Download Capacity of Epstein–Barr virus to infect monocytes and inhibit their

Journal of General Virology (2004), 85, 2767–2778
DOI 10.1099/vir.0.80140-0
Capacity of Epstein–Barr virus to infect
monocytes and inhibit their development
into dendritic cells is affected by the cell
type supporting virus replication
Andre Ortlieb Guerreiro-Cacais,13 LiQi Li,13 Daria Donati,2
Maria Teresa Bejarano,1,2 Andrew Morgan,3 Maria G. Masucci,1
Lindsey Hutt-Fletcher4 and Victor Levitsky1
Correspondence
Victor Levitsky
[email protected]
1,2
Microbiology and Tumor Biology Center1 and Center for Infectious Medicine, Huddinnge
Hospital2, Karolinska Institutet, Nobels väg 16, S-17177 Stockholm, Sweden
3
Department of Pathology and Microbiology, School of Medical Sciences, University
of Bristol, UK
4
Department of Microbiology and Immunology, Louisiana State University, Health Science
Center, Shreveport, LA, USA
Received 28 March 2004
Accepted 24 June 2004
Epstein–Barr virus (EBV) is a ubiquitous human herpesvirus that is involved in the
pathogenesis of a wide spectrum of malignant and non-malignant diseases. Strong evidence
implicates T lymphocytes in the control of EBV replication and tumorigenesis, but cellular
components of the innate immune system are poorly characterized in terms of their function in
the development of EBV-specific immunity or interaction with the virus. This study demonstrates
that EBV virions produced in epithelial cells surpass their B cell-derived counterparts in the
capacity to enter monocytes and inhibit their development into dendritic cells (DCs). Different
ratios of the gp42 and gH glycoproteins in the envelope of virions that were derived from
major histocompatibility complex class II-positive or -negative cells accounted primarily for
the differences in EBV tropism. EBV is shown to enter both monocytes and DCs, although the
cells are susceptible to virus-induced apoptosis only if infected at early stages of DC differentiation.
The purified gH/gL heterodimer binds efficiently to monocytes and DCs, but not to B cells,
suggesting that high expression levels of a putative binding partner for gH contribute to virus
entry. This entry takes place despite very low or undetectable expression of CD21, the
canonical EBV receptor. These results indicate that the site of virus replication, either in B cells
or epithelial cells, alters EBV tropism for monocytes and DCs. This results in a change in the
virus’s immunomodulating capacity and may have important implications for the regulation of
virus–host interactions during primary and chronic EBV infection.
INTRODUCTION
Epstein–Barr virus (EBV), a ubiquitous human herpesvirus, causes infectious mononucleosis and is associated
with a number of malignancies of different cellular origins
(Rickinson & Kieff, 1996). EBV establishes latent infection predominantly, if not exclusively, in B lymphocytes
(Thorley-Lawson et al., 1996). B lymphocytes also represent
the only documented site of EBV replication in healthy
virus carriers (Tao et al., 1995). Whether EBV replicates in
oropharyngeal epithelial cells during primary infection
remains a matter of controversy (Sixbey et al., 1984;
3These authors contributed equally to this work.
0008-0140 G 2004 SGM
Niedobitek et al., 1997, 2000). It is clear, however, that EBV
can replicate in polarized epithelial cells in vitro and in
immunocompromised individuals with hairy leukoplakia,
a benign, hyperplastic, epithelial lesion that is composed of
EBV-infected cells producing large amounts of the virus
(Greenspan et al., 1985; Conant, 1987).
Specific T lymphocytes inhibit growth of EBV-transformed
B cells in vitro. In vivo, T cells suppress virus reactivation and EBV-induced tumorigenesis, as demonstrated by
the effects that they mediate upon adoptive transfer to
immunodeficient patients (Rooney et al., 1995; Heslop
et al., 1996; Heslop & Rooney, 1997; Gustafsson et al.,
2000). Little is known, however, about the mechanisms
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2767
A. O. Guerreiro-Cacais and others
that are responsible for the induction of EBV-specific
immunity and the capacity of the virus to interfere with this
process.
Antigen presentation by dendritic cells (DCs) is believed to
be required for primary T-cell stimulation. Consistent with
the critical function of DCs in anti-virus immunity, many
viruses are known to infect different subsets of DCs and
affect their differentiation, survival, migration and/or T cellstimulatory capacity (reviewed by Becker, 2003; Kobelt et al.,
2003; Schneider-Schaulies et al., 2003; Sevilla et al., 2003;
Steinman et al., 2003). DCs were recently shown to crosspresent virus antigens that are derived from EBV-infected
cells and to be necessary for inhibition of EBV-induced Bcell transformation in lymphocyte cultures that were
established from EBV-seronegative, but not -seropositive,
individuals (Subklewe et al., 2001; Bickham et al., 2003).
These findings support a critical role for DCs in the
induction of primary T-cell anti-virus responses. Whilst
other human herpesviruses, such as cytomegalovirus
(CMV) and herpes simplex virus, have been shown to
inhibit the T cell-stimulatory function of DCs by a number
of mechanisms, the capacity of EBV to interact with, and
affect the function of, DCs or their precursors is poorly
characterized. We have recently shown that EBV infection
inhibits DC development from monocyte precursors in the
presence of granulocyte macrophage colony stimulating
factor (GM-CSF) and interleukin-4 (IL4), in a manner that
is apparently independent of de novo expression of viral
genes (Li et al., 2002). It remains unclear, however, whether
the virus is capable of entering monocytes or developing
DCs and how the tropism of the virus for these cells is
regulated at the molecular level.
