Epstein-Barr Virus Polypeptides: Identification of Early Proteins and Their Synthesis and Glycosylation

JOURNAL OF VIROLOGY, Aug. 1981, p. 651-655 0022-538X/81/080651-05$02.00/0 Vol. 39, No. 2 Epstein-Barr Virus Polypeptides: Identification of Early Proteins and Their Synthesis and Glycosylation ROBERT J. FEIGHNY,1* BERCH E. HENRY II,' AND JOSEPH S. PAGANO2 The Cancer Research Center' and Departments ofmedicine and Bacteriology and Immunology,2 The University of North Carolina at Chapel Hill School ofmedicine, Chapel Hill, North Carolina 27514 Received 10 October 1980/Accepted 17 April 1981 We have identified at least six early polypeptides induced by Epstein-Barr virus in cells or under conditions which are nonpermissive for Epstein-Barr virus DNA replication ranging in molecular weight from 140,000 to 26,000. Insights into the biochemistry of Epstein-Barr virus (EBV) infection have come mainly from two general systems, each with advantages and limitations. The first involves endogenously virus-producing cell lines, such as P3HR-1 and B95-8, in which virus replication can be induced (18) and the effect of these inducing agents can be studied. In the second system, exogenous EBV is used to superinfect the latently infected Raji cell line, resulting in copious viral DNA replication (14), but largely abortive virus production (17). There are, however, at least two significant drawbacks to analysis of virus-induced events in these systems. First, in both, the viral genome is continuously present (12). Hence, induction or inhibition of virus-producing cell lines leads only to a change in the number of EBV genomes and associated synthesis of macromolecules; a corresponding cell line in which the virus enzymes, proteins, DNA, or antigens are not present before treatment is not available. In the superinfection system, whether the observed changes are due to the infecting virus, activation of the latent viral episome, or stimulation of host genes remains indeterminate. Both systems have the additional limitation that the cells have already been transformed by the virus, which makes attempts to probe molecular steps in oncogenic transformation somewhat after the fact. The initial events that have led the viral genome to become a stable component of the cell and to maintain the transformed state have already transpired. The cell line BJA-B, established from a Burkitt's lymphoma, does not express EBV antigens, nor does it contain the viral genome (10). This line cannot be chemically induced (18). Infection of BJA-B cells with EBV leads to expression of early antigen (EA) within 6 h after infection, but without viral DNA synthesis. The viral proteins that are expressed in the absence of vi,ral DNA replication may play a key role in the transformation of the cell. In Raji cells superinfected with EBV, 7 of the 40 polypeptides induced by infection have been classified as early (5, 7). However, inasmuch as 33 to 34 EBV-associated polypeptides are still made ultimately in superinfected Raji cells treated with acyclovir, a focus on fewer polypeptides would be valuable. Although no in vitro system is totally permissive for EBV with production of fully infectious virus, some cell lines can be infected in vitro, resulting in viral DNA replication and formation of transforming virus (17). Available data on the early class of polypeptides detectable in superinfected Raji cells are summarized in Table 1. Although it is difficult to make precise comparisons, there appears to be a consensus that at least five polypeptides can be designated as early. The variability in both the number of early proteins and their immunoprecipitability was due to variations in sera and the permissive systems employed. The BJA-B cell line is nonpermissive for EBV DNA replication. Upon infection with EBV, intermediate and late EBV polypeptide synthesis does not occur, prevented apparently by the host cell itself, and only early proteins seem to be made. Moreover, a better definition of the polypeptide components of the EA complex is possible since, in contrast to superinfected Raji cells, virus capsid antigen (VCA) is not made in EBV-infected BJA-B cells. One of the early polypeptides defined in this way comigrated with a glycoprotein and may be glycosylated. Maintenance of cells and virus production were as previously described (5). Infection of cells, electrophoresis, and immunoprecipitation were as before (7). We first confirmed that BJA-B cells infected with EBV from P3HR-1 cells do not replicate EBV DNA (1, 10) (data not shown). The synthesis of early polypeptides precedes and does 651

