1 CR-UK Institute for Cancer Studies, The University of Birmingham, Vincent Drive, Birmingham B15 2TT, UK
2 German Cancer Centre, Department of Virus Associated Tumours, Im Neuenheimer Feld 242, D-69120 Heidelberg, Germany
Correspondence
Alan Rickinson
A.B.Rickinson{at}bham.ac.uk
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Details of virus quantification procedures and of experiments confirming the DNase resistance of EBV genomes in virus preparations are given in the Supplementary Information and the results of the quantification of EBV preparations in Supplementary Fig. S1, available as supplementary material in JGV Online.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The events of B-cell transformation in vitro have been studied by many groups, almost all using high-titre virus preparations in an attempt to synchronize events in as large a number of cells as possible (Allday et al., 1989; Finke et al., 1987
; Henderson et al., 1977
; Hurley & Thorley-Lawson, 1988
; Mark & Sugden, 1982
; Moss et al., 1986
; Sugden & Mark, 1977
; Thorley-Lawson & Mann, 1985
). Apart from early studies using Gardella gel analysis to track the fate of input virus genomes and to measure their delivery to the nucleus (Hurley & Thorley-Lawson, 1988
), most published work has concentrated on events as they later unfold in the successfully infected cell. With the high virus doses routinely used in such experiments, most if not all viral antigens are detectable in B-cell cultures within 2 days of infection and are accompanied by upregulation of the B-cell surface-activation marker CD23; cellular DNA synthesis is initiated by day 3, and microscopic colonies of proliferating B lymphoblastoid cells are visible within 1 week (Allday et al., 1989
; Finke et al., 1987
; Moss et al., 1986
; Thorley-Lawson & Mann, 1985
).
However, many quantitative aspects of B-cell infection and transformation still remain to be determined, particularly following exposure to lower virus doses that are more likely to reflect those experienced in the natural situation in vivo, in which the virus is transmitted to an infant early in life, usually from its mother, who as a long-term carrier herself will be shedding low levels of EBV in the saliva. New approaches to these questions become possible with the development of quantitative PCR-based assays of EBV genome number (Junying et al., 2003; Murray et al., 2003
), which can in principle be used to determine the genome content of virus preparations, and of fluorescence in situ hybridization (FISH) techniques capable of visualizing the successful delivery of virus genomes into cells (Delecluse et al., 1993
). Here, we use these approaches alongside single-cell-based assays of virus latent antigen expression, B-cell activation, cell cycle transit and outgrowth to look again at the rate-limiting steps in the process of B-cell transformation.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Quantification of virus binding to primary B and T cells.
Adult peripheral blood mononuclear cells were prepared from buffy coats (kindly donated from the National Blood Service, Birmingham) and B cells positively selected using M-450 CD19 Dynabeads (Dynal); primary T cells were prepared by negative selection using anti-CD19, -CD16, -CD14, -CD11b (OKM1) and -glycophorin monoclonal antibodies (MAbs) combined with M-450 sheep anti-mouse immunoglobulin G (IgG) Dynabeads (Dynal). B-cell preparations were routinely >99 % CD20+ and T cell preparations >99 % CD3+. From each population, 106 cells were exposed for 3 h at 4 °C to 104108 virions in 1 ml volumes, equivalent to m.o.i.s of 0·01100, then the cells were washed extensively in PBS, total cellular and bound viral DNA was extracted, and the EBV-DNA copy-number per cell was determined using the Q-PCR assay (Junying et al., 2003), which simultaneously quantitates both viral and cellular genome copies. Some experiments used virus preparations first incubated for 1 h at 37 °C with varying dilutions of an anti-gp350 MAb known to block gp350/CR2-dependent virus binding (Thorley-Lawson & Geilinger, 1980
); other experiments used a gp350 knockout recombinant EBV strain (Janz et al., 2000
) as the virus source.
Quantification of virus genome delivery.
