Cell Host & Microbe
Article
An ATM/Chk2-Mediated DNA Damage-Responsive
Signaling Pathway Suppresses Epstein-Barr Virus
Transformation of Primary Human B Cells
Pavel A. Nikitin,1,4 Christopher M. Yan,1,4 Eleonora Forte,1,5 Alessio Bocedi,1 Jason P. Tourigny,1 Robert E. White,2
Martin J. Allday,2 Amee Patel,3 Sandeep S. Dave,3 William Kim,3 Katherine Hu,1 Jing Guo,1 David Tainter,1 Elena Rusyn,1
and Micah A. Luftig1,*
1Department
of Molecular Genetics and Microbiology, Center for Virology, Duke University School of Medicine, Durham, NC 27712, USA
of Virology, Faculty of Medicine, Imperial College London, Norfolk Place, London W2 1PG, UK
3Duke Institute for Genome Sciences and Policy, Duke University, Durham, NC 27712, USA
4These authors contributed equally to this work
5Present address: Division of Infectious Diseases, Department of Internal Medicine, University of Utah, Salt Lake City, UT 84132, USA
*Correspondence: micah.luftig@duke.edu
DOI 10.1016/j.chom.2010.11.004
2Department
SUMMARY
Epstein-Barr virus (EBV), an oncogenic herpesvirus
that causes human malignancies, infects and immortalizes primary human B cells in vitro into indefinitely
proliferating lymphoblastoid cell lines, which represent a model for EBV-induced tumorigenesis. The
immortalization efficiency is very low, suggesting
that an innate tumor suppressor mechanism is operative. We identify the DNA damage response (DDR) as a
major component of the underlying tumor suppressor
mechanism. EBV-induced DDR activation was not
due to lytic viral replication, nor did the DDR marks
colocalize with latent episomes. Rather, a transient
period of EBV-induced hyperproliferation correlated
with DDR activation. Inhibition of the DDR kinases
ATM and Chk2 markedly increased transformation
efficiency of primary B cells. Further, the viral latent
oncoprotein EBNA3C was required to attenuate the
EBV-induced DDR. We propose that heightened
oncogenic activity in early cell divisions activates
a growth-suppressive DDR that is attenuated by viral
latency products to induce cell immortalization.
INTRODUCTION
Epstein-Barr virus (EBV) is an oncogenic herpesvirus causally
implicated in several malignancies including African endemic
Burkitt’s lymphoma (BL), posttransplant lymphoproliferative
disease, nasopharyngeal carcinoma, and HIV-associated lymphomas (Kieff and Rickinson, 2006). EBV infection in vitro drives
primary human B cells into indefinitely proliferating lymphoblastoid cell lines (LCLs), providing a model for tumorigenesis. This
process of growth transformation depends on a subset of viral
latent oncoproteins and noncoding RNAs collectively termed
‘‘latency III.’’ The proteins expressed include the Epstein-Barr
nuclear antigens, EBNA1, -2, -3A, -3B, -3C, and -LP as well as
three latent membrane proteins, LMP1, -2A, and -2B. EBNALP and EBNA2 are the first viral proteins expressed following
primary B cell infection (Alfieri et al., 1991) and upregulate cellular
genes inducing a transition of resting B cells into the cell cycle
(Sinclair et al., 1994; Wang et al., 1991). EBNA2 also induces
expression of the remaining EBNA proteins (Zimber-Strobl
et al., 1993) and subsequently the viral latent membrane
proteins, LMP1 and LMP2A/2B (Wang et al., 1990).
While the initial burst of viral and cellular gene expression
leads to the proliferation of infected cells in vitro, only a small
percentage of infected cells become indefinitely proliferating
lymphoblasts (Henderson et al., 1977; Sugden and Mark,
1977). The study of EBV-induced innate tumor suppressor pathways has been limited. EBV infection of primary B cells induces
the p53 protein concomitant with EBNA-LP expression early
after infection (Szekely et al., 1995). However, it remains unclear
whether this innate response to EBV-induced proliferation has
any long-term functional consequence or what pathways activate p53.
Innate tumor suppressor responses have been better characterized in other systems. The DNA damage response (DDR) has
recently been appreciated as an important tumor suppressor
pathway in vitro and in vivo (Bartkova et al., 2005; Gorgoulis
et al., 2005). The DDR is triggered by aberrant replication structures generated by activated oncogenes attempting to constitutively fire new origins and inappropriately enter S phase
(Halazonetis et al., 2008). The DDR limits aberrant proliferation
by mediating oncogene-induced senescence and apoptosis
(Bartkova et al., 2006; Di Micco et al., 2006). Signaling downstream of oncogenic stress involves activation of the singlestranded DNA-dependent ATR pathway and the doublestranded break-induced ATM pathway. These DDR kinases
relay downstream signals to critical repair factors and other
checkpoint kinases including Chk1 and Chk2 with extensive
crosstalk ultimately resulting in suppression of oncogeneinduced proliferation (Halazonetis et al., 2008; Stiff et al., 2006).
Genetic experiments have identified critical roles for ATM and
Chk2 in mediating oncogene-induced senescence and tumor
suppression (Bartkova et al., 2006; Pusapati et al., 2006;
Stracker et al., 2008). Given these observations and the low
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ATM/Chk2 Pathway Suppresses EBV Transformation
Figure 1. EBV Induced a DNA Damage Response in Primary B Cells
(A) Indirect IF images of EBNA-LP (green) and g-H2AX (red) in uninfected B cells, B cells 4 and 7 days after infection with EBV B95-8 (moi 5), the recently derived
LCL EF3D, and uninfected g-irradiated B cells (0.2, 1, and 5 Gy, 1 hr). DNA is stained with DAPI. These images are representative of infections in five different
normal donors.
(B) Ser1981 phosphorylated ATM (pATM, red) in uninfected B cells and B cells 7 days after EBV B95-8 infection. EBNA-LP or other EBV latent antigen staining was
not possible in these samples due to antibody source; however, we know that the majority of these infected cells are EBNA-LP positive.
(C) EBNA-LP (green) and 53BP1 (red) in uninfected B cells and B cells 7 days after infection.
(D) EBNA-LP (green) and g-H2AX (red) in B cells 4 days after infection with UV-inactivated EBV B95-8 (UV EBV) or the nontransforming EBV strain P3HR1 (P3).
efficiency of EBV transformation, the intriguing question remains
as to whether the host DDR senses EBV-induced oncogenic
stress and, importantly, if this is responsible for the block to
long-term outgrowth of the majority of infected cells.
RESULTS
Epstein-Barr Virus Infection of Primary B Cells Activates
a Cellular DNA Damage Response
We first sought to determine whether EBV infection of primary
B cells might drive an oncogenic stress leading to the activation
of the DDR. Purified CD19+ B cells were infected with the proto-
typical transforming EBV strain B95-8 at a multiplicity of infection
(moi) of 5. Nearly all cells were EBV genome positive as determined by fluorescence in situ hybridization (FISH) (see Figure S1A available online). Infected cells were initially assayed
for the expression of the earliest viral latency gene product,
EBNA-LP (LP), and the DNA damage marker, g-H2AX, at
different times postinfection. g-H2AX activation was not evident
prior to 4 days postinfection, was robust from 4 to 7 days postinfection, and declined after 7 days to the low levels observed in
LCLs (Figure 1A and data not shown). Approximately 60% of the
infected cells were g-H2AX positive at 7 days postinfection.
Corroborating our findings of g-H2AX activation, EBV infection
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ATM/Chk2 Pathway Suppresses EBV Transformation
induced additional hallmarks of the DDR including autophosphorylation of the H2AX kinase ATM (pATM Ser1981), and
punctate localization of the damage adaptor 53BP1 (Figures
1B and 1C).
EBV gene expression was important for virus-induced DDR
activation. Cells infected with UV-inactivated B95-8 virus did
not show g-H2AX staining at any point within the first week after
infection (Figure 1D and data not shown). Importantly, UV-inactivated EBV B95-8 genomes reached the nucleus, and these
infections induced interferon-responsive genes (Figures S1A
and S1B). EBNA2 and latency III gene expression was specifically necessary to induce the DDR, as B lymphocytes infected
with the EBNA2-deleted, transformation-incompetent P3HR1
strain of EBV did not contain g-H2AX foci (Figure 1D), despite
similar levels of infection compared to B95-8 (Figures S1A and
S1C). These data collectively demonstrate that EBV latent
gene expression, rather than simply virion binding or nucleic
acid deposition into the nucleus, was required to induce
g-H2AX activation.
The EBV-Induced DNA Damage Response in Primary
B Cell Infection Is Not Associated with Viral Episomes
or Lytic Replication
We reasoned that either viral or cellular DNA may activate the
DDR. Since evidence in the literature suggested that either viral
lytic DNA replication (Kudoh et al., 2005) or latent viral episome
replication (Dheekollu et al., 2007) may be capable of inducing
a DDR, we first assayed viral DNA as a possible source of the
damage. Incoming linear viral DNA was not the source of the
damage, since UV-irradiated and EBNA2-deleted P3HR1 virus
infections did not induce the DDR (Figure 1). We next used
a FISH-based assay to assess the possible role of lytic DNA
replication. The B95-8 Z-HT cell line was used as a positive
control where lytic EBV DNA was recognized as a brightly staining FISH signal rather than the punctate foci of episomal
genomes (Figure S1D). Less than 1% of EBV-infected cells contained evidence of lytic viral DNA 5 days postinfection, while
1%–5% of infected cells were spontaneously undergoing lytic
replication by 14 days, similar to that found in LCLs (Figure S1E
and Kieff and Rickinson, 2006). Since far greater than 1% of
EBV-infected cells were g-H2AX positive early after infection,
we conclude that viral lytic DNA replication is not responsible
for DDR activation.
Next we assessed the possibility that latent viral episomes
activate the DDR. The mean episome number per cell as
assessed by FISH did not increase during the period when
g-H2AX activity was high early after infection (Figure S1F).
