Early Events Associated with Infection of Epstein-Barr Virus Infection of Primary B-Cells

Abstract

Epstein Barr virus (EBV) is closely associated with the development of a vast number of human cancers. To develop a system for monitoring early cellular and viral events associated with EBV infection a self-recombining BAC containing 172-kb of the Epstein Barr virus genome BAC-EBV designated as MD1 BAC (Chen et al., 2005, J.Virology) was used to introduce an expression cassette of green fluorescent protein (GFP) by homologous recombination, and the resultant BAC clone, BAC-GFP-EBV was transfected into the HEK 293T epithelial cell line. The resulting recombinant GFP EBV was induced to produce progeny virus by chemical inducer from the stable HEK 293T BAC GFP EBV cell line and the virus was used to immortalize human primary B-cell as monitored by green fluorescence and outgrowth of the primary B cells. The infection, B-cell activation and cell proliferation due to GFP EBV was monitored by the expression of the B-cell surface antigens CD5, CD10, CD19, CD23, CD39, CD40 , CD44 and the intercellular proliferation marker Ki-67 using Flow cytometry. The results show a dramatic increase in Ki-67 which continues to increase by 6-7 days post-infection. Likewise, CD40 signals showed a gradual increase, whereas CD23 signals were increased by 6-12 hours, maximally by 3 days and then decreased. Monitoring the viral gene expression pattern showed an early burst of lytic gene expression. This up-regulation of lytic gene expression prior to latent genes during early infection strongly suggests that EBV infects primary B-cell with an initial burst of lytic gene expression and the resulting progeny virus is competent for infecting new primary B-cells. This process may be critical for establishment of latency prior to cellular transformation. The newly infected primary B-cells can be further analyzed for investigating B cell activation due to EBV infection.

Full text

Early Events Associated with Infection of Epstein-Barr
Virus Infection of Primary B-Cells
Sabyasachi Halder, Masanao Murakami, Subhash C. Verma, Pankaj Kumar, Fuming Yi, Erle S. Robertson*
Department of Microbiology and Abramson Comprehensive Cancer Center, Tumor virology Program, University of Pennsylvania School of Medicine, Philadelphia,
Pennsylvania, United States of America
Abstract
Epstein Barr virus (EBV) is closely associated with the development of a vast number of human cancers. To develop a system
for monitoring early cellular and viral events associated with EBV infection a self-recombining BAC containing 172-kb of the
Epstein Barr virus genome BAC-EBV designated as MD1 BAC (Chen et al., 2005, J.Virology) was used to introduce an
expression cassette of green fluorescent protein (GFP) by homologous recombination, and the resultant BAC clone, BAC-
GFP-EBV was transfected into the HEK 293T epithelial cell line. The resulting recombinant GFP EBV was induced to produce
progeny virus by chemical inducer from the stable HEK 293T BAC GFP EBV cell line and the virus was used to immortalize
human primary B-cell as monitored by green fluorescence and outgrowth of the primary B cells. The infection, B-cell
activation and cell proliferation due to GFP EBV was monitored by the expression of the B-cell surface antigens CD5, CD10,
CD19, CD23, CD39, CD40 , CD44 and the intercellular proliferation marker Ki-67 using Flow cytometry. The results show a
dramatic increase in Ki-67 which continues to increase by 6–7 days post-infection. Likewise, CD40 signals showed a gradual
increase, whereas CD23 signals were increased by 6–12 hours, maximally by 3 days and then decreased. Monitoring the viral
gene expression pattern showed an early burst of lytic gene expression. This up-regulation of lytic gene expression prior to
latent genes during early infection strongly suggests that EBV infects primary B-cell with an initial burst of lytic gene
expression and the resulting progeny virus is competent for infecting new primary B-cells. This process may be critical for
establishment of latency prior to cellular transformation. The newly infected primary B-cells can be further analyzed for
investigating B cell activation due to EBV infection.
Citation: Halder S, Murakami M, Verma SC, Kumar P, Yi F, et al. (2009) Early Events Associated with Infection of Epstein-Barr Virus Infection of Primary B-Cells. PLoS
ONE 4(9): e7214. doi:10.1371/journal.pone.0007214
Editor: Linqi Zhang, Comprehensive AIDS Reseach Center, China
Received May 29, 2009; Accepted August 27, 2009; Published September 28, 2009
Copyright: ß2009 Halder et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: 1R01CA137894-01 and 5R01CA108461-05 The funders had no role in study design, data collection and analysis, decision to publish, or preparation of
the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: erle@mail.med.upenn.edu
Introduction
Epstein-Barr virus (EBV) is a ubiquitous human herpesvirus that
establishes latent infection in B lymphocytes. EBV is associated
with various lymphoid and epithelial malignancies, such as
Burkitt’s lymphoma, nasopharyngeal carcinoma, lymphoprolifer-
ative diseases in immunosuppressed patients, and gastric carcino-
ma [1] . The principal target cells for EBV infection are human
primary B lymphocytes, but the virus can also infect other
lymphocytes and epithelial cells [2,3,4,5,6]. EBV has two
alternative lifestyles: latent (non-productive) infection, and lytic
(productive) replication [7]. Following primary infection, EBV
persists within memory B lymphocytes in a latent state for the life
of the host. A low level of reactivation during the lytic cycle allows
viral shedding into the saliva and transmission of the virus in vivo
[7]. EBV binds to B-lymphocytes through interaction of the
glycoprotein gp350/220 with the complement receptor CD21[8].
In vitro, EBV transforms peripheral human B lymphocytes into
indefinitely proliferating lymphoblastoid cell lines (LCLs) that
allows for genetic manipulation of the virus [7].
Latently infected B cells maintain EBV genomes as 184-kb
circular plasmids, referred to as episomes, and express only a limited
number of viral gene products [7]. At present, four patterns of EBV
latency are recognized [9,10]. In type I latency, represented mainly
by Burkitt lymphoma (BL) cells, viral gene expression is restricted to
the two EBER genes, the BART transcripts, and EBNA1 (EBV
nuclear antigen 1) [9]. In latency II additional expression of three
latent-membrane proteins, LMP-1, LMP-2A and LMP-2B is
observed and is most frequently seen in Hodgkin’s lymphoma.
Latency III is seen in lymphoproliferative diseases developed in
immunocompromised individuals and EBV-transformed lympho-
blastoid cell lines [7]. In this group all six EBNAs, all three LMPs
and the two EBERs are expressed [9,10]. Type IV latency is less
strictly defined and pertains to infectious mononucleosis patients
and patients with a post-transplant lymphoproliferative disease [9].
Some individuals also presents with the so called putative latency
program (latency 0), in which no detectable latent gene expression is
detected [10]. The principal mediators of EBV-induced growth and
cellular transformation of B lymphocytes in vitro include EBNA2,
EBNA3A, 3C and LMP1 proteins [11]. The EBNA genes are
important for transformation of primary B lymphocytes, leading to
transactivation and regulation of other cellular and viral genes
[4,12]. These proteins are involved in augmentation of the
expression of genes coding for CD21, CD23, LMP1 and LMP2
proteins in B lymphocytes [4,11,13].
The lytic cascade of Epstein-Barr virus infection is divided into
three phases of regulated gene expression, immediate early, early
and late [14]. Synthesis of the viral encoded transactivator,
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BZLF1(also referred to as Zta or ZEBRA) serves as a checkpoint
for initiation of the replicative cycle [15]. BZLF1 is a DNA-
binding protein, and its expression precedes the switch from latent
to lytic infection [4]. BZLF1 is a viral transactivator protein known
to be directly involved in triggering expression of the lytic genes
and downregulation of latent genes, culminating in cell death and
release of infectious virions [15]. This protein up-regulates
expression of other immediate early genes as well as its own
expression [16]. This immediate early expression in turn up-
regulates the expression of early genes such as viral DNA
polymerase (BALF5) and thymidine kinase [4,17]. The major
proteins of the lytic phase are the EBV DNA polymerase, BALF5
[18] and the late lytic cascade, major capsid protein, BcLF1[14].
Two small RNAs (EBER-1 and EBER-2) represent the most
abundant EBV RNA expressed during latent infection and
undergo continuous expression in EBV-positive tumors, indepen-
dently of the latency type [19,20].