652 NOTES J. VIROL. TABLE 1. Early polypeptides in EB V-superinfected Raji ceilsa Sensitivity to: Time of ap- Polypep- Mol wt Immunoprecipitation pearance tide tide (X (x 103) with EA' sera after super- Reference srapaa arac infection (h) 3 140 - - + 6-9 7,9 155 - NDb + 7.5 3 130 (late) ND - + 24-28 18 120 - ND ND 4-8 2 152C ND - + NAb 11 4 125 (glycoprotein) - - + 6-9 7, 9 130d _ ND + 7.5 3 115d _ ND ND 24-28 2 134 (glycoprotein)c ND - + NA 11 130 (glycoprotein) ND ND +e ND 13 11 100 - - + 6-9 7,9 100 - ND + 7.5 3 85 ND - + 24-28 18 90 - ND ND 4-8 2 103 ND - + NA 11 26 45 - - + 6-9 7,9 45 - ND + 7.5 3 35 ND - + 24-28 18 45 - ND ND 4-8 2 51i ND - + NA 11 27 26 - - + 6-9 7,9 30 - ND + 7.5 3 31c ND _ + NA 11 a Molecular weights of additional polypeptides that have been designed as early are as follows (x 103): 98 and 75 (7, 9); 55 (3); 85, 80, and 40 (2); and 138, 90, 70, 65, and 37 (11)c. PAA, Phosphonoacetic acid; arac, 1-fl-Darabinofuranosylcytosine. b ND, Not done; NA, not applicable. c Polypeptides from P3HR-1 cells stimulated by n-butyrate. d Glycosylation was not examined in these studies. e Immunoprecipitation with MA' serum. not require viral DNA synthesis. Since host protein synthesis continues in BJA- B cells throughout infection, we immunoprecipitated cell extracts to help to identify virus-specific proteins. Figure 1 shows the gel patterns obtained when the cell extracts were immunoprecipitated with human serum containing antibodies to EA and VCA (EA+/VCA+ serum). Three early polypeptides, 3, 4, and 11, were confirned as present in the infected BJA-B cells. Additionally, polypeptide 26 was resolved in some experiments. The early polypeptide, 27, which was immunoprecipitated from extracts of superinfected Raji cells, could not be detected in extracts of infected BJA-B cells. The intermediate polypeptides, 1 and 2, and the late polypeptides, 14, 18, and 29, which were not present in the immunoprecipitated infected BJA-B cell extracts, could be immunoprecipitated from superinfected Raji cell extracts with EA+/VCA+ antiserum. The polypeptides are numbered as described previously (5, 7). A glycoprotein comigrated with polypeptide 4, one of the early class, and was labeled both in the superinfected Raji cells and in the infected BJA-B cells (Fig. 1). The superinfected Raji cells also showed an increased incorporation of label into a high-molecular-weight polypeptide that was present before infection (molecular weight of about 260 x 103), whereas other glycoproteins present in the uninfected Raji cells showed decreased levels of incorporation. Additional virusinduced glycoproteins were faintly visible in the superinfected Raji cells; these are presumably late polypeptides since they were not detected in infected BJA-B cells. The infected BJA-B cells expressed only a single virus-induced glycoprotein comigrating with polypeptide 4, with no apparent decrease in host glycoprotein levels. This result was expected since superinfection of

VOL. 39, 1981 A B.:"" C NOTES 653 i I 12 3 4 1 2 3 4 1 2 3 4 FIG. 1. Proteins synthesized in Raji cells and BJA-B cells after infection with EBV. Cells were labeled continuously from 9 to 24 h postinfection. After harvesting, protein extracts were subjected to electrophoresis in a 7.5% polyacrylamide gel. (A) [3S]methionine-labeled cells. (B) Immunoprecipitation ofpolypeptides from (A) with EA'/VCA' antiserum. (C) [3H]glucosamine-labeled cells. Lanes: (1) mock-infected Raji cells; (2) EBV-infected Raji cells; (3) mock-infected BJA-B cells; (4) EBV-infected BJA-B cells. Raji cells, but not infection of BJA-B cells, leads to shutoff of host protein synthesis. All of the early viral proteins were present at 9 h after infection in the acyclovir-treated cells before viral DNA replication (Fig. 2) (4, 5, 16). After viral DNA replication began, late polypeptides could be detected that were not present in the acyclovir-treated cells. None of the early polypeptides was affected by the blockage of viral DNA replication. However, as previously reported (7), the shutoff of early protein synthesis occurred slightly sooner in the drug-treated cells. Working criteria for classification of polypeptides as early were three. (i) The polypeptides were synthesized in a nonpermissive system for viral DNA replication. (ii) The polypeptides were not affected by addition of an inhibitor of viral DNA synthesis. (iii) The