A-type and B-type Epstein Barr virus differ in their ability to spontaneously enter the lytic cycle

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1 Journal of General Virology (1999), 80, Printed in Great Britain... SHORT COMMUNICATION A-type and B-type Epstein Barr virus differ in their ability to spontaneously enter the lytic cycle M. Buck, S. Cross, K. Krauer, N. Kienzle and T. B. Sculley Queensland Institute of Medical Research, Bancroft Centre, 300 Herston Road, Herston, Brisbane 4029, Australia In this study replication of A-type and B-type Epstein Barr virus (EBV) strains has been assessed. A-type and B-type lymphoblastoid cell lines (LCLs) were established by infecting B lymphocytes, isolated from five EBV-seropositive donors, with different A-type and B-type virus isolates. The presence of viral capsid antigens (VCA) in these LCLs was determined by immunofluoresence assay and by immunoblotting. All of the B-type EBV strains were capable of spontaneously generating virus regardless of the origin of the donor cells. In contrast the A-type strains, other than strain IARC-BL36, did not readily produce VCA in any of the different donor lymphocytes used. This study demonstrates another biological difference between the two virus types: their ability to spontaneously enter the lytic cycle. Epstein Barr virus (EBV) is a herpesvirus with a DNA genome of approximately bp. Worldwide, the virus infects more than 90% of the human population, and following primary infection with EBV all individuals retain the virus for life. In addition, in vitro infection of B lymphocytes with EBV leads to their immortalization, and the outgrowth of lymphoblastoid cell lines (LCL). Following in vitro infection of B lymphocytes the EBNA gene products are expressed in a sequential fashion (Alfieri et al., 1991). EBNA 2 and EBNA LP appear by 12 h after infection and reach maximum levels by 32 h. Expression of EBNAs 1, 3, 4, 6 and LMP is evident by 36 h, reaching maximum levels by 46 h. EBV strains can be categorized into one of two types (Atype or B-type) which show sequence divergence within their BamHI WYH and HindIII E gene regions reflecting divergence in the EBNA 2, 3, 4 and 6 gene products (Adldinger et al., 1985; Dambaugh et al., 1984; Sculley et al., 1989). Differences in a number of other regions of the genome have also been identified. These include differences in EBNA LP (Sample et al., 1986; Dambaugh et al., 1984) and the EBERs (Arrand et al., 1989). Differences in the proteins expressed during EBV- Author for correspondence: Tom Sculley. Fax toms qimr.edu.au induced transformation of B-lymphocytes are reflected in biological differences between A-type and B-type EBV. The two types of EBV display distinct growth phenotype characteristics with B-type transformants demonstrating lower transformation efficiency, growth rate and saturation density as well as greater sensitivity to seeding at limiting dilutions, as compared to A-type transformants (Rickinson et al., 1987). LCLs are largely nonpermissive for virus replication; however, in producer cell lines (usually BL and marmoset lines) a small but variable proportion of the cells supports the viral lytic cycle with the resulting synthesis of early antigens (EA) and viral capsid antigen (VCA), leading to production of progeny virus. The viral lytic cycle can be induced in latently infected cells by superinfection with P3HR1 EBV (Henle et al., 1970), addition of chemical inducers (Luka et al., 1979; zur Hausen et al., 1978) or transfection with the transactivator BZLF1 (Countryman et al., 1987). The VCA complex of EBV consists of at least five components, with molecular masses of 150, 110, 40 and kda. Serio et al. (1996) showed that the Table 1. Virus isolates used to establish transformed cell lines Virus Origin of virus* Reference IARC-BL74 BL Zimber et al. (1986) IARC-BL36 BL Sculley et al. (1988) Ag876 BL Dambaugh et al. (1984) B95-8 IM patienta Miller & Lipman (1973) QIMR-KTu IM patient Unpublished QIMR-MBl Spontaneous LCLb Unpublished QIMR-JSt Spontaneous LCLb Sculley et al. (1989) QIMR-Gor BL Young et al. (1987) QIMR-JSm Spontaneous LCLc Moss et al. (1988) QIMR-L19 Spontaneous LCLd Young et al. (1987) QIMR-L4 Spontaneous LCLd Young et al. (1987) WAN-BL BL Sculley et al. (1988) Af12 Spontaneous LCLe Unpublished * a, Cell line derived by passage of EBV (from an IM patient) in a marmoset; b, spontaneous cell line from normal healthy donor in Australia; c, spontaneous cell line from rheumatoid arthritis patient; d, spontaneous cell line from healthy donor in Papua New Guinea; e, spontaneous cell line from healthy donor in Africa SGM EEB