Conventionally, herpesvirus mutants are generated by homol-
ogous recombination in infected cells with DNA fragments or
plasmids carrying the mutant allele as described almost 30 years
ago [21,22,23]. As a consequence, recombination between the
herpesvirus genome and the mutant allele gives rise to a mixed
population that consists of the wild-type and mutant virus, such
that their separation is necessary and important for evaluation of
the phenotype. This approach has been proven to be quite tedious
with gammaherpesviruses, (i.e. EBV), because so far no host cell
type has been shown to fully support the lytic, productive phase of
these viruses. In the case of EBV, to study latent genes it is first
essential to obtain an immortalized cell line latently infected with
the mutant virus, which takes place often in combination with
wild-type virus if the gene is essential. To separate these viruses in
a second step, the latently infected cell needs to support the lytic
phase to produce infectious virions important for establishment of
another latently infected, immortalized B cell line exclusively
carrying the viral mutant or can be passed into an already
immortalized cell line like Ramos or BL41[13]. Because B cell
immortalization is a prerequisite to establishment of a mutant
EBV LCL, this approach excludes the genetic analysis of genes
that are essential for B cell immortalization in vitro [24,25,26].
The introduction of the bacterial artificial chromosome (BAC)
system into the genetics of herpesviruses brought a new dimension
to the field [27]. In the BAC system, the entire viral genome can
be propagated in Escherichia coli, and mutations can be rapidly and
precisely introduced into viral genes [27]. To facilitate the
generation of recombinant viruses, the EBV genome was first
cloned as a bacterial artificial chromosome (BAC). F-plasmid
sequences for prokaryotic replication [28], kanamycin resistance
marker for prokaryotic selection, and a cytomegalovirus promoter-
driven puromycin resistance cassette for eukaryotic selection were
inserted into the BamHI site of a plasmid containing EBV BamW
DNA and then transfected into B95-8 cells [29]. Following the
successful cloning of other herpesviruses, the B95-8 strain of EBV
was cloned in a BAC vector [29]. The system employed an
epithelial cell background as the virus producing cell and virus
production was induced by transfecting an expression vector
encoding a viral immediate protein BZLF1 [30,31] .
In vitro EBV infection results in human B-lymphocytes
activation and perpetual proliferation [32,33]. EBV infected cells
grow in tight clumps and express a number of B-cell activation
molecules including CD5, CD23, CD39, CD40 and CD44 and
proliferation surface antigen marker CD10 as well as intracellular
proliferation marker Ki-67 [34]. CD5 expression on B-cell can be
up-regulated by various activating agents, which indicate that
CD5 is a B-cell activation antigen [35]. CD5 has also been shown
to be important for the apoptosis of antigen-receptor induced
lymphocytes and for the maintenance of tolerance by B cells [36].
Additionally, previous work reported that CD5 expression is
down-regulated by EBV during transformation of CD19 positive
B-cells [37]. The B-cell activation marker CD23 has been shown
to be upregulated by EBV infection and induced at high level in
EBV-transformed lymphocytes [38,39]. EBNA-2 and LMP-1
cooperatively induces CD23 [13] as well as the human CD40
antigen which is a 50-kD glycosylated phosphoprotein [40]. LMP-
1 is a transmembrane protein with a structure reminiscent of G
protein coupled receptors, but its signaling activities are similar to
that of CD40 which belongs to the TNF receptor family [41]. In
addition, depending on the cellular context, LMP1 can induce
specific B-cell activation antigens such as CD39, CD23, CD21,
CD40 and CD44 [13,42]. Ki-67 is a monoclonal antibody that
recognizes a proliferation related human nuclear antigen
expressed during G1, S, and G2/M phase of the cell cycle but
not in resting (G0 phase) cells [43,44].
Gp350 is the glycoprotein found most abundantly on the surface
of EB virions as well as on the surface of EBV-infected cells in
which EBV is lytically replicating [45]. It is the glycoprotein of the
virion that binds to the EBV receptor CD21 (or complement
receptor type II, CR2) and initiates infection ([8,46].The another
glycoprotein, gp110 encoded by EBV BALF4 ORF is expressed
during the lytic phase of EBV not only at nuclear membrane but
also at the cellular membrane and has been shown genetically to
be essential for virus maturation [47,48,49].
Acyclovir [ACV; 9-(2-hydroxyethoxy methyl) guanine] is one of
a class of antiviral compounds effective in curbing a variety of
herpesvirus infection in vitro and in animal models [50]. In the
EBV system ACV is an effective inhibitor of viral DNA replication
in productively infected cells but is essentially devoid of any effect
on the replication of viral DNA in latently infected cells, where
cellular control mechanisms regulate EBV DNA synthesis[51,52].
In this report, we generated a recombinant EBV containing a
GFP cassette cloned in the BAC vector backbone designated as
BAC GFP-EBV in an effort to study the molecular changes during
early infection of primary B-cell induced by the virus. [53].
Previous studies by other groups which generated fluorescence
tagged EBV proved difficult in our hands [54,55]. However, the
MD1 system generated by Wang and collegues [29] proved to be
manipulatable in our hands and so we decided to introduce a
fluorescence marker for monitoring infection in this system. The
green fluorescent protein is a suitable marker because it fluoresces
strongly and stably in mammalian cells and can be monitored by
noninvasively strategies in living cells [56,57]. In this study, a GFP,
puromycin and ampicillin cassette was introduced into the EBV-
BAC [29] to establish the BAC based recombinant EBV GFP
designated as GFP-EBV by homologous recombination, and the
recombinant virus DNA was shuttled from E.coli to mammalian
cells. The induced GFP-EBV virus was used for the infection of
Peripheral Blood Mononuclear cells (PBMC) and establishment of
lymphoblastoid cell lines (LCLs). Using the GFP-EBV infected
PBMCs, we monitored a range of immunophenotypic changes.
Several B-cell surface antigen markers such as CD5, CD10, CD19,
CD23, CD39, CD40 and CD44 [40], as well as the intercellular
proliferation protein Ki-67 were used during initial infection of
EBV. We also analyzed the latent and lytic the protein profiles
during early infection of primary B-cell by recombinant EBV. Our
results suggested that EBV infection to B-cells involves an initial
burst of lytic replication which may be critical for the many
signaling events involving anticrine and paracrine factors which
eventually leads to B-cell transformation and establishment of
latency after 2–4 weeks in culture.
Early Events in EBV Infection
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Materials and Methods
Cells and virus cultures
BJAB was used as EBV negative cell line and LCL1 & LCL2
were used as EBV positive cell lines [58]. BAC-EBV was
propagated in EL350 [29] and GFP-Amp cassette was incorpo-
rated into BAC-EBV by homologous recombination. BAC GFP-
EBV was transferred into HEK 293T cells and the BAC GFP-
EBV infected Lymphoblastoid cell lines (LCLs) were established
from primary B-cell (Immunology core of UPENN). PBMCs were
obtained from UPENN immunology core from de-identified
different donors for multiple infection studies. All B-cells were
grown in RPMI1640 with 10% fetal bovine serum, and adherent
cells in Delbecco’s Modified Essential Medium (DMEM) supple-
mented with 10% fetal bovine serum (FBS), 50 mg/ml
streptomycin, and 50U penicillin (medium) (Bio-Whittaker,
Walkersville, MD). 1 mg/ml final concentration of Puromycin
was used for selection of cells transfected or infected with the BAC
GFP-EBV.
The construction of BAC GFP-EBV genome
For incorporation of GFP in the BAC EBV genome, we
considered the region of EBV genome from 149,116 to
154,747 bp. The GFP DNA was introduced into the site of the
B95.B deletion at 152,008 and the GFP cassette contained a
BamHI site and was flanked by 50 bp downstream and upstream
sequence from 152,008 bp [59]. 3646 bp of the GFP cassette
(GFP-Puro-Amp) with one BamHI site was electroporated into the
bacterial cells EL350 with BAC EBV (MD1BAC) and the cassette
was incorporated by homologous recombination where positive
clones were screened for ampicillin resistance. The amplicon was
transfected into electrocompetent bacterial cells EL350 MD1BAC
by electroporation at 1.75 kV. BAC GFP-EBV DNA was
extracted from the positive clones and subjected to BamHI
digestion overnight. The digested products were resolved on
0.65% agarose gel by running for 16 h–20 h at 40V and visualized
by ethidium bromide staining and UV exposure. The digestion
pattern was analyzed and compared with wild type MD1BAC.