Furthermore, we failed to observe significant colocalization of
EBV episomes with g-H2AX foci in these cells (Figure S1G). In
fact, the number of g-H2AX foci per cell was consistently much
greater than the number of EBV genomes (Figure S1G). Therefore, our data collectively suggest that the observed EBVinduced DDR is not activated by viral DNA.
The EBV-Induced DNA Damage Response Is Associated
with a Transient Period of Hyperproliferation
We next focused our studies on changes in cellular DNA that may
induce a DDR. The period of time postinfection when the DDR
was active correlates with the initiation of B cell proliferation
(Kieff and Rickinson, 2006). Analysis of CD19+ B cells using
the proliferation-tracking dye CFSE at different days after infection indicated that (1) proliferating cells appeared at day 3 (Figure 2A), (2) between days 3 and 4 there were always cells that
had divided more than once or even twice in 24 hr, and (3) at later
days postinfection cells appeared to proliferate at a slower rate
as judged by the less-pronounced shift of the CFSE profile to
the left.
A more rigorous kinetic analysis of EBV-induced B cell expansion highlighted the biphasic nature of the proliferation rate
(Figure 2B). Infected CD19+ B cell CFSE profiles from five normal
donors were analyzed at time points prior to and during the first
seven cell divisions. The mean division number (MDN) at each
time point was determined by fitting the precursor-normalized
number of cells in each division to a Gaussian distribution (Figure S2A and Hawkins et al., 2007). The slope of the function
relating MDN to time postinfection inversely correlates with the
proliferation rate. Consistent with the data in Figure 2A, we
observed that EBV induced an early phase of hyperproliferation
that was attenuated over time (Figure 2B). The proliferation rate
of initially proliferating cells was approximately once per 8–12 hr,
while later cycles were 24–30 hr, similar to the 24–28 hr rate of
LCLs. These findings were corroborated by cell-sorting experiments where cells from earlier divisions proliferated more quickly
than those in later divisions (Figure S2B). Thus, EBV-mediated B
cell expansion proceeds through an initial period of hyperproliferation followed by slower cell divisions typical of emergent
LCLs.
We next asked whether the DDR was activated specifically
during the hyperproliferative divisions independent of time postinfection. EBV-infected B cells sorted based on population
doubling (PD) were subjected to immunofluorescence (IF)
for EBNA-LP and g-H2AX (Figure 2C). Sorted cells were >85%
EBNA-LP positive in cells not yet dividing (PD0) and >95%
EBNA-LP positive in all later PDs. We observed a robust
increase in LP+/g-H2AX+ cells during the early PDs (1–2 and
3–4) relative to uninfected cells or infected cells not yet proliferating (PD0) (Figures 2C and 2D). Importantly, this response was
attenuated through later PDs and in LCLs. Moreover, g-H2AX
intensity per cell was significantly higher in PD3-4 than PD0
(p < 0.0001) and LCL (p < 0.0001). We also observed a transient
activation and attenuation of the ATM-specific phosphorylation
of Chk2 on Thr68 (Figure 2E) as well as accumulation of
53BP1 into DDR foci (Figure 2F). These data strongly support
the notion that the EBV-induced DDR is caused by an early
period of hyperproliferation and is attenuated during LCL
outgrowth.
Proliferation and DNA Damage-Responsive Genes
Are Highly Induced Early after EBV Infection,
then Attenuated during LCL Outgrowth
Our cell-based findings were corroborated by mRNA microarray
studies of (1) uninfected B cells, (2) EBV-infected early proliferating cells (Prolif), and (3) monoclonal LCLs from four normal
donors (Figure 3). We first asked in an unbiased manner
which genes were significantly changed upon proliferation and
then, subsequently, during LCL outgrowth (two-way ANOVA,
p < 0.01). As expected, the most enriched gene ontology (GO)
category for genes induced from resting B cells to EBV-infected,
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Figure 2. EBV Induced a Period of Hyperproliferative Early after Infection that Was Associated with Activation of the DNA Damage Response
(A) Histograms show CD19+ B cell division as measured by CFSE staining at different days after EBV infection. Mock, mock infected cells.
(B) The MDN based on precursor cohort analysis for EBV-infected B cells is plotted at different times post infection. Vertical dashed lines estimate the hyperproliferation period. Data are presented from five normal donors.
(C) IF of g-H2AX (red) and EBNA-LP (green) in uninfected cells, infected cells that have yet to divide (PD0), infected cells after one or two divisions (PD1-2), or
seven or more divisions (PD7+) and LCLs. DNA is stained with DAPI.
(D) The percentage of EBNA-LP-positive cells with g-H2AX signal >53 over background is graphed from uninfected B cells, sorted PDs, and LCLs. Uninfected
B cells following 0.2, 1, and 5 Gy (1 hr) g-irradiation are also shown as a positive control. These data are representative of similar experiments from three independent normal donors.
(E) Immunoblot of p-Chk2 Thr68 and Chk2 in sorted cells as in (D) including an LCL following 5 Gy g-irradiation (1 hr).
(F) The percentage of EBNA-LP-positive cells containing four or more 53BP1 foci per cell in sorted populations as in (D) are shown along with uninfected irradiated
B cell controls. PD3-4 contained significantly more 53BP1 foci per cell than uninfected B cells (p < 0.0001), PD0 (p < 0.0001), PD7 (p < 0.01), and LCL (p < 0.0001).
proliferating B cells was ‘‘Cell Proliferation’’ (Figure 3A; GO,
0008283; Bayes factor, 51, p < 0.0001 [Chang and Nevins,
2006]). Genes associated with the ‘‘Response to DNA Damage
Stimulus’’ were also highly induced (Figure 3B; GO, 0006974;
Bayes factor, 17, p < 0.0001). Notably, we observed that the
majority of genes involved in cell proliferation and the DDR
were consistently repressed as cells transitioned from early
proliferating to established LCLs (Figure 3A, ‘‘Cell Proliferation,’’
Bayes factor, 63, p < 0.0001 and Figure 3B, ‘‘Response to DNA
Damage Stimulus,’’ Bayes factor, 22, p < 0.0001). Consistently,
the expression of genes in an independently derived set of DNA
damage responsive and ATM-dependent p53 targets (Elkon
et al., 2005) was also increased in early proliferating cells and
subsequently attenuated during LCL outgrowth (Figure 3C and
Figure S3). Collectively, these global gene expression analyses
corroborate our findings of a period of hyperproliferation and
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Figure 3. Transcriptional Changes Correlate with an EBV-Induced Early Period of Hyperproliferation and DNA Damage Response Followed
by Attenuation upon LCL Outgrowth
(A) Heat map of average expression data across four normal donors for the GO category ‘‘Cell Proliferation’’ in uninfected resting B cells (B cell), EBV-infected
early proliferating B cells (Prolif), and monoclonal LCLs (LCL). The genes presented were derived from GATHER analysis of all genes with significant expression
changes (two-way ANOVA, p < 0.01) where the expression level increased from B cell to Prolif at least 1.5-fold and decreased from Prolif to LCL at least 1.2-fold
(left). Heat map of individual samples of top 20 ‘‘Cell Proliferation’’ genes (right).
(B) Heat map of ‘‘Response to DNA Damage Stimulus’’ GO genes across individual samples.
(C) Gene Set Enrichment Analysis (GSEA) of known DNA damage induced ATM and p53-dependent genes in the context of B-Prolif-LCL expression data. The
reference list of ATM/p53 target genes was derived from clusters 2 and 3 of Elkon et al. (2005) and compared with a preranked list (by fold) of global average gene
expression changes from B cell to Prolif (left) and Prolif to LCL (right). Statistical scores are inset into the top right of analysis images (NES, normalized enrichment
score; and FWER, familywise error rate).
activation of an ATM-dependent DDR early after infection that is
attenuated during LCL outgrowth.
The EBV-Induced Hyperproliferation-Associated DNA
Damage Response Is Growth Suppressive
To further analyze the consequences of the activated DDR in
early rapidly proliferating cells, we designed a sorting strategy
to assess the relative growth potential and DDR activation in
cells derived from early or late divisions after infection (Figure 4A).
We initially stained cells with the proliferation tracking dye
PKH26 and sorted cells after infection for PD1-4 and PD6+ populations (Figure 4B). Subsequent staining with CFSE enabled the
analysis of proliferation from these populations. Supporting our
hypothesis, the cells in early hyperproliferating divisions
(PD1-4) were, in fact, more prone to growth arrest and cell death
than those in later divisions (PD6+) and LCLs (Figure 4C). Consistently, arrested PD1-4 cells displayed more intense g-H2AX
staining than their proliferating counterparts (Figure 4D).
ATM and Chk2 Kinases Suppress EBV-Mediated
Transformation and Initial B Cell Proliferation
To determine if the activation of the DDR restricts EBV-mediated
long-term outgrowth, we simultaneously infected peripheral
blood mononuclear cells (PBMCs) with EBV and treated
them with an inhibitor of either ATM (ATMi [Hickson et al.,
2004]) or its downstream effector kinase Chk2 (Chk2i [Arienti
et al., 2005]); both are critical kinases in the DDR checkpoint responding to DNA double-stranded breaks and oncogenic stress.
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Figure 4. Growth Suppression and DNA Damage Enrichment in Early Cell Divisions
(A) A flowchart shows the separation of arrested and proliferating EBV-infected CD19+ PBMCs used for IF and FACS. PBMCs were first infected with EBV and
labeled with the red fluorescent dye PKH26.
(B) Then, 8 days postinfection, proliferating CD19+ B cells were sorted for PD1-4 and PD6+ based on PKH26 intensity, labeled with CFSE, and cultured for 2 days.
(C) Sorted cells were then analyzed in a FACS-based growth assay where cells in the CFSElow population were considered proliferating and cells in the CFSEhi
population were considered arrested. Forward scatter (FSC) low reflects dying cells. Results are representative of three normal donors.