Cellular tropism of herpesviruses is determined by virusencoded proteins in the virion envelope that interact with
various cellular receptors (reviewed by Spear & Longnecker,
2003). Recent studies have shown that the canonical EBV
receptor, CD21, which interacts with the major glycoprotein of the EBV envelope, gp350, is neither sufficient
nor essential for B-cell infection and that fusion of the
EBV envelope with the cell membrane requires components of the trimolecular complex, which includes
glycoproteins gp85, gp42, and gp25 (Li et al., 1997; Wang
& Hutt-Fletcher, 1998; Haan et al., 2000). Abundance of
another EBV envelope glycoprotein, gB (also referred to as
gp110), has recently been shown to influence EBV tropism
for different cell types (Neuhierl et al., 2002). Whilst major
histocompatibiliy complex (MHC) class II molecules have
been identified as the cellular ligand of gp42, the interaction
partners of gp85 and gp25 are currently unknown. These
EBV proteins are homologous, respectively, to the gH and
gL components of the herpes simplex virus and CMV
membrane fusion complexes (Yaswen et al., 1993), are noncovalently associated in the virus envelope and are often
referred to as the gH/gL heterodimer. The importance of
the trimolecular assembly in EBV infection is demonstrated
by the inability of recombinant viruses that are devoid of
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gp42 or gH to infect B cells (Wang & Hutt-Fletcher, 1998;
Oda et al., 2000). However, the presence of gp42 in the
virus envelope impedes infection of epithelial cells that are
infected efficiently by gp42-deficient virions (Wang et al.,
1998). These data suggest that different modes of receptor–
ligand interactions are operational during EBV infection
of different cell types (Wang et al., 1998). Thus, the
bimolecular gH/gL complex mediates epithelial cell infection, whereas the trimolecular gp42/gH/gL complex is
required for infection of B cells. The content of the biversus trimolecular complexes in the EBV envelope and
the tropism of EBV virions can be affected by the type
of cell that is supporting virus replication. Expression of
human leukocyte antigen class II results in reduced levels
of gp42 in the virion, due to association of these two
proteins inside the cell and degradation of gp42 in the
endosomal/lysosomal compartment. This may play an
important role in the virus life cycle. Thus, EBV replication
in epithelial cells generates virions that infect B cells preferentially, thereby establishing the EBV-carrier state in
the infected individual, whereas EBV that is produced in B
cells may be more efficient at mediating virus spread, due
to more efficient infection of epithelial cells (Borza &
Hutt-Fletcher, 2002). The effect of EBV envelope composition on its interaction with monocytes or DCs has not
been analysed.
In this study, we demonstrate that, compared to B
lymphocyte-produced virions, EBV from epithelial cells
exhibits a significantly increased capacity to infect monocytes and inhibit their development into DCs. These
findings may have important implications for our understanding of the immunoregulation and pathogenesis of
EBV infection.
METHODS
Recombinant virus strains and virion isolation. All EBV virions
used in the study were derived from the EBV strain originating from
Akata, a Burkitt’s lymphoma cell line. Generation of viruses carrying
a cassette that encodes both neomycin resistance and green fluorescent protein (GFP), inserted into the open reading frame of the
non-essential tyrosine kinase gene, has been described previously
(Molesworth et al., 2000). EBV-negative Akata and gastric carcinoma AGS cells infected with recombinant viruses were grown in
medium that was supplemented with 500 mg geneticin sulphate
ml21 (G418) (Sigma Aldrich). AGS cells expressing the MHC class
II transactivator (AGS CIITA cells) were grown in the presence of
0?4 mg puromycin ml21 (Borza & Hutt-Fletcher, 2002). Recombinant
Akata virus (B-EBV) was produced by inducing the EBV lytic cycle
in Akata cells with anti-human IgG, as described previously (Wang
& Hutt-Fletcher, 1998). Virus lytic cycle in AGS (E-EBV) and AGS
CIITA (E-II-EBV) cells was induced by incubating the cells for 24 h
with 30 ng 12-O-tetradecanoylphorbol-13-acetate ml21 and 0?5 mM
sodium butyrate (Calbiochem). These chemicals were removed by
exchange of culture medium and virus-containing supernatants were
harvested 5 days later. Viruses were concentrated from filtered
supernatants (0?8 mM) by centrifugation at 16 000 g for 90 min at
4 uC and the pellets were resuspended in RPMI medium with 100 mg
bacitracin ml21 (Sigma). Mock preparations were produced from
EBV-negative AGS cells, following the same procedures.
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Journal of General Virology 85
Altering tropism of EBV for monocytes and DCs
Quantification of EBV virions. DNA slot-blot analysis and
measurement of cell-associated virus by fluorescence-activated cell
sorting (FACS) analysis or real-time PCR were used to equalize
virus stocks for virion content. Details of the virus-binding assay
and FACS analysis for both virus quantification and comparison of
envelope glycoproteins are described below. Slot-blot analysis was
performed as described previously (Wang & Hutt-Fletcher, 1998).
