author.comparison, in patients with acquired immunodeficiencysyndrome (AIDS) the plasma concentration of AZT reacheslevels of 6 to 10 ,uM 1 h after an intravenous dose of 5 mg/kg.We considered that the EBV-inhibitory effect of AZTcould be the consequence of selective killing of the produc-tive cells in P3HR-1 (LS) populations, and therefore, wemonitored cell growth and viability during drug treatment.Cell growth was not significantly affected at drug concentra-tions as high as 50 p.M. At 100 ,uM AZT, approximately 30%growth inhibition was observed; however, the viability of thecells remained the same as that of the control. These resultsindicate that AZT at concentrations as high as 100 ,uM iscytostatic but not cytotoxic.Since all herpesviruses induce the formation of new viralDNA polymerases, we thought that drugs inhibitory to EBVmight also inhibit HSV, VZV, and HCMV replication (13).We therefore tested the effect of AZT on the replication ofthese viruses by using plaque-reduction assays (3); 9-(1,3-dihydroxy-2-propoxymethyl)guanine (DHPG) was used as apositive drug control. As expected, DHPG exhibited potentanti-HCMV, -HSV-1 and -HSV-2 activities, with ED50sranging from 0.05 to 1.5 puM (Table 1). Surprisingly, AZThad no appreciable effect on HCMV, VZV, or HSV-1 andHSV-2.-J-iwI-)wa-L0C-)w0zLiCDLiEDso= 3pMED90= 30,uM400300200100ED50- \ ED900 0.1 10DRUG CONC. (SM)FIG. 1. Dose-dependent inhibition of EBV genome replicationby AZT. Exponentially growing P3HR-1 (LS) cells were seeded at adensity of 106/ml and incubated in various concentrations of drugsfor 14 days. EBV genome copy numbers per cell represent theaverage of two determinations at each drug concentration.265100ANTIMICROB. AGENTS CHEMOTHER.TABLE 1. Effects of AZT and DHPG on herpes-group virusesVirus ED50 (,uM)(strain) DHPG AZTHSV-1 (KOS) 0.05 >100HSV-2 (333) 0.2 >100HCMV (AD169) 1.5 >100VZV (Oka) ND" >250a ND, Not determined.To assess whether the inhibitory effect of AZT on EBVreplication was reversible, we treated P3HR-1 (LS) cellswith 50 ,uM AZT for 14 days to reduce the viral genome copynumbers down to the residual levels (30 copies per cell) andthen released the cells into drug-free medium. Figure 2shows that the inhibitory effect was slowly reversed uponremoval of the drug. Replication of approximately 50% ofthe viral genome numbers was recovered by 14 days afterdrug removal. It required 21 days for the level of viralgenomes to be restored to the control level; this result is incontrast to that obtained with acyclovir (ACV), the effect ofwhich was completely abolished 11 days after removal of thedrug (16). Slow kinetics of recovery have been demonstratedwith other anti-EBV agents such as DHPG, E-5-(2-bromo-vinyl)-2'-deoxyuridine (BVDU), 1-(2-deoxy-2-fluoro-P-D-arabinofuranosyl)-5-iodocytosine, and 1-(2-deoxy-2-fluoro-,-D-arabinofuranosyl)-5-methyluracil (14).Because patients with AIDS have toxic reactions to AZT,combinations of drugs that are potentially synergistic andwould permit reduction of AZT dosage are being sought.Synergistic inhibition of HIV replication in vitro by suraminand ACV (22), by phosphonoformate and alpha-A-interferon(11), and by ACV and AZT (19) has been reported, and acombination AZT-ACV therapy in AIDS is being evaluatedclinically (S. Broder, personal communication). We lookedfor evidence of a potentiating effect between AZT and ACVon the replication of EBV but found only an additive effectwhen the two drugs were used in combination in suboptimalconcentration (Table 2). The ED50 and ED90 of ACV alonefor EBV replication are 0.3 and 9 ,uM, respectively (16).The mechanism of selective inhibition by AZT on EBVreplication is not understood. Initial preferential phosphor-ylation in virus-infected cells is a prerequisite for selectiveactivity of several nucleoside analogs (for a review, seereference 13). The monophosphorylated compounds areconverted by host cellular kinases to triphosphates, which,in turn, become preferential substrates for virus-specificDrug Removed3000W 2000z100mw~~~TIMEt(day) 0 8 14 4 8 14 21DIRuGAZT) - + + - - - - -5Op"MFIG. 2. Kinetics of inhibition and reversibility of EBV DNAreplication in P3HR-1 (LS) cells treated with AZT.TABLE 2. Combined effects of AZT and ACVon EBV replicationConcn of EBV genome copy no. at AZT concn (,uM)ACV (,uM) 0 2.5 100 465 382 1161 216 133 805 