(D) PKH26low (PD1-4) cells were subsequently labeled with CFSE as above, sorted after 48 hr in culture into CFSEhi (arrested) and CFSElow (proliferating) populations, and analyzed by IF for g-H2AX (red).
EBV-mediated B cell transformation efficiency increased in a
dose-dependent manner in response to the inhibitors where 2 mM
ATMi increased efficiency by 2-fold and 5 mM ATMi by 6-fold
over DMSO control-treated cells (Figure 5A). Similarly, Chk2 inhibition increased EBV transformation efficiency 3-fold for 2 mM
Chk2i and 9-fold for 5 mM Chk2i (Figure 5B). Therefore, an
ATM- and Chk2-dependent DDR restricts EBV transformation.
We next assessed whether ATM and Chk2-mediated suppression of EBV transformation was due to limiting initial B cell proliferation. The continuous presence of either ATM or Chk2 inhibitor
led to a dose-dependent increase in infected B cell number at
2 weeks postinfection (Figure 5C). Importantly, ATM or Chk2
inhibitor did not induce B cell proliferation in the absence of
EBV, suggesting that these compounds act to alleviate a block
to proliferation rather than stimulating B cells per se (Figure 5D).
ATM and Chk2 Suppress B Cell Growth 4–8 Days
after EBV Infection
Since the DDR peaked during the first week after infection, we
assessed when ATM and Chk2 inhibition enhanced proliferation
and transformation. To that end, PBMCs infected with EBV were
transiently exposed to ATMi and Chk2i from the start of infection
or the compounds were added at different days postinfection.
EBV-induced B cell proliferation was most sensitive to the inhibitors between 4 and 8 days after infection when cells were
present in the hyperproliferative period (Figure 5E). For example,
when either inhibitor was added within the first 4 days of infection, we observed as pronounced an effect on proliferation as
if the inhibitor was added at day 0. However, if we added inhibitors after day 8, there was no effect on proliferation. Conversely,
if the inhibitors were removed prior to 4 days after infection, then
increased proliferation was not observed.
Similar results were obtained in long-term transformation
assays. Addition of either compound within 4 days of infection
increased transformation efficiency, while adding the compounds at 12 days postinfection had little effect (Figures 5F
and 5G). The inhibitors also did not increase LCL growth rates
at normal or limiting density (Figure S4 and data not shown).
Therefore, during a critical period 4–8 days following infection,
EBV induced an ATM- and Chk2-dependent growth suppressive
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Figure 5. Inhibition of ATM and Chk2 Kinases Increased EBV Transformation Efficiency and Proliferation of B Cells during a Critical Period
4–8 Days Postinfection
(A) Quantification of EBV-induced B cell outgrowth following PBMC infection in the presence of 0.1% DMSO (black), 2 mM ATMi (green), or 5 mM ATMi (red). The
percentages of wells positive for LCLs at 5 weeks postinfection are plotted relative to the transforming units (TU) of B95-8 virus per well. Results shown are the
average of experiments with at least four independent normal donors. Error bars represent standard error of the mean (SEM). Dotted line represents 62.5% positive wells, which, based on a Poisson’s distribution, indicates outgrowth from the virus amount in the x axis of a single LCL per well.
(B) Similar experiments were performed as in (A), except using DMSO (black), 2 mM Chk2i (green), or 5 mM Chk2i (red).
(C) CFSE-stained PBMCs were infected with EBV in the presence of increasing amounts of ATMi or Chk2i (0, 1, 2, 5, and 10 mM). The percentage of CD19+/CFSElow
cells of total PBMCs at 14 days postinfection are plotted. The data shown are the average values from two different donors ± SEM. These data are representative of more than five independent experiments.
(D) Dot plots show CFSE- and CD19-stained PBMCs that were treated with DMSO, 5 mM ATMi, or 5 mM Chk2i or infected with EBV for 6 days.
(E) This table summarizes when ATM and Chk2 suppressed EBV-mediated proliferation at different times following infection. CFSE-stained PBMCs were infected
with EBV at day 0. ATMi or Chk2i (5 mM) was added at different times after infection (top bars) or at day 0 and washed out at different times after infection (bottom bars).
EBV-mediated B cell proliferation was detected by FACS at day 14 postinfection using CD19-PE and CFSE as in (C). A more than 2-fold increase in treated cells
versus DMSO is represented by a green plus, a less than 2-fold increase is represented by a yellow plus, and no increase is represented by a red dash. The lines
indicate the period of incubation and are colored with the proliferation phenotype after ATMi and Chk2i treatment. Average values from two independent donors
are shown.
(F) EBV-induced outgrowth following PBMC infection was measured as in (A) in the presence of 5 mM Chk2i (blue), 5 mM ATMi (red), or DMSO (black) added at day 0,
day 4, or day 12 after EBV infection. Results shown are the average of four independent normal donors ± SEM.
(G) Efficiency of EBV outgrowth from (F) was calculated, and the average ratio of inhibitor-treated to DMSO-treated infections ± SEM for four normal donors is plotted.
signaling pathway that limited initial B cell proliferation and,
consequently, long-term outgrowth into LCLs.
EBV Latent Gene Expression Changes and
Consequences in Early Infected Cell Divisions
The dynamic changes in proliferation and DDR-associated gene
expression support our cell-based assays indicating an early
period of ATM/Chk2-mediated growth suppression that is
attenuated in later divisions enabling long-term LCL outgrowth.
However, to determine whether these changes correlated with
viral gene expression, we queried viral transcripts and proteins
associated with the latency III growth program in sorted PDs after
infection (Figure 6 and Figure S5). Wp-associated transcripts were
expressed at a markedly higher level than Cp transcripts prior to
the first infected cell division (PD0) (Figure 6A). However, this ratio
shifted such that Cp levels were greater after three to four cell
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Figure 6. EBV Latency and Consequential
Host Gene Expression Changes from Initial
B Cell Proliferation through LCL Outgrowth
(A) Expression of Wp (filled triangles)- and Cp
(open circles)-derived mRNAs in EBV-infected
cells sorted by PD and monoclonal LCLs. Relative
mRNA abundance normalized to a b-actin control
is plotted versus PD. These data are representative of two normal donors and consistent with published time course experiments (Woisetschlaeger
et al., 1989, 1990).
(B) The ratio of Wp to Cp mRNA expression levels
from (A) is plotted versus division and through LCL
outgrowth.
(C) Protein expression of EBNA-LP, EBNA2,
EBNA3A, and EBNA3C are shown from sorted
infected PDs and a polyclonal LCL from the
same donor.
(D) Proteins detected by western blotting from
three independent normal donors similar to those
in (C) were quantified. The average ratio of total
EBNA-LP protein (i.e., all isoforms) relative to total
EBNA3A or EBNA3C ± SEM is plotted versus PD
through LCL.
(E) Average CD23 surface expression as mean
fluorescence intensity (MFI) is plotted versus
PD ± SEM for two donors.
(F) (Left) The expression level of c-Myc mRNA is
plotted versus sorted PD. (Right) The activity of
the c-Myc target gene expression signature (Bild
et al., 2006) is plotted from the average expression
of targets in microarray samples from four independent donors of resting B cells (B), early proliferating B cells (Prolif), and monoclonal LCLs. Error
bars represent SEM.
divisions and through LCL outgrowth consistent with previous
observations (Figure 6B and Schlager et al., 1996; Woisetschlaeger et al., 1989; Woisetschlaeger et al., 1990). The consequence of the high Wp/Cp ratio was heightened levels of
EBNA-LP protein as well as a heightened EBNA-LP to EBNA3A
and -3C protein ratio in early divisions that waned through LCL
outgrowth (Figures 6C and 6D). Thus, the initial cell divisions characterized by hyperproliferation display a distinct EBNA gene
expression equilibrium that may affect EBNA2 target gene
expression.
To more rigorously assess this, we analyzed EBNA2 targets
including CD23 (Wang et al., 1991) and c-Myc (Kaiser et al.,
1999). In both cases, these EBNA2 targets were highly induced
in early cell divisions and then attenuated through LCL
outgrowth, still remaining significantly higher than resting B cell
levels (Figures 6E and 6F). The consequences of the transient
increase in c-Myc mRNA was manifested in an increase of the
c-Myc target gene expression signature (Bild et al., 2006) during
early proliferation that was attenuated in LCLs, though still
greater than resting B cell levels (Figure 6F). Given the importance in titrating this potentially genotoxic oncoprotein and the
known role of ATM in suppressing c-Myc oncogenesis (Hong
et al., 2006; Murphy et al., 2008; Pusapati et al., 2006), these
findings strongly support a model of acute oncogenic stress
early after EBV infection that is modulated through the welldescribed Wp to Cp switch enabling modest EBNA2 activity critical for indefinite EBV-infected cell outgrowth.
EBNA3C Is Required to Attenuate the EBV-Induced DNA
Damage Response
While the induction of the DDR after EBV infection requires
latent gene expression and proliferation, a definitive role for viral
latent genes in attenuating this response was not demonstrated.
