Epstein-Barr virus transformed human lymphocytes despite the presence of up to 500 microM acyclovir [9-(2-hydroxyethoxymethyl)guanine], a viral DNA polymerase inhibitor. The transformed cells contained multiple Epstein-Barr virus genome copy numbers. Functional viral DNA polymerase is probably not required for cell transformation and the initial amplification of the viral genome

Pagano, Joseph S.

Sixbey, John W.

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Vol. 53, No. 1JOURNAL OF VIROLOGY, Jan. 1985, p. 299-3010022-538X/85/010299-03$02.00/0Copyright © 1985, American Society for MicrobiologyEpstein-Barr Virus Transformation of Human B LymphocytesDespite Inhibition of Viral PolymeraseJOHN W. SIXBEYt* AND JOSEPH S. PAGANOLineberger Cancer Research Center and Departments of Medicine and Microbiology and Immunology, University ofNorth Carolina, Chapel Hill, North Carolina 27514Received 21 May 1984/Accepted 21 September 1984Epstein-Barr virus transformed human lymphocytes despite the presence of up to 500 ,uM acyclovir[9-(2-hydroxyethoxymethyl)guanine], a viral DNA polymerase inhibitor. The transformed cells containedmultiple Epstein-Barr virus genome copy numbers. Functional viral DNA polymerase is probably not requiredfor cell transformation and the initial amplification of the viral genome.Epstein-Barr virus (EBV) is a transforming herpesviruswhich, on infection of human B lymphocytes in vitro, iscapable of producing permanent lymphoblastoid cell lines.This effect mirrors the consequence of infection of B lym-phocytes in vivo (14). In its latent state, the viral genomeconfers on the host cell the potential for unlimited cellgrowth. A critical step in EBV-induced cell transformationprobably involves early EBV replication Within the infectedtarget cell. Human umbilical cord lymphocytes that havebeen transformed by EBV generally contain several copiesof the EBV genome in its episomal form but do not freelysynthesize viral DNA or produce virus (1, 12). A relatedquestion, therefore, is whether EBV-induced DNA polymer-ase is needed for transformation. These basic questions arealso important because of the interest in applying antiviraldrugs in the treatment of the EBV-induced polyclonal lym-phoproliferative diseases that occur in irnmunocompromisedpersons (13, 18).In vitro use of the viral DNA polymerase inhibitorphosphonoacetic acid (PAA) as a probe of early transforma-tional events has produced results with conflicting interpre-tations. Thorley-Lawson and Strominger (19, 20) and Lemonet al. (8) have reported the absence of cell outgrowth inlymphocytes infected with EBV and maintained on PAA.However, Lemon et al. did note increased cellular nucleicacid synthesis consistent with beginning transformation (8).Moreover, the EBV nuclear antigen (EBNA), a marker ofEBV-induced cell transformation, was detected in somePAA-treated cells (20). Rickinson and Epstein have de-scribed not only the establishment of permanent lymphoblas-toid cell lines after comparable exposures of cells to PAA,but also apparent amplification of the incoming viral genome(16). These discrepancies may be due in part to dose-depend-ent inhibition by PAA not only of viral (10) but also ofcellular DNA polymerase (7, 10). To eliminate any ambigu-ity introduced in these results by the lack of specificity ofPAA, we have used the acyclic nucleoside analog acyclovir[9-(2-hydroxyethoxymethyl)guanine] (ACV) as a highly se-lective inhibitor of EBV-specific DNA polymerase to inves-tigate EBV-induced transformation of human umbilical cordlymphocytes.ACV was a gift of the Burroughs Wellcome Co. Clinicalisolates of EBV from saliva of persons with acute infectious* Corresponding author.t Present address: Division of Infectious Diseases, St. JudeChildren's Research Hospital, Memphis, TN 38101.mononucleosis were obtained by throat washings with phos-phate-buffered saline solution. Filtered samples capable ofproducing permanent, EBNA-positive cell lines from umbil-ical cord lymphocytes were selected for use in the