The quantity of EBV DNA in virion particles was measured by
hybridization with a 32P-labelled BamHI W fragment of EBV DNA
by means of a random-primed DNA labelling kit (Boehringer
Mannheim). Samples (20 ml of a 1006 concentrated virus stock)
were digested for 10 min at room temperature with DNase I. 0?5 M
EDTA (10 ml) was added to stop digestion and virus particles were
sedimented for 1 h at 16 000 g. Sedimented virus was digested overnight at 56 uC with proteinase K (2?5 mg ml21 in 0?2 M EDTA) and
serial dilutions were made in PBS. Sample DNA was denatured by
the addition of 0?1 vol. 5 N NaOH, neutralized with 2 M ammonium acetate, applied to a nylon membrane and cross-linked by UV
irradiation. Hybridizations were carried out as described previously
(Mackett et al., 1990) and quantified by scanning with a Storm
PhosphorImager (Molecular Dynamics).
EBV-negative Akata cells were incubated with different dilutions of
virus stocks for 1 h at 4 uC to determine the number of virions that
were capable of binding to CD21-positive cells. Cells were washed three
times in cold PBS and DNA was extracted with a QIAamp DNA blood
mini kit (Qiagen), eluted in sterile water and stored at 220 uC. The
TaqMan PCR reagent system and ABI PRISM 7700 sequence detection system (Applied Biosystems) were used to measure EBV DNA
(Heid et al., 1996). The EBV target gene chosen was latent membrane
protein (LMP)1 and the amplification primer sequences were: forward
primer, 59-AAGGTCAAAGAACAAGGCCAAG-39; reverse primer, 59GCATCGGAGTCGGTGGG-39; probe, 59-AGGAGCGTGTCCCCGTGGAGG-39. The probe was labelled with 6-carboxyfluorescein (FAM;
reporter) and 6-carboxytetramethylrhodamine (TAMRA; quencher).
A standard curve was created to calculate the number of EBV genomes
(3) by using serial dilutions of DNA extracted from the EBV-positive
human cell line Namalwa, which carries two integrated EBV copies
per cellular genome (Klein et al., 1972). The standard and the DNA
samples were run in triplicate. Amplification mixtures (25 ml) contained 16 TaqMan Universal PCR master mix (Applied Biosystems)
and 900 nM of each EBV primer, 200 nM EBV probe, water and 5 ml
sample DNA. Cycling parameters were as follows: 50 uC for 2 min,
95 uC for 10 min and 45 cycles of 95 uC for 15 s and 60 uC for 1 min.
Preparation of monocytes and DCs and EBV infection.
Peripheral blood mononuclear cells (PBMCs) were isolated from
the blood of healthy donors by centrifugation on Ficoll density
gradients. Monocytes were purified from PBMCs by centrifugation
on Percoll density gradients, as described by Kouwenhoven et al.
(2001). Alternatively, CD14+ monocytes were purified by using a
monocyte-negative isolation kit (Dynal Biotech) or MACS CD14
MicroBeads (Miltenyi Biotec) according to the manufacturers’
instructions. The negatively selected population contained >98 %
CD14+ cells, as determined by staining and FACS analysis.
Monocytes were resuspended in culture medium (RPMI 1640 medium
supplemented with 10 % heat-inactivated fetal calf serum, 2 mM
21
L-glutamine, 100 U penicillin ml
and 100 mg streptomycin ml21) at
a density of 16106 cells ml21 and cultured for the indicated time
in medium supplemented with 1000 U recombinant GM-CSF ml21
and 1000 U recombinant IL4 ml21 (a kind gift from the ScheringPlough Research Institute, New Jersey, USA). Changes in cell morphology were monitored daily by visual inspection using an inverted
microscope.
EBV infection was performed by resuspending 16106 monocytes in
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culture medium containing 1 arbitrary unit (AU) (see below) of the
indicated EBV preparation. Cells were incubated for 2 h at 37 uC,
washed and cultured in medium that contained IL4 and GM-CSF to
induce DC development.
EBV-specific antibodies and binding assays. The mAbs F-2-1
(Strnad et al., 1982), which is specific for the EBV envelope glycoprotein gp42 (Li et al., 1995), E1D1 (Oba & Hutt-Fletcher, 1988)
[reacting with the EBV gH/gL complex (Li et al., 1995)], 72A1
(Hoffman et al., 1980), specific for the EBV gp350/220 glycoprotein,
and CL59 (Molesworth et al., 2000), specific for gH, were purified
from culture supernatants of the corresponding hybridoma cell
lines. The mAb 5B2, which is specific for gB, was purchased from
Virusys. FACS analysis of virus bound to the surface of Burkitt’s
lymphoma Raji cells, which express high levels of the CD21 molecule, was used to measure virus in different preparations and analyse
the content of different glycoproteins in the EBV envelope. To compare virus stocks, Raji cells were fixed in 0?1 % paraformaldehyde,
resuspended in 100 ml RPMI medium that contained the indicated
EBV preparation and incubated for 1 h. Cells were washed in PBS
and incubated in 50 ml 2L10 gp350-specific mAb or isotype control
antibody diluted in PBS. Antibody binding was revealed by allophycocyanin (APC)-conjugated goat anti-mouse antibody (BD Biosciences) or R-phycoerythrin (R-PE)-conjugated rabbit anti-mouse
antibody (Dako). All procedures were carried out on ice. Cells that
had not been exposed to EBV were used as a control. The amount
of virus that generated a mean fluorescence intensity (MFI) value
with gp350-specific antibody in the binding assay that was comparable to that obtained with 1 ml B95.8 supernatant was expressed as
1 AU of EBV virions. B95.8 virus (0?5 AU) was used in binding
assays that were performed for equalization of virus stocks. To
analyse the protein composition of the envelope, EBV was reduced
to 0?25 AU, virus glycoprotein-specific antibodies were used at
10 mg per sample (2?5 mg per sample for mAb 5B2) and the biotinylated anti-mouse antibody and PE- or APC-conjugated streptavidin
were used at a 1 : 100 dilution (all from BD Biosciences).