EBV genome delivery to the nucleus of virus-exposed cells was assayed between 1 and 3 days post-exposure using FISH with an EBV genome-specific probe generated by nick translation of the EBV cosmid cm302-21 of 51 kb (Polack et al., 1984), using methods previously developed for Marek's disease virus genome detection (Delecluse et al., 1993
). Cells of the EBV-negative BJAB cell line served as negative controls, and cells of the Namalwa (two EBV genome copies per cell) and Raji (up to 50 genome copies per cell) BL cell lines served as positive controls.
Quantification of viral gene expression.
Following infection, primary B cells were cultured in RPMI 1640 medium with 10 %, v/v, fetal calf serum (culture medium) at 106 cells per 2 ml well. Cells were harvested at varying time points up to 6 days post-infection, washed in PBS, dropped onto glass slides, air-dried and then fixed in methanol/acetone (1 : 1, v/v) at 20 °C. Slides were stained with the EBNA2-specific MAb PE2, as described by Young et al. (1989), followed by a Cy3-conjugated anti-mouse IgG secondary antibody (Stratech).
Quantification of cell activation and cell-cycle entry.
Freshly isolated primary B cells were extensively washed, resuspended at 2x107 cells ml1 in PBS and exposed to an equal volume of 5 µM 5-(and-6)-carboxyfluorescein diacetate (CFSE) for 10 min at 37 °C, after which labelling was stopped by adding an equal volume of pre-warmed culture medium and incubation at room temperature for 1 min. After further extensive washing in PBS, cells were infected with EBV preparations at m.o.i.s of 0·550 and cultured as above. Cultures were harvested daily up to 6 days post-infection and stained for CD23 expression with a phycoerythrin (PE)-conjugated anti-CD23 MAb (Pharmingen), and both CD23 levels and CFSE content were determined by fluorescence-activated cell sorting (FACS) analysis. In some experiments, cells were sorted on day 5 post-infection into CFSE high (i.e. non-divided) and CFSE low (i.e. divided) cell fractions by FACS, and aliquots of cells from the two fractions were assayed separately for expression of the CD23 marker and for expression of EBNA2 by MAb staining, as above.
Quantification of outgrowth to LCLs.
Primary B cells were infected with EBV at m.o.i.s of 1100 and cultured as above. The cultures were harvested at 3 days post-infection, an aliquot stained for EBNA2, and the remainder seeded into U-bottomed microtitre plate wells at cell densities calculated to deliver 1, 3, 10, 20, 60 and 200 EBNA2-positive cells per well. In each well, -irradiated human embryo fibroblasts and 103
-irradiated cells of an allogeneic LCL (transformed with a BZLF1 knockout virus and therefore incapable of virus production) served as a feeder layer (Feederle et al., 2000
). Cultures were maintained for 46 weeks and successful outgrowth to an LCL visually scored.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
|
Initiation of cell proliferation
In further experiments we stained resting B cells with CFSE before exposing the cells to EBV at known m.o.i.s, and then followed the progress of cells through subsequent mitotic divisions by tracking the reduction of CFSE label. Fig. 6 shows the results of such an experiment, conducted with m.o.i.s of 0·5, 5 and 50, where MAb staining on day 3 identified, respectively, 3, 22 and 56 % of cells as EBNA2-positive. Note that on day 3 there was still no evidence of cell division in any of the infected cultures, each CFSE profile showing a single peak like that of the uninfected B cells. By day 6, cell division had occurred in all these cultures and there was no evidence that using low doses of virus in any way delayed the response to infection. Thus, cells that were actively infected at an m.o.i. of 0·5 or 5·0 had completed between two and five cell cycles by day 6. Interestingly, at the higher m.o.i. of 50, although more cells had become actively infected, cell proliferation monitored on day 6 was slightly less advanced, with CFSE peaks showing that the cells had completed between one and four cell cycles only (see Fig. 6
). We consistently found that the first cell division was delayed by 24 h until day 5 post-infection at high virus doses (data not shown).