assaysdescribed below. Mixed populations of umbilical cord lym-phocytes were prepared by centrifugation of heparinizedcord blood on Ficoll-Isopaque (Pharmacia Fine Chemicals)(2). B-cell enrichment of adult peripheral blood lymphocyteswas accomplished by erythrocyte rosetting (21). EBV-in-duced cell transformation was confirmed by detection ofEBNA (15). Numbers of EBV genome equivalents in dupli-cate lines from ACV- and mock-treated groups were deter-mined by nucleic acid hybridization as previously described(11).A transformation centers assay similar to that describedby Henderson et al. (6) was performed to study the effects ofACV on the induction of lymphocyte transformation byEBV isolates (Table 1). We obtained cell outgrowth andestablishment of permanent EBNA-positive lytnphoblastoidcell lines with two different preparations of umbilical cordlymphocytes after exposure to EBV in the presence of up to500 ,uM ACV, a concentration at least five times thatrequired for complete inhibition of EBV DNA synthesis insuperinfected Raji cells (4, 9). Efficiency of transformation inACV-treated cells was identical to that in control cultures atlevels of up to 250 ,uM, 25 times the 90% effective dose of thedrug for EBV (4, 9). There was no significant delay inestablishing transformation (Table 1). Only at 500 pLM didtransformation efficiency decrease, presumably due to drugtoxicity.Drug-induced enhancement of transformation did not oc-cur. This provides evidence against a cytolytic stage in asubset of lymphocytes shortly after infection which, ifinhibited by the drug, would increase the chances for virallatency, cell survival, and eventual transformation (8). Thispoint is worth noting given the possibility that the clinicalisolates used might contain both lytic and transformingstrains of virus (17). None of the 48 wells containing umbil-ical cord lymphocytes maintained in 100 ,uM ACV sponta-neously transformed in the absence of EBV exposure.ACV requires phosphorylation by either viral or cellularenzymes to be effective against EBV-specific DNA polymer-ase (3). EBV-positive and -negative lymphoid cell lines andfresh peripheral lymphocyte preparations from both EBV-seropositive and -seronegative adult donors are capable ofphosphorylating drug in vitro (3). To ensure that our resultswere not a consequence of inadequate phosphorylation of299300 NOTESACV in umbilical cord lymphocytes, we also studied a Blymphocyte-enriched preparation from the peripheral bloodof an EBV-serotlegative adult whose uninfected cells werepreviously shown to be capable of ACV phosphorylation invitro (3). EBV-induced cell transformation in the presence ofACV occurred in all cases regardless of the source of thelymphocytes and with all three viral isolates used.Previous determinations have established that most celllines produced by in vitro EBV infection of umbilical cordblood cells contain 10 to 15 copies of the EBV genome percell (12). Because we achieved cell transformation at ACVconcentrations capable of completely inhibiting viral poly-merase-directed EBV replication, we measured the copynumber of EBV genomes in cell lines established in ACV-containing and control medium (Table 2). EBV DNA ge-nome equivalents were estimated from levels of hybridiza-tion to total cellular DNA with a tritiated cRNA probederived from the cloned BamHI V fragment of EBV DNA.The probe was first calibrated by hybridization with knownamounts of purified viral DNA from Raji cells (11). Umbili-cal cord lymphocytes transformed in ACV-free mediumcontained an average of seven genome equivalents per cell,whereas cells transformed in the presence of 100 FM ACVcontained an average of six.TABLE 1. Transformation of human umbilical cord lymphocytesby EBV in the presence of the viral DNA polymerase inhibitorACV"ACV No. of EBV Wells transformed/ No. of Mean time to(>M) exposed cells total wellsb transformants transformationper well per 105 cells (days)0 l0o 10/12 0.8 46104 4/12 53l03 0/1210 i05 10/12 0.8 46104 3/12 43103 0/12100 105 10/12 0.8 