In order to determine which latent genes are critical for DDR
attenuation during late divisions after infection, we chose to
interrogate the EBNA3 proteins, EBNA3A and EBNA3C, as
they are known to modulate EBNA2 activity. Infection of primary
B cells with EBV B95-8, EBNA3A knockout (KO), or EBNA3C KO
virus (Anderton et al., 2008) supported early B cell proliferation
(Figures S6A–S6C). However, upon sorting these early proliferating cells we observed that EBNA3C KO virus-infected cells
displayed increased activation of the DDR, while EBNA3A KO-infected cells were similar to WT B95-8 infection in DDR activation
(Figures 7A and 7B). Indeed, greater than 80% of EBNA3C
KO-infected cells were g-H2AX positive relative to 50% of
WT or EBNA3A KO-infected cells (Figure 7C). Similarly, EBNA3C
KO-infected cells accumulated 53BP1 DDR foci to a greater
extent than WT or EBNA3A KO-infected cells (p < 0.001, 3C
KO versus WT; p > 0.1, 3A KO versus WT). Thus, while B cells
infected with either EBNA3A KO or EBNA3C KO virus were crippled for long-term outgrowth (Figures S6B and S6C), these
experiments define a critical role for EBNA3C in attenuating
the host DDR to EBV infection early after infection. These data
strongly support our model of a latent gene expression-triggered
hyperproliferation-induced DDR, followed by proper expression
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ATM/Chk2 Pathway Suppresses EBV Transformation
Figure 7. EBNA3C Attenuates the EBV-Induced DNA Damage Response
(A) Representative IF images are shown of g-H2AX staining (red) from WT, EBNA3A KO (D3A), and EBNA3C KO (D3C) infected and sorted PD1-4 B cells. DAPI
DNA stained (blue) and DAPI/g-H2AX merged images are also shown.
(B) Representative IF images are shown of 53BP1 staining (red) from WT, D3A, and D3C infected and sorted PD1-4 B cells.
(C) Quantification of IF data from (A) is plotted as percentage g-H2AX-positive cells. Average values are plotted for infected cells, uninfected B cells, and 5 Gy
g-irradiated B cells.
(D) Model for EBV-induced DDR/hyperproliferative period and attenuation during LCL outgrowth. Early in infection EBNA2 and EBNA-LP associate with cellular
transcription factors (TF) to potently upregulate expression of growth control genes and B cell activation markers, including c-Myc and CD23, activating the host
DDR (left). Later in infection, the activity of the EBNA3 proteins, in particular EBNA3C, downregulate EBNA2 function, as LMP1 and LMP2 are upregulated and
may cooperate in the constitutive but attenuated expression of host growth control genes and enhanced cell survival (right).
of the EBNA3 proteins, in particular EBNA-3C, in order to attenuate a potentially genotoxic and growth-suppressive signaling
pathway (Figure 7D).
DISCUSSION
It has long been recognized that EBV transformation efficiency is
on the order of 1%–10% of infected primary human B cells
(Henderson et al., 1977; Sugden and Mark, 1977). However, little
is known about the molecular mechanism responsible for this
low efficiency. We hypothesize that a robust innate tumor
suppressor response is activated by latent viral oncoproteins
and blocks outgrowth of the majority of infected cells. Recent
evidence suggests that activated oncogene expression is sufficient to trigger a growth-suppressive DNA damage responsive
signaling pathway (Halazonetis et al., 2008), and other oncogenic viruses, including the Kaposi’s sarcoma-associated
herpesvirus, have been shown to induce the DDR after infection
or when viral oncoproteins are expressed in primary cells (Dahl
et al., 2005; Koopal et al., 2007). Therefore, in this study we
asked whether EBV was capable of inducing a DDR in primary
B cells and, importantly, whether this response resulted in the
low transformation efficiency. We observed that as EBV-infected
cells initiated proliferation, a transient DDR was activated as
evidenced by phosphorylation of ATM Ser1981, H2AX Ser139
(g-H2AX), Chk2 Thr68, and accumulation of 53BP1 in nuclear
foci. Modulation of this signaling pathway by chemical antagonism of ATM and its downstream target Chk2 markedly
increased EBV-mediated B cell polyclonal expansion and transformation efficiency, thereby demonstrating that the DDR
518 Cell Host & Microbe 8, 510–522, December 16, 2010 ª2010 Elsevier Inc.
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ATM/Chk2 Pathway Suppresses EBV Transformation
contributes to an EBV-induced innate tumor suppressor
pathway. This study identifies a molecular pathway that restricts
EBV transformation.
The Source of DNA Damage
Toward characterizing the EBV-induced DNA damage signal, we
reasoned that either viral or cellular DNA was important for ATM
activation. In addition to oncogenic stress, replication intermediates of DNA viruses and retroviruses contain double-stranded
DNA ends that activate ATM (Lilley et al., 2007). In fact, EBV lytic
replication induces a DDR which is suppressed by inhibition of
downstream transcriptional activation of p53 (Kudoh et al.,
2005; Mauser et al., 2002). In our studies of primary B cell infection, however, we found no evidence of viral lytic DNA replication
or viral DNA associated with DDR activation. First, we did not
observe DDR activation within the first 3 days after EBV infection,
nor did we observe activation using UV-inactivated virus or
the nontransforming EBV variant P3HR1, suggesting that the
incoming linear DNA genome and tegument proteins within the
virion were not responsible for this signal. Second, lytic viral
DNA was not responsible for DDR activation, as less than 1%
of infected cells were undergoing lytic DNA replication when
greater than 50% of infected cells were g-H2AX positive. Third,
we asked whether the DNA damage signal was derived from viral
episomes since DNA repair factors are recruited to the episome
to ensure proper resolution of Holliday junctions following
episome replication (Deng et al., 2002; Dheekollu et al., 2007).
We observed little increase in episome number per cell and
found that viral episomes and g-H2AX did not colocalize during
the period of DDR activation. These data collectively demonstrate that viral DNA is not the source of DNA damage. Our
experiments cannot rule out the possibility, however, that viral
lytic gene expression downstream of BZLF1 in the absence of
lytic DNA replication (Kalla et al., 2010) plays a role in the transient DDR early after infection. Despite this possibility, we
inferred from our data that viral latent gene expression causes
an oncogenic stress response leading to cellular DNA damage.
The initiation of cell proliferation defines the period after EBV
infection when ATM and Chk2 were active in suppressing transformation. Rigorous analysis of infected cell division rates
uncovered a period of hyperproliferation where early population
doublings (PDs) were every 8–12 hr leading to DDR activation,
while later divisions displayed an attenuated rate of 24–30 hr
per division, similar to LCLs, and had little evidence of DDR activation. Microarray analysis of gene expression during the transition from resting B cell to early EBV-induced hyperproliferation
and through LCL outgrowth strongly supported our cell-based
observations. Specifically, genes involved in proliferation and
the DDR, including ATM/p53-dependent targets (Elkon et al.,
2005), were highly induced early after infection and then attenuated during the transition to LCL. We propose that aberrant
induction of cellular DNA replication early after EBV infection
activates a DDR that is dependent on EBNA2- and EBNA-LPmediated upregulation of S phase promoting oncoproteins
including c-Myc, cyclin D2, and E2F1 (Kaiser et al., 1999; Sinclair
et al., 1994; and Figure 6). Indeed, we observed increased
expression of c-Myc and its gene activation signature in hyperproliferating cells relative to LCLs. Furthermore, EBNA-LP
protein levels and Wp-derived transcripts were heightened
during this early period relative to EBNA3 proteins and Cp transcripts, consistent with previous analysis of the initial cascade of
viral latent gene expression at different days postinfection
(Schlager et al., 1996; Woisetschlaeger et al., 1989, 1990).
Finally, EBNA3C- but not EBNA3A-deleted virus-infected cells
displayed a significantly stronger DDR during early proliferation.
Thus, while both EBNA3A and EBNA3C likely mitigate growth
arrest in LCLs through p16 suppression (Hertle et al., 2009; Skalska et al., 2010), during early outgrowth EBNA3C is also required
to modulate the DDR. Collectively, our data support a model in
which initial EBV-driven hyperproliferation leads to an oncogenic
stress that is ultimately attenuated as EBNA3 proteins moderate
EBNA2 driven c-Myc expression and its genotoxic and growth
suppressive consequences. This ultimate balance in viral and
host gene expression enables constitutive S phase induction
without driving selection of cells with genomic instability.
In Vivo Implications
Our findings have implications pertaining to the germinal center
model for EBV infection (Roughan and Thorley-Lawson, 2009) in
the context of B cell lymphomagenesis. In particular, our
observed hyperproliferative phase early after infection in vitro
may be similarly induced by EBV in vivo and is reminiscent of
B cell proliferation rates in the germinal center (MacLennan,
1994). Bcl-6 downregulation of the DDR mitigates the consequences of centroblast hyperproliferation in the germinal center
(Ranuncolo et al., 2007), while EBV potently suppresses Bcl-6
early after infection, leaving DDR checkpoints intact (Siemer
et al., 2008). In vivo, an EBV-induced hyperproliferative period
after primary infection may promote extrafollicular B cell maturation or drive EBV-infected naive B cells into GCs. However, a
critical balance must be struck between the aberrant latent
oncoprotein-driven proliferation early after infection and the
stable proliferative signals found in LCLs to maintain an activated, immortalized state. Perturbations in this balance in vivo
may select for mutations driving lymphomagenesis. For example, the IgH/c-myc translocation common in BL may be the
consequence of such an event. Given our findings, it is plausible
that imbalances in EBV latent gene expression may provide
a milieu of cells with an increased potential for genomic instability. Recent work in BL cell lines suggests that this is likely
the case.
EBV infection of BL cell lines or heterologous expression of
EBNA1, EBNA3C, or LMP1 in BL cell lines increased the
frequency of nonclonal chromosomal aberrations (Gruhne
et al., 2009a, 2009b). EBNA1 increased reactive oxygen species
(ROS) through transcriptional upregulation of NOX2, EBNA3C
perturbed mitotic spindle checkpoints through BubR1 downregulation, and LMP1 attenuated ATM protein levels and
decreased DNA repair. EBNA3C has also been shown to modulate the activity of Chk2 (Choudhuri et al., 2007). However, these
three viral proteins are constitutively expressed in LCLs in the
absence of overt genomic aberrations. Therefore, we expect
that these findings unmask activities that may link the aforementioned potential for imbalanced gene expression to tumorigenesis. In our system, we did not observe increased transformation
in the presence of antioxidants including N-acetyl cysteine or
citric acid (data not shown), suggesting that EBNA1-induced
ROS was not responsible for the EBV-induced DDR. We also
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ATM/Chk2 Pathway Suppresses EBV Transformation
did not observe changes in BubR1 or ATM expression through
LCL outgrowth (data not shown). However, we anticipate that
genomic instability may ensue in the setting of aberrant latent
oncoprotein expression that may exist in BL and other EBVassociated tumors. Consistent with this notion and our findings,
a recent report suggests that while LCLs maintain a stable karyotype, early DNA damaging events may lead to nonclonal chromosomal aberrations including telomere fusions (Lacoste
et al., 2009). This report supports our findings of an early hyperproliferation-associated oncogenic stress that may induce such
structures leading to ATM activation (Karlseder et al., 1999) and
suppression of long-term outgrowth. Thus, only cells with the
ability to maintain a stable karyotype emerge as LCLs.