polypeptides were made before the onset of viral DNA replication (6 to 9 h after superinfection). Based on all three criteria, three of the polypeptides in Table 1 were defined as early virus-induced EBV polypeptides. Another four met some of the criteria and were tentatively classified as early. At least three of the early proteins could be positively identified and two could be tentatively identified in the nonpermissive, infected BJA-B cells by immunoprecipitation (Table 2). An additional early polypeptide, 16, could be identified in the same cells by radiolabeling. A single early polypeptide, 27, did not appear in the infected BJA- B cells; however, it did meet the other two criteria. Since viral DNA replication did not occur, early proteins, such as 27, might be required for viral DNA synthesis. The sequence of EBV-induced early protein synthesis seems to occur in two stages. There are three polypeptides, tentatively designated as immediate-early, synthesized before the remainder of the early viral proteins. These immediateearly polypeptides are seen most clearly in EBVinfected Raji cells treated with 1-ft-D-arabinofuranosylcytosine where they are oversynthes-

654 NOTES 3-4- 7A- 11> lla 19-24- 26-27- 9 13 17 21 25 Hours After Infection FIG. 2. Time course of appearance of virus-induced proteins in superinfected Raji cells in the presence (+) and absence (-) of 100 plm acyclovir. Cells were infected and pulse-labeled at the times indicated. After extraction, the polypeptides were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Numbers correspond to virus-inducedpolypeptides. ized (7). These data suggest that the immediateearly proteins are in a class different from the early polypeptides or are a subset of them. The three immediate-early polypeptides are not detectable by immunoprecipitation in either virus-infected Raji or BJA-B cells, perhaps because their transient appearance and confinement intracellularly do not permit them to act as effective antigens. The other herpesviruses, herpes simplex virus and cytomegalovirus, show a similar pattern of early protein synthesis. In herpes simplex virus TABLE 2. J. VIROL. Early EBVpolypeptides Detection by method Early polypep- Mol wt Direct radio- Immunopretide (x 103) labeling cipitation Raji BJA-B Raji BJA-B 3 140 + + + + 4 (GP)a 125 + + + + 11 100 + + + + 11A 98 + ± ± ± 16 75 + + - - 26 45 + ± + ± 27 26 + - + - a GP, Glycoprotein 4 detected by labeling with [3H]glucosamine. infection, nonstructural proteins are initially synthesized, followed by late structural proteins. However, the transition to late protein synthesis does not require viral DNA replication (8), whereas in cytomegalovirus infection, this transition does appear to require viral DNA replication (15). Both the present and previous work (7) indicate that EBV has some similarity to cytomegalovirus in that the synthesis of some EBV polypeptides seems to require viral DNA replication. The initiation of viral DNA replication may allow synthesis of intermediate proteins. After viral DNA replication is partially completed, late viral proteins appear. Through the use of infected BJA-B cells, the polypeptides comprising the EA complex may be defined without confusion with VCA or membrane antigen. The inability to detect Epstein- Barr nuclear antigen in this system may be related to poor immunoprecipitability of the polypeptides comprising the antigen. Alternatively, since the detection of Epstein-Barr nuclear antigen has not been reported in superinfected Raji cells, perhaps synthesis of early proteins interferes with its expression. Although the functions of the early EBV proteins remain unknown, a novel DNA polymerase found in EBV-infected Raji cells (6) is probably an essential early protein. Early proteins may also have DNA-binding activity (2). The Epstein-Barr nuclear antigen is detectable in the nucleus of transformed cells and may be an early protein involved with initiation or maintenance of the transformed state. R.J.F. is a recipient of a postdoctoral fellowship award (1- F32-CA06500-01) from the National Cancer Institute. B.E.H. was supported by National Research Service Training Fellowship Grant 5-T32-CA09156 and the American Cancer Society Fellowship Grant PF-1736. This study was supported in part by a Public Health Service grant from the National Cancer Institute (CA19014) and by a pilot project award from the American Cancer Society Institutional Research Grant to the University of North Carolina (IN-150).