The resolved gel was transferred onto the Genescreen tranfer
membrane (PerkinElmer, Waltham, MA, USA) and the cassette
was visualized by hybridizing with the EBV BamHI I fragment as
a probe.
Transformation of recombinant BAC GFP-EBV into
bacteria
Oligonucleotide primers corresponding to the different region of
EBV genome were synthesized 59to 39. The sequence of
oligonucleotides and the product length are shown in table 1.
The genomic DNA was prepared from approximately 5610
4
cells. Following centrifugation to remove medium, cells were
resuspended in 0.26phosphate buffered saline, boiled for 10 min,
and mixed with 0.1 volume of 10-mg/ml proteinase K (Sigma,
Marlborough, MA), and the mixture was incubated for 30 min at
55uC. Proteinase K was inactivated by incubation at 95uC for
20 min. PCR analysis was performed by using Perkin-Elmer
thermal cycler with 5 ml of DNA in a 50 ml reaction. PCR-
amplified DNA was analyzed by electrophoresis using 2% agarose
gels and visualized by staining with ethidium bromide and UV
exposure.
Tranfection of GFP BAC-EBV into 293T cells
HEK293T cell were transfected with 5 to 10 mg of BAC GFP-
EBV DNA by lipofectamine 2000 (Invitrogen, Inc., Carlsbad, CA)
according to manufacturer instruction. After 2 days post
transfection, cells were trypsinised and plated at 10
4
cells per well
in 96-well tissue culture plates in DMEM medium and the
medium was replaced with fresh 1 mg/ml puromycin-containing
DMEM medium every three days. Puromycin-resistant clones
(shown in Table 2) were screened for the presence of episomally
maintained BAC GFP-EBV by visualization of GFP protein.
Table 1. Primers used in PCR analysis[59].
Primers Product size Oligonucleotide sequence EBV genome
BamHIT sense 350 bp 59-CCCCCTTTTCCGCATCAG-39140112-140461 bp
BamHIT antisense 59-AGTCCGGATTGGGCACCA-39
BamHIK sense 341 bp 59-GCTGCTTTCCTCGGATGCC-39112231-112571 bp
BamHIK antisense 59-CTGGGATGGGGAGCGGAG-39
BamHI E sense 1128 bp 59-TACTGCCACCAGTACCACAACA-3997001-98128 bp
BamHI E antisense 59-GGCCGACATTCTCCAAGATAA-39
BamHI H sense 194 bp 59-CTCTGCCACCTGCAACACTA-3949117-49310 bp
BamHI H antisense 59-ATTTGGGGTGCTTTGATGAG-39
Fragment C sense: 761 bp 59-GCAGGGCTCGCAAAGTATAG-3911095-11855 bp
Fragment C antisense 59-TGCGGAAGTGACACCAAATA-39
Puro sense 220 bp 59-CGTGCAGTGCTTCAGCCGCTACCCC-39152856-153075 bp
Puro antisense 59-CTTGTGCCCCAGGATGTTGCCGTCC-39
EGFP Sense 552 bp 59-GACGTAAACGGCCACAAGTT-39152716-153267 bp
EGFP antisense 59-CTGGGTGCTCAGGTAGTGGT-39
E3Ct1t2 sense 152 bp 59-AGAAGGGGAGCGTGTGTTGT-3999939-100091 bp
E3Ct1t2 antisense 59-GGCTCGTTTTTGACGTCGGC-39
EBVGFP sense 820 bp 59-GGGCTCGTTTAAACAAAGTCTCATC-39151921-152740 bp
EBVGFP antisense 59-CGCTGAACTTGTGGCCGTTTACGTC-39
doi:10.1371/journal.pone.0007214.t001
Early Events in EBV Infection
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Induction of virus from 293T cells containing GFP EBV
BACmid
Cells harboring BAC GFP-EBV were induced to release virus
by culturing for 5 days in complete RPMI 1640 medium
containing phorbol ester TPA (12-O-tetradecanoylphorbol-13-
acetate, 20 ng/ml) and butyric acid (BA, 3 mM; both from
Sigma). Cell suspensions were centrifuged at 1,800 rpm for 10 min
and the supernatant was filtered through a 0.45 micron cellulose
acetate filter. The viral particle were concentrated by ultracentri-
fugation at 27K rpm at 4uC and stored at 280uC.
Infection of primary human B cells with GFP-EBV
Lymphoblastoid cell lines were generated by infections of 1610
6
primary B-cells incubating with virus suspension in 1 ml of RPMI
1640 (10% Fatal Bovine Serum) medium in the presence of
cyclosporin A (Sigma, Marlborough, MA) and incubated for 4 hrs
in 37uC. Cells were centrifuged for 5 min at 1500 rpm, the
supernatant discarded, pelleted cells were resuspended in fresh
RPMI 1640 (10% FBS) medium in 96 well plates. The infection was
checked by the visualization of GFP expression using fluourescence
microscopy. The transfected green cells were enriched by selection
with puromycin. The infected cells were then transferred to 48 well
plates and expanded to larger well plates until the cultures
continued to grow continuously in complete media. The number
of clones generated for BAC GFP-EBV LCL was also shown in
table 2. To monitor the early stage of infection after 4 hrs
incubation with EBV and PBMC, the cells were then washed two
times with fresh RPMI 1640 (10% FBS) medium to remove excess
virus and fresh medium was added. This infection step is designated
as Infection I. The supernatant from primary infection (Infection I)
of 2610
7
PBMC by GFP-EBV were used infection of fresh PBMC
(1610
6
) in a similar manner and infection further monitored by
GFP fluorescence. This step of infection was designated as Infection
II. Infection I was also monitored by adding 25 mM acyclovir and
the supernatant from infection I were also used for infection II to
determine if virus produced was due to replication during lytic
replication during lytic infection in Infection I and not due to virus
passed on from the initial infection .
Western Blotting
Cell lysates were electrophoresed on SDS-PAGE gels and
transferred to 0.45 micron nitrocellulose membranes. Blots were
then probed using specific primary antibodies (S12) for LMP-1
[60] and human serum (KJ) for EBNA-1 and A10 for EBNA-3C
[61] with required dilutions. This was followed by incubation with
fluorescence labeled secondary antibodies, Alexa Fluor 680 and
Alexa Fluor 800 (Molecular Probes Inc., Carlsbad, CA; and
Rockland Inc., Gilbertsville, PA, respectively) diluted at 1:20,000.
Blots were visualized and analyzed using LICOR Odyssey
imaging system and Odyssey software (Li-Cor, Lincoln, NE).
Flow cytometry analysis of EBV infected peripheral blood
mononuclear cells
Peripheral Blood Mononuclear Cells (PBMCs) (procured from
Immuology core, University of Pennsylvania Medical School,
Philadelphia, PA) were infected with BAC GFP- EBV and the
infected cells were fixed with 0.5% paraformaldehyde at 6 h, 12 h,
24 h, 48 h, 72 h, 96 h, 120 h and 168 h post-infection for 1 hour
at 4uC and washed three times with buffer W (1X PBS with 0.1%
BSA and 0.001% NaN3). A broad panel of fluorochrome-
conjugated monoclonal antibodies was used for detection of the
following cell surface markers: CD3, CD5, CD10, CD19, CD23,
CD39, CD40, CD44 and Ki-67, respectively. The surface antigen
markers contained different conjugates such as Phycoerythrin (PE),
Phycoerythrin-Cy7 (PE-Cy7), Per CP, PercpCy 5.5 and Allophy-
cocyanin (APC).