58104 0/12103 1/12 66250 105 10/12 0.8 50104 0/12103 0/12500 105 5/12 0.4 63104 1/12 66103 0/12" Human umbilical cord lymphocytes (2 x 106) were incubated for 1 h at37°C with 0.2 ml of filtered throat washings from patients with infectiousmononucleosis. Control or ACV-containing medium (RPMI 1640, 20% fetalcalf serum) was added to cells, and cultures were incubated for 24 h at 37°C in5% CO,. The tnedium was removed, and the cells were incubated for 1 h inhuman serum containing EBV-neutralizing antibodies (diluted 1:10 in controlor ACV medium) to eliminate all extracellular virus. Cells were washed twiceand diluted to concentrations of 106, 105, and 104 lymphocytes per ml. One-tenth milliliter of each lymphocyte dilution was added to 12 replicate wells of amicroassay plate containing feeder cells prepared from autochthonous, nonin-fected lymphocytes to give a final cell number in all wells of 2 x 105 cells. Thecultures were incubated at 37°C in 5% CO2 and fed twice weekly with controlor ACV medium by replacing half of the medium. Controls consisted of mock-infested umbilical cord lymphocytes and lymphocytes exposed to freeze-thawed extracts and washings of EBV-exposed cells after treatment with EBVneutralizing antibody.b Transformation was judged microscopically on the basis of obvious cellgrowth and clumping. Only wells producing permanent EBNA-positive celllines on passage were called transformed. Final results were scored at 10weeks. Efficiency of transformation was calculated as (number of transformedwells)/[(total number of cells per well) (number of wells)l.TABLE 2. EBV genomes in newly transformed umbilical cordlymphocytes'Transformed No. of EBV genome equivalents in:lymphocytesfrom cell line: Control medium ACV medium1 9 32 6 9"Duplicate cell lines 1 and 2 from mock- and ACV-treated cultures wereestablished. Total cellular DNA was extracted from 4 x 107 ACV- and mock-treated cells and affixed to nitrocellulose filters. EBV DNA sequences wereestimated from levels of hybridization with a 3H-labeled cRNA probe whichhad been calibrated by hybridization with various known amounts of purifiedviral DNA. Total DNA per filter was quantitated by a diphenylamine assay(11).The presence of multiple genome copy numbers in cellstransformed in the presence of high concentrations of a viralpolymerase inhibitor confirms the findings of Rickinson andEpstein with PAA (16). The almost identical EBV genomenumber in cells transformed in ACV containing medium andin control medium makes it unlikely that viral DNA polymer-ase is involved in any amplification of the incoming viralgenome. The rnultiple genome number may reflect replica-tion of the EBV genome by the host polymerase, which is atleast 100-fold less sensitive to ACV triphosphate than isEBV DNA polymerase (5). Replication of EBV genomes inthe episomal form is unaffected by ACV and other potentinhibitors of herpesvirus DNA polymerases (9). Althoughtransformation is believed to occur according to single-hitkinetics, another possibility is that the transformed cellsrepresent progeny of lymphocytes infected at a multiplicityof infection greater than 1, an event made unlikely by thevirus concentrations present in the throat washings used.These results suggest that functional viral DNA polymer-ase may not be necessary for induction of cell transforma-tion, but the relationship of early viral replication to thetransformation process remains unknown. The replicationand maintenance of a limited copy number of EBV episomesmay be crucial to transformation, but the apparent role ofhost rather than viral DNA polymerase in this processmakes the use of viral polymerase-active drugs for treatmentof EBV-transformed cells of questionable value.We thank Carolyn Smith for technical assistance.This work was supported by Public Health Service grant 5-POl-CA-19014 from the National Cancer Institute and grant 1N-15Wfrom the American Cancer Society. J.W.S. was supported by aVenereal Disease Research Fund fellowship from the AmericanSocial Health Association.LITERATURE CITED1. Anvret, M., and G. Miller. 