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Remarkably, combining the FISH assay with other markers of viral infection showed that a large majority of the cells in which an EBV genome reaches the nucleus will go on both to initiate viral antigen expression, as determined by EBNA2 staining, and to activate cell growth, as determined by high-level CD23 staining and stepwise reduction in CFSE labelling. Thus, at all three m.o.i.s tested, the number of EBNA2-positive cells detected at day 3 post-infection, immediately before the first round of cell division, almost exactly matched the number of EBV genome-positive cells detected by FISH. Furthermore, on day 3, most if not all of these EBNA2-positive cells were morphologically identifiable as large lymphoblasts and clearly distinct from the small EBNA2-negative population. We found that CD23 itself is a less reliable marker of active infection in this early period since, as already described (Gordon et al., 1986; Roberts et al., 1996
), EBV binding to CR2 on the resting B-cell surface will itself induce transient low-level CD23 expression. This almost certainly explains our findings that the percentage of CD23-positive cells appearing within the first few days of infection exceeds that of EBNA2-positive cells. Indeed, we carried out a number of control experiments using Q-PCR-titrated doses of P3HR1 virus, an EBNA2-deleted EBV strain which is non-transforming and which does not induce any transit through the cell cycle (Miller et al., 1974
; Rooney et al., 1989
). P3HR1 virus binding induced significant levels of CD23 which were detectable on days 1 and 2 post-exposure (data not shown).
Importantly, we found no evidence for the existence, as reported in earlier studies (Thorley-Lawson & Mann, 1985), of a substantial population of EBNA-positive cells which failed to activate CD23 expression or to move into cycle. However, in agreement with that earlier study, we found that the population of EBNA (in our case EBNA2)-positive, CD23 high cells did indeed consist of those destined to proliferate. Indeed, if these cells (identified in the earlier studies by CD23 positivity and in our case by EBNA2 staining) are reseeded in limiting dilution on day 3 post-infection before their first cell division, then their capacity for outgrowth to an established line is the equal of that shown by limiting-dilution seedings of recently established LCL cells themselves. The cloning efficiency of both cell populations, measured as 510 % under the conditions used in our assays, is likely to be an underestimate, since optimization of cell-culture conditions with auxiliary activation signals has been reported to achieve even more efficient outgrowth from limiting-dilution cultures (Traggiai et al., 2004
). The important point is that the outgrowth potential of cells in their first cell cycle following EBV infection is essentially indistinguishable from that of established LCL cells. We infer that EBV confers the capacity for unlimited growth on B cells very early post-infection.
An important objective in the present study was to follow infection at low m.o.i.s, rather than at the high, non-physiologic virus doses used in most earlier work. Interestingly, we found that at the single-cell level, growth induction occurred at least as quickly at m.o.i.s of 0·51 as it did at m.o.i.s of 510, and indeed slightly quicker than at m.o.i.s of 50100. The 24 h delay in movement through the first cell cycle seen at higher viral doses could reflect competition between cells immediately post-infection for extracellular factors required for activation from G0, or possibly an effect caused by the acquisition of multiple EBV genomes per nucleus. Defining the conditions under which only a single genome (visualized by FISH) is delivered per infected cell also allowed us to follow genome load over time in progeny cells. Transformation assays carried out at such low m.o.i.s, in the presence of acyclovir to prevent any subsequent EBV lytic replication and reinfection in vitro, showed that cells of the resultant LCL harboured multiple EBV episomes per cell (data not shown). This confirms that amplification of the episomal EBV genome does occur in latently infected cells as part of the transformation process (Hurley & Thorley-Lawson, 1988; Sugden et al., 1979
), albeit through as-yet unknown mechanisms. Indeed, our results showing equivalent efficiencies of outgrowth from EBNA2-positive cells on day 3 post-infection at an m.o.i. of 1 (before genome amplification) and of cells from recently established LCLs (after genome amplification) indicate that the amplification process itself is not rate-limiting for transformation.