Flow cytometry was carried out on an 8-color flow cytometry
instrument CYANADP (Wistar Institute, Philadelphia, PA) with
Cell-Quest software (Becton-Dickinson, San Jose, CA) used in
accordance with the manufacturer’s instructions. Instrument
settings were adjusted so that fluorescence of cells from uninfected
controls, in the case of GFP readings, or negative controls (i.e.,
with antibody omitted in antibody labeling) fell within the first
decade of a four decade logarithmic scale on which emission is
displayed. Flow cytometry plots showed at least 20000 events. The
data were analyzed by FlowJo software (Becton-Dickinson, San
Jose, CA). The expression levels of different surface antigen
markers as well as an intracellular proliferating marker were
analyzed from GFP positive EBV infected cells. The extent of
infected B-cell and T-cell population from total cell populations
were analyzed from CD19 and CD3 positive cells.
Real time PCR
Total RNA were prepared from 5610
6
GFP EBV infected
PBMCs after different times post-infection (6h, 12h, 24 h, 48h, 72h,
96h, 120h and 168 hours) using Trizol reagent (Invitrogen, Inc.,
Carlsbad, CA) according to manufacturer’s instructions. cDNA was
synthesized using a Superscript II RT kit (Invitrogen, Inc.,
Carlsbad, CA) according to the manufacturer’s instructions. The
specific primers used for the amplification of latent genes (EBNA-1,
EBNA-2, LMP-1), lytic genes (BZLF1, BcLF1 and BALF5) and
internal control GAPDH are shown in Table 3. The lytic gene,
BALF5, was also amplified in the presence of 25 mM ACV.
Table 2. No. of clones generated in HEK293T and LCLs of BAC
GFP-EBV.
Cell No. of clones
293T 4,10,11,12,15
BAC GFP-EBV
LCL 10,11,14,15
BAC GFP-EBV
doi:10.1371/journal.pone.0007214.t002
Table 3. Primers used in q-Real time PCR[59].
Primer Name DNA Sequence (59-39) Product length
EBNA-1 59-CATTGAGTCGTCTCCCCTTTGGAAT-39150 bp
59-TCATAACAAGGTCCTTAATCGCATC-39
EBNA-2 59-GAGACCAGAGCCAAACACCTCCAGT-39150 bp
59-TTAGGGGTTGCCGTGTGTGAATTTC-39
LMP-1 59-CCCGCACCCTCAACAAGCTACCGAT-39150 bp
59-TTGTCAGGACCACCTCCAGGTGCGC-39
BZLF1 59-AACCGCTCCGACTGGGTCGTGGTTT-39150 bp
59-CCAGGTTGAGGTGCTTCTCCCCCGG-39
BcLF1 59-CCTCTTGGAATGCAGCTGGGGCCAG-39150 bp
59-CCAATTATGACCTGCTGCGGCTGGA-39
BALF5 59-GCTGGCCTTGAGGGCGCTGAGGACT-39259 bp
59-CACCCACGGAAGCCCTCTGGACTTC-39
doi:10.1371/journal.pone.0007214.t003
Early Events in EBV Infection
PLoS ONE | www.plosone.org 4 September 2009 | Volume 4 | Issue 9 | e7214

The target gene was amplified from cDNA using SYBR green
real-time master mix (MJ Research Inc.,Waltham,MA), 1 mM
each primer and 5 ml of the cDNA product in a total volume of
20 ml. Thirty-five cycles of 1 min at 94uC, 1 min at 55uC and
1 min at 72uC, followed by 10 min at 72uC, were performed in an
MJ Research Opticon II thermocycler (MJ Research Inc.,
Waltham, MA). Each cycle was followed by two plate reads, with
the first at 72uC and the second at 85uC. A melting curve analysis
was performed to verify the specificity of the amplified products,
and the values for the relative quantitation were calculated by the
DDCt method [62]. All experiments were performed in triplicate.
Immunofluorescence
GFP infected PBMC cells after different time intervals of
postinfection were dried onto slides and fixed using a 1:1 mixture
of acetone and methanol. After fixation cells were extensively
washed in PBS and incubated in blocking buffer [PBS supplement-
ed with 0.1% Triton-X 100, 0.2% fish skin gelatin (Sigma)] at room
temperature for 30 min. Endogenous expression of gp350 and
gp110 were detected using mouse monoclonal antibody (1:500
dilution), and rabbit (1:250 dilution) respectively. Primary antibod-
ies were diluted in blocking buffer and incubated with fixed cells for
1 h at RT. Slides were washed three times (5 min each) with PBS
and incubated with appropriate secondary antibody (1:2000) for 1 h
at RT followed by three times washes (5 min each) with PBS.
The last wash contained 49,69-diamidino-2-phenylindole (DAPI;
Promega Inc., Madison, WI) for nuclear staining. Goat anti-mouse
antibody Alexa Fluor 594 and goat anti-rabbit antibody Alexa Fluor
594 were purchased from Molecular Probes Inc.(Carlsbad, CA).
Slides were then washed in PBS and mounted using Prolong anti-
fade (Molecular Probes Inc, Carlsbad, CA). Fluorescence was
viewed by confocal microscopy and analyzed with Fluoview 300
software from Olympus Inc. (Melville, NY).
Results
Incorporation of GFP into BAC EBV (MD1BAC) and
selection in mammalian epithelial cells
To incorporate the GFP cassette into the BAC vector sequences
containing the entire EBV genome, designated as MD1BAC [29],
we transfected a GFP cassette which contained GFP, puromycin
and ampicillin resistance genes under the control of the CMV
promoter into EL350 by homologous recombination between the
viral genome (Fig. 1A). Ampicillin was introduced along with the
GFP cassette into EBV genome with the BAC vector backbone for
selection in E.coli [29]. Viral genome position 152,008 was chosen
for insertion of the cassette as there is no identifiable open reading
frame from 151,959 to 152,058 i.e. 100 bp is unique sequence in
B95-B EBV genome [59].
The GFP cassette was amplified from GFP-pBSpuro cassette
[63] with 50 nucleotides of EBV genome (151,959-152,008 and
152,008-152,058) at either terminal of the cassette (Figure 1A); the
amplified cassette was then transfected in EL350 with MD1BAC
for homologous recombination and the clones were screened on
amp-kan plate (kanamycin was already inserted into BAC EBV in
MD1BAC [29]). The DNA was extracted from amp-kan resistant
EL350 colonies and digested with BamHI. The digestion pattern
of BAC GFP-EBV is shown in Figure 1B (left panel). The GFP
cassette had a BamHI site, therefore the BamHI fragment
containing the 152,008 site split into two fragments of 4,981 bp
and 4,297 bp after BamHI digestion (Figure 1B, arrow heads).
The digestion profile compared with MD1BAC clearly indicated
that there were full EBV genomes with the GFP cassette
incorporated at the desired site (Figure 1B, left panel). Generation
of two bands after cassette introduction was confirmed by
Southern blot analysis using the BamHI I fragment as a probe
(Figure 1B, right panel). Inclusion of the GFP cassette at this site
was also confirmed by amplifying the junction region of EBV gene
and GFP cassette. The amplification of an 820 bp size PCR
product, which is shown in Figure 2, is identical with the predicted
size shown in schematic diagram. The GFP incorporated
MD1BAC was then designated as BAC GFP-EBV.
To test whether the BAC GFP-EBV clone was competent for
viral replication and B-cell immortalization, the BAC GFP-EBV
was induced for replication in 293T cells [29,54]. Highly pure
BAC GFP-EBV DNA was prepared and transfected into HEK
293T cell. Two days after transfection, 10–20% of the cells
typically showed GFP expression. Transfected cells were selected
by puromycin in 96 well plates. After Four to six weeks of
puromycin selection, 5 stable HEK 293T clones of BAC GFP-
EBV expressing GFP were selected for further analysis. The GFP
signals for two such clones are shown in Figure 3B. Persistence of
the full length EBV genome was determined by PCR analysis of
hirt extracted DNA from these stable cell lines using 5 primer sets
across the entire genome (Figure 3A). The PCR results suggested
the presence of full length intact EBV containing GFP and
Puromycin markers within the BAC GFP-EBV. Amplification of
the different regions across the EBV genome confirmed that these
stable cells were able to maintain the intact BAC GFP-EBV.