2 M. Buck and others Fig. 1. Immunoblot detection of the p21 component of VCA in cell lines infected with different strains of EBV. B lymphocytes isolated from donors JBL (A), DJM (B) and ISM (C) were infected with different isolates of A-type and B-type EBV. Extracts from each of these cell lines were subjected to immunoblotting using a human serum (MCr) which contained antibodies to the EBNAs and p21 VCA. The EBV strains used are indicated at the top of each lane and the type of each isolate is indicated at the bottom of each lane. VCA was also determined by IFA on cell lines established from donor JBL and the percentage of VCA positive cells is shown at the bottom of panel (A). The positions of the EBNAs and of the p21 VCA component are indicated. BFRF3 open reading frame encoded an kda protein which was an immunodominant marker of EBV infection. EBVseropositive human sera consistently detected these proteins in the 21 kda range on immunoblots of B cells in which EBV had been induced into the lytic cycle. In this study we have assessed A-type and B-type EBV strains for their permissiveness for virus replication. To overcome problems associated with differences in cellular backgrounds on the ability of each strain to undergo lytic replication (Sculley et al., 1987), virus isolates were obtained from parental cell lines and were used to infect and transform B lymphocytes from different healthy individuals. To establish the A- and B-type LCLs, B lymphocytes, isolated from five EBV-seropositive donors (JBL, JST, DJM, ISM and MBA), were infected with different A-type and B-type virus isolates (Table 1). After infection cells were seeded in threefold dilutions from 10 cells to 10 cells per well. After 4 weeks cell lines were established by subculturing the cells seeded at the lowest concentration at which transformation was visible. This procedure was important to ensure that the established cell lines arose by infection with exogenous virus rather than by outgrowth of endogenous EBV-infected B lymphocytes. The virus type in each of the cell lines was checked by Western blotting and Southern analysis of viral DNA and in all cases the transformed cell lines were of the same type as the exogenous virus (results not shown). The LCLs were maintained in RPMI 1640 supplemented with 10% foetal calf serum, benzylpenicillin (0 7 mg ml) and streptomycin (1 mg ml) at 37 Cin a5%co atmosphere. The cell lines were not stimulated in any way to induce virus production and were harvested while the cells were in the exponential growth phase. Initially six LCLs (three A-type and three B-type), established from donor JBL, were assayed by immunoblot for expression of the p21 VCA component and by immunofluorescence (IFA) to determine the proportion of cells expressing VCA. For IFA, cells were allowed to grow to approximately 1 10 cells ml and VCA expression in these cells was measured according to the method of Henle & Henle (1966), using a known VCA-positive serum (STh). For immunoblotting cells were collected by centrifugation and the cell pellets were dispersed by sonication in 2% SDS 1% 2- mercaptoethanol 0 1 mm PMSF 10 mm sodium phosphate (ph 6 8). The samples were then placed in a boiling water bath for 2 min, allowed to cool, and centrifuged at g for 5min. Samples (20 µl; 150 µg protein) were separated on 12% polyacrylamide gels. Electrophoresis was performed at 100 V while the transfer of proteins from polyacrylamide gels to nitrocellulose paper (Amersham) was performed essentially as described by Burnette (1981). Detection of EBV antigens with human serum (MCr) was performed using either radioiodinated protein A ( Ci µg) (New England Nuclear) or horseradish peroxidase-conjugated sheep anti-human IgG (Amersham) and an ECL Western blotting detection system (Amersham). EEC