Summary
Our study provides the characterization of an innate tumor
suppressor pathway that regulates EBV immortalization of B
cells. This pathway depends on ATM and Chk2, which are activated early after infection during a period of hyperproliferation.
The initial high level expression of EBNA2 and EBNA-LP target
genes such as c-Myc leads to DDR activation. Following this
initial period, the activity of additional viral latent proteins,
including EBNA3C and possibly LMP1 and LMP2, is important
for attenuating early gene expression targets, limiting activation
of the DDR, and ensuring cell survival. The end result of these
dynamic changes in viral and cellular gene expression is
outgrowth of a constitutively activated LCL harboring a stable
karyotype. However, perturbations in this gene expression
program through loss of upstream control by viral latent proteins
may lead to the progression of EBV-associated lymphomas. Our
studies provide a model for the study of EBV transformation
accounting for dynamic viral and host changes during the early
period following primary B cell infection.
EXPERIMENTAL PROCEDURES
Antibodies
Primary antibodies to gH2AX, pATM Ser1981, and 53BP1 (Cell Signaling
#2577, #4526, and #4937) were used for IF at 1:50. Alexa488 goat a-mouse
and Alexa 568 goat a-rabbit were used as secondary antibodies (Molecular
Probes #A-11029 and #A-11011). Rabbit a-human Chk2 or pChk2 Thr68
(Cell Signaling #2662 or #2661) and mouse a- EBNA-LP (JF-186, 1:250),
EBNA2 (PE2, 1:100), EBNA3A (Exalpha #F115P, 1:500), and EBNA3C (A10,
gift of E. Johannsen 1:2500) were used for western blotting.
Fluorescence Microscopy
Immunofluorescence
3 3 105 B cells were pelleted, washed in PBS, resuspended in 25 ml of PBS,
spread on a microscope slide and dried at 37 C for 20 min then fixed in 4%
PFA in PBS for 15 min, permeabilized in PBS containing 0.5% Tween-20 for
20 min then blocked in PBS with 0.2% Tween-20 containing 5% normal
goat serum for 1 hr. Indirect IF was performed as described in Bridger and
Lichter (1999). Slides were mounted in Vectashield containing DAPI (Vector
Laboratories).
Fluorescence In Situ Hybridization
Cells were fixed in methanol acetic acid as described previously (Sullivan and
Warburton, 1999). The EBV genome containing bacterial artificial chromosome
MD-1 (kindly provided by F. Wang, Harvard Medical School) was labeled with
fluorescent green-dUTP using a nick translation kit (Abbott Molecular)
following the manufacturer’s instructions. Slides were denatured in 70%
formamide, hybridized, and washed as described previously (Sullivan and
Warburton, 1999) except with using fluorescent probes, slides were not
blocked then incubated with a fluorescent secondary antibody.
IF/FISH
Slides were fixed for IF in 2% paraformaldehyde in PBS. Antibodies were
added to slides and denatured as previously described (Sullivan and Warburton, 1999) except that slides were denatured for 6 min. Slides were hybridized
and washed as described above.
Data analysis was performed as described in the Supplemental Experimental Procedures.
Flow Cytometry Analysis
B Cell Proliferation Assays
6-carboxyfluorescein succinimidyl ester (CFSE, Sigma, #21888)-stained or
CellTrace Violet (Invitrogen, #C34557)-stained PBMCs were infected with
EBV and incubated with different concentrations of ATMi, Chk2i, or DMSO
during different periods. Proliferation of CD19+ cells was assayed by flow cytometry as a ratio of CFSElow cells to total PBMCs at 14 days after infection.
Detailed kinetics of EBV proliferation was determined as described in Figure S3
(Hawkins et al., 2007).
ACCESSION NUMBERS
Exon array data have been deposited in the Gene Expression Omnibus (GEO)
as GSE20200.
SUPPLEMENTAL INFORMATION
Supplemental Information includes six figures, Supplemental Experimental
Procedures, and Supplemental References and can be found with this article
at doi:10.1016/j.chom.2010.11.004.
ACKNOWLEDGMENTS
We thank E. Cahir-McFarland and R. Valdivia for critical reading of the manuscript and E. Johannsen for critical discussion. We thank N. Mukherjee for
discussions on microarray analysis. We thank M. Garcia-Blanco for use of
the Olympus fluorescent microscope. We thank L. Martinek, N. Martin, and
M. Cook for extensive help and discussions in flow cytometry-based experiments. We thank F. Wang for providing the MD1-BAC for our FISH experiments and E. Kieff for JF186 and PE2 antibodies. This work was supported
by ACS, Duke CFAR, and Golfers Against Cancer pilot awards to M.A.L.
Received: February 9, 2010
Revised: June 25, 2010
Accepted: November 4, 2010
Published: December 15, 2010
REFERENCES
Alfieri, C., Birkenbach, M., and Kieff, E. (1991). Early events in Epstein-Barr
virus infection of human B lymphocytes. Virology 181, 595–608.
Anderton, E., Yee, J., Smith, P., Crook, T., White, R.E., and Allday, M.J. (2008).
Two Epstein-Barr virus (EBV) oncoproteins cooperate to repress expression of
the proapoptotic tumour-suppressor Bim: clues to the pathogenesis of
Burkitt’s lymphoma. Oncogene 27, 421–433.
Arienti, K.L., Brunmark, A., Axe, F.U., McClure, K., Lee, A., Blevitt, J., Neff,
D.K., Huang, L., Crawford, S., Pandit, C.R., et al. (2005). Checkpoint kinase
inhibitors: SAR and radioprotective properties of a series of 2-arylbenzimidazoles. J. Med. Chem. 48, 1873–1885.
Bartkova, J., Horejsi, Z., Koed, K., Kramer, A., Tort, F., Zieger, K., Guldberg, P.,
Sehested, M., Nesland, J.M., Lukas, C., et al. (2005). DNA damage response
as a candidate anti-cancer barrier in early human tumorigenesis. Nature 434,
864–870.
Bartkova, J., Rezaei, N., Liontos, M., Karakaidos, P., Kletsas, D., Issaeva, N.,
Vassiliou, L.V., Kolettas, E., Niforou, K., Zoumpourlis, V.C., et al. (2006).
Oncogene-induced senescence is part of the tumorigenesis barrier imposed
by DNA damage checkpoints. Nature 444, 633–637.
520 Cell Host & Microbe 8, 510–522, December 16, 2010 ª2010 Elsevier Inc.
Cell Host & Microbe
ATM/Chk2 Pathway Suppresses EBV Transformation
Bild, A.H., Yao, G., Chang, J.T., Wang, Q., Potti, A., Chasse, D., Joshi, M.B.,
Harpole, D., Lancaster, J.M., Berchuck, A., et al. (2006). Oncogenic pathway
signatures in human cancers as a guide to targeted therapies. Nature 439,
353–357.
Bridger, J.M., and Lichter, P. (1999). Analysis of mammalian interphase chromosomes by FISH and immunofluorescence. In Chromosome Structural
Analysis, W.A. Bickmore, ed. (Oxford: Oxford University Press), pp. 103–121.
Chang, J.T., and Nevins, J.R. (2006). GATHER: a systems approach to interpreting genomic signatures. Bioinformatics 22, 2926–2933.
Choudhuri, T., Verma, S.C., Lan, K., Murakami, M., and Robertson, E.S. (2007).
The ATM/ATR signaling effector Chk2 is targeted by Epstein-Barr virus nuclear
antigen 3C to release the G2/M cell cycle block. J. Virol. 81, 6718–6730.
Dahl, J., You, J., and Benjamin, T.L. (2005). Induction and utilization of an ATM
signaling pathway by polyomavirus. J. Virol. 79, 13007–13017.
Deng, Z., Lezina, L., Chen, C.J., Shtivelband, S., So, W., and Lieberman, P.M.
(2002). Telomeric proteins regulate episomal maintenance of Epstein-Barr
virus origin of plasmid replication. Mol. Cell 9, 493–503.
Dheekollu, J., Deng, Z., Wiedmer, A., Weitzman, M.D., and Lieberman, P.M.
(2007). A role for MRE11, NBS1, and recombination junctions in replication
and stable maintenance of EBV episomes. PLoS ONE 2, e1257. 10.1371/
journal.pone.0001257.
Di Micco, R., Fumagalli, M., Cicalese, A., Piccinin, S., Gasparini, P., Luise, C.,
Schurra, C., Garre, M., Nuciforo, P.G., Bensimon, A., et al. (2006). Oncogeneinduced senescence is a DNA damage response triggered by DNA hyperreplication. Nature 444, 638–642.
Elkon, R., Rashi-Elkeles, S., Lerenthal, Y., Linhart, C., Tenne, T., Amariglio, N.,
Rechavi, G., Shamir, R., and Shiloh, Y. (2005). Dissection of a DNA-damageinduced transcriptional network using a combination of microarrays, RNA
interference and computational promoter analysis. Genome Biol. 6, R43.
Gorgoulis, V.G., Vassiliou, L.V., Karakaidos, P., Zacharatos, P., Kotsinas, A.,
Liloglou, T., Venere, M., Ditullio, R.A., Jr., Kastrinakis, N.G., Levy, B., et al.