82 62 62DNA polymerase (5). In HIV-infected cells, initial phos-phorylation of AZT is carried out by cellular thymidinekinase rather than viral enzyme, and the levels of AZT-monophosphate measured in uninfected and infected cellsare similar (7). Thus, EBV-encoded thymidine kinase isunlikely to be required for the selective inhibition of EBVreplication by AZT.Of special interest are the findings that AZT has virtuallyno effect on HCMV, VZV, and HSV-1 and HSV-2, despitethe fact that all herpesviruses encode novel DNA polymer-ases that are quite distinct from cellular DNA polymerasesand which share unique biochemical properties such asstimulation by high-salt concentrations in vitro. Moreover,there is extensive conservation of sequence of herpesvirusDNA polymerase genes (2). Indeed, in the predicted se-quence ofEBV and HSV-1 polymerases, there is 45% homol-ogy in the C-terminal two-thirds of the polypeptides (8). All ofthe known antiherpetic drugs generally affect several of theherpes-group viruses, although HCMV is insensitive to ACV(18) and HSV-2 is insensitive to BVDU (4).Since AZT-triphosphate is a potent inhibitor of HIVreverse transcriptase (7), the question arises whether EBVDNA polymerase shares with the reverse transcriptase somecommon binding sites for the phosphorylated drug. If so,these sites are not available in oa-polymerase, since AZT-triphosphate has low affinity for this cellular enzyme (7). Inaddition, treatment of EBV-infected cells with AZT maycause a reduction in the intracellular level of dTTP, which isa competing substrate for EBV DNA polymerase. This effectwould facilitate the binding of AZT-triphosphate to viralpolymerase. The structural characteristics of AZT with its3'-azido group would also cause termination of DNA elon-gation if the nucleoside moiety were incorporated into viralDNA. In any case, the unique susceptibility of EBV to AZTmay provide a lead toward ascertaining active sites on HIVreverse transcriptase for binding of the drug.EBV has been suggested as one of the cofactors whichdetermine whether AIDS will result from infection by HIV(9, 23). Since opportunistic viral infections, especially EBVand HCMV, are prevalent in AIDS (1, 24) treatment ofsymptomatic HIV infection with AZT may have an addi-tional benefit in reducing possible facilitative effects orcomplications resulting from reactivated or primary EBVinfection; however, this possibility has not yet been studieddirectly. Although the combination of ACV and AZT is notsynergistic against EBV, neither is it antagonistic (25).Whether the EBV-associated B-lymphocytic lymphomasthat are relatively common in AIDS would be affected is amatter of speculation (10).We thank Carolene P. Griffin and Arthur Stock for typing themanuscript.This work was supported by Public Health Service grants 5-POI-CA-19014 and AI-17205 from the National Cancer Institute and theNational Institute of Allergy and Infectious Diseases and by a grantfrom the Burroughs Wellcome Co.266 NOTESNOTES 267LITERATURE CITED1. Andiman, W. A., K. Martin, A. Rubinstein, S. Pawha, R.Eastman, B. Z. Katz, J. Pitt, and G. Miller. 1985. Opportunisticlymphoproliferations associated with Epstein-Barr viral DNA ininfants and children with AIDS. Lancet i:1390-1393.2. Baer, R., A. T. Bankier, M. D. Biggin, P. L. Deininger, P. J.Farrell, T. J. Gibson, G. Hatful, G. S. Hudson, S. C. Satchwell,C. Seguin, P. S. Tuffnell, and B. G. Barrell. 1984. DNAsequence and expression of the B95-8 Epstein-Barr virus gen-ome. Nature (London) 310:207-211.3. Biron, K. K., and G. B. Elion. 1980. In vitro susceptibility ofvaricella-zoster virus to acyclovir. Antimicrob. Agents Chemo-ther. 18:443-447.4. DeClercq, E., J. Descamps, G. Verhelst, R. T. Walker, A. S.Jones, P. F. Torrence, and D. Shugar. 1980. Comparativeefficacy of different antiherpes drugs against different strains ofherpes simplex virus. J. Infect. Dis. 141:563-574.5. Elion, G. B., P. A. Furman, J. A. Fyfe, P. de Miranda, L.Beauchamp, and P. A. Shaeffer. 1977. Selectivity of action of anantiherpetic agent, 9-(2-hydroxyethoxy-methyl) guanine. Proc.Natl. Acad. Sci. USA 74:5716-5720.6. Ernberg, I., and J. Anderson. 1986. Acyclovir efficiently inhibitsoropharyngeal excretion of Epstein-Barr virus in patients withacute infectious mononucleosis. J. Gen. Virol. 