The two most significant findings of the present study both concern early events in the transformation process. One is the identification of nuclear genome delivery as a key rate-limiting step. Thus we estimate that for EBV infection of small resting B cells, some 1015 % of all bound viruses deliver the genome to the cell nucleus. Although equivalent studies have not been carried out in any other herpesvirus system, the above estimate is in the same range as might be predicted for herpes simplex virus (HSV) infecting permissive cell lines in vitro, based on the observation that the virus particle : plaque-forming unit ratio in permissive cells is typically between 1 in 10 and 1 in 50 (Frenkel et al., 1975; McLauchlan et al., 1992
; Smith, 1964
; Watson et al., 1963
) and that plaque-forming units themselves underestimate the number of cells initiating HSV infection by two- to threefold (Everett et al., 2004
). However, the main obstacles limiting efficient intracellular herpesvirus transport and genome delivery to the nucleus are not known. One of the few quantitative studies involving electron microscopic analysis of HSV infecting Vero cells at very high m.o.i.s (Sodeik et al., 1997
) observed fusion of the viral envelope with the plasma membrane and suggested that the majority (60 %) of capsids thus entering the cytosol were transported to the nuclear membrane within 4 h, although the efficiency of subsequent genome release into the nucleus was not determined. Direct parallels between HSV and EBV need to be drawn with caution, however, since EBV entry into small resting B cells occurs not by plasma-membrane fusion but by endocytosis of the virus bound to its receptor CR2 (Nemerow & Cooper, 1984
; Tanner et al., 1987
), followed presumably by a fusion event at the endosomal membrane releasing the capsids into the cytosol. It will need a detailed study of these events in resting B cells to identify where in the process input viruses are lost; we cannot exclude the possibility that some virions within our EBV preparations, though carrying the viral genome and capable of binding target cells, were nevertheless defective in virion components required for virus entry and transport.
A second significant finding of the present work is the remarkable efficiency with which a single EBV genome, having reached the B-cell nucleus, then drives the cell growth transforming programme. Early studies demonstrating the single-hit kinetics of transformation made it clear that a single EBV genome could initiate the transformation process (Henderson et al., 1977; Sugden & Mark, 1977
), but it was never appreciated that essentially every cell that receives one virus genome in its nucleus is then driven into growth. We ascribe this efficiency, at least in part, to the nature of the BamH1 W promoter, which is responsible for initiating viral gene transcription in resting B cells (Woisetschlaeger et al., 1990
). This promoter is highly dependent upon B-cell-specific factors that are present in resting B cells (Tierney et al., 2000
), and its activity may be further augmented by factors induced as a result of CR2 binding (Bohnsack & Cooper, 1988
; Sugano et al., 1997
). Moreover, multiple copies of the promoter are present within the EBV genome through its representation in each BamHI W repeat. If, as available evidence suggests (Finke et al., 1987
; Rooney et al., 1989
), the incoming virus genome uses all available copies of the promoter to drive EBNA transcription, then this would serve to optimize the chances of an introduced viral genome initiating the transformation process.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Anagnostopoulos, I., Hummel, M., Kreschel, C. & Stein, H. (1995). Morphology, immunophenotype, and distribution of latently and/or productively EpsteinBarr virus-infected cells in acute infectious mononucleosis: implications for the interindividual infection route of EpsteinBarr virus. Blood 85, 744750.
Babcock, G. J., Decker, L. L., Volk, M. & Thorley-Lawson, D. A. (1998). EBV persistence in memory B cells in vivo. Immunity 9, 395404.[CrossRef][Medline]
Bohnsack, J. F. & Cooper, N. R. (1988). CR2 ligands modulate human B cell activation. J Immunol 141, 25692576.