Detection of apoptosis and GFP expression. Efficiency of
Annexin V binding to the surface of EBV-infected and control cells
was measured by using an Annexin V–PE apoptosis detection kit I
(Pharmingen). GFP expression in cells infected by the recombinant
EBV strains was monitored either by measuring the intensity of green
fluorescence in infected cells using FACS analysis or by immunoblotting of total cell lysates using a mixture of two GFP-specific
mAbs, 7.1 and 13.1 (Roche).
gH/gL binding assay. gH was co-expressed with gL in SF9
insect cells and precipitated as a gH/gL heterodimer. SF9 cells
were infected at an m.o.i. of 3 with baculovirus expressing gH/gL
(Pulford et al., 1995). Five days later, the culture medium was
clarified by low-speed centrifugation to remove cells and then
centrifuged at 16 000 g for 90 min to remove virus. PEG 3500 was
added to a final concentration of 20 % (w/v), the solution was
stirred for 1 h at 40 uC and then centrifuged at 14 000 g for 20 min.
The pellet was resuspended in 0?1 vol. PBS and the precipitated
gH/gL was dialysed against PBS in dialysis tubing with a molecular
mass cut-off of 25 000 Da, as described previously (Pulford et al.,
1995). The indicated cells were resuspended at 36105 cells in 200 ml
PBS containing 10 % serum from an EBV-seronegative individual
and were incubated on ice for 1 h in the presence of 50 mg purified
gH/gL. Where indicated, 100 mg E1D1 mAb or isotype control antibody were added to the reaction. Cells were then washed in PBS and
gH/gL binding was revealed by CL59 mAb staining, followed by
incubation with anti-mouse IgG fluorescein isothiocyanate (FITC)labelled secondary antibody. Cells were washed and analysed by
FACS.
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A. O. Guerreiro-Cacais and others
RESULTS
EBV virions produced in different cell types
differ in their capacity to induce apoptosis in
developing DCs
The same recombinant EBV strain, which was generated
by using virus that was originally derived from the EBVpositive Burkitt’s lymphoma cell line Akata, has been
produced in either Akata cells, gastric carcinoma AGS cells
or AGS CIITA cells that express MHC class II molecules
constitutively. These virus preparations, referred hereafter
to as B-EBV (for B cells), E-EBV (for epithelial cells) or
E-II-EBV, respectively, were normalized according to the
intensity of gp350-specific staining in a virus-binding assay
using CD21+ Raji cells. The number of virions in each virus
stock was expressed in AU, as described in Methods. The
amount of gp350 has previously been shown to correlate
with viral DNA content (Neuhierl et al., 2002). In confirmation, we detected comparable amounts of viral DNA
in the normalized virion preparations by using DNA slotblot analysis (data not shown).
Purified monocytes were infected for 2 h (1 AU in 16106
monocytes) with E-EBV, E-II-EBV or B-EBV and cultured
in the presence of IL4 and GM-CSF to stimulate their
differentiation into DCs. To assess the pro-apoptotic
activity of different EBV preparations, cells were stained
with Annexin V and analysed by flow cytometry. After
infection with E-EBV, the proportion of apoptotic cells in
developing DC cultures reached 50 % on day 3 and >70 %
on day 5 post-infection. In contrast, only 9?9 and 6?4 % of
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We set out to analyse the expression of virus-encoded
proteins in the envelope of EBV virions, in order to uncover
why there were differences in the pro-apoptotic activity of
EBV preparations. We could not exclude, however, that
the presence of gp350-containing membrane-like structures
that were devoid of viral genomes, non-enveloped capsids
in concentrated virus preparations or variability in the
number of repeats present in the BamHI W genome fragment affected our evaluation of virion content in EBV
stocks. To circumvent these potential problems, we used
an EBV-specific real-time PCR to compare the numbers
of EBV genomes that were associated with EBV-negative
Akata cells after preincubation with different virion preparations that had been equalized for gp350 content. This
analysis revealed that the number of virions that were able
to bind to CD21 was similar in E-EBV and E-II-EBV
preparations, whereas B-EBV stocks contained approximately 2?5-fold more virus genomes, suggesting that these
virions have a relatively decreased amount of gp350
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Composition of virion envelope in different EBV
preparations
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cells were apoptotic in control cultures at the same time
points. Infection with B-EBV only slightly increased the
proportion of Annexin V-positive cells, whilst E-II-EBV
exhibited an intermediate activity, as compared with BEBV and E-EBV (Fig. 1). Similar relative pro-apoptotic
activities of different EBV virions were revealed by monitoring the recovery of viable cells in DC cultures at day 6 or
7 post-infection (Table 1).
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Fig. 1. EBV virions produced in different
cell lines differ in their capacity to induce
apoptosis in developing DCs. Viruses from
supernatants of AGS (E-EBV), AGS-CIITA
(E-II-EBV) and Akata (B-EBV) cells were
concentrated by ultracentrifugation and numbers of virions in the resulting preparations
were equalized by using a binding assay to
CD21+ cells and slot-blot analysis. Purified
monocytes were infected with 1 AU of the
indicated virions, cultured in the presence of
GM-CSF and IL4 and tested for their capacity to bind PE-conjugated Annexin V on
day 3 or 5 post-infection. Control cells were
mock-infected with preparations obtained
from EBV-negative AGS cells as described
in Methods. The results shown are one
representative of five experiments performed
with cells isolated from different donors.