67:2267-2272.7. Furman, P. A., J. A. Fyfe, M. H. St. Clair, K. Weinhold, J. L.Rideout, G. A. Freeman, S. N. Lehrman, D. P. Bolognesi, S.Broker, H. Mitsuya, and D. W. Barry. 1986. Phosphorylation of3'-azido-3'-deoxythymidine and selective interaction of the 5'-triphosphate with human immunodeficiency virus reverse tran-scriptase. Proc. Natl. Acad. Sci. USA 83:8333-8337.8. Gibbs, J. S., H. C. Chiou, J. D. Hall, D. W. Mount, M. J.Retondo, S. K. Weller, and D. M. Coen. 1985. Sequence andmapping analyses of the herpes simplex virus DNA polymerasegene predict a C-terminal substrate binding domain. Proc. Natl.Acad. Sci. USA 82:7969-7973.9. Greenspan, J. S., D. Greenspan, E. T. Lennette, D. I. Abrams,M. A. Conant, V. Peterson, and V. K. Freese. 1985. Replicationof Epstein-Barr virus within the epithelial cells of oral "hairy"leukoplakia, an AIDS-associated lesion. N. Engl. J. Med. 313:1564-1571.10. Grooppman, J. E., J. L. Sullivan, C. Mulder, D. Ginzborg, S. H.Orkin, C. J. O'Hara, K. Falchuk, F. Wong-Staal, and R. C.Gallo. 1986. Pathogenesis of B cell lymphoma in a patient withacquired immunodeficiency syndrome. Blood 67:612-615.11. Hartshorn, K. L., E. G. Sandstrom, D. Neumeyer, T. J. Paradis,T.-C. Chou, R. T. Schooley, and M. S. Hirsch. 1986. Synergisticinhibition of human T-cell lymphotropic virus type III replica-tion in vitro by phosphonoformate and recombinant alpha-Ainterferon. Antimicrob. Agents Chemother. 30:189-191.12. Lin, J.-C., D. J. Nelson, C. U. Lambe, and E. I. Choi. 1986.Metabolic activation of 9([2-hydroxy-1-(hydroxymethyl)eth-oxy]methyl) guanine in human lymphoblastoid cell lines in-fected with Epstein-Barr virus. J. Virol. 60:569-573.13. Lin, J.-C., and J. S. Pagano. 1985. Cellular transformation bythe herpesvirus and antiviral drugs. Pharmacol. & Ther. 28:135-161.14. Lin, J.-C., M. C. Smith, Y. C. Cheng, and J. S. Pagano. 1983.Epstein-Barr virus: inhibition of replication by three new drugs.Science 221:578-579.15. Lin, J.-C., M. C. Smith, E. I. Choi, E. DeClercq, A. Verbruggen,and J. S. Pagano. 1985. Effect of (E)-5-(2-bromovinyl)-2'-deoxyuridine on replication of Epstein-Barr virus in humanlymphoblastoid cell lines. Antiviral Res. Suppl. 1:121-126.16. Lin, J.-C., M. C. Smith, and J. S. Pagano. 1984. Prolongedinhibitory effect of 9-(1,3-dihydroxy-2-propoxymethyl)guanineagainst replication of Epstein-Barr virus. J. Virol. 50:50-55.17. Lin, J.-C., M. C. Smith, and J. S. Pagano. 1985. Comparativeefficacy and selectivity of some nucleoside analogs againstEpstein-Barr virus. Antimicrob. Agents Chemother. 27:971-973.18. Mar, E. C., P. C. Patel, and E. S. Huang. 1982. Effect of9-(2-hydroxyethoxymethyl)guanine on viral-specific polypep-tide synthesis in human cytomegalovirus-infected cells. Am. J.Med. 73:82-85.19. Mitsuya, H., M. Matsukura, and S. Broder. 1987. Rapid in vitrosystems for assessing activity of agents against HTLV-III/LAV,p. 303-333. In S. Broder (ed.), AIDS: modem concepts andtherapeutic challenges. Marcel Dekker, Inc., New York.20. Mitsuya, H., K. J. Weinhold, P. A. 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Gallo, and B. Wahren. 1986. Restricted IgGsubclass responses to HTLV-III/LAV and to cytomegalovirusin patients with AIIDS and lymphadenopathy syndrome. J.Infect. Dis. 153:9702973.25. Vogt, M. W., K. L. Hartsihorn, P. A. Furman, T. C. Chow, J. A.Fyfe, L. A. Coleman, C. Crumpacker, R. T. Schooley, and M. S.Hirsch. 1987. Ribaviran antagonizes anti-HIV effect of azidothy-midine (AZT) by phosphorylation inhibition. Science 235:1376-1379.26. Yarchoan, R., R. W. Klecker, K. J. Weinfold, P. D. Markham,H. K. Lyerly, D. T. Durack, E. Gelmann, S. N. Lehrman, R. M.Blum, D. W. Barry, G. M. Shearer, M. A. Fischl, H. Mitsuya,R. C. Gallo, J. M. Collins, D. P. Bolognesi, C. E. Myers, and S.Broder. 1986. Administration of 3'-azido-3'deoxythymidine, aninhibitor of human T lymphotropic virus III/lymphoadenopathy-associated virus replication, to patients with acquired immuno-deficiency syndrome or acquired immunodeficiency syndrome-related complex. Lancet i:575-580.VOL. 32, 1988

Anti-human immunodeficiency virus agent 3'-azido-3'-deoxythymidine inhibits replication of Epstein-Barr virus.

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Anti-human immunodeficiency virus agent 3'-azido-3'-deoxythymidine inhibits replication of Epstein-Barr virus.

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