1981. Copy number and location ofEpstein-Barr viral genomes in neonatal human lymphocytestransformed after separation by size and treatment with mito-gens. Virology 111:47-55.2. Boyum, A. 1968. isolation of mononuclear cells and granulo-cytes from human blood. Scand. J. Clin. Lab. Invest. Suppl.97:77-89.3. Colby, B. M., P. A. Furman, J. E. Shaw, G. B. Elion, and J. S.Pagano. 1981. Phosphorylation of acyclovir [9-(2-hydroxy-ethoxymethyl)guanine] in Epstein-Barr virus-infected lym-phoblastoid cell lines. J. Virol. 38:606-611.4. Colby, B. M., J. E. Shaw, G. B. Elion, and J. S. Pagano. 1980.Effect of acyclovir [9-(2-hydroxyethoxymethyl)guanine] on Ep-stein-Barr virus DNA replication. J. Virol. 34:560-568.5. Datta, A. K., B. M. Colby, J. E. Shaw, and J. S. Pagano. 1980.Acyclovir inhibition of Epstein-Barr virus replication. Proc.Natl. Acad. Sci. U.S.A. 77:5163-5166.J. VIROL.NOTES 3016. Henderson, E., G. Miller, J. Robinson, and L. Heston. 1977.Efficiency of transformation of lymphocytes by Epstein-Barrvirus. Virology 76:152-163.7. Huang, E.-S., C. H. Huang, S. M. Huong, and M. Selgrade.1976. Preferential inhibition of herpes-group viruses byphosphonoacetic acid: effect on virus DNA synthesis andvirus-induced DNA polymerase activity. Yale J. Biol. Med.49:93-98.8. Lemon, S. M., L. M. Hutt, and J. S. Pagano. 1978. Cytofluo-rometry of lymphocytes infected with Epstein-Barr virus: effectof phosphonoacetic acid on nucleic acid. J. Virol. 25:138-145.9. 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. 50:50-55.10. Miller, R. L., R. Glaser, and F. Rapp. 1977. Studies of anEpstein-Barr virus-induced DNA polymerase. Virology 76:494-502.11. Nonoyama, M., and J. S. Pagano. 1971. Complementary RNAspecific to the DNA of the Epstein-Barr virus. Detection ofEpstein-Barr viral genome in non-productive cells. Nature(London) New Biol. 233:103-106.12. Pagano, J. S. 1975. Diseases and mechanisms of persistent DNAvirus infection: latency and cellular transformation. J. Infect.Dis. 132:209-223.13. Pagano, J. S., J. W. Sixbey, and J.-C. Lin. 1983. Acyclovir andEpstein-Barr virus infection. J. Antimicrob. Chemother.12(Suppl. B):113-121.14. Pope, J. H. 1979. Transformation by the virus in vitro, p.205-223. In M. A. Epstein and B. G. Achong (ed.), TheEpstein-Barr virus. Springer-Verlag, Berlin.15. Reedman, B. M., and G. Klein. 1973. Cellular localization of anEpstein-Barr virus (EBV)-associated complement fixing antigenin producer and non-producer lymphoblastoid cell lines. Int. J.Cancer 11:499-520.16. Rickinson, A. B., and M. A. Epstein. 1978. Sensitivity of thetransforming and replicative functions of Epstein-Barr virus toinhibition by phosphonoacetate. J. Gen. Virol. 40:421-431.17. Sixbey, J. W., J. G. Nedrud, N. Raab-Traub, R. A. Hanes, andJ. S. Pagano. 1984. Epstein-Barr virus replication in oropharyn-geal epithelial cells. N. Engl. J. Med. 310:1225-1230.18. Sixbey, J. W., and J. S. Pagano. 1984. New perspectives on theEpstein-Barr virus in the pathogenesis of lymphoproliferativediseases, p. 146-176. In J. S. Remington and M. N. Swartz(ed.). Current clinical topics in infectious diseases, Vol. 5.McGraw-Hill Book Co., New York.19. Thorley-Lawson, D., and J. L. Strominger. 1976. Transforma-tion of human lymphocytes by Epstein-Barr virus is inhibited byphosphonoacetic acid. Nature (London) 263:332-334.20. Thorley-Lawson, D., and J. L. Strominger. 1978. Reversibleinhibition by phosphonoacetic acid of human B-lymphocytetransformation by Epstein-Barr virus. Virology 86:423-431.21. Weiner, M. S., C. Bianco, and V. Nussenzweig. 1973. Enhancedbinding of neuraminidase-treated sheep erythrocytes to humanT-lymphocytes. Blood 42:939-946.VOL. 53, 1985

Epstein-Barr virus transformation of human B lymphocytes despite inhibition of viral polymerase.

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Epstein-Barr virus transformation of human B lymphocytes despite inhibition of viral polymerase.

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