(2005). Activation of the DNA damage checkpoint and genomic instability in
human precancerous lesions. Nature 434, 907–913.
Gruhne, B., Sompallae, R., Marescotti, D., Kamranvar, S.A., Gastaldello, S.,
and Masucci, M.G. (2009a). The Epstein-Barr virus nuclear antigen-1
promotes genomic instability via induction of reactive oxygen species. Proc.
Natl. Acad. Sci. USA 106, 2313–2318.
Gruhne, B., Sompallae, R., and Masucci, M.G. (2009b). Three Epstein-Barr
virus latency proteins independently promote genomic instability by inducing
DNA damage, inhibiting DNA repair and inactivating cell cycle checkpoints.
Oncogene 28, 3997–4008.
Halazonetis, T.D., Gorgoulis, V.G., and Bartek, J. (2008). An oncogene-induced
DNA damage model for cancer development. Science 319, 1352–1355.
Hawkins, E.D., Hommel, M., Turner, M.L., Battye, F.L., Markham, J.F., and
Hodgkin, P.D. (2007). Measuring lymphocyte proliferation, survival and differentiation using CFSE time-series data. Nat. Protoc. 2, 2057–2067.
Henderson, E., Miller, G., Robinson, J., and Heston, L. (1977). Efficiency of
transformation of lymphocytes by Epstein-Barr virus. Virology 76, 152–163.
Hertle, M.L., Popp, C., Petermann, S., Maier, S., Kremmer, E., Lang, R.,
Mages, J., and Kempkes, B. (2009). Differential gene expression patterns of
EBV infected EBNA-3A positive and negative human B lymphocytes. PLoS
Pathog. 5, e1000506. 10.1371/journal.ppat.1000506.
Hickson, I., Zhao, Y., Richardson, C.J., Green, S.J., Martin, N.M., Orr, A.I.,
Reaper, P.M., Jackson, S.P., Curtin, N.J., and Smith, G.C. (2004).
Identification and characterization of a novel and specific inhibitor of the
ataxia-telangiectasia mutated kinase ATM. Cancer Res. 64, 9152–9159.
Hong, S., Pusapati, R.V., Powers, J.T., and Johnson, D.G. (2006). Oncogenes
and the DNA damage response: Myc and E2F1 engage the ATM signaling
pathway to activate p53 and induce apoptosis. Cell Cycle 5, 801–803.
Kaiser, C., Laux, G., Eick, D., Jochner, N., Bornkamm, G.W., and Kempkes, B.
(1999). The proto-oncogene c-myc is a direct target gene of Epstein-Barr virus
nuclear antigen 2. J. Virol. 73, 4481–4484.
Kalla, M., Schmeinck, A., Bergbauer, M., Pich, D., and Hammerschmidt, W.
(2010). AP-1 homolog BZLF1 of Epstein-Barr virus has two essential functions
dependent on the epigenetic state of the viral genome. Proc. Natl. Acad. Sci.
USA 107, 850–855.
Karlseder, J., Broccoli, D., Dai, Y., Hardy, S., and de Lange, T. (1999). p53- and
ATM-dependent apoptosis induced by telomeres lacking TRF2. Science 283,
1321–1325.
Kieff, E., and Rickinson, A. (2006). Epstein-Barr virus and its replication. In
Fields Virology, D.M. Knipe and P.M. Howley, eds. (Philadelphia: Lippincott,
Williams, and Wilkins), pp. 2603–2654.
Koopal, S., Furuhjelm, J.H., Jarviluoma, A., Jaamaa, S., Pyakurel, P.,
Pussinen, C., Wirzenius, M., Biberfeld, P., Alitalo, K., Laiho, M., et al. (2007).
Viral oncogene-induced DNA damage response is activated in Kaposi
sarcoma tumorigenesis. PLoS Pathog. 3, 1348–1360.
Kudoh, A., Fujita, M., Zhang, L., Shirata, N., Daikoku, T., Sugaya, Y., Isomura,
H., Nishiyama, Y., and Tsurumi, T. (2005). Epstein-Barr virus lytic replication
elicits ATM checkpoint signal transduction while providing an S-phase-like
cellular environment. J. Biol. Chem. 280, 8156–8163.
Lacoste, S., Wiechec, E., Dos Santos Silva, A.G., Guffei, A., Williams, G.,
Lowbeer, M., Benedek, K., Henriksson, M., Klein, G., and Mai, S. (2009).
Chromosomal rearrangements after ex vivo Epstein-Barr virus (EBV) infection
of human B cells. Oncogene 29, 503–515.
Lilley, C.E., Schwartz, R.A., and Weitzman, M.D. (2007). Using or abusing:
viruses and the cellular DNA damage response. Trends Microbiol. 15, 119–126.
MacLennan, I.C. (1994). Germinal centers. Annu. Rev. Immunol. 12, 117–139.
Mauser, A., Saito, S., Appella, E., Anderson, C.W., Seaman, W.T., and Kenney,
S. (2002). The Epstein-Barr virus immediate-early protein BZLF1 regulates p53
function through multiple mechanisms. J. Virol. 76, 12503–12512.
Murphy, D.J., Junttila, M.R., Pouyet, L., Karnezis, A., Shchors, K., Bui, D.A.,
Brown-Swigart, L., Johnson, L., and Evan, G.I. (2008). Distinct thresholds
govern Myc’s biological output in vivo. Cancer Cell 14, 447–457.
Pusapati, R.V., Rounbehler, R.J., Hong, S., Powers, J.T., Yan, M., Kiguchi, K.,
McArthur, M.J., Wong, P.K., and Johnson, D.G. (2006). ATM promotes
apoptosis and suppresses tumorigenesis in response to Myc. Proc. Natl.
Acad. Sci. USA 103, 1446–1451.
Ranuncolo, S.M., Polo, J.M., Dierov, J., Singer, M., Kuo, T., Greally, J., Green,
R., Carroll, M., and Melnick, A. (2007). Bcl-6 mediates the germinal center
B cell phenotype and lymphomagenesis through transcriptional repression
of the DNA-damage sensor ATR. Nat. Immunol. 8, 705–714.
Roughan, J.E., and Thorley-Lawson, D.A. (2009). The intersection of EpsteinBarr virus with the germinal center. J. Virol. 83, 3968–3976.
Schlager, S., Speck, S.H., and Woisetschlager, M. (1996). Transcription of the
Epstein-Barr virus nuclear antigen 1 (EBNA1) gene occurs before induction of
the BCR2 (Cp) EBNA gene promoter during the initial stages of infection in
B cells. J. Virol. 70, 3561–3570.
Siemer, D., Kurth, J., Lang, S., Lehnerdt, G., Stanelle, J., and Kuppers, R.
(2008). EBV transformation overrides gene expression patterns of B cell differentiation stages. Mol. Immunol. 45, 3133–3141.
Sinclair, A.J., Palmero, I., Peters, G., and Farrell, P.J. (1994). EBNA-2 and
EBNA-LP cooperate to cause G0 to G1 transition during immortalization of
resting human B lymphocytes by Epstein-Barr virus. EMBO J. 13, 3321–3328.
Skalska, L., White, R.E., Franz, M., Ruhmann, M., and Allday, M.J. (2010).
Epigenetic repression of p16(INK4A) by latent Epstein-Barr virus requires the
interaction of EBNA3A and EBNA3C with CtBP. PLoS Pathog. 6, e1000951.
Stiff, T., Walker, S.A., Cerosaletti, K., Goodarzi, A.A., Petermann, E.,
Concannon, P., O’Driscoll, M., and Jeggo, P.A. (2006). ATR-dependent phosphorylation and activation of ATM in response to UV treatment or replication
fork stalling. EMBO J. 25, 5775–5782.
Stracker, T.H., Couto, S.S., Cordon-Cardo, C., Matos, T., and Petrini, J.H.
(2008). Chk2 suppresses the oncogenic potential of DNA replication-associated DNA damage. Mol. Cell 31, 21–32.
Sugden, B., and Mark, W. (1977). Clonal transformation of adult human leukocytes by Epstein-Barr virus. J. Virol. 23, 503–508.
Sullivan, B.A., and Warburton, P.E. (1999). Studying progression of vertebrate chroosomes through mitosis by immunofluorescence and FISH. In
Cell Host & Microbe 8, 510–522, December 16, 2010 ª2010 Elsevier Inc. 521
Cell Host & Microbe
ATM/Chk2 Pathway Suppresses EBV Transformation
Chromosome Structural Analysis, W.A. Bickmore, ed. (Oxford: Oxford
University Press), pp. 81–100.
Szekely, L., Pokrovskaja, K., Jiang, W.Q., Selivanova, G., Lowbeer, M.,
Ringertz, N., Wiman, K.G., and Klein, G. (1995). Resting B-cells, EBV-infected
B-blasts and established lymphoblastoid cell lines differ in their Rb, p53 and
EBNA-5 expression patterns. Oncogene 10, 1869–1874.
Wang, F., Tsang, S.F., Kurilla, M.G., Cohen, J.I., and Kieff, E. (1990). EpsteinBarr virus nuclear antigen 2 transactivates latent membrane protein LMP1.
J. Virol. 64, 3407–3416.
Wang, F., Kikutani, H., Tsang, S.F., Kishimoto, T., and Kieff, E. (1991). EpsteinBarr virus nuclear protein 2 transactivates a cis-acting CD23 DNA element.
J. Virol. 65, 4101–4106.
Woisetschlaeger, M., Strominger, J.L., and Speck, S.H. (1989). Mutually exclusive use of viral promoters in Epstein-Barr virus latently infected lymphocytes.
Proc. Natl. Acad. Sci. USA 86, 6498–6502.
Woisetschlaeger, M., Yandava, C.N., Furmanski, L.A., Strominger, J.L., and
Speck, S.H. (1990). Promoter switching in Epstein-Barr virus during the
initial stages of infection of B lymphocytes. Proc. Natl. Acad. Sci. USA 87,
1725–1729.