Infection of primary B-cell with BAC GFP-EBV and
establishment of LCLs
BAC GFP-EBV virus from the stable 293T cells were induced
by treating with chemical inducers, TPA and butyric acid for 4
days [29]. The supernatant containing GFP EBV was used to
infect PBMC after concentrating by ultracentrifugation. Human
primary B-cells were infected by using different volumes of the
concentrated virus. The infected cells were incubated at 37uC and
grown overnight. The cells having high GFP expression and B-cell
clumping were then diluted and plated into 96 well plates and
incubated for 3–4 weeks. The infection was monitored by GFP
expression. Approximately, 20–30% of the cells were positive for
green flourescence after 36 hours of infection. Four weeks later,
these green cells were clearly transformed to LCLs. The GFP
content in LCL was enhanced by puromycin screening, and stable
LCLs with GFP expression were obtained from the above pool of
cells as shown in Figure 4B. To monitor whether or not the GFP-
EBV positive LCLs were intact, the genome was analyzed by PCR
amplification across different regions of the EBV genome
(Figure 4A). PCR analysis of the different region of the EBV
genome in LCLs suggested that the LCL maintained an intact
EBV genome along with GFP cassette (Figure 4A).
Analysis of EBV latent proteins expression in GFP-EBV
positive LCLs
In latency type III, six nuclear antigens (EBNA1, EBNA2,
EBNA3A, EBNA3B, EBNA3C, and EBNA-LP), three latent
membrane proteins (LMP1, LMP2A, and LMP2B), two small
nonpolyadenylated RNAs (EBER-1 and EBER-2), and transcripts
from the BamHI-A region (BARTs) are expressed [64]. To further
investigate the establishment of latent infection due to GFP-EBV,
the latent protein expression profile of the critical latent antigens in
these GFP-EBV transformed LCLs were analysed. The protein
expression in HEK 293T containig BAC GFP-EBV and GFP-
EBV infected LCLs were analysed by immunoblotting along with
BJAB as negative control and previously created LCLs as a
positive control (Figure 5). Antibodies detecting EBNA3C and
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EBNA1 and the oncoprotein LMP-1 were used to detect the
specific antigens. The expression patterns of these proteins are
shown in Figure 5. Detection of EBNA-1, LMP-1 and EBNA-3
proteins clearly showed that the recombinant GFP-EBV was
capable of transforming primary B-cells into LCLs and was also
able to maintain a type III latency program.
EBV infection induces B-cell activation and cell
proliferation
To investigate the immunophenotypic effects of EBV infection
on B-cell during early infection, we analyzed the expression profile
of several B-cell activation and proliferation surface antigen
markers (CD5, CD10, CD23, CD39, CD40, and CD44) as well as
the intracellular proliferation marker (Ki-67) at different times
post-infection.
Since EBV also infects T-cells along with B-cell, we investigated
EBV infected T-cell population by analyzing the expression of
CD3 antigen at different times postinfection. The expression of
CD3 in GFP positive cells showed that 20% of the CD3 positive T
cells were positive by 6 hours infection increasing to 38% by
24 hours. However, this signal was dramically decreased by
48 hours and went down further to 8% after 168 hrs (Figure 6A,
left panel). Of significance, the extent of B-cell infectivity measured
by GFP positive CD19 expression was very high within 6 hours
Figure 1. Strategy for insertion of the GFP cassette into BAC EBV (MD1BAC). (A) BamHI region of EBV genome (149116 bp -154747) [59]
was targeted for homologous recombination. A DNA fragment was generated by PCR amplification of a GFP/AMP cassette with a BamHI site using
primers incorporating 50 nucleotides of the EBV genome upstream of the 152008 nucleotide and downstream of the 152009 nucleotide at the 59
termini. The fragment was electroporated into E.coli EL350 carrying EBV bacmid (MD1BAC) and expressing recombinase to allow homologous
recombination. After recombination the BamHI site splits to two fragments compared to BACMD1. (B) The BamHI digestion pattern of BAC GFP-EBV
and MD1BAC was visualised in 0.65% agarose (left panel). The fragments which split in to two fragments are indicated by white arrows. BamHI I
fragment was used as a probe for southern blot analysis (left panel). The southern blot analysis showed that the 5.6 bp fragment from MD1BAC
(white arrow) and two BAC GFP-EBV fragments ,5 kb and ,4.3 kb.
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postinfection increasing to above 90% after 7 days postinfection
(Figure 6A, right panel).
PBMCs were infected by GFP-EBV and the infected cells were
subjected to flow cytometry analysis at different times post-
infection. The expression of the indicated surface antigen markers
were determined up to 7 days for GFP positive EBV infected cells.
The expression of the CD5 cell surface marker did show a
gradual change during the course of early infection. By 6 hours
post-infection only 28% of the GFP positive cells were expressing
CD5 (Figure 6B). This was increased to 54% by 5 days later.
Interestingly the percent of GFP positive cells that were CD5
positive were dramatically less by 6–7 days suggesting that this
initial increase was not sustained after 5 days with development of
LCLs (Figure 6A, left panel). However, the CD10 activation
marker showed that about 30% of the GFP-positive cells expressed
CD10 by 6 hours postinfection and this was relatively unchanged
by 7 days postinfection (Figure 6A, middle panel). The expression
pattern of CD23 during early infection with GFP-EBV was
interesting with an increase in the percentage of GFP positive cells
about 30% by 6 hours postinfection increasing to about 50% by 72
hours post-infection and then showed a rapid decline immediately
after which continued to 7 days later (Figure 6C, left panel).
Importantly, a well-known B-cell activation and proliferation
marker CD40 was detected in about 15% of the GFP-positive cells
and gradually increased to over 50% by 7 days postinfection
(Figure 6C, midle panel). The expression of CD44 was unchanged
throughout the events of early infection (Figure 6C, right panel).
However, the percentage of EBV positive cells as determined by
GFP expressing CD39 was significant, approximately 90%, but
gradually decreased to 42% by 7 days postinfection (data not
shown).
To study proliferation due to infection of primary B-cell by
GFP-EBV on B-cell, we monitored expression of the intracellular
proliferation marker Ki-67 [34] by Flow Cytometry. Ki-67 was
expressed at 48 hrs of post-infection of GFP EBV and its signal was
consistently increased up to 7 days post-infection (Figure 6B, right
panel). Importantly, analysis of LCLs showed extremely high levels
of Ki-67 suggesting a requirement for Ki-67, or that it has a
Figure 2. Verification of recombinant BAC GFP-EBV. (A) The DNA from bacterial colonies screened by kanamycin-ampicillin together and
sequence analysis of the inserted GFP/amp into MD1BAC genome by PCR amplification, with one primer of EBV genome and another primer from
GFP. Amplification of the 820 bp fragment was shown from DNA observed from 4 independent isolates (2–5 lanes) with MD1BAC as negative control.
(B) Schematic of EBV showing position of the markers and GFP.
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Figure 3. Transfection of Full length BAC GFP-EBV into 293T cells. (A) BAC GFP-EBV DNA was transfected to 293T and stable cell line
generated as screened by puromycin. Different genes encoding by EBV (BamHI T, BamHI K, BamHI E, BamHI H and BamHI C) as well as puromycin and
GFP were analyzed by PCR amplification taking 5 different transfected clones (4, 10, 11, 12 and 15). BJAB and LCL were taken as negative and positive
control, respectively. (B) Phase contrast (left panel) and Flourescence images (right panel) showed two indifferent (number 4 and 12) containing BAC
GFP-EBV transfected into 293T stable cell line screened by puromycin.
doi:10.1371/journal.pone.0007214.g003
Figure 4. LCLs established by infection with GFP-EBV. (A) PBMC cells were infected by GFP- EBV and made GFP positive LCLs selected with
puromycin. The proliferating cells are clustered and GFP positive. BAC GFP-EBV transfected 293T cell clones and LCL established EBV encoded
different region of EBV (E3CT1T2, BamHI H, BamHI K, BamHI T and BamHI C) as well as puromycin and GFP were checked by PCR amplification taking
2 different infected LCL clones (11 and 14). BJAB was used as negative control and LCL1 & LCL2 were used as positive controls, respectively. (B) Phase-
contrast (left) and fluorescence (right) images of 2 different established (11 and 14) GFP-EBV LCLs.
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critical role in initiating and maintaining the transformed state
induced by the virus.