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Journal of General Virology 85
Altering tropism of EBV for monocytes and DCs
Table 1. Recovery of DCs in cultures of monocytes infected with EBV virions produced in different cell lines
Purified monocytes were infected with the indicated preparations of EBV (the cell lines used to produce the relevant virus are indicated in
parentheses) and cultured in the presence of GM-CSF and IL4. Recovery of viable cells was monitored at day 6 or 7 of culture. NT, Not
tested.
EBV virion type
B-EBV (Akata)
E-EBV (AGS)
E-II-EBV (AGSCIITA)
DC recovery (%) in experiment no.*:
1
2
3
4
5
6
Mean±SD
NT
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34?8
46?4
100
35?5
63?2
89?3
10?7
38?1
50?3
14?4
31
88?5
26?2
56?8
81±19
22?6±11
43?2±15?3
14?3
22?5
*Results of six independent experiments are shown as a percentage of viable cells relative to the number of cells in cultures of mock-infected
monocytes.
(Fig. 2a). In fact, B-EBV, E-EBV and E-II-EBV exhibited
similar gB contents after normalization according to the
number of genomes, as measured by real-time PCR. Some
of the stocks of B-EBV contained up to twofold higher
levels of gH, as compared to E-EBV or E-II-EBV (Fig. 2b),
but this difference was not observed with every virus preparation (Fig. 2d and data not shown). In contrast, the
level of gp350-specific signal was decreased consistently
in B-EBV virions by two- to threefold. Normalization of
virus stocks by using gp350 signal intensity resulted in a
corresponding increase of fluorescence intensity obtained
with gH or gB specific antibody in a binding assay with
B-EBV (Fig. 2b). Analysis of independent experiments
demonstrated that the ratio between the intensities of gHand gp350-specific signals was, on average, 2?5-fold higher
in B-EBV than in E-EBV or E-II-EBV (Fig. 2d). In addition,
E-EBV and E-II-EBV contained comparable amounts of
gp350 and gH, whereas the level of gp42 was significantly
decreased in E-II-EBV, resulting in an approximately
twofold increase in the gH : gp42 ratio. B-EBV expressed a
barely detectable level of gp42 that was reflected in a fivefold increase in the gH : gp42 ratio (Fig. 2c, e). Collectively,
these data demonstrated that the three types of EBV
virions contained comparable amounts of gH and gB in the
virion envelope, the content of gp350 was approximately
2?5-fold lower in B cell-derived virions than in E-EBV- and
E-II-EBV-derived virions and the content of gp42 was
decreased to a different extent in B-EBV and E-II-EBV, as
compared to E-EBV.
Decreased gp42 content and decreased
pro-apoptotic activity of EBV virions correlates
with their impaired capacity to enter monocytes
To test to what extent the lower inhibitory activity of B-EBV
and E-II-EBV can be compensated for by increasing the
m.o.i., monocytes were infected with E-EBV (1 AU) or with
the indicated increasing amounts of the other two virus
preparations. Induction of apoptosis in developing DCs
was monitored by Annexin V staining on day 4 postinfection. In this and all subsequent experiments, numbers
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of virions were normalized by using the gp350 staining
intensity in the virus-binding assay, which underestimated
the number of B-EBV genomes by approximately 2?5-fold.
We have chosen to use gp350-based normalization as
purified gp350 was shown to mediate a number of biological
and biochemical changes in human monocytes (D’Addario
et al., 1999). Infection of monocytes with 1 AU B-EBV did
not induce any significant increase in the proportion of
Annexin V-positive cells in DC cultures. The proportion of
apoptotic cells increased gradually with increasing amounts
of B-EBV used for infection, but even cultures that were
infected with 9 AU B-EBV (approx. 20–25-fold more EBV
genomes than in 1 AU E-EBV) contained relatively fewer
apoptotic cells than cultures that were infected with 1 AU EEBV (Fig. 3a). Infection of monocytes with 1 AU E-II-EBV
resulted in only a slight increase in the percentage of apoptotic cells (1?4-fold), whereas the numbers increased by
1?7- and 2?2-fold after infection with 2 or 3 AU E-II-EBV,
respectively. The latter was comparable with the proapoptotic activity of 1 AU E-EBV (Fig. 3b). A similar
pattern of relative pro-apoptotic activities in the three virus
preparations was observed by monitoring the recovery of
viable cells in infected DC cultures (data not shown).