Zimber-Strobl, U., Kremmer, E., Grasser, F., Marschall, G., Laux, G., and
Bornkamm, G.W. (1993). The Epstein-Barr virus nuclear antigen 2 interacts
with an EBNA2 responsive cis-element of the terminal protein 1 gene
promoter. EMBO J. 12, 167–175.
522 Cell Host & Microbe 8, 510–522, December 16, 2010 ª2010 Elsevier Inc.
Cell Host & Microbe, Volume 8
Supplemental Information
An ATM/Chk2-Mediated DNA Damage-Responsive
Signaling Pathway Suppresses Epstein-Barr Virus
Transformation of Primary Human B Cells
Pavel A. Nikitin, Christopher M. Yan, Eleonora Forte, Alessio Bocedi, Jason P. Tourigny,
Robert E. White, Martin J. Allday, Amee Patel, Sandeep S. Dave, William Kim,
Katherine Hu, Jing Guo, David Tainter, Elena Rusyn, and Micah A. Luftig
Supplemental Information Inventory
1. Supplemental figures (6)
Figure S1. FISH and quantitative RT-PCR data (A-C) demonstrating that P3HR1 and UVinactivated EBV B95-8 infected primary B cells as efficiently as B95-8. These controls are
important to assess the relevance of the data indicating that UV-inactivated B95-8 and P3HR1
infections shown in Figure 1 were unable to induce the DNA damage response. Panels D-G
demonstrate the ability of our FISH assay to distinguish latent from lytically infected cells,
characterization of latent/lytic DNA in early infected B cells, quantitation of viral episomes per
cell during early infection, and analysis of the association between DDR signals and EBV
genomes by IF/FISH.
Figure S2. This figure shows first (A) a schematic of the flow for experimentally deriving the
mean division number using CFSE-based proliferation assays. In part B, the proliferation of cells
sorted by CFSE labeling indicates that early divisions contain a higher capacity to divide (i.e.
faster rate) than later divisions. These data corroborate our findings in Figure 2.
Figure S3. This figure provides a schematic flowchart for how the experiments present in Figure
3C were performed.
Figure S4. This figure demonstrates that inhibition of ATM or Chk2 in established LCLs does
not increase proliferation. These data support those presented in Figure 5F where addition of
the inhibitors 12 days after infection did not impact long-term outgrowth.
Figure S5. Expression of viral latency genes measured by qPCR supports expression data in
Figure 6.
Figure S6. Detection of viral latency proteins in WT, EBNA3A KO, and EBNA3C KO infections
as well as early and late proliferation data from these infection. These data support Figure 7.
2. Supplemental Experimental Procedures
A description of all experimental procedures and data analysis are provided here in full.
3. Supplemental References
Figure S1, related to Figure 1. Nuclear genome deposition and the analysis of viral and host gene
expression following primary B cell infection with EBV B95-8, UV-inactivated B95-8, P3-HR1 (A-C). EBV
genomes in infected primary B cells are independent of γ-H2AX foci (D-G). (A) Fluorescence in situ
hybridization (FISH) of viral genomes 2 days post B cell infection with EBV B95-8, UV-inactivated B95-8
(UV B95-8), and P3HR1 (P3) at a multiplicity of infection (MOI) of ~5. (B) Quantitative RT-PCR of the
interferon responsive mRNAs ISG15 and IFIT4 24h following PBMC infection with B95-8 and UVinactivated B95-8. mRNA levels were normalized to GAPDH and primers were as described previously
(Martin et al., 2007) (C) Quantitative RT-PCR for Wp-initiated mRNAs 48h after PBMC infection with B958, UV-B95-8, or P3 virus strains. Analysis of the relative levels of W0 – W1/W2 containing mRNAs
indicate that infection was equivalent between B95-8 and P3, while UV-inactivated virus was essentially
unable to produce Wp-initiated transcripts. mRNAs were normalized to GAPDH and primers for these
experiments were previously described (Bell et al., 2006). (D) Fluorescence in situ hybridization (FISH) of
EBV genomes (green) in B95-8 Z-HT cells (Johannsen et al., 2004). Uninduced cells (-HT) mostly
contained latent episomes (left). A representative cell undergoing lytic replication following exposure to
4-hydroxytamoxifen (+HT) is shown on the right. (E) EBV FISH in B cells 5 days and 14 days after
infection. Intense lytic staining was rarely observed 5 days after infection (left). Approximately 1-5% of
cells were undergoing lytic DNA replication by FISH at 14 days (right). (F) Left, Representative images of
EBV genomic FISH in primary CD19+ B cells at 1 and 5 days post infection with B95-8 (MOI ~5), steady
state 8-10 genomes in a LCL, and 50 genomes per Raji cell. Right, Episome number as determined by
FISH at different times after infection. The y-axis represents the average number of episomes per cell,
the x-axis represents days after infection when cells were collected and fixed for hybridization (5, 7, 10,
14, 21, and 35 days). Vertical dashed lines show the beginning and end of the period in which the DNA
damage response (DDR) was observed. Error bars represent SEM. These data were collected from
greater than 50 nuclei at each time point for three independent normal donors. (G) Left, IF/FISH of
γH2AX (red) and EBV genomes (green) in B cells 7 days post infection. Left cell shows IF/FISH, right cell
shows only IF for γH2AX with white arrows representing EBV genome location. The right panels are
additional representative IF/FISH and IF images.
Figure S2, related to Figure 2. CFSE-based kinetic analysis of EBV-induced proliferation. (A) Analysis of
EBV-infected PBMC stained by CFSE was performed by FACS at different time of infection (141 hour post
infection point is shown, left). CFSE-based population doublings (PDs) of CD19+ B cells were determined
using the “Proliferation” tool in the FlowJo program. Number of cells in each PD (N) was normalized to
averaged division number i (i=i+0.5) and fit to a Gaussian distribution for each time point. Mean division
number was determined as shown (MDN=3.09 for 141 hour p.i. for a given donor, middle). Then, MDN
over time post infection plot was generated for each time point (right). (B) CD19+ cells from population
doublings 0, 1, 2 and 3 were double-sorted by flow-cytometry and incubated separately for 48 hours.
Then, proliferative potency of cells from each population doubling was measured by FACS as a percent
of cells divided more than once, twice or thrice. The data shown is the average for 2 donors. Error bars
represent SEM.
Figure S3, related to Figure 3. Schematic diagram of GSEA analysis performed on B cells, Proliferating
cells, and LCL microarray data with ATM/p53 target genes. We imported our average mRNA expression
changes from either B to Proliferating (Prolif) or Prolif to LCL as molecular profiles. Then we assigned the
ATM and p53 target genes as identified from (Elkon et al., 2005) as a gene set into GSEA. The
enrichment of this gene set relative to 1000 random permutations within the B to Prolif samples was
plotted in the data in Fig. 3C, which indicated that EBV-induced proliferation was associated with an
activation of the ATM/p53 gene expression signature, while from proliferation through LCL outgrowth
this gene set was depleted indicating attenuation of ATM/p53 target gene expression.
Figure S4, related to Figure 5. ATM and Chk2 inhibition did not increase the growth of LCLs. Three
recently-derived LCL lines were incubated at 2 x 105 cells/mL with DMSO, 2 μM ATMi, 5 μM ATMi, 2 μM
Chk2i or 5 μM Chk2i for 6 days. Cell number was counted microscopically using the Trypan Blue
exclusion method at days 3 and 6 and plotted against time. The results shown are the average of the
three independent lines with error bars representing standard error of the mean.
Figure S5, related to Figure 6. Expression of viral latency genes during early cell divisions through LCL
outgrowth. The expression of (A) EBNA-3A, EBNA-3B, and EBNA-3C, (B) EBNA-1, (C) LMP1, and (D)
LMP2A was detected by quantitative real-time PCR. EBV-infected PBMC were sorted 5 and 7 days post
infection by CD19 and CFSE for population doublings and also a polyclonal LCL was derived from the
same infected donor (day 35 post infection). RNA was extracted and SYBR green-based qRT-PCR was
performed using primers spanning exons as previously described (EBNA1 Y3/U/K, LMP1 exon 2/3, and
LMP2A exon 1/2 from (Bell et al., 2006) and EBNA-3A (exon1/2), EBNA-3B (exon 1/2), and EBNA-3C
(exon 1/2) from (Sengupta et al., 2006).Data from one normal donor are shown in the graphs which are
representative of three independent infections. The relative mRNA abundance is plotted normalized to
an HPRT mRNA control (primers from (Ranuncolo et al., 2007)). We note that for each mRNA, an
increase is observed from PD5-6 through LCL outgrowth, which is likely due to an increase in genome
copy number.
Figure S6, related to Figure 7. Characterization of EBNA-3A KO and EBNA-3C KO viruses. (A) The
protein expression of EBNA-3A, -3C, -2, -LP and LMP-1 was analyzed 10 days after B cell infection with
EBV by western blotting. EBV-negative BJAB and EBV positive EF-3D LCL were used as controls. (B) (Top)
Dot plot analysis of EBV-driven B cell proliferation is depicted using the CD19 B-cell marker (y-axis) and
CellTrace Violet dye (x-axis). Both EBNA-3A and 3C KO viruses drive B cell proliferation at 7 days post
infection. (Bottom) Similar dot plots of CD19/Violet indicate that EBNA-3A and EBNA-3C KO viruses are
impaired in driving B cell proliferation through 14 days after infection compare to WT virus. (C)
Quantification of proliferating CD19+ B cells, infected with WT, EBNA-3A KO and EBNA-3C KO EBV at day
7 and day 14 post infection. Average for two normal donors is shown +/- SEM.