For further investigation of the immunophenotypic effects of
EBV infection on B-cell during early infection, we also analyzed
the expression profiles of several B-cell activation and proliferation
surface antigen markers (CD5, CD10, CD23, CD39, CD40, and
CD44) as well as the intracellular proliferation marker (Ki-67) at
different times post-infection in non-infected B-cells i.e. GFP
negative cells. Flowcytometry analysis from GFP-negative cells
showed that there was no significant change in surface antigen
markers (CD5, CD10, CD23, CD40 and CD44) or the
intercellular marker Ki-67 during early infection as shown in
figure 6D and figure 6E.
Rapid induction of EBV lytic replication during early
infection of human primary B-cells
EBV establishes different types of latency characterized by
differential expression of a group of latency proteins [14]. To
evaluate the gene expression pattern during early events of
infection, GFP-EBV was used to infect PBMCs and latent and lytic
gene expression were analyzed. Latent expression of EBNA-1,
EBNA-2 and LMP-1 and the lytic genes immediate early
transactivator (BZLF1), major capsid protein (BcLF-1) and DNA
polymerase (BALF5) were monitored. The transcript levels of
these genes were determined from the total RNA extracted from
GFP-EBV infected PBMCs at different time points post-infection
up to 7 days (Figure 7).
The copies of latent gene transcripts at different time points
post-infection of GFP-EBV was determined by semi-quantitative
real time PCR as shown in Figure 7A. Expression of EBNA-1 was
barely detectable at 24 hrs, but was clearly detected at 48 hrs and
peaked by 120 hrs. EBNA2 signals were evident by 6 hrs and
plateaued by 24 hrs post-infection (Figure 7A, middle panel).
EBNA-2 transcript was consistently detected through 7 days of
infection at similar levels up to 7 days throughout the course of the
study. Similarly, LMP-1 transcript levels was detectable at 6 h and
reached at maximum level by 24 hours of post-infection
(Figure 7A, right panel). Interestingly, the level of LMP-1
transcripts remained consistent up to 7 days post infection.
The mRNA levels of lytic genes BZLF1, BcLF1 and BALF5
were also determined using real time qPCR at different time points
post infection. We found that the BZLF-1 gene was expressed
during the initial stages of infection i.e. it came on at 6 hrs post-
infection and peaked at 24 hrs (Figure 7B, left panel). Interestingly,
the BZLF1 transcripts decreased after 24 hrs but began to increase
again by 96–120 hours post-infection. Another lytic gene BcLF-1
was detected at 6 hrs post-infection and continued to increase
throughout the course of study. The expression of DNA
polymerase (BALF5) during early infection peaked by 12 hours
post-infection followed by a gradual reduction to 96 hrs. However,
the level of BALF5 was also increased again by 7 days (Figure 7B).
Interestingly, the levels of lytic transcripts as determined by Real
time PCR was greater than that compared to the latent genes
during early stage of B-cell infection by GFP-EBV.
The progeny GFP-EBV produced during early infection is
infectious
To investigate further our hypothesis that early lytic infection
produces viral progeny capable of infecting new cells, we used
supernatant collected during the early time points to infect new
cells. PBMCs were infected with supernatant collected at the
different times and GFP-EBV was monitored by GFP expression
using a fluorescence microscope as well as FACS to 7 days
(Figure 8A and B, left panel). After 24 hours post-infection, GFP
signals and clumping of the primary B-cells were visualized. FACS
analysis also showed that 5% of the cells were GFP positive by 12
hours and increased to 20% after 72 hrs post infection (Figure 8A
and 8B). Importantly, we showed that the lytic genes were
expressed at a higher level when compared to latent genes
suggesting a burst of lytic infection and particle release. To
examine whether the lytic replication cycle produces virion
particles released into the supernatant at each time point post
infection, the supernatant was collected and used to infect fresh
PBMCs. The infection of fresh PBMCs was monitored for GFP
expression. The result showed that the supernatant from 72 hours
post infection was capable of infecting fresh PBMCs followed by
GFP expression and cell clumping (Figure 8B, right panel). The
percentage of GFP expressing cells as determined by FACS was 1–
2% from 72 h to 168 h. This result strongly suggested a burst of
lytic replication and release of virion particles during the initial
stages of EBV infection. However, the number of infected cells was
significantly higher when the supernatant from 7 days of post-
infection was used to infect new cells.
Production of progeny GFP-EBV particles during early
infection is inhibited by acyclovir
To investigate lytic gene expression as well as the release of
virion particles during early stages of infection, we monitored
GFP-EBV infection to PBMCs in the presence of 25 mM ACV.
PBMCs were infected with GFP-EBV with and without 25 mM
ACV at the different times post-infection and infection was
monitored by GFP expression using a fluorescence microscope as
well as FACS analysis up to 7 days (Figure 9A, left panel and 9C).
After 24 hours post-infection, GFP signals and clumping of the
primary B-cells were clearly observed. FACS analysis also showed
that 5% of the cells were GFP positive by 24 hours and increased
to 20% after 72 hrs post infection, whereas in the presence of
25 mM ACV, the extent of GFP expression decreased (Figure 9A,
right panel and 9B). It was observed that GFP expression was 13–
15% in presence of ACV, whereas in absence of ACV it was
,20%. The proliferation marker Ki-67 was also measured by flow
cytometry in presence of ACV (Figure 9, right panel). The results
showed that in the presence of 25 mM ACV, the expression of Ki-
67 decreased by 1.5 fold. To test the inhibition effect of ACV on
viral DNA polymerase synthesis, we checked the level of viral
Figure 5. Latent gene expression. Latent proteins expressions were
analyzed in cells infected with in BAC GFP-EBV stably transfected 293T
and LCLs made from BAC GFP-EBV infection. Expression of EBV EBNA-1,
EBNA-3C and LMP-1 were determined by Western blotting.
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Figure 6. Expression of B-cell proliferation and activation markers due to GFP-EBV early infection. 2610
6
PBMC were infected by GFP-
EBV and (A) describe CD3 and CD19 GFP population. (B) the expression of proliferation markers (CD5, CD10 and Ki-67) and (C) activation markers
(CD23, CD40 and CD44) were measured by Flow cytometry after different time post-infection (6h, 12h, 24h, 48h, 96h, 120h and 168h) with GFP-EBV
infected cells (i.e. GFP-positive cells). (D) The expression of proliferation markers (CD5, CD10 and Ki-67) and (E) activation markers (CD40, CD44 and
CD23) were measured by Flow cytometry after different time post-infection (6 h, 12 h, 24 h, 48 h, 96 h, 120 h and 168 h) with GFP-EBV non-infected B-
cells (i.e. GFP-negative B-cells).
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DNA polymerase (BALF5) gene expression by Real Time PCR in
the presence of ACV shown in Figure 9D. The results clearly
showed that in the presence of 25 mM ACV, the expression of
BALF5 mRNA decreased by 4–5 fold decreased compared to the
absence of ACV. These results suggest that in the presence of
ACV, the rate of lytic replication is inhibited which results in lower
infection of GFP-EBV and B-cell proliferation.
To examine the effect of ACV on the lytic replication cycle as
well as the production of virion particles released into the
supernatant at each time point postinfection, the supernatants
from infection-I were collected and used to infect fresh PBMCs
(infection-II). Infection-II was monitored by visualization GFP
using fluorescence microscopy (Figure 9B). The GFP results
showed that the release of virion particle was inhibited in presence
of ACV at Infection-I. This data strongly supports the conclusion
that virion particles produced due to burst of lytic replication
during early stages of infection.
To support the above results that lytic replication occurred in
EBV infected cells , we checked the expression of late genes
glycoproteins gp110 and gp350 by detection of the protein sing
fluorescence microscopy. PBMCs were infected with GFP-EBV.
The expressions of glycoprotein (gp110 and gp350) were
monitored by immunoflourescence analysis in the presence and
absence of ACV at different times post-infection (Figure 10A and
10B). The results showed that in absence of ACV gp110 was
expressed at 96 h whereas in presence of ACV, the gp110
expression was dramatically decreased. At 168h post-infection, the
level of gp110 expression was higher in the absence of ACV as
compared to the levels in the presence of ACV. The same result
was also seen for gp350 expression (Figure 10B). Thus, the late
protein expression profiles in ACV suggested that the productive
cycle was inhibited by acyclovir and further supportive our
conclusions that the bursts of replication and virion particle
production during the early stages of infection is likely to be crucial
for establishment of latency and transformation of the infected
primary B-cells.