Borza, C. M. & Hutt-Fletcher, L. M. (2002). Alternate replication in B cells and epithelial cells switches tropism of EpsteinBarr virus. Nat Med 8, 594599.[CrossRef][Medline]
Delecluse, H. J., Schuller, S. & Hammerschmidt, W. (1993). Latent Marek's disease virus can be activated from its chromosomally integrated state in herpesvirus-transformed lymphoma cells. EMBO J 12, 32773286.[Medline]
Delecluse, H. J., Hilsendegen, T., Pich, D., Zeidler, R. & Hammerschmidt, W. (1998). Propagation and recovery of intact, infectious EpsteinBarr virus from prokaryotic to human cells. Proc Natl Acad Sci U S A 95, 82458250.
Everett, R. D., Boutell, C. & Orr, A. (2004). Phenotype of a herpes simplex virus type 1 mutant that fails to express immediate-early regulatory protein ICP0. J Virol 78, 17631774.
Feederle, R., Kost, M., Baumann, M., Janz, A., Drouet, E., Hammerschmidt, W. & Delecluse, H. J. (2000). The EpsteinBarr virus lytic program is controlled by the co-operative functions of two transactivators. EMBO J 19, 30803089.[CrossRef][Medline]
Fingeroth, J. D., Weis, J. J., Tedder, T. F., Strominger, J. L., Biro, P. A. & Fearon, D. T. (1984). EpsteinBarr virus receptor of human B lymphocytes is the C3d receptor CR2. Proc Natl Acad Sci U S A 81, 45104514.
Finke, J., Rowe, M., Kallin, B., Ernberg, I., Rosen, A., Dillner, J. & Klein, G. (1987). Monoclonal and polyclonal antibodies against EpsteinBarr virus nuclear antigen 5 (EBNA-5) detect multiple protein species in Burkitt's lymphoma and lymphoblastoid cell lines. J Virol 61, 38703878.[Medline]
Frenkel, N., Jacob, R. J., Honess, R. W., Hayward, G. S., Locker, H. & Roizman, B. (1975). Anatomy of herpes simplex virus DNA. III. Characterization of defective DNA molecules and biological properties of virus populations containing them. J Virol 16, 153167.[Medline]
Gardella, T., Medveczky, P., Sairenji, T. & Mulder, C. (1984). Detection of circular and linear herpesvirus DNA molecules in mammalian cells by gel electrophoresis. J Virol 50, 248254.[Medline]
Gordon, J., Walker, L., Guy, G., Brown, G., Rowe, M. & Rickinson, A. (1986). Control of human B-lymphocyte replication. II. Transforming EpsteinBarr virus exploits three distinct viral signals to undermine three separate control points in B-cell growth. Immunology 58, 591595.[Medline]
Henderson, E., Miller, G., Robinson, J. & Heston, L. (1977). Efficiency of transformation of lymphocytes by EpsteinBarr virus. Virology 76, 152163.[CrossRef][Medline]
Hurley, E. A. & Thorley-Lawson, D. A. (1988). B cell activation and the establishment of EpsteinBarr virus latency. J Exp Med 168, 20592075.
Janz, A., Oezel, M., Kurzeder, C., Mautner, J., Pich, D., Kost, M., Hammerschmidt, W. & Delecluse, H. J. (2000). Infectious EpsteinBarr virus lacking major glycoprotein BLLF1 (gp350/220) demonstrates the existence of additional viral ligands. J Virol 74, 1014210152.
Junying, J., Herrmann, K., Davies, G. & 8 other authors (2003). Absence of EpsteinBarr virus DNA in the tumor cells of European hepatocellular carcinoma. Virology 306, 236243.[CrossRef][Medline]
Kieff, E. & Rickinson, A. (2001). EpsteinBarr virus and its replication. In Fields Virology, 4th edn, pp. 25112573. Edited by D. M. Knipe & P. M. Howley. Philadelphia: Lippincott, Williams & Raven.
