Infection of human epithelial cells by Epstein-Barr Virus (EBV)

Virus Research,

3 (1985)

207

207-212

Elsevier

VRR 00207

Infection of human epithelial cells by Epstein-Barr virus ( EBV) II. Biochemical characterization of EBV-determined proteins synthesized in epithelial cells *

Faruk Sinangil, Barbara Volsky and David J. Volsky ** Department

ofPathology

and Laboratory

University of Nebraska

Medicine,

and Eppley Institute for Research

Medical Center, Omaha, NE 68105,

(Accepted

on Cuncer,

U.S.A.

30 May 1985)

Summary

Primary cultures of epithelial cells were grown from tonsils of patients with diseases not related to EBV. The cells were implanted with EBV receptors and exposed to EBV of the transforming (B95-8, AG-876) and nontransforming (P3HR-1) strains. The EBV-infected and control cells were pulsed with [35S]methionine at 18-24 h after infection, and cell extracts were prepared for immunoprecipitation with anti-EBV sera and analysis by gel electrophoresis and autoradiography. About 20 EBV-determined proteins ranging from 22 to 185 kDa were detected in P3HR-1 virus-infected epithelial cells. Only a few polypeptides were detected in extracts of cells infected with AG-876 virus while no EBV-specific proteins were immunoprecipitated from extracts of B95-8 virus-infected cells. These results demonstrate that the system of EBV receptor-implanted normal human epithelial cells can be used for direct biochemical analysis of EBV infection in the epithelial tissue.

human nasopharyngeal receptor-implantation

epithelial

cells,

* The first article in this series is cited as Shapiro ** To whom correspondence should be addressed.

016%1702/85/$03.30

Epstein-Barr

virus,

immunoprecipitation,

and Volsky. 1983

0 1985 Elsevier Science Publishers

B.V. (Biomedical

Division)

208 EBV has been implicated in the etiology of two human malignancies, Burkitt’s lymphoma (BL) and nasopharyngeal carcinoma (NPC) (reviewed in Miller, 1980. and Klein, 1979). Despite the apparent dual tropism of EBV to lymphocytes and epithelial cells in vivo, most of the studies on the interaction between the virus and host cells in vitro have been limited to human B lymphocytes (reviewed in Miller, 1980, and Kieff et al., 1982). Data obtained in these studies thus far are consistent with the role of EBV in infectious mononucleosis and other lymphoproliferative diseases, and with the presumed contribution of the virus to the development of BL (Miller, 1980; Kieff et al., 1982). The experimental proof for the association between EBV and nasopharyngeal carcinoma, or oropharynx epithelium in general, has been less forthcoming. In contrast to B lymphocytes (Jondal and Klein, 1973) normal epithelial cells explanted from oropharynx do not express EBV receptors, and are generally nonsusceptible to infection by most of the known EBV strains (Glaser et al., 1980; Shapiro and Volsky, 1983). Even in systems which permit direct infection by certain isolates of EBV, such as the primary explant cultures of human ectocervix, the infection is transient and limited to about 0.05% of the cells (Sixbey et al., 1983). In order to develop more efficient systems for studying the course of EBV infection in epithelial cells in vitro, several groups including ours used DNA transfection (Stoerker et al., 1981; Griffin and Karran, 1984; Miller et al., 1981) microinjection (Glaser et al.. 1983) and receptor transplantation (Shapiro and Volsky, 1983; Seshi et al., 1984) as a means for introducing EBV genome into cultured epithelial cells. Two different lines of evidence have been obtained in these experiments. Infection of human nasopharyngeal and thymic epithelial cells with EBV of B95-8 strain, following implantation of EBV receptors (Volsky et al., 1980) resulted in the induction of EB virus nuclear antigen (EBNA) but not of the early (EA) or virus capsid (VCA) antigens, or virus replication (Shapiro and Volsky, 1983; Seshi et al., 1984). Similarly, transfection of monkey epithelial cells with cloned recombinant EBV DNA fragments from B95-8 virus induced foci of morphologically transformed cells without evidence of viral DNA replication (Griffin and Karran, 1984). On the other hand, transfection of EBV DNA into placental epithelial cells (Miller et al., 1981) explanted tumor epithelium (Stoerker et al., 1981) or direct infection of human ectocervix epitheliu’m with fresh isolates of EBV (Sixbey et al., 1983) resulted in viral replication rather than cell transformation. The question whether human oropharynx epithelium is the site of viral replication in vivo. or the target tissue for viral transforming activity, or both, remains unresolved. Further studies are required to determine the precise nature of the interaction between EBV and human epithelial cells. We report here on the application of our system of EBV receptor-implanted human nasopharyngeal epithelial cells (Shapiro and Volsky, 1983) for the first direct biochemical anal,ysis of EBV infection in these cells. Primary cultures of human epithelial cells were explanted as described previously from tonsils of patients with diseases unrelated to Epstein-Barr virus (Shapiro and Volsky, 1983). Falcon Primaria tissue culture flasks were used to facilitate tissue adherence and to minimize fibroblast growth. The cells grew as monolayers and had the typical morphology of epithelial cells. Four to six weeks after initiation of