VOL. 39, 1981 We thank Joel Kostyu for technical help, Barbara Leonard and Fumi Bostic for typing, Sue Feighny for artwork, and G. Ehon for the gift of acyclovir. LITERATURE CITED 1. Adams, A. 1980. Molecular biology of the Epstein-Barr virus, p. 683. In G. Klein (ed.), Virology oncology. Raven Press, New York. 2. Bayliss, G. J., and M. Nonoyama. 1978. Mechanisms of infection with Epstein-Barr virus. III. The synthesis of proteins in superinfected Raji cells. Virology 87:204-207. 3. Bodemer, W. W., W. C. Summers, and J. C. Niederman. 1980. Detection of virus-specific antigens in EB- (P3HR-1) virus-superinfected Raji cells by immunoprecipitation. Virology 103:340-349. 4. Colby, B. M., J. E. Shaw, G. B. Elion, and J. S. Pagano. 1980. Effect of acyclovir [9-(2-hydroxyethoxymethyl)guanine] on Epstein-Barr virus DNA replication. J. Virol. 34:560-568. 5. Feighny, R. J., M. P. Farrell, and J. S. Pagano. 1980. Polypeptide synthesis and phosphorylation in Epstein- Barr virus-infected cells. J. Virol. 34:455-463. 6. Feighny, R. J., B. E. Henry, A. K. Datta, and J. S. Pagano. 1980. Induction of DNA polymerase activity after superinfection of Raji cells with Epstein-Barr virus. Virology 107:415-423. 7. Feighny, R. J., B. E. Henry, and J. S. Pagano. 1981. Epstein-Barr virus polypeptides: effect of inhibition of viral DNA replication on their synthesis. J. Virol. 37: 61-71. 8. Honess, R. W., and B. Roizman. 1974. Regulation of herpesvirus macromolecular synthesis. I. Cascade regulation of the synthesis of three groups of viral polypep- NOTES 655 tides. J. Virol. 14:8-19. 9. Kallin, B., J. Luka, and G. Klein. 1979. Immunochemical characterization of Epstein-Barr virus-associated early and late antigens in n-butyrate-treated P3HR-1 cells. J. Virol. 32:710-716. 10. Miller, G. 1980. Biology of Epstein-Barr virus, p. 713. In G. Klein (ed.), Viral oncology. Raven Press, New York. 11. Mueller-Lantzsch, N., N. Yamamoto, and H. zur Hausen. 1979. Analysis of early and late Epstein-Barr virus associated polypeptides by immunoprecipitation. Virology 97:378-387. 12. Pagano, J. S. 1979. Extrachromosomal DNA, p. 235-248. Academic Press, Inc., New York. 13. Qualtiere, L. F., and G. R. Pearson. 1979. Epstein-Barr virus-induced membrane antigens: immunochemical characterization of Triton X-100 solubilized viral membrane antigens from EBV-superinfected Raji cells. Int. J. Cancer 23:808-817. 14. Shaw, J. E., T. Seebeck, J.-L. H. Li, and J. S. Pagano. 1977. Epstein-Barr virus DNA synthesized in superinfected Raji cells. Virology 77:762-771. 15. Stinski, M. F. 1978. Sequence of protein synthesis in cells infected by human cytomegalovirus: early and late virus-induced polypeptides. J. Virol. 26:686-701. 16. Summers, W. C., and G. Klein. 1976. Inhibition of Epstein-Barr virus DNA synthesis and late gene expression by phosphonoacetic acid. J. Virol. 18:151-155. 17. Yajima, Y., and M. Nonoyama. 1976. Mechanisms of infection with Epstein-Barr virus. I. Viral DNA replication and formation of noninfectious particles in superinfected Raji cells. J. Virol. 19:187-194. 18. zur Hausen, H., F. J. O'Neill, U. K. Freese, and E. Hecker. 1978. Persisting oncogenic herpesvirus induced by the tumor promoter TPA. Nature (London) 272:373-375.