GSF-Research Center for Environment and Health, Institute for Clinical Molecular Biology and Tumor Genetics, Marchioninistrasse 25, D-81377 Munich, Germany
Correspondence
Barbara Adler
adlerb{at}lmb.uni-muenchen.de
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ABSTRACT |
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Present address: MorphoSys AG, Martinsried, Germany.
Present address: Max von Pettenkofer Institute, Genecenter, Feodor-Lynen-Strasse 25, D-81377 Munich, Germany.
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INTRODUCTION |
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LMP2A is an integral membrane protein with 12 hydrophobic transmembrane domains and cytoplasmic N and C termini (Laux et al., 1988, 1989
; Rowe et al., 1990
). Similarly to the intracellular domain of the B-cell receptor, the N terminus of LMP2A contains a number of motifs involved in proteinprotein interactions with different phosphotyrosine kinases and motifs involved in immunoreceptor tyrosine-based activation motif formation (Longnecker, 2000
) and it resides in lipid rafts (Higuchi et al., 2001
). In transgenic mice, LMP2A provides signals for B-cell survival allowing B-lymphocyte development to proceed in the absence of a functional B-cell receptor (Caldwell et al., 1998
). Studies with chimeric proteins expressing the LMP2A N terminus have shown that the N terminus can induce cytokine expression and calcium release (Alber et al., 1993
; Beaufils et al., 1993
). Recently, it has additionally been shown that expression of LMP2A in epithelial cells leads to activation of the extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) pathways (Chen et al., 2002
).
Cross-linking of the B-cell receptor leads to activation of a complex signalling cascade resulting in calcium mobilization and activation of the ERK and JNK pathways and of protein kinase C (Campbell, 1999). It has long been known that stimulation of the B-cell receptor induces EBV reactivation in virus-infected B cells (Tovey et al., 1978
; Takada, 1984
). Even though LMP2A mimics a functional B-cell receptor, it has been shown that in LMP2A-expressing B cells, B-cell receptor-dependent activation of the lytic cycle is inhibited (Miller et al., 1993
, 1994
, 1995
; Dykstra et al., 2001
). LMP2A expression interferes with calcium mobilization and EBV reactivation after cross-linking of surface Ig. This is presumably due to a dominant-negative effect on phosphotyrosine kinase activation and interference with the transfer of the B-cell receptor to lipid rafts. The inhibitory effect is dependent on the level of expression of LMP2A (Konishi et al., 2001
).
We have recently shown that expression of LMP1 can inhibit induction of the EBV lytic cycle (Adler et al., 2002). Comparing in a conditional lymphoblastoid cell line (LCL) the inhibitory effect of LMP1 with the long-known effect of LMP2A on virus reactivation by 12-O-tetradecanoyl-phobol-13-acteate (TPA) or anti-Ig, we previously showed that, in this cell system, LMP2A also interferes with virus reactivation, although to a lesser extent than LMP1. Here, we have demonstrated that, in EBV-positive cells that are permissive for virus reactivation, expression of LMP2A alone induced entry into the lytic cycle in the absence of other inducers. LMP2A thus has a dual role with respect to lytic cycle induction: it induces entry into the lytic cycle in the absence of B-cell receptor stimulation and it impedes lytic-cycle induction through the B-cell receptor when both signals act simultaneously.
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METHODS |
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Plasmids.
GFP was expressed from pEGFP-C1 (BD Clontech). pHEBop5 is derived from pHEBo (Sugden et al., 1985) and contains the simian virus 40 (SV40) promoter (SalI fragment from pSG5; Stratagene). pHEBop5 is the backbone for the SVLMP1 and the SVLMP2A plasmids. SVLMP1 expresses EBV LMP1 (Zimber-Strobl et al., 1996
), SVLMP1
C expresses a C-terminal deletion mutant of LMP1 (Kieser et al., 1999
) and SVLMP2A expresses EBV LMP2A (EcoRI fragment of U516/1) (Zimber-Strobl et al., 1991
). The plasmid p92.11 expresses a truncated (aa 1279) human nerve growth factor receptor (NGFR), which lacks the intracellular signal-transduction domain (Brielmeier et al., 2001
).
Transfection and cell stimulation.
Cells (1x107) were co-transfected with 5 µg pEGFP-C1 or p92.11 and 5 µg pHEBo-based plasmids by electroporation using a Bio-Rad Gene Pulser II. Akata and P3HR1 cells were electroporated at 250 V and 975 µF, and EREB2-1 cells were electroporated at 220 V and 975 µF. The mitogen-activated protein kinase (MAPK) kinase inhibitor PD98059 (50 µM; Calbiochem) was added to the cell-culture medium directly after transfection. Cells co-transfected with pEGFP-C1 were harvested 72 (BZLF1 detection) or 96 [virus capsid antigen (VCA) detection] h after transfection. For additional stimulation with anti-Ig antibodies, EREB2-1 and Akata cells were stimulated 48 h after transfection with plate-bound anti-IgM (Sigma) or plate-bound anti-IgG (ICN Biomedicals), respectively. Anti-IgM was used at 20 µg per well and anti-IgG at 72 µg per well in six-well plates. Cells co-transfected with NGFR were purified by microbead-assisted cell sorting (MACS; Miltenyi Biotec) using an anti-NGFR antibody (HB8737; ATCC) and anti-mouse IgG microbeads. Purification was performed 48 h after transfection and before additional stimulation.
FACS analysis.