The virus used in this study contains a cassette that confers
both neomycin resistance and GFP expression; the latter
can be used for monitoring virus entry. Total cell lysates
of monocytes that were infected with 1 AU of the indicated
viruses were tested by immunoblotting with GFP-specific
antibodies to compare the efficiency of virus entry 48 h
after infection. At this time point, the highest intensity of
GFP fluorescence was detected in E-EBV-infected monocytes and Raji cells by using time kinetics experiments
(data not shown). GFP-specific band intensity was significantly stronger in E-EBV-infected than in E-II-EBVinfected monocytes, whereas the level of GFP expression
was below the detection limit in B-EBV-infected cells
(Fig. 3c). Expression of GFP in monocytes that were
infected with B-EBV increased with the increaing virus
dose that was used for infection. However, the level of GFP
expression in monocytes that were infected with 9 AU
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B-EBV was still significantly lower than that observed in
cells infected with 1 AU E-EBV (Fig. 3d). Next, we used
FACS analysis to determine the proportion of monocytes
that were susceptible to EBV entry. In different preparations of monocytes, a large proportion (>60 %) of cells
that were infected with 1 AU E-EBV became positive for
green fluorescence, as assessed by FACS analysis. A lower
proportion of positive cells with a lower MFI was detected
in E-II-EBV-infected cells, whereas monocytes that were
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infected with B-EBV at the same m.o.i. were negative or
only marginally positive for GFP expression (Fig. 3e,f).
Immature DCs remain permissive for EBV entry
In our previous study, we showed that immature DCs
become resistant to the pro-apoptotic effect of the virus at
day 5–6 of their development in culture in vitro (Li et al.,
2002). To analyse whether the capacity of EBV virions
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Altering tropism of EBV for monocytes and DCs
Fig. 2. Protein composition of the envelope differs in EBV virions isolated from different cell lines. Raji cells were preincubated with each of the EBV virions indicated. The gp350, gH and gp42 contents in the virus envelope were estimated by
using the relevant specific mAbs. Binding of antibodies to cell surface-bound EBV was revealed by sequential incubation with
biotinylated anti-mouse antibody and APC-conjugated streptavidin. (a) The left panel shows histogram plots obtained by
FACS analysis of Raji cells pre-incubated with the indicated preparations of virions and detected by gp350-specific antibody.
MFIs are indicated and the numbers in parentheses are the same values expressed as percentages relative to the MFI
obtained with E-EBV. The same virus stocks were diluted, as indicated, in the graph on the right panel and pre-incubated with
EBV-negative Akata cells for 1 h at 4 6C. DNA was extracted from cells after washing away unbound virus and the number of
cell-associated virus genomes was evaluated by EBV-specific real-time PCR. (b) gp350, gH and gB contents were analysed
for E-EBV (X), E-II-EBV (&) and B-EBV (m), using the relevant specific antibodies in the EBV-binding assay. B-EBV was
used at 0?5 and 0?2 AU, respectively corresponding to the virion normalization based on either gp350 content or genome
copy numbers. (c) The histogram plots show comparative analysis of the gp350 (dotted line), gH (thin line) and gp42 (thick
line) content in the indicated virion preparations. (d) Ratios between MFIs obtained in the virus-binding assay with the gH- or
gp350-specific antibody and calculated for each of the indicated virion preparations are shown as the mean±SD of four
independent experiments. (e) Ratio of MFIs obtained with the gH- or gp42-specific antibodies was calculated and presented
as described for (d).
to inhibit DC development correlates with the capacity
of EBV virions to enter cells, we infected monocytes or
developing DCs with B-EBV, E-EBV, or E-II-EBV at days
0, 3 or 6 of culture in vitro in the presence of GM-CSF and
IL4. Efficiency of EBV entry was assessed by monitoring
green fluorescence intensity in monocytes 48 h after infection. Infection with 1 AU B-EBV resulted in only a slight
increase in the MFI of infected cells, compared to controls
120
Counts
100
80
60
40
20
0
http://vir.sgmjournals.org
(Fig. 4). In contrast, the majority of cells that were infected
with either E-EBV or E-II-EBV became positive for green
fluorescence. Infection with E-EBV was more efficient than
with E-II-EBV, as judged by GFP signal intensity in infected
cells (Figs 3 and 4), which was consistent with the differences in their capacity to inhibit DC development. This
pattern was observed in cells that were infected at different
time points.
Fig. 3. Comparative analysis of EBV virions
for their pro-apoptotic activity in developing
DCs and capacity to enter monocytes.
Freshly isolated monocytes were infected
with the indicated amounts of E-EBV, E-IIEBV or B-EBV and cultured in the presence
of GM-CSF and IL4. Pro-apoptotic activity
of EBV virions was assessed by Annexin V
staining on day 5 of culture (a, b) and their
capacity to enter monocytes was evaluated
by monitoring GFP expression, either in total
cell lysates by immunoblotting with GFPspecific antibodies (c, d) or by direct measurement of GFP-dependent fluorescence
(e, f). In (c), B95.8 virus was used as an
additional negative control. The histograms
in (e) show green fluorescence of monocytes non-infected or infected with 1 AU of
the indicated viruses. (f) Percentages of
cells falling into the M1 region and the numbers above the graph represent MFIs of
non-gated populations shown in (e). Each
panel shows the results of one representative of two or three experiments.
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2773
60
50
40
30
20
10
0
(a)
500
400
Day 0
0
10
101
102
FL1-H
103
Counts
Counts
A. O. Guerreiro-Cacais and others
104
101
Day 3
103
104
103
104
103
104
(b)
20
10
500
100
101
102
FL1-H
103
400
104
gH+E1D1
300
gH
200
100
40
30
0
100
Day 6
20
101
10
0
102
FL1-H
Counts
Counts
0
0
10
50
40
30
60
50
Counts
200
100
60
0
gH+E1D1
gH
300
100
101
102
FL1-H
103
102
FL1-H
(c)
104
500
Fig. 4. Immature monocyte-derived DCs remain permissive for
EBV entry. E-EBV (thick line), E-II-EBV (grey line) or B-EBV
(thin line) were used to infect monocytes at the indicated days
of culture in the presence of GM-CSF and IL4. GFP expression
levels were monitored in mock-infected (control; grey shading)
or EBV-infected cells by FACS analysis. Results are one representative of three experiments.