3 Replicative capacity of EBV types Table 2. Relative intensity of VCA in cell lines transformed with A- or B-type EBV p21 VCA Donor cells Infecting virus Virus type rel. intensity* QIMR-JST QIMR-JSm B 11 Ag876 B 21 2 Af12 B 19 8 QIMR-L4 B 7 B95-8 A 0 1 IARC-BL36 A 9 8 IARC-BL74 A 0 1 QIMR-DJM QIMR-KTu B 13 8 QIMR-L4 B 5 1 Ag876 B 23 QIMR-JSm B 9 4 IARC-BL36 A 3 8 IARC-BL74 A 0 4 B95-8 A 0 7 QIMR-JBL QIMR-L4 B 24 5 QIMR-Gor B 5 4 QIMR-JSm B 9 5 IARC-BL74 A 1 5 B95-8 A 1 8 IARC-BL36 A 3 8 QIMR-MBA Ag876 B 7 6 WAN-BL B 14 QIMR-L19 B 11 2 B95-8 A 1 IARC-BL74 A 0 6 IARC-BL36 A 22 QIMR-MBl A 1 QIMR-ISM QIMR-L4 B 22 8 IARC-BL74 A 0 6 IARC-BL36 A 0 6 QIMR-JSt A 0 6 *Relative intensity was determined using a Molecular Dynamics densitometer and ImageQuant software. Values can only be compared within each donor group. The results obtained from cell lines established from donor JBL are presented in Fig. 1(A). All of the B-type cell lines expressed VCA by IFA, with 20% of the cells transformed with the QIMR-L4 strain expressing VCA. Of the A-type transformed cell lines only cells containing the IARC-BL36 virus strain showed any VCA expression. Immunoblotting detected the p21 VCA component in each of the cell lines that were shown to be VCA positive by IFA. Quantification (using a Molecular Dynamics densitometer and ImageQuant software) of the p21 protein expressed in each of these cell lines (Table 2) compared favourably with the IFA results. The QIMR- JBL L4 cell line expressed the highest level of the p21 VCA protein, followed by QIMR-JBL JSm and QIMR-JBL Gor; the A-type line QIMR-JBL IARC-BL36 expressed a small amount of p21. These results indicated that VCA expression as determined by immunoblotting correlated well with the percentage of IFA VCA-positive cells. A number of cell lines was also assayed for the presence of BZLF1 by IFA using an anti-bzlf1 mouse monoclonal supplied by DAKO. All of the cell lines expressed much higher levels of BZLF1 than VCA, with the A-type cell lines QIMR-JST B95-8 and QIMR-JST IARC-BL36 displaying 10% positive cells and QIMR-JST IARC-BL74 2% positive cells. Of the B-type lines QIMR-JST Ag876 had 3% positive cells, QIMR- JST QIMR-L4 15% positive and QIMR-JST QIMR-JSm 50% positive cells. Five of these cell lines were also tested for induction of virus replication using the phorbol ester TPA (phorbol 12-myristate 13-acetate) (Sigma). Cells were seeded at 5 10 cells ml and TPA added to a concentration of 40 ng ml. Samples were taken prior to the addition of TPA and at 3 and 7 days after treatment. IFA VCA-positive cells were then determined at each time-point. Each virus strain reacted differently to addition of TPA. The A-type cell line QIMR-JST IARC-BL74 had 0 5% VCA-positive cells on day 0 and 10% VCA-positive cells by day 7 whereas TPA did not cause an increase in virus production in the QIMR-JST IARC- BL36 cell line which had 1% VCA-positive cells at all timepoints. Likewise, the B-type cell line QIMR-JST QIMR-L4 displayed 5% VCA positive cells at all time-points and was not induced by TPA. The other two B-type cell lines tested were induced, QIMR-JST Ag876 going from 1 to 5% and QIMR- JST QIMR-JSm from 5 to 70% VCA-positive cells by day 7. A further 25 LCLs were established by infection of B lymphocytes from four different donors and expression of the p21 VCA component was determined by immunoblotting. Immunoblots of the LCLs established from donors DJM and ISM are shown in Fig. 1(B, C). The relative amounts of p21 VCA protein in each cell line were determined and results from all of the cell lines analysed are shown in Table 2. In each group of LCLs the B-type EBV-infected cell lines showed expression of VCA, whereas of the A-type-infected cells only cell lines containing strain IARC-BL36 demonstrated any VCA, and then not in all donor cells (Fig. 1C). One possibility for the increased virus production in B-type cell lines could be related to the rate of growth of these cell lines. To examine this three A-type (B95-8, IARC-BL36, IARC-BL74) and four B-type (Ag876, QIMR-JSm, QIMR-L4, Af12) LCLs, established from donor JST, were seeded at 10 cells ml and were cultured for 5 days in the presence of 1 or 10% FCS with cell counts being taken at 24 h intervals. All cell lines grew at a slower rate in 1% FCS; however, there was very little difference in the growth rate of any of the cell lines (at either 1 or 10% FCS) except for the A-type QIMR-JST IARC-BL36 line, which remained in stationary phase for the 5 day period. These results indicate that the level of spontaneous replication observed in the B- type cell lines was not related to their growth rate. It has been known for a long time that different cell phenotypes can affect the permissive status of EBV strains (Sculley et al., 1987), as is the case with the B95-8 virus which EED