For staining of GFP-positive cells for EBV antigens, cells were fixed in 4 % paraformaldehyde, washed in staining buffer (PBS with 1 % BSA and 0·03 % saponin) and incubated with a mouse mAb to VCA (gp125, BALF4; Chemicon) or a mouse mAb to BZLF1 (B21; a kind gift of M. Rowe, University of Wales, Cardiff). Phycoerythrin-conjugated goat anti-mouse IgG (Dianova) was used as a secondary antibody for staining positive cells. Isotype controls were either mouse IgG1 (Dianova) or mouse IgG2a (Pharmingen). LMP2A-expressing cells were stained with a rat mAb to LMP2A (Fruehling et al., 1996) and an anti-rat IgG Cy3-conjugated secondary antibody (Dianova). Cells were analysed using a Becton Dickinson FACScan with CELLQUEST analysis software.
Immunoblot analysis.
Cellular extracts were prepared by sonication in sample buffer (0·13 M Tris/HCl pH 6·8, 6 % SDS, 10 % -thioglycerol), separated on 10 % polyacrylamide gels and transferred to nitrocellulose (Hybond ECL; Amersham Pharmacia). Filters were blocked in 5 % low-fat milk in PBS and then incubated with mouse mAb to BZLF1 or a human antiserum recognizing VCA. Immunoreactive proteins were detected by peroxidase-conjugated goat anti-mouse IgG antibody or rabbit anti-human Igs (Sigma) and enhanced chemiluminescence (ECL system; Amersham Pharmacia). An anti-actin mAb (C-2; Santa Cruz Biotechnology) was used to stain the blots for equal loading.
Luciferase reporter assays.
Hygromycin B-resistant Raji DR-LUC cells (2·5x105) were co-incubated for 72 h with supernatants of P3HR1 cells transfected with SVLMP2A or pHEBop5. Cells were lysed in luciferase lysis buffer (100 mM potassium phosphate pH 7·8, 1 mM DTT, 1 % Triton X-100). Luciferase activity was measured in cleared lysates. Luciferase assays were performed in luciferase assay buffer [25 mM glycylglycine pH 7·8, 15 mM MgSO4, 5 mM ATP and 15 µg D-luciferin (Roche Diagnostics) per sample]. Inducibility of luciferase activity was controlled by treatment of the cells with 20 ng TPA ml1.
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RESULTS |
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DISCUSSION |
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Stimulation of EBV-infected B cells through the B-cell receptor is one of the classical modes of virus reactivation in permissive B cells (Tovey et al., 1978; Takada, 1984
). Extrapolating from the extensive similarities between LMP2A and the B-cell receptor, one might postulate that LMP2A should also induce the lytic cycle in permissive B cells in a similar fashion to B-cell receptor stimulation. However, the opposite finding has been reported. LMP2A interferes with B-cell receptor signalling and the LMP2A signalling inhibits rather than induces EBV reactivation (Miller et al., 1993
, 1994
, 1995
). We have now resolved this apparent paradox by providing evidence that LMP2A, when acting alone, can indeed induce viral reactivation in permissive B cells. However, when an activated B-cell receptor and LMP2A act simultaneously, LMP2A does not augment but rather attenuates the signal from the B-cell receptor although it does not block it completely. We additionally showed that LMP2A and the B-cell receptor act through the MEK/MAPK pathway, since both B-cell receptor- and LMP2A-mediated virus reactivation can be inhibited by the MEK1 inhibitor PD98059.
We have described previously that ligands activating the same receptor and the same pathway can interfere with each other if one ligand is providing a much weaker signal than the other (Adler et al., 2001). If, for instance, the effect of the strong inducer on a given phenotype is maximal, any competition with a weak inducer will result in an attenuation of the phenotype imposed by the strong inducer. LMP2A-mediated EBV induction is indeed much weaker than anti-Ig-dependent virus reactivation. Inhibition of anti-Ig-mediated virus reactivation by LMP2A may thus be the result of sequestration of signalling molecules from the B-cell receptor. The finding that EBV co-opts lipid rafts through LMP2A and thus excludes the B-cell receptor from the intracellular signalling compartment is consistent with this notion (Dykstra et al., 2001
). It remains an open question whether the interference of LMP2A with B-cell receptor signalling plays a role in EBV infection. In cells in which LMP2A and LMP1 are expressed simultaneously, e.g. in EBV-infected memory B cells in lymphoid organs, LMP1 should suppress LMP2A-dependent virus reactivation. In resting memory B cells in the peripheral blood, LMP2A has been detected by RT-PCR (Qu & Rowe, 1992
; Tierney et al., 1994
; Miyashita et al., 1997
; Babcock et al., 1998
, 1999
), yet newer findings indicate that there is no latent gene expression at all and that previous reports are based on an experimental artefact (Hochberg et al., 2004a
, b
). In immunocompetent individuals, LMP2A-expressing cells are supposed to be eliminated by LMP2A-specific cytotoxic T cells unless specific mechanisms would spare these B cells. In immunocompromised patients, however, LMP2A expression could contribute to spontaneous EBV reactivation. LMP2A thus might play a dual role in EBV infection: it may on the one hand provide a signal for entry of the virus into the memory compartment necessary for long-term survival and persistence of EBV in infected memory B cells, while on the other hand providing a signal that favours sporadic virus reactivation. This spontaneous reactivation should be under the control of antigen-specific cytotoxic T cells in immunocompetent individuals, but might contribute significantly to the strong increase in virus load in immunocompromised patients (Lam et al., 1991
; Babcock et al., 1999
). It will be of interest to find out when and where in the course of an EBV infection LMP2A expression might contribute to EBV reactivation in immunocompromised patients in vivo. A detailed knowledge of the signalling pathways involved in spontaneous virus reactivation is a prerequisite for the identification of drugs that interfere with this process and that may be used as antiviral agents in immunocompromised individuals.
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ACKNOWLEDGEMENTS |
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Received 14 July 2004;
accepted 29 November 2004.