Discussion
Using the BAC system, the viral genome can be propagated in
Escherichia coli, and mutations can be rapidly and precisely
introduced into any of the viral genes. To facilitate the generation
of recombinant viruses, the EBV genome was first cloned into the
bacterial artificial chromosome (BAC) [28,29]. The resultant BAC
based EBV genome was able to make virus and was capable of B-
cell immortalization [29]. To monitor infection of primary B-cell
by EBV, we introduced the GFP ORF into the EBV BAC by
homologous recombination and the resultant construct was
designated as BAC GFP-EBV [29]. The construction of BAC
GFP-EBV was confirmed by exhaustive restriction enzyme
digestion pattern, PCR analysis of the junctions, selected regions
of EBV as well as southern blot analysis. In addition, the new BAC
Figure 7. Latent and lytic gene expression during GFP-EBV early infection. 5610
6
of PBMC cells were infected with GFP-EBV. (A) The
designated time of GFP-EBV postinfection (6h, 12h, 24h, 48h, 72h, 96h, 120h and 168h) of early infection, the expression of latent genes EBNA-1,
EBNA-2 and LMP-1 mRNAs were examined by qReal Time PCR. (B) The lytic genes mRNAs BZLF1 which is the immediate early transcriptional and
replication protein, major capsid protein BcLF1 and DNA polymerase BALF5 mRNA were also examined by qReal Time PCR after GFP-EBV infection at
similiar intervals stated above. To determine quality of the RNA, GAPDH mRNA was also amplified by RT-PCR. The fold change was calculated by the
DDCt method. Each data point shown is the average of three identical experiments. 6SD was shown in error bar.
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GFP-EBV clone was also competent for virus replication and B-
cell immortalization. The induction of 293T cells stably main-
taining the BAC GFP-EBV by chemical inducers produced
progeny recombinant virus which was used to infect primary B-
cells leading to the generation of LCLs expressing GFP. These
studies strongly suggested that the recombinant GFP-EBV had
similar infectious properties when compared to the wild type EBV.
The advantage of this GFP expressing BAC-EBV system is that
EBV infection and propagation in mammalian cells can now be
monitored from the early stages post-infection to transformation of
B-cells and generation of LCLs.
The infection of human B lymphocytes by EBV in vitro results in
immortalization of the infected cells and augmentation of
numerous B-cell surface antigens[65]. We wanted to obtain a
more detailed picture of the early events after EBV infection and
to monitor changes in expression of cell surface markers as a result
of infection. We used infection of PBMCs by GFP-EBV to monitor
the early stages post-infection. We used a panel of surface antigen
markers CD5, CD10, CD19, CD23, CD39, CD40 & CD44 and
the intracellular marker Ki-67 to correlate B-cell activation with
proliferation during the early stages of infection up to 7 days. GFP
positive cells were evaluated for expression of the surface proteins
indicated. CD19, a specific surface antigen marker for B-cells
[66,67], was detected in greater than 90% of the GFP positive
cells. This strongly suggested that the GFP-EBV infected cells were
predominantly B-cells among the mixed population of PBMCs.
The cell surface protein CD5 expression on B cell can be up-
regulated by a number of agents which results in B-cell activation
[35]. CD5 has been shown to be important for apoptosis of
antigen-receptor induced B lymphocytes [68]. The expression of
CD5 was also shown to be regulated by EBV [37]. CD5
expression was suppressed in EBV transformed cells suggesting
that that the virus may down-regulate its expression to prevent
apoptosis of the transformed cell. Our data during the early stages
Figure 8. Lytic burst of EBV virus during early infection of GFP-EBV. PBMC cells were infected with GFP-EBV and at specific times
postinfection the supernanat was collected and used infect fresh PBMC cells. (A) Phase-contrast (left) and fluorescence (right) images of GFP-EBV
infected PBMC cells are shown after specific times postinfection (24 hrs, 72 hrs, 120 hrs and 168 hours) (left panel). Phase-contrast (left) and
fluorescence (right) images of PBMC cells infected with supernatant from the above mentioned times post-infection (from 24hrs, 72hrs, 120hrs and
168h) (right panel). B. Flow cytometry analysis of GFP expression at post-infection of different time intervals (6h, 12h, 24h, 48h, 72h, 96h, 120h and
168h) (left panel), and after infection of fresh PBMC from supernatant of infected cells (right panel).
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of EBV infection showed that CD5 levels increased from 6 hrs to 5
days but was depressed after 5 days to relatively low levels seen in
LCLs. Our data supports previous studies monitoring CD5 levels
in transformed B-cell [37]. However, during the initial days of
EBV infection (up to 5 days), the increased expression of CD5 is
likely to be due to signaling as a result of virus-host interaction and
resulting B-cells activation. Also, the infection of primary B-cell by
EBV leads to killing of a large percentage of cells due to lytic
replication during the early stage of infection. This resulting cell
death may also be a reason for up-regulation of CD5 expression
during the early stages of infection. CD10, another cell surface
protein, preferentially expressed in germinal center is an activation
marker for B-cells as the germinal center is the site for activation
and proliferation of B-cells [69]. The expression of CD10 was
unchanged after 6 hrs post-infection during the course of early
infection of EBV. This suggests that the initial interaction between
EBV and B-cells led to a rapid change in CD10 expression, which
was maintained after 6 hrs followed by activation and cell
proliferation.
The B-cell activation markers CD23, CD40 and CD44 have
been shown to be associated with EBV infection [13]. Addition-
ally, the viral proteins EBNA-2 and LMP-1 cooperatively induce
the cell surface protein, CD23 [13]. CD23 expression induced by
infection of B-cell with GFP-EBV showed an interesting trend in
that CD23 expression increased and reached maximum levels by
72 hrs but then dramatically decreased by 7 days. However,
EBNA-2 was expressed at the initial stage of early infection and
reached maximum within 24 hours. These results suggest that the
expression of EBNA-2 at the initial stage activates the CD23
expression which continued to increase up to 72 hrs. In addition,
LMP-1 expression was detected after 48 hours which is also known
to up-regulate CD23 expression [70]. Moreover, the increase in
lytic replication by EBV during the early stages post infection is
also expected to lead to cell death. This provides a possible
Figure 9. The progeny virus produced in the primary infection is inhibited by acyclovir. PBMCs were infected with GFP-EBV in
presence and absence of 25 mM of ACV (infection-I) and at specific times postinfection the supernatant was collected and used
infect fresh PBMCs cells (infection II). (A) Phase-contrast (left) and fluorescence (right) images of GFP-EBV infected PBMC cells are shown after
specific times postinfection (24 hrs, 72hrs, 120 hrs and 168 hours) in absence of ACV (left panel) and in presence of 25 mM ACV (right panel). (B) Phase-
contrast (left) and fluorescence (right) images of PBMC cells infected with supernatant from the above mentioned times post-infection from 24hrs,
72hrs, 120hrs and 168h (infection-II) in absence (left panel) and presence of 25 mM ACV (right panel). (C) Flow cytometry analysis of GFP (left panel)
and Ki-67(right panel) expression at post-infection of different time intervals (6h, 12h, 24h, 48h, 72h, 96h, 120h and 168h) in absence and presence of
25 mM ACV. D. DNA polymerase BALF5 mRNA was also examined by qReal Time PCR after GFP-EBV infection at similiar intervals stated above in
absence and presence of 25 mM ACV. To determine quality of the RNA, GAPDH mRNA was also amplified by RT-PCR. The fold change was calculated
by the DDCt method. Each data point shown is the average of three identical experiments. 6SD was shown in error bar.