Developing DCs express a binding partner for
soluble gH/gL
Experiments with gp42- and gH-deficient EBV strains
demonstrated that these two components of the virus
fusion complex are required for EBV-induced apoptosis
of monocyte-derived DCs (Li et al., 2002). Whilst MHC
class II molecules (which are expressed abundantly on
monocytes) have been characterized as the binding
partner of gp42, the identity and cellular distribution
of structures that interact with gH are unknown. To test
whether an interacting partner of gH is expressed on
developing DCs, AGS cells, Akata cells and monocytes
cultured overnight in GM-CSF and IL4 were incubated with
soluble gH/gL. Protein binding to the cell surface was
revealed by the gH-specific CL59 mAb and the specificity
of the binding was confirmed by the ability of E1D1 gHspecific blocking mAb, but not control mAb, to reduce the
2774
Counts
400
gH+E1D1
gH
300
200
100
0
100
101
102
FL1-H
Fig. 5. Soluble gH/gL binds efficiently to AGS cells and developing DCs. EBV-negative Akata (a) and AGS (b) cells and
purified monocytes cultured in the presence of GM-CSF and
IL4 (developing DCs) (c) were incubated with purified soluble
gH/gL for 30 min on ice (0?256106 cells in 50 ml, containing
5 mg gH/gL) in the absence (solid dark line) or the presence
(solid grey line) of E1D1 antibody (50 mg). Binding of gH/gL
was revealed by incubation with CL59 antibody followed by
incubation with anti-mouse FITC-conjugated antibody. Filled
and dotted histograms represent control samples cultured without gH/gL or with gH/gL-specific antibody alone, respectively.
One representative of three experiments is shown.
MFI of gH/gL-stained cells. As shown in Fig. 5, both AGS
cells and developing DCs bound soluble gH/gL with comparable efficiency, whereas binding of the gH/gL protein to
Akata cells was barely detectable.
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Altering tropism of EBV for monocytes and DCs
DISCUSSION
The results of this study demonstrate that the capacity of
EBV to inhibit development of monocytes into DCs in the
presence of GM-CSF and IL4 is influenced strongly by the
cell type supporting virus replication. Virions that were
produced in the Burkitt’s lymphoma cell line Akata (B-EBV)
exhibited significantly reduced pro-apoptotic activity on
developing DCs, as compared to EBV virions that were
derived from a cell line of epithelial origin (E-EBV). In
addition, our data show that E-EBV efficiently enters the
majority of monocytes and developing DCs, as demonstrated by the expression of the GFP gene, which was
inserted into the virus genome. These results seem to contradict those of our previous publication, which suggested
that the inhibitory activity of the virus is independent of
viral gene expression (Li et al., 2002). However, it should be
taken into account that GFP expression in EBV-infected
monocytes is driven by a heterologous CMV promoter and
EBV promoters may remain silent in monocytes/DCs. In
agreement with our previous data, short-UV inactivation of
B-EBV or E-EBV abrogated induction of EBV nuclear
antigens in B cells following virus infection, but did not
affect the capacity of the virions to inhibit DC development (data not shown). Although we cannot exclude the
possibility that not all open reading frames of the EBV
genome were inactivated by UV treatment, in our previous
study we failed to detect viral gene expression in infected
monocytes or DCs by using RT-PCR analyses (Li et al.,
2002). Collectively, our results suggest that the effect of the
virus is mediated by virion components during virus entry
or post-entry events. In agreement with this hypothesis, the
inhibitory activity of EBV virions correlated directly with
the m.o.i. Therefore, accurate determination of virion
content in different virus preparations was critical for
estimating their relative activities. A good correlation was
observed between the measurements of virion content,
based either on the levels of gp350 in cell surface-associated
virus or the amount of viral DNA in concentrated virus
stocks, as assessed by DNA slot-blot analysis and according to previously published data (Neuhierl et al., 2002).
However, some dissociation between these parameters was
revealed when the number of CD21-bound genomes was
estimated by EBV-specific real-time PCR. After adjustment
for the intensity of gp350-specific staining, preparations
of B-EBV were shown to contain two- to threefold higher
numbers of EBV genomes than preparations of E-EBV or
E-II-EBV. Further analysis of protein composition of the
virus envelope demonstrated that all EBV preparations
had comparable amounts of gH and gB, whereas gp350
content was two- to threefold lower in B cell-derived virions
(Fig. 2). The reasons for this decrease remain unclear, but
it is conceivable that gp350 may be partially retained in B
cells, due to an interaction with CD21, as has been described
for the interaction of gp42 with MHC class II molecules
(Borza & Hutt-Fletcher, 2002). Nevertheless, it is unlikely
that decreased amounts of gp350 contribute to the differences in virus tropism and activity described in this study.