209 cultures the cells were removed, implanted with functional EBV receptors as described (Shapiro and Volsky, 1983; Seshi et al., 1984) and exposed to EBV of various strains. The different virus preparations used were diluted to contain in the approximately the same number of virions, as judged by viral infectivity respective host cells. In the long-term infection experiments, the cells were then cultured under standard conditions for prolonged periods of time. Induction of EBNA, but not of EA or VCA, was regularly observed in 3-5% of cells 2-3 days after infection with EBV of the B95-8 or AG-876 strains, as reported previously (Shapiro and Volsky, 1983). However, no cell transformation has been achieved in this system as yet. About 0.5% of P3HR-l-infected epithelial cells were positive for EA 24 h after infection. No EBNA-positive cells were found in P3HR-1 virus-infected cultures at any stage. To identify individual EBV-determined proteins synthesized in epithelial cells during the first 24 h of the infection, the EBV-exposed and control cells were labelled with [ “Slmethionine 16-18 h post-infection and extracted 6-8 h later. The labelled polypeptides were precipitated from cell extracts with EBV-positive sera, resolved electrophoretically on SDSpolyacrylamide gels and visualised by autoradiography. For comparison, normal mouse lymphocytes were receptor-implanted, infected with the same batch of EBV and analysed like the epithelial cells. Uninfected cells and extracts of infected cells treated with EBV-negative sera served as controls. 24 h post-infection was chosen as the time point for EBV protein pattern analysis since recent reports showed that EBV-specific early protein synthesis peaks at 12-24 h in superinfected Raji cells (Bayliss and Wolf, 1981) and in EBV-infected mouse lymphocytes (Sinangil and Volsky, 1984). Figs. 1A and B show the results of two typical experiments. About 20 EBV-specific polypeptides could be detected in P3HR-1 virus-infected epithelial cells after immunoprecipitation with EBV-positive serum. No proteins were precipitated by serum from an EBV-negative individual. The polypeptides ranged from 22 to 185 kDa. Polypeptides of 48, 65, 86 and 95 kDa were particularly evident in the P-virus infected cells. EBV-infected normal mouse lymphocytes (Fig. 1C) served as a system of reference in these experiments. As we demonstrated recently, mouse cells undergo a full replicative primary infection when implanted with functional EBV receptors and exposed to EBV (Sinangil and Volsky, 1984; Volsky et al., 1981). EBV-determined proteins can be detected by immunoprecipitation or immunofluorescence 12224 h after infection (Sinangil and Volsky, 1984). Newly replicated viral particles can be detected in the cells by electron microscopy, or can be collected from the culture medium, 36-48 h post-infection (Volsky et al., 1981). It was thus conceivable that the expression of EBV genome in this experimental EBV permissive system may be similar to that in the presumably productively infected (Sixbey et al., 1983, 1984) human epithelial cells. Indeed, comparison of Figs. 1A (lane 2) and B (lane 2) with Fig. 1C (lane 2) shows significant similarity of EBV-specific protein patterns in P3HR-1 virus-infected epithelial and mouse cells. The main translational products of the P3HR-1 virus genome in both cells, i.e., polypeptides 38K. 48K, 50K, 65K, 78K and 95K, seem to have identical electrophoretic mobility as determined by

%

2

Fig. 1. Pattern of EBV-determined proteins synthesized in normal human epithelial ceils 24 h after infection with EBV. Tonsils removed by surgery were extensively washed in phosphate-buffered saline (PBS) supplemented with penicillin (2500 pU/ml). streptomycin (2.5 mg/ml) and gentamycin (2.5 mg/ml). Pieces measuring 2 mm2 were submerged in fetal calf serum (FCS) and then placed in Falcon Primaria tissue culture flasks (No. 3813, 25 cm’ area}. Cultures were grown for 68 weeks in the Dulbecco modified Eagle medium supplemented with defeted fetal calf serum and antibiotics ( x 150). Epithelial cells were removed from flasks by rubber policemen and repetitive washings, suspended in a fusion buffer (160 mM NaCI/IO mM Tris-HCI. pH 7.4) and implanted with EBV receptors as previously described (Shapiro and Volsky. 1983: Miller et al.. 1981). I gg of the receptor-containing reconstituted Sendai virus envelopes (RSVE/EBV-R, see Shapiro and Volsky. 1983; VoIsky et al., 1980) wab used for epithelial cells wet weight equivalent of 1 X 10h Raji cells. EBV receptors, Sendai virus and RSVE/EBV-R were prepared as described previously (Volsky et al., 1980). The receptor-implanted cells (wet weight equivalent of 20X 10h Raji cells) were exposed to EBV of an indicated strain in 0.5 ml and cultured for 16-18 h. The infectivity of EBV strains used for infection was as follows: P3HR-I virus at 1 : 200 dilution induced EA in 5% of Raji cells 24 h after infection: B95-8 and AG-876 viruses at 1 : 200 dilution induced EDNA in 3% of Loukes cells 48 h after infectton. EBV-infected epithelial cells were pulsed in [ “Slmethionine (80 pCi/ml, New, England Nuclear. 1166.5 Ci/mmol) in methionine-free medium for 6-8 h, and harvested at the 24th hour since the beginning of infection. The cells were then extracted in the inlmunoprecipitation buffer (20 mM Tris-HCI, pH = 9/0.137 M NaCI/l mM CaC12/1 mM MgC12/1% Triton X-100/0.1% SDS/IO% glycerol/O.Ol’% NaN?/l pg/ml PMSF), and immunoprecipitated with EA+/VCA+ (titer: EA 1 : 10240. VCA 1 : 40960, EBNA 1 : 80. obtained from W. Henle) or EBV-negative (S.E.) serum. The immunoprecipitates were resolved in 10% SDS- PAGE gels (Laemmli. 1970) followed by autoradiography using Kodak XAR-2 paper. The figure shows protein patterns in human epitheliai cells (A and 8) and mouse lymphocytes (C) following infection with EBV of P3HR-I strain (lanes A2, B2, C2). 895-8 strain (A3) or AG-876 (Bl). Al and Cl: EBV-negative serum. C3: low EA/high VCA serum (EA 1 : 1280, VCA 1 : 20480, EBNA 1 : 40). The molecular weight protein standards were purchased from the Bio-Rad Laboratory (CA 94804) and contained the following (M,): Soybean trypsin inhibitor (21500). carbonic anhydrase (31000). ovalbumin (45000). bovine serum albumin (66200). phosphorylase h (92 500). &galactosidase (116 250) and myosin (200000).

