Department of Veterinary Pathology, University of Edinburgh, Summerhall EH9 1QH, UK1
Author for correspondence: Bernadette Dutia.Fax +44 131 650 6511. e-mail B.M.Dutia{at}ed.ac.uk
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Abstract |
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Introduction |
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The association of EBV with malignant disease in vivo is reflected in the ability of the virus to effect growth transformation of human B cells in vitro. Resting B cells are the primary target for growth transformation in vitro but the virus can also transform progenitor B lymphocytes, pre-B lymphocytes and cells with fully rearranged and mature immunoglobulin chains (Ernberg et al., 1987 ). Growth transformation of marmoset and human T cells by HVS is readily achieved (Biesinger et al., 1992
; Schirm et al., 1984
; Fickenscher & Fleckenstein, 1998
) and recent experiments have shown that HHV-8 can transform primary human endothelial cells in vitro (Flore et al., 1998
).
For EBV, the process of growth transformation in vitro has been widely studied and is the primary experimental system used to identify the viral proteins involved in transformation and to characterize their functions (Raab-Traub, 1996 ). The initial events in the process of infection of primary B cells are mediated by the binding of the virus to its receptor and mirror the changes that occur during mitogen- or antigen-induced activation (Gordon et al., 1986
; Sinclair & Farrell, 1995
). Within 1216 h of infection, circularized EBV genomes are detected and virus gene transcription commences (Hurley & Thorley-Lawson, 1988
). EBNA2 and EBNA-LP are the first genes transcribed. By 32 h post-infection (p.i.), transcription of the nine genes expressed in EBV-growth-transformed B cells can be detected, while transcription of the EBER RNAs lags by 24 h and does not reach substantial levels until 70 h p.i. (Alfieri et al., 1991
). Over the next 5 days, cell proliferation begins and colonies can be detected from 1 week p.i. (Stuart et al., 1995
).
In order to further our understanding of the biology of MHV-68, we have studied the effect of infection of murine splenocytes and purified B lymphocytes in vitro.
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Methods |
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Virus and cells.
MHV-68 was originally obtained from D. Blaskovic (Blaskovic et al., 1980 ). Working stocks of MHV-68 were prepared by infection of BHK-21 cells with MHV-68 clone g2.4 (Efstathiou et al., 1990
) at low multiplicity (0·001 p.f.u. per cell) as described previously (Sunil-Chandra et al., 1992 a
). S11 is a B cell tumour line derived from a BALB/c mouse and was grown as described previously (Usherwood et al., 1996b
). The CD40L-expressing cell line K47 was a generous gift from A. Schhimpl, Institut für Virologie und Immunobiologie, Universit ät Wü rzburg, Würzburg, Germany.
Purification of B cells.
B lymphocytes were purified from total splenocytes by negative selection by using CD43 MACS beads (Miltenyi Biotec) according to the manufacturer's directions. This method routinely gave preparations that were 95% pure.
In vitro infection of lymphocytes.
Cells were teased out of spleens and mononuclear lymphocytes were purified by centrifugation on Ficoll-Hypaque gradients (Histopaque 1077, Sigma). Lymphocytes were washed extensively with RPMI containing 10% foetal calf serum, 50 µM 2-mercaptoethanol, 2 mM glutamine, 100 µg/ml streptomycin, 100 U/ml penicillin and 2 µg/ml fungizone, resuspended at a concentration of 2x107 cells/ml and infected with appropriate amounts of virus or mock- infected with BHK cell lysate by incubating on a shaker at 37 °C for 1 h. Cells were diluted to 2x106 cells/ml, plated in 24 well tissue culture plates and incubated at 37 °C in a CO2 incubator for the required time. For long-term experiments, cell were fed weekly with fresh medium and cytokines as described in Results.
In vitro survival of lymphocytes.
Lymphocytes were purified, infected at varying m.o.i. and 2 ml samples containing 2x106 cells/ml were plated in triplicate. Cells were resuspended thoroughly with a pipette prior to counting. On days 0 and 1 p.i., 50 µl samples were stained with 1% trypan blue and duplicate counts were made of live cells. At later time-points, cells were concentrated 1030-fold by centrifugation prior to counting to ensure adequate numbers of viable cells for accurate counting.
Lymphocyte proliferation.
Lymphocyte proliferation was measured by using the Biotrak cell proliferation ELISA system 2 (Amersham). B lymphocytes were purified by positive selection with rat anti-mouse CD19 antibody (Pharmingen) and goat anti-rat MACS microbeads according to the manufacturer's instructions. Infected and mock-infected cells were plated at 107 cells/ml in 96 well flat-bottomed plates, 100 µl per well. After 72 h, 5-bromo-2'-deoxyuridine (BrdU) was added to a final concentration of 20 µM and cells were cultured for a further 24 h. Cells were then pelleted by centrifugation, the medium was removed and the plates were dried at 60 °C for 1 h. The plates were fixed and stained according to the manufacturer's instructions with the following modifications: fixation time was increased to 1 h, incubation with anti-BrdU antibody was carried out at 37 °C rather than at room temperature and the antibody concentration was increased 4-fold.