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explanation for the observed down-regulation of CD23 after 72
hrs. However, other possibilities exist in that additional latent
antigens may also contribute to CD23 regulation and that the
early increase in CD23 levels are important for persistence or
signaling events that eventually leads to cell proliferation and
transformation. The Cell surface protein CD40 is mainly
expressed in antigen presenting cells, and plays a critical role in
B-cell activation by providing cell survival signals via interaction
with the CD40 ligand (CD40L) expressed on the surface of
activated T-cells [71]. It is reported that signaling CD40 and its
ligand CD40L contributes and is likely to be critical for the
antiapoptotic function of EBV and B cell transformation in the
presence of LMP1 after EBV infection [72]. Our results showed
that the expression level of CD40 from GFP positive cells (infected
cells) increased from 6 hrs post-infection and continued to increase
up to 7 days, and are maximally expressed in transformed EBV
positive LCLs. The increased expression of CD40 due to EBV
infection also activates the CD40 signaling pathway which
suppresses apoptosis and promotes proliferation of infected cells
[73]. CD44 is a cell adhesion molecule which exists in multiple
isoforms associated with tumorigenesis and metastasis [74]. The
level of CD44 was detected by 6 hrs post-infection and was
maintained throughout our study suggesting an important
contribution to the proliferation and transformation process.
Ki-67 is a nuclear antigen that is expressed in proliferating cells
during the different phases of the cell cycle and its expression is used
as a marker for cell proliferation [34]. It is reported that Ki-67 is
expressed in CD19 positive cells in B-CLL [42]. It was also observed
that Ki-67 positive cells infected with EBV express EBNA2 [42]. We
showed that expression of Ki-67 post-infection of B-cell by GFP-EBV
was detected by 48 hrs and consistently increased after 7 days as well
as in EBV transformed cells. Therefore, B-cell proliferation is most
likely initiated a few hours after EBV infection eventually leading to
B-cell transformation and LCLs. The delayed expression of Ki-67 (at
48 hours) is likely to be due to lytic replication of infected cells at the
early stages of infection where the infected cells may not survive but
produces progeny capable of infecting new cells that eventually
persists and establishes latency. This result strongly supports the
hypothesis that during the early stages of EBV infection primary B-
cells undergo lytic replication important for persistence of the virus,
latency and transformation to LCLs. It is also possible that the lytic
genes may contribute and is important to the upregulation of cellular
genes important for driving cell proliferation and transformation.
In an effort to understand the latent and lytic gene expression
profile during early stage of EBV infection we used semi-
quantitative real time PCR to determine the levels of transcript
for the latent genes EBNA-1, EBNA-2 and LMP-1 [7] as well as
the immediate early, early and late lytic genes BZLF1, BALF5 and
BcLF1 [14]. Data from the Real time PCR showed that the latent
genes EBNA-1, EBNA-2 and LMP-1 were expressed along with
the lytic genes BZLF1, BALF5 and BcLF1.
Alfieri et al.[75] prevoiusly showed latent gene expression by
immunostainig during initial stages of infection (up to 3 days) of
PBMCs by EBV. They showed that EBNA-1 expressed at 20–32 hrs
post-infection and reached levels seen in LCLs at 46–70 hrs post-
infection, whereas EBNA-2 expressed at 16 hrs post-infection and
LMP-1 expressed in 48 h post-infection. Expression of EBNA-1 and
EBNA-2 was similiar in our studies. However, we observed that
LMP-1 was expressed at an earlier time post-infection (maximum at
24 hours) when compared to the previous report (maximum at 48
hours) [75]. However, Yuan et al. [76] reported that induction of
lytic infection by IgG crosslinking, EBNA and LMP mRNA were
expressed which supports our result. Since the gene expression
profile of our results suggests that after infection with GFP-EBV,
PBMCs establishes latent infection as well as lytic replication at
during the early stages of post-infection. As infected cells are
undergoing latent and lytic replication at the same time and are in
the overall cell population, it would be difficult to measure the
precise number of cells in the lytic or latent phase of infection and the
cells expressing LMP-1 during the early stages of infection. Further,
studies are ongoing to determine whether or not the expression
patterns we see are directly related to latent or lytic replication.
Since BZLF1 was strongly expressed during the early phase of
infection the lytic genes are then induced resulting in progeny and
infection of new cells. Expression of the DNA polymerase (BALF5)
and major capsid protein BcLF1, also indicate that virion particles
were produced which was confirmed by infection of fresh PBMCs
using supernatant from primary infection. The infection of fresh
PBMCs strongly showed that virion particles were produced
during the early stages of infection (Figure 8). To determine if the
production of virion particles occurred during the early stages of
Figure 10. Glycoprotein expression during early stage of
infection in presence of acyclovir. Endogenous expression of (A)
gp110 and (B) gp350 were detected using mouse monoclonal antibody
(1:200 dilution), and rabbit respectively (1:250 dilution). Primary
antibodies were diluted in blocking buffer and incubated with fixed
cells for 1 h at RT. Slides were washed three times (5 min each) with PBS
and incubated with appropriate secondary antibody (1:2000) for 1 h at
RT followed by three times washes (5 min each) with PBS. The last wash
contained 49,69-diamidino-2-phenylindole (DAPI; Promega Inc., Madi-
son, WI) for nuclear staining. Goat anti-mouse antibody Alexa Fluor 594
and goat anti-rabbit antibody Alexa Fluor 594 were purchased from
Molecular Probes Inc. (Carlsbad, CA). Slides were then washed in PBS
and mounted using Prolong anti-fade (Molecular Probes Inc, Carlsbad,
CA). Fluorescence was viewed by confocal microscopy and analyzed
with Fluoview 300 software from Olympus Inc. (Melville, NY). The
images were sequentially captured using an Olympus confocal
microscope. All panels are representative pictures from similar repeat
experiments.
doi:10.1371/journal.pone.0007214.g010
Early Events in EBV Infection
PLoS ONE | www.plosone.org 14 September 2009 | Volume 4 | Issue 9 | e7214

infection, we used acyclovir in the course of infection. The
inhibition of viral DNA polymerase expression as well as the late
lytic protein (gp110 and gp350) expression due to addition of ACV
strongly supported our conclusion that virion particles produced
were due lytic replication and not from virus produced on from the
initial infection (see Figure 9 and Figure 10). Additionally, the
insertion of the cassette in the B95-8 deletion site was the least
deleterious in affecting changes in lytic replication as BZLF1 is
located at the distant position in the genome from the insertion site
and that induction of the other late genes are dependent on
BZLF1 expression. Thus, we were confident that the early lytic
replication is important to contributing to establishment of latency
and transformation of primary infected B-cells. Additionally, since
latent genes were also produced during the early infection, there is
most likely a finely tuned mechanism for early induction of lytic
genes and viral progeny production important for triggering cell
proliferation and a switch from a lytic type infection to a latent
infection in the newly infected cells.
LCL were initially believed to arise from direct outgrowth of in
vivo-infected EBV–carying B-cells (One step mechanism)[77].
However, Rickinson et al. [78,79] showed that cell lines can also
be generated in two steps: release of virus from infected cells
during the initial period of in vitro cultivation, followed by the
secondary immortalization of normal B-cells in vitro. Lewin et
al.[80] showed experimentally that the 2-step mechanism is more
common. Our results also suggest that the 2-step mechanism (the
virus particles, due to productive cycle at the early stage of
infection, competent for infection of uninfected B-cells and
immortalization of B-cells) is the most probable for infected
primary B-cells to be driven to transformation by EBV infection in
vitro (see Figure 11). However, studies are ongoing to carefully
address these questions and will provide a more detailed molecular
mechanism of this process. Additionlly, the induction of lytic genes
may also be committed to a small population of the infected cells
that eventually dies but are critical for induction of the
proliferative genes and switching to a latent type infection. It
would certainly be important to determine whether the infected
cells can undergo same level of lytic replication, survive and is
eventually switch to latent infection and transformation or whether
only a committed number of cells in the population goes lytic
replication, dies but is critical for the remainder of the population
to survive and B transformed.
Acknowledgments
The authors would like to thank Prof. Fred Wang, Harvard Medical
School, MA for the gift of the MD1BAC which allowed us to successfully
generate the functional BAC GFP-EBV.
Author Contributions
Conceived and designed the experiments: SH ESR. Performed the
experiments: SH FY. Analyzed the data: SH. Contributed reagents/
materials/analysis tools: SH MM SCV PK ESR. Wrote the paper: SH
ESR.
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