SUPPLEMENTAL EXPERIMENTAL PROCEDURES
Viruses, Cells and Reagents. B95-8 virus was produced from the B95-8 Z-HT cell line as previously
described (Johannsen et al., 2004). P3HR1 virus was generated from P3HR1 Z-HT cells (kindly provided
by Ellen Cahir-McFarland (Harvard Medical School)). Buffy coats were obtained from normal donors
through the Carolina Red Cross and peripheral blood mononuclear cells (PBMC) were isolated by Ficoll
Hystopaque gradient (Sigma #H8889). CD19+ B cells were either purified from PBMC using the BD iMag
Negative Isolation Kit (BD, cat #558007) or from buffy coats using the RosetteSep kit (Stem Cell #15024).
Purity was routinely greater than 90% as determined by flow cytometry. Primary cells were cultured in
RPMI 1640 plus 15% fetal calf serum (FCS), 2 mM L-glutamine, and penicillin/streptomycin (R15), while
LCLs were cultured in similar media containing only 10% FCS (R10).
Kinases inhibitors. ATM was inhibited using KU-55933, or ATMi (Hickson et al., 2004) (EMD
#118500). Chk2 was inhibited using the 2-arylbenzamidazole compound Chk2i II (Arienti et al., 2005)
(EMD #220486).
Cell infection. All bulk infections were performed by incubating cells with B95-8 Z-HT
supernatants or GFP-expressing B95-8 (WT), EBNA-3A KO B95-8 or EBNA-3C KO concentrated
supernatant for 1h at 37°C in a CO2 incubator followed by washing twice in PBS and resuspending cells in
R15 media. For IF studies, each 106 primary CD19+ B cells were infected with 500 μL of B95-8 Z-HT
supernatant, or 107 PBMC were infected with 100,000 Green Raji Units of WT, EBNA-3A KO or EBNA-3C
KO B95-8 EBV and sorted for CD19+ proliferating B cells at 7 days post infection. For proliferation assays,
500 μL of B95-8 Z-HT supernatant virus was used per 107 PBMC (~106 CD19+ cells).
Antibodies. Monoclonal antibodies to EBNA-LP were purified from the supernatant of the JF-186
hybridoma cell line (Finke et al., 1987). Mouse anti-human CD19 antibodies conjugated with either APC
(BD Bioscience, #555415) or PE (BD, #555413) were used as surface B cell markers in flow cytometry.
Transformation assays. Infection of human PBMC by B95-8 EBV was performed in the presence of 0.1%
DMSO, 2 μM or 5 μM ATMi or Chk2i added at different times post infection. B95-8 Z-HT supernatant
was titrated from 500 μL/107 PBMC to 0.05 μL/107 PBMC. 2.5 x 106 infected PBMC were seeded in 24
wells of a 96-well plate for each infection point. The percentages of wells positive for B cell outgrowth
(LCL) at five weeks post infection were plotted relative to the amount of virus used per well. The
efficiency of transformation was determined as published (Henderson et al., 1977) where the amount of
B95-8 virus necessary to yield 62.5% of positive wells was considered 1 transforming unit (TU) per well.
Flow cytometry analysis. B cell proliferation assay. A secondary proliferation assay was done using the
dye PKH26 (Sigma, #MINI-26) similar to that described above. However, upon sorting CD19+ B infected
cells into early (PD1-4) and late (PD6+) divisions, cells were secondarily stained with CFSE and followed
daily for proliferation (CFSElow cells) and viability. A population of cells with low FSC signal, which was
also found to be largely 7-AAD positive (data not shown), was measured as a proxy for loss of cell
viability.
Cell Sorting. CD19+ B cells were sorted for population doublings PD0-PD7 based on CFSE
intensity at different times after EBV infection on FACSVantage Diva or MoFlo sorters using two, three or
four way sorts. Sorted populations of B cells were either examined by IF/FISH, Western blot, or
microarray analysis or returned to culture for further analysis.
Fluorescence microscopy data analysis. Images were captured using a Nikon Eclipse TS100 microscope
with a 60X Nikon objective and a Photometrix Cool Snap camera, using the Nikon F imaging software
provided. All IF images were taken using the same capture settings between samples. Images were
pseudo-colored in NIS Basic Research (BR, Nikon). Quantification of the fluorescence intensity was
performed in BR by identifying each nucleus as a region of interest (ROI) in a field using the DAPI image.
The ROIs were then overlaid on the fluorescein and rhodamine images from the same field where mean
pixel intensity was assigned to each ROI and exported to MS Excel for additional analysis. For episome
number analysis in BR, ROIs were assigned as before. The ROIs would then be overlaid on the FISH
image of the same field. Using the object count option, the number of fluorescent objects in each
region of interest was assigned and counts were exported to MS Excel for further analysis.
Gene expression analysis. Quantitative real-time RT-PCR. RNA was extracted using QIAgen RNeasy from
purified primary B cells, infected cells sorted using CFSE on cell division, and LCLs derived from the same
donor. Reverse transcription followed by random cDNA priming was as described (Forte and Luftig,
2009). Real-time PCR was performed either using Quanta SYBR Green or specific Taqman-based probes
in an Applied Biosystems Step One Plus instrument. Wp- and Cp-derived transcripts were detected using
primers flanking the W0-W1/W2 and C0-W1/W2 boundaries, respectively, and a probe from within W1
as described (Bell et al., 2006). EBNA1, 3A, 3B, 3C and LMP1, and 2A mRNAs were detected by SYBR
green with primers described in the supplementary information.
Western blotting. Samples were lysed in a 1% TritonX-100 containing buffer (20 mM Tris, pH 7.5,
100 mM NaCl, 10% glycerol, 1% Triton X-100, 1 mM EDTA, 1 mM DTT, 20 mM NaF, 10 mM sodium
pyrophosphate, and and Complete protease inhibitors without EDTA [Roche]) and normalized to total
protein content by BCA assay. All samples were run on Novex 4-12% Bis-Tris gels and blotted using
standard procedures. Antibodies used were described above.
Microarray analysis and gene enrichment. Total mRNA was prepared from four normal donors in
three conditions: i) uninfected purified CD19+ B cells, ii) infected PBMC, sorted at 6 days post infection
for PD1-PD4, iii) monoclonal LCL derived by limiting virus dilution on PBMC. cDNA preparation, labeling,
and fragmentation was performed using the Gene Chip wt cDNA synthesis and amplification kit
(Affymetrix # 900673) and Exon Array labeling kits (Affymetrix # 900671). 12 samples (4 of each
condition) were hybridized to HuEx 1.0ST Exon Arrays (Affymetrix # 900650) and the chips were scanned
in the Duke Microarray Facility. The resultant CEL files were RMA normalized and the data was analyzed
with GenePattern (Reich et al., 2006) and GSEA v2 (Subramanian et al., 2005) to analyze the ATM/p53
target data set (Elkon et al., 2005). Differential expression of genes was evaluated by applying 2-way
ANOVA or SAM (Tusher et al., 2001). Gene ontology categories were assessed using the GATHER
algorithm (Chang and Nevins, 2006).
Myc signature analysis. Affymetrix HuEx Exon array data was RMA normalized using Expression
Console software obtained from Affymetrix website. To enable the use of Myc pathway signature
generated on the U133A chip (Bild et al., 2006), a bridging file was generated by aligning U133 plus 2
chip probeset target sequences to Mar 2006 genome and comparing their genomic coordinates with the
Affymetrix provided coordinates of the HuEx probesets. The resulting bridging file was used to merge
the two datasets by using FileMerger (http://www.filemerger.genome.duke.edu). The training data, as
represented by Myc or GFP was designated 1 or 0, respectively, while the validation data was labeled 2
to assess the probability of the individual samples with the Myc pathway activation using Bayesian
binary regression analysis (BINREG) in MATLAB. The details of the parameters used to assess the Myc
pathway probability (i.e. gene numbers, metagenes) as well as the validation and optimization strategy
is described elsewhere (Gatza et al., 2010).
SUPPLEMENTAL REFERENCES
Bell, A.I., Groves, K., Kelly, G.L., Croom-Carter, D., Hui, E., Chan, A.T., and Rickinson, A.B. (2006). Analysis
of Epstein-Barr virus latent gene expression in endemic Burkitt's lymphoma and nasopharyngeal
carcinoma tumour cells by using quantitative real-time PCR assays. J Gen Virol 87, 2885-2890.
Elkon, R., Rashi-Elkeles, S., Lerenthal, Y., Linhart, C., Tenne, T., Amariglio, N., Rechavi, G., Shamir, R., and
Shiloh, Y. (2005). Dissection of a DNA-damage-induced transcriptional network using a combination of
microarrays, RNA interference and computational promoter analysis. Genome Biol 6, R43.
Finke, J., Rowe, M., Kallin, B., Ernberg, I., Rosen, A., Dillner, J., and Klein, G. (1987). Monoclonal and
polyclonal antibodies against Epstein-Barr virus nuclear antigen 5 (EBNA-5) detect multiple protein
species in Burkitt's lymphoma and lymphoblastoid cell lines. J Virol 61, 3870-3878.
Martin, H.J., Lee, J.M., Walls, D., and Hayward, S.D. (2007). Manipulation of the toll-like receptor 7
signaling pathway by Epstein-Barr virus. J Virol 81, 9748-9758.
Ranuncolo, S.M., Polo, J.M., Dierov, J., Singer, M., Kuo, T., Greally, J., Green, R., Carroll, M., and Melnick,
A. (2007). Bcl-6 mediates the germinal center B cell phenotype and lymphomagenesis through
transcriptional repression of the DNA-damage sensor ATR. Nat Immunol 8, 705-714.
Sengupta, S., den Boon, J.A., Chen, I.H., Newton, M.A., Dahl, D.B., Chen, M., Cheng, Y.J., Westra, W.H.,
Chen, C.J., Hildesheim, A., et al. (2006). Genome-wide expression profiling reveals EBV-associated
inhibition of MHC class I expression in nasopharyngeal carcinoma. Cancer Res 66, 7999-8006.