Kurth, J., Spieker, T., Wustrow, J., Strickler, G. J., Hansmann, L. M., Rajewsky, K. & Kuppers, R. (2000). EBV-infected B cells in infectious mononucleosis: viral strategies for spreading in the B cell compartment and establishing latency. Immunity 13, 485495.[CrossRef][Medline]
Li, Q., Turk, S. M. & Hutt-Fletcher, L. M. (1995). The EpsteinBarr virus (EBV) BZLF2 gene product associates with the gH and gL homologs of EBV and carries an epitope critical to infection of B cells but not of epithelial cells. J Virol 69, 39873994.[Abstract]
Li, Q., Spriggs, M. K., Kovats, S., Turk, S. M., Comeau, M. R., Nepom, B. & Hutt-Fletcher, L. M. (1997). EpsteinBarr virus uses HLA class II as a cofactor for infection of B lymphocytes. J Virol 71, 46574662.[Abstract]
Mark, W. & Sugden, B. (1982). Transformation of lymphocytes by EpsteinBarr virus requires only one-fourth of the viral genome. Virology 122, 431443.[CrossRef][Medline]
McLauchlan, J., Addison, C., Craigie, M. C. & Rixon, F. J. (1992). Noninfectious L-particles supply functions which can facilitate infection by HSV-1. Virology 190, 682688.[CrossRef][Medline]
Miller, G., Robinson, J., Heston, L. & Lipman, M. (1974). Differences between laboratory strains of EpsteinBarr virus based on immortalization, abortive infection, and interference. Proc Natl Acad Sci U S A 71, 40064010.
Moss, D. J., Sculley, T. B. & Pope, J. H. (1986). Induction of EpsteinBarr virus nuclear antigens. J Virol 58, 988990.[Medline]
Murray, P. G., Lissauer, D., Junying, J. & 11 other authors (2003). Reactivity with a monoclonal antibody to Epstein-Barr virus (EBV) nuclear antigen 1 defines a subset of aggressive breast cancers in the absence of the EBV genome. Cancer Res 63, 23382343.
Nalesnik, M. A. (1997). Posttransplant lymphoproliferative disease of donor origin. Arch Pathol Lab Med 121, 665666.[Medline]
Nemerow, G. R. & Cooper, N. R. (1984). Infection of B lymphocytes by a human herpesvirus, EpsteinBarr virus, is blocked by calmodulin antagonists. Proc Natl Acad Sci U S A 81, 49554959.
Neuhierl, B., Feederle, R., Hammerschmidt, W. & Delecluse, H. J. (2002). Glycoprotein gp110 of EpsteinBarr virus determines viral tropism and efficiency of infection. Proc Natl Acad Sci U S A 99, 1503615041.
Niedobitek, G., Agathanggelou, A., Herbst, H., Whitehead, L., Wright, D. H. & Young, L. S. (1997). EpsteinBarr virus (EBV) infection in infectious mononucleosis: virus latency, replication and phenotype of EBV-infected cells. J Pathol 182, 151159.[CrossRef][Medline]
Polack, A., Hartl, G., Zimber, U., Freese, U. K., Laux, G., Takaki, K., Hohn, B., Gissmann, L. & Bornkamm, G. W. (1984). A complete set of overlapping cosmid clones of M-ABA virus derived from nasopharyngeal carcinoma and its similarity to other EpsteinBarr virus isolates. Gene 27, 279288.[CrossRef][Medline]
Roberts, M. L., Luxembourg, A. T. & Cooper, N. R. (1996). EpsteinBarr virus binding to CD21, the virus receptor, activates resting B cells via an intracellular pathway that is linked to B cell infection. J Gen Virol 77, 30773085.[Abstract]
Rooney, C., Howe, J. G., Speck, S. H. & Miller, G. (1989). Influence of Burkitt's lymphoma and primary B cells on latent gene expression by the nonimmortalizing P3J-HR-1 strain of EpsteinBarr virus. J Virol 63, 15311539.[Medline]
Seigneurin, J. M., Vuillaume, M., Lenoir, G. & De-The, G. (1977). Replication of EpsteinBarr virus: ultrastructural and immunofluorescent studies of P3HR1-superinfected Raji cells. J Virol 24, 836845.[Medline]
Smith, K. O. (1964). Relationship between the envelope and the infectivity of herpes simplex virus. Proc Soc Exp Biol Med 115, 814816.