In situ hybridization.
Non-radioactive in situ hybridizations were carried out on MHV-68-infected and mock-infected lymphocytes 72 h p.i. essentially as described previously (Stewart et al., 1998 ). After recovery of viable lymphocytes by centrifugation over Ficoll-Hypaque, cells were washed twice in PBS, fixed in PLP (75 mM l-lysine hydrochloride, 37·5 mM sodium phosphate buffer, pH 7·4, 2% paraformaldehyde, 10 mM sodium metaperiodate) for 5 min, washed once more and then resuspended in PBS at a concentration of 106 cells/ml. Cells (100 µl) were transferred onto Biobond-treated slides by using a cytocentrifuge (Shandon) and fixed again in PLP. S11 cells for use as positive controls were treated similarly except that the cells were assumed to be 95% viable. Probes were digoxigenin-labelled RNAs generated with an RNA-labelling kit (Boehringer Mannheim) and were detected by using a combination of alkaline phosphatase-conjugated anti-digoxigenin antibody and BCIP/NBT tablets (Sigma), followed by counter-staining with neutral red. Sense and anti-sense transcripts spanning MHV-68 tRNAs 14 were generated from pEH1.4 (Bowden et al., 1997
). The anti-sense probe for the overlapping thymidine kinase/glycoprotein H (tk/gH) mRNAs has been described previously (Stewart et al., 1998
). The M2 probe was generated from a DNA fragment containing the complete ORF that had been cloned into pKS (-) (Stratagene).
Gardella gel analysis.
Viable cells were recovered by centrifugation over Ficoll-Hypaque. S11 cells were harvested from exponentially growing cultures. Gardella gel electrophoresis was performed in a horizontal format as described previously (Decker et al., 1996 a ). Southern blots were hybridized with 32P-labelled probes directed against MHV-68 sequences. DNase I digestion was performed as described previously (Jamieson et al., 1995
).
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Results |
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As it appeared that the virus was not expressing lytic cycle- associated antigens in infected cultures, we investigated virus latent gene expression by RNA in situ hybridization. The first set of probes detected the transcription of the MHV-68-encoded, tRNA-like molecules. These unique tRNA molecules are transcribed from an RNA polymerase III promoter and are found in both lytically and latently infected cells (Bowden et al., 1997 ). The second probe was directed against the M2 gene, which is transcribed only in latently infected cells (Husain et al., 1999
). Both these probes show similar staining of the S11 tumour cell line (Husain et al., 1999
). The third probe was designed to detect the overlapping transcript that detects both the early thymidine kinase mRNA and the late gH message (tk/gH). Fig. 4
shows results of in situ hybridization carried out on purified B cells 3 days p.i. The tRNA anti- sense probe detected transcripts in 90% of cells in the infected B cells (Fig. 4a
). No viral transcripts were detected in these cells with the M2 or tk/gH probes (Fig. 4c
d
), although both these were positive on the S11 cell line (Fig. 4g
h
). No positive signal was obtained with the tRNA sense probe on the infected cells (Fig. 4b
) or with any probe on mock- infected cells (Fig. 4e
f
). The tk/gH probe confirmed the results obtained with the polyclonal anti-MHV- 68 antiserum, that no early or late virus gene expression was occurring in the infected cultures. Similar results were obtained from cultures infected for 2 or 4 days.
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To eliminate the possibility that the linear DNA was not indicative of a failure to circularize but rather a failure of virus capsids to uncoat, we examined the susceptibility of nuclear DNA to DNase I treatment. Encapsidated DNA is resistant to DNase I, whereas uncoated, productively replicating or episomal DNA should be susceptible. In this experiment, we examined four lymphocyte cultures. These included total murine splenocytes (infected or mock-infected), lymphocytes lacking the CD43 cell-surface marker (resting B cells) and CD43-positive cells (T lymphocytes, macrophages and activated B cells). Nuclei from these cells and from the S11 cell line were analysed by using Gardella gels before (untreated) or after treatment with DNase I (Fig. 5b). Only linear virus DNA was detected in all the untreated, in vitro-infected nuclei. Thus, the virus behaved the same in purified B cells as it did in total lymphocytes. In addition, the virus was able to infect the CD43-positive population readily, indicating that the virus was infecting cell types other than resting B cells. After treatment of nuclei with DNase I, linear genome was no longer present in the in vitro-infected cell cultures, whereas, in the S11 cell line, while episomal DNA was absent after DNase I treatment, linear DNA was still present and hence nuclease resistant. This shows that, in this experiment, naked DNA was readily digested whereas the encapsidated DNA present in the 12% of S11 cells that support productive virus replication was nuclease resistant. The resistance of encapsidated virus DNA to nuclease digestion in our assay was further confirmed by similar analysis after spiking mock- infected splenocytes with intact virus (results not shown). This experiment therefore confirms that MHV-68 was infecting cultured lymphocytes and that virus DNA was uncoated but that it failed to circularize to any significant extent.