http://vir.sgmjournals.org
Firstly, we compared the biological activity and entry
efficiency of different virions by using normalization of
virus stocks based on gp350 content, which did not compensate for the lower activity of B-EBV. We failed to detect
expression of CD21 on monocytes (Li et al., 2000) and to
block virus entry or its pro-apoptotic activity by using a
gp350-specific blocking antibody (Li et al., 2002). Finally,
virions produced from AGS cells that constitutively express
MHC class II molecules, E-II-EBV, had the same gp350
content, but were less efficient at entering monocytes and
inhibiting DC development than E-EBV. The only molecular change detected that could explain the difference
between E-EBV and E-II-EBV was the decreased gp42
content in the latter type of virions. Although E-II-EBV
contained significantly higher amounts of gp42 in the
virion envelope than B-EBV, its pro-apoptotic activity and
tropism for monocytes were significantly lower than those
of E-EBV. Notably, virions that were derived from the
B95.8 EBV-producing cell line resemble E-EBV virions in
their trimolecular complex composition and inhibitory
activity on DC development [data not shown and Li et al.,
(2002)], although the reason for high gp42 content in B95.8
virions remains unclear, as the virus is produced from
monkey B cells. These results indicate that the trimolecular
fusion complex, which is composed of gp42, gH and gL,
plays a critical role in mediating EBV entry into monocytes
and in the pro-apoptotic effects of the virus on DC
development. The data suggest that, as in B-cell infection,
the trimolecular gp42/gH/gL fusion complex mediates
EBV infection of monocytes/immature DCs by involving
the interaction of gp42 with MHC class II molecules.
High expression levels of a putative binding partner for
the gH/gL heterodimer, revealed by binding assays with
purified proteins (Fig. 5), are likely to contribute to the
efficient entry of E-EBV into monocytes.
Previous studies have demonstrated that EBV infection of
monocytes results in a number of biological effects (Gosselin
et al., 1991, 1992; Roberge et al., 1997; Shimakage et al.,
1999; Savard et al., 2000a, b; Masy et al., 2002; Tardif et al.,
2002) that have been attributed either to virus entryindependent effects or infection of a small proportion of
cells. In this respect, our finding that E-EBV can infect
virtually all cells in monocyte cultures is quite remarkable.
The relatively high capacity of EBV from epithelial cells to
inhibit DC development from monocyte precursors efficiently may play an important role in the regulation of virus–
host interactions. EBV spreads in the human population
through the oral route of infection and cells of the oral or
nasopharyngeal epithelium represent its most likely entry
site. This hypothesis is supported by a recent demonstration of EBV replication in polarized in vitro cultures of
non-transformed human epithelial cells (Tugizov et al.,
2003). In vivo, replication of the virus in epithelial cells
is observed in hairy leukoplakia [reviewed by Rickinson &
Kieff (1996)], as well as in histologically normal epithelium
of severely immunocompromised patients (Herrmann
et al., 2002), and is likely to occur during primary EBV
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2775
A. O. Guerreiro-Cacais and others
infection in individuals lacking virus-specific immunity.
One arbitrary unit of EBV used in our experiments
corresponded to 20 virions per cell on average, an m.o.i.
that seems to be easily achievable at a site of EBV replication in vivo. Although strong humoral and cellular immune
responses are eventually elicited against the virus, EBV
infection is asymptomatic in the majority of infected
individuals. Even in rare cases of infectious mononucleosis,
a lag period of several weeks is frequently observed between
the infection episode and appearance of clinical symptoms
of the disease, which may be caused by the pathological
side effects of extensive anti-virus immunity (Peter & Ray,
1998). DCs are critical for efficient induction of primary
immune responses and inhibition of their function is likely
to delay the development of both cellular and humoral
specific immunity in EBV-infected individuals. In agreement with this model, sites of hairy leukoplakia lack
morphological and histological signs of inflammation and
have a decreased content of Langerhans cells (Daniels et al.,
1987; Walling et al., 2004); this may be explained by the
ability of EBV virions, which are produced abundantly in
leukoplakia lesions, to exert an inhibitory effect on DCs.
In contrast, reactivation of the virus in latently infected B
cells of immune, chronic virus carriers should result in the
production of EBV virions with a significantly decreased
capacity to infect and modulate the function of monocytes
and DCs, which would be more compatible with asymptomatic and non-pathogenic EBV persistence.
DCs that develop from monocyte precursors in the presence of IL4 and GM-CSF gradually lose their sensitivity
to the pro-apoptotic effects of EBV infection. Whether this
reflects decreased infectivity of immature DCs by EBV, their
decreased general sensitivity to apoptosis or changes in
the regulation of signalling pathways mediated by EBV
infection was unclear. Here, we show that immature
DCs that become resistant to EBV-induced apoptosis still
support virus entry, as demonstrated by GFP expression
conferred by infection with recombinant EBV strains
(Fig. 4). Therefore, resistance to EBV-induced apoptosis
can be accounted for by differentiation-associated changes
in developing DCs. However, the ability of EBV to infect
immature DCs raises the possibility that the virus can
influence phenotypic and functional characteristics of these
cells without affecting their lifespan.
affected in a given disease. Our results suggest that EBV
replication can be involved in disease pathogenesis
indirectly through effects that are mediated by EBV virions
independently of viral gene expression. In addition, the cell
type supporting EBV replication at, or in close proximity
to, the site affected by the disease may significantly influence
the activity and type of effects that are induced by EBV
virions.
ACKNOWLEDGEMENTS
This work was supported by grants awarded by the Swedish Cancer
Society, the Swedish Paediatric Cancer Foundation, the Swedish
Foundation of Strategic Research, the Petrus and Augusta Hedlund
Foundation and the Karolinska Institutet, Stockholm, Sweden. The
LHF laboratory was supported by NIH grant no. AI20662 from the
Institute of Allergy and Infectious Diseases.
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