Sodeik, B., Ebersold, M. W. & Helenius, A. (1997). Microtubule-mediated transport of incoming herpes simplex virus 1 capsids to the nucleus. J Cell Biol 136, 10071021.
Sugano, N., Chen, W. P., Roberts, M. L. & Cooper, N. R. (1997). EpsteinBarr virus binding to CD21 activates the initial viral promoter via NF-kappa B induction. J Exp Med 186, 731737.
Sugden, B. & Mark, W. (1977). Clonal transformation of adult human leukocytes by EpsteinBarr virus. J Virol 23, 503508.[Medline]
Sugden, B., Phelps, M. & Domoradzki, J. (1979). EpsteinBarr virus DNA is amplified in transformed lymphocytes. J Virol 31, 590595.[Medline]
Tanner, J., Weis, J., Fearon, D., Whang, Y. & Kieff, E. (1987). EpsteinBarr virus gp350/220 binding to the B lymphocyte C3d receptor mediates adsorption, capping, and endocytosis. Cell 50, 203213.[CrossRef][Medline]
Thorley-Lawson, D. A. & Geilinger, K. (1980). Monoclonal antibodies against the major glycoprotein (gp350/220) of EpsteinBarr virus neutralize infectivity. Proc Natl Acad Sci U S A 77, 53075311.
Thorley-Lawson, D. A. & Mann, K. P. (1985). Early events in EpsteinBarr virus infection provide a model for B cell activation. J Exp Med 162, 4559.
Tierney, R. J., Kirby, H., Nagra, J., Rickinson, A. & Bell, A. I. (2000). The EpsteinBarr virus promoter initiating B-cell transformation is activated by RFX proteins and the B-cell-specific activator protein BSAP/Pax5. J Virol 74, 1045810467.
Traggiai, E., Becker, S., Subbarao, K., Kolesnikova, L., Uematsu, Y., Gismondo, M. R., Murphy, B. R., Rappuoli, R. & Lanzavecchia, A. (2004). An efficient method to make human monoclonal antibodies from memory B cells: potent neutralization of SARS coronavirus. Nat Med 10, 871875.[CrossRef][Medline]
Wang, X. & Hutt-Fletcher, L. M. (1998). EpsteinBarr virus lacking glycoprotein gp42 can bind to B cells but is not able to infect. J Virol 72, 158163.
Wang, F., Gregory, C. D., Rowe, M., Rickinson, A. B., Wang, D., Birkenbach, M., Kikutani, H., Kishimoto, T. & Kieff, E. (1987). EpsteinBarr virus nuclear antigen 2 specifically induces expression of the B-cell activation antigen CD23. Proc Natl Acad Sci U S A 84, 34523456.
Wang, F., Gregory, C., Sample, C., Rowe, M., Liebowitz, D., Murray, R., Rickinson, A. & Kieff, E. (1990). EpsteinBarr virus latent membrane protein (LMP1) and nuclear proteins 2 and 3C are effectors of phenotypic changes in B lymphocytes: EBNA-2 and LMP1 cooperatively induce CD23. J Virol 64, 23092318.[Medline]
Watson, D. H., Russell, W. C. & Wildy, P. (1963). Electron microscopic particle counts on herpes virus using the phosphotungstate negative staining technique. Virology 19, 250260.[CrossRef][Medline]
Woisetschlaeger, M., Yandava, C. N., Furmanski, L. A., Strominger, J. L. & Speck, S. H. (1990). Promoter switching in EpsteinBarr virus during the initial stages of infection of B lymphocytes. Proc Natl Acad Sci U S A 87, 17251729.
Young, L., Alfieri, C., Hennessy, K. & 7 other authors (1989). Expression of EpsteinBarr virus transformation-associated genes in tissues of patients with EBV lymphoproliferative disease. N Engl J Med 321, 10801085.[Abstract]
Received 29 April 2005;
accepted 22 June 2005.