Effect of culture conditions on growth-transforming potential of MHV-68
Although initial experiments showed that MHV-68 did not readily elicit growth transformation of murine lymphocytes, it remained possible that the culture conditions used in these experiments were not optimal. Primary murine lymphocyte cultures have a very limited half- life in the absence of growth factors and activation signals. In an attempt to elicit growth transformation, we designed culture conditions to prolong the viability of the cells and to provide growth signals that might normally be present in vivo. Modifications to the growth medium that were used included addition of murine IL-2, IL-4 or IL-10, addition of mitogens such as phytohaemagglutinin (PHA) and addition of conditioned medium from the tumour cell line S11 or from mitogen-stimulated lymphocytes. Infected lymphocytes cultures were plated onto irradiated, CD40L-expressing fibroblasts to provide viability signals or onto irradiated murine splenocytes. We also investigated the possible requirement for activation prior to infection by activating cells with PHA and infecting after the onset of cellular activation. The growth of infected cultures was prolonged under all of these conditions, but we were still unable to demonstrate growth transformation.
To investigate whether the potential of MHV-68 to initiate growth transformation was dependent on predisposing factors in the cell phenotype, we infected splenocytes from p53-/- and Rb-1+/- mice. Mice with either of these phenotypes have a predisposition to tumorigenesis, p53-/- because of defects in normal induction of apoptosis and Rb-1+/- because of defects in cell cycle control (Clarke, 1995 ). Use of these cells did not lead to the derivation of cell lines. We therefore surmised that, although MHV-68 initially affected the phenotype of infected lymphocytes in vitro, it was not capable of initiating growth transformation of these cells.
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Discussion |
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The prevailing model for herpesvirus infection proposes that the input viral DNA circularizes soon after infection (Roizman & Sears, 1996 ). This is supported by studies with a BHK cell temperature-sensitive mutant cell line, tsBN2, that undergoes premature condensation of chromatin at the non-permissive temperature. Failure to circularize in these cells shows that, at least in herpes simplex virus type 1, this process is dependent on a cellular gene product (Umene & Nishimoto, 1996
). In addition, evidence from latent infections of the gammaherpesviruses EBV, HVS and HHV-8 indicates that the viral DNA is maintained and replicated as an episome (Decker et al., 1996b
; Gardella et al., 1984
). Our studies indicate a failure of the MHV-68 genome to circularize to any great extent in splenocytes infected in vitro . While we cannot exclude the possibility that circularization occurs in a limited number of cells, below the limit of detection in our assay, it is clear that the majority of the viral DNA is present in a linear, uncoated form. MHV-68 DNA circularizes in vivo (Stewart et al., 1998
) and in myeloma cell lines such as the NS0 cell line infected in vitro (Sunil-Chandra et al. , 1993
). Our studies support the hypothesis that, during infection in vivo, specific activation of infected lymphocytes is required to induce synthesis of a cellular gene that allows circularization of the virus genome and allows infection to proceed. Thus, while the virus is capable of infecting and initiating limited transcription in all B cells in vitro, this infection is clearly abortive, since the expression of known lytic and latency- associated genes is absent. The rapid expansion of latently infected B cells seen in the spleen after MHV-68 infection is clearly dependent upon CD4+ T cell `help' (Ehtisham et al. , 1993
; Usherwood et al., 1996a
). It is therefore likely that, in vivo, infection of B cells only results in latent or productive infection after the delivery of specific activation signals to the B cell. Such a signal(s) may be delivered by MHV-68-specific T cells. An additional restraint may be that infection can only proceed in a specific B cell; for example, a B cell with a defined cell phenotype or, indeed, an MHV-68-specific B cell.
It is clear from the work described here that, under a wide range of conditions that might activate and support cellular proliferation, MHV- 68 does not readily transform growth of murine lymphocytes in vitro. We cannot, however, discount the association of MHV-68 with cellular transformation in vivo or rule out the possibility that, given the required conditions, MHV-68 is capable of growth transformation of murine lymphocytes in vitro. Circularization of the MHV-68 genome, which is a prerequisite for productive and persistent infections, was not detectable in virus-infected unstimulated lymphocyte cultures or in infected resting B cells, indicating that these conditions did not mimic in vivo events. While we have not examined the form of the genome under a wide range of conditions, the experiments suggest that it may be necessary to investigate culture conditions that support circularization of the virus genome before conclusions on the growth-transforming ability of MHV-68 can be drawn.
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Acknowledgments |
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References |
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Received 6 April 1999;
accepted 17 June 1999.