211 SDS-PAGE. Polypeptides of 39, 65, 95 kDa and others probably belong to the EA complex because their appearance was markedly diminished after precipitation with a low EA/high VCA serum (Fig. lC, lane 3). The similarity of EBV protein band patterns in productively infected mouse lymphocytes and in P3HR-1 virus-infected epithelial cells indicates that human nasopharynx epithelium may serve as a target tissue for the primary replicative infection with P-EBV. It has been recently reported by Sixbey and colleagues that human ectocervix epithelial cells could be productively infected, albeit with a very low efficiency, with fresh isolates of EBV from patients with acute mononucleosis but not with laboratory strains, such as the B95-8 virus (Sixbey et al., 1983). The authors did not report about similar experiments with nasopharynx epithelium. It is therefore interesting that few if any EBV-specific polypeptides indicative of the viral replicative cycle could be detected in immunoprecipitates of B95-8 virus-infected nasopharyngeal epithelial cells (Fig. lA, lane 3). Only few proteins were detected in epithelial cells infected with AG-876 virus, a natural isolate from Burkitt’s lymphoma cells, indicating that the genome of this isolate was not fully transcribed and translated in the epithelial cells either (Fig. IB, lane 1). Experiments are now in progress to determine whether or not EBV isolates from patients with acute infectious mononucleosis would induce polypeptides indicative of a replicative cycle similar to P3HR-1 virus (Fig. lA, B). Nasopharyngeal epithelium is not only the proposed site of EBV replication in vivo (Sixbey et al., 1983, 1984) but also the well-characterized target site for the development of NPC (Klein, 1979). Usually, herpesviruses cannot induce cell transformation and self-replication in the same target cells (Spear, 1980). Cell transformation is concomitant with, if not the result of, the block of viral replication (Miller, 1980; Kieff et al., 1982). Our results can be thus interpreted in two ways: (1) Human epithelial cells explanted from tonsils are permissive for P3HR-1 but non-permissive for B95-8 or AG-876 viruses. Our failure to establish EBNA-positive cell lines after infection with B95-8 or AG-876 viruses may be due to technical difficulties in long-term culture of nasopharyngeal epithelial cells, as is the case with epithelial cells of NPC tumor origin (Klein, 1979). (2) Alternatively, two types of nasopharynx epithelial cells might be involved in the interaction with EBV. Cells of one type, possibly the differentiated ones, do not have the mechanisms to control viral replication, as demonstrated by the infection with P3HR-1 virus in our experiments (Figs. lA, B) and by infection with the natural EBV isolates by Sixbey et al. (1983). The other cell type, possibly poorly differentiated, has the capability to block viral replication and to undergo transformation as a result. These are probably the cells which, in our experiments, responded with the induction of EBNA (Shapiro and Volsky, 1983; Seshi et al., 1984) but not of the proteins indicative of the lytic cycle of the virus (Figs. lA, B), following infection by B95-8 or AG-876 virus. In summary, we have demonstrated that the system of EBV receptor-implanted normal human epithelial cells from nasopharynx can be used for direct biochemical analysis of EBV infection. The cells can either synthesize polypeptides associated with the lytic cycle of EBV, or completely block the synthesis of these proteins, depending on the viral strain used for infection. The system will be useful for studying the mechanisms involved in the control of EBV genome expression in epithelial cells.

212 Acknowledgements

The authors would like to thank Drs. Purtilo and Younkers for help in obtaining the human epithelial tissue used in these experiments. Dr. Henle for supplying the EBV-positive serum, and S. Blum for typing the manuscript. This work was supported in part by the NIH-NC1 grants CA37465 and CA33386 and also by the Nebraska Department of Health (LB506).

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