4 M. Buck and others in the context of marmoset cells is permissive, but when used to establish an LCL from human B cells is largely nonpermissive. To overcome this problem different A-type and B- type strains of EBV were used to establish LCLs from B cells obtained from different healthy individuals. In this way it has been possible to compare the intrinsic ability of each virus strain to spontaneously enter the lytic cycle. The results show that the A-type strains, other than strain IARC-BL36, did not result in significant VCA production in any of the different donor B lymphocytes used. In contrast, all of the cell lines infected with B-type EBV strains were spontaneously producing VCA regardless of the origin of the donor cells. Remarkably, 50% of the QIMR-JSm virus-infected B cells from donor JST were positive for BZLF-1 expression and 70% were of cells were VCA positive after induction by TPA. This high degree of replication is reminiscent of P3HR-1 cells, which were found to contain a deleted and rearranged genome (termed het DNA) that disrupts latency and induces EBV to replicate in vitro (Jenson & Miller, 1987). It is possible that cells infected with B-type EBV may contain het DNA and this could account for their ability to spontaneously enter the lytic cycle. A condition in which EBV has been found to be actively replicating is hairy leukoplakia, an oral lesion that occurs in patients infected with the human immunodeficiency virus. The presence of both A-type and B-type EBV has been demonstrated in these lesions (Walling et al., 1992), as well as the presence of het DNA (Patton et al., 1990), raising the possibility that the replicative nature of these lesions could be driven to some extent by the B-type EBV (or possibly het DNA associated with the B-type EBV). It is widely believed that virus in circulating B-lymphocytes and in B-cell malignancies is stringently latent. However, Gutierrez et al. (1993) demonstrated by Southern blot analysis that replicative forms of virion DNA could be detected in 14 5% (8 of 55) of EBV-positive Burkitt s lymphoma biopsies. The percentage of BL biopsies containing replicative EBV is similar to the percentage of individuals in Africa and Papua New Guinea that were found to be infected with B-type EBV (Young et al., 1987). Given the replicative nature of B-type EBV it is possible that those biopsies identified as containing replicative forms of EBV may also have contained B-type EBV. Studies on immunocompromised patients have demonstrated a high incidence of B-type as well as A-type B-type coinfections (Sixbey et al., 1989; Sculley et al., 1988, 1990; Kyaw et al., 1992). The high incidence of B-type EBV in immunocompromised patients could be a consequence of the ability of B-type EBV to replicate. Disruption to the immune system in immunocompromised patients, restricting CTLs directed against lytic antigens, could result in the B-typeinfected cells actively replicating virus and high levels of B lymphocytes becoming infected with B-type EBV in these patients. The most significant difference between the A-type and B- type EBV strains is a reduction in the efficiency of transformation together with clearly defined phenotypic changes in the emerging transformed cells (Rickinson et al., 1987). This study demonstrates another biological difference between the two virus types, namely their ability to spontaneously enter the lytic cycle. This increased lytic ability may compensate for the reduced transforming ability of B-type EBV and allow this virus type to survive in vivo. A- and B-type EBV are characterized by sequence divergence in regions of the genome encoding EBNAs 2, 3, 4 and 6 (Sculley et al., 1989; Sample et al., 1990; Adldinger et al., 1985; Dambaugh et al., 1984); however, none of these gene products is involved in the lytic cycle. The increased lytic ability of B-type EBV suggests that other genes or their promoter regions may also be divergent between these two EBV types. This work was supported by grants from the National Health and Medical Research Council of Australia, the University of Queensland and from the Queensland Cancer Fund. N. Kienzle was a fellow of the German Infektionsforschung AIDS-Stipendiumsprogramm, DKFZ, Heidelberg. References Adldinger, H. K., Delius, H., Freese, U. K., Clarke, J. & Bornkamm, G. W. (1985). A putative transforming gene of Jijoye virus differs from that of Epstein Barr virus prototypes. Virology 141, Alfieri, C., Birkenbach, M. & Kieff, E. (1991). 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