Epstein-Barr virus induction of recombinase-activating genes RAG1 and RAG2.
ABSTRACT In experimental B-cell infections, Epstein-Barr virus induced sustained expression of V(D)J recombinase-activating genes RAG1 and RAG2, whose aberrant activity has been implicated in chromosomal translocations in B-cell neoplasms. In cell lines in which RAG1 and RAG2 were detected, virus integrated into cellular DNA rather than assumed the configuration of extrachromosomal episomes. Expression of the Epstein-Barr virus nuclear antigen 1 in transient transfection assays was sufficient to induce both recombinase-activating genes.
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ABSTRACT: EBNA1, a nuclear protein expressed in all EBV-associated neoplasms is indispensable for the maintenance of the viral episomes in latently infected cells. EBNA1 may induce genetic alterations by upregulating cellular recombinases, production of reactive oxygen species (ROS) and affecting p53 levels and function. All these changes may contribute to tumorigenesis. In this overview we focus, however, on the epigenetic alterations elicited by EBNA1 by drawing a parallel between EBNA1 and the FoxA family of pioneer transcription factors. Both EBNA1 and FoxA induce local DNA demethylation, nucleosome destabilization and bind to mitotic chromosomes. Local DNA demethylation and nucleosome rearrangement mark active promoters and enhancers. In addition, EBNA1 and FoxA, when associated with mitotic chromatin may "bookmark" active genes and ensure their reactivation in postmitotic cells (epigenetic memory). We speculate that DNA looping induced by EBNA1-EBNA1 interactions may reorganize the cellular genome. Such chromatin loops, sustained in mitotic chromatin similarly to the long-distance interactions mediated by the insulator protein CTCF, may also mediate the epigenetic inheritance of gene expression patterns. We suggest that EBNA1 has the potential to induce patho-epigenetic alterations contributing to tumorigenesis.Pathogens. 12/2012;
JOURNAL OF VIROLOGY, Dec. 1995, p. 8155–8158
Copyright ? 1995, American Society for Microbiology
Vol. 69, No. 12
Epstein-Barr Virus Induction of Recombinase-Activating Genes
RAG1 and RAG2
SHAMALA K. SRINIVAS AND JOHN W. SIXBEY*
Departments of Infectious Diseases and Virology & Molecular Biology, St. Jude’s Children’s Research Hospital,
and University of Tennessee College of Medicine, Memphis, Tennessee
Received 6 June 1995/Accepted 11 September 1995
In experimental B-cell infections, Epstein-Barr virus induced sustained expression of V(D)J recombinase-
activating genes RAG1 and RAG2, whose aberrant activity has been implicated in chromosomal translocations
in B-cell neoplasms. In cell lines in which RAG1 and RAG2 were detected, virus integrated into cellular DNA
rather than assumed the configuration of extrachromosomal episomes. Expression of the Epstein-Barr virus
nuclear antigen 1 in transient transfection assays was sufficient to induce both recombinase-activating genes.
Epstein-Barr virus (EBV), a common human herpesvirus,
infects 95% of the world’s population by adolescence. Infec-
tion may be silent, manifest acutely as infectious mononucle-
osis, or be associated with tumors of lymphoid, epithelial, and
myocytic origin during the lifelong virus carrier state. In the
infected cell, EBV is maintained extrachromosomally in a cir-
cular (latent) or linear (replicating) molecular configuration
(1, 12, 29). Recent detection in human lesions of EBV DNA
rearrangement (31, 39, 43) and chromosomal integration (20,
35) suggests that recombination events are a central feature of
EBV biology and pathogenesis (41, 46). Because internal re-
arrangements in the viral genome exhibit general sequence
specificity (9, 16, 31, 41), we questioned whether site-specific
recombinases involved in diversification of the host immune
response mediate DNA rearrangements in this lymphotropic
virus. V(D)J recombinase activity is restricted to immature
lymphoid cells, whereas EBV normally targets mature B lym-
phocytes. Thus, in order to become substrate, EBV would have
to activate RAG1 and RAG2, whose concerted expression has
been shown sufficient for V(D)J recombination (30).
To determine if EBV infection stimulates RAG expression
in mature B lymphocytes, we first examined established cell
lines derived from sporadic (EBV-negative) Burkitt’s lympho-
mas (sBL) paired with their in vitro-infected, EBV-positive
counterpart (provided by C. Rooney, St. Jude Children’s Re-
search Hospital) (7). Sporadic Burkitt’s tumors do not express
RAG1 or RAG2 (5), and biopsy-derived cell lines have a
relatively mature, surface immunoglobulin-positive cell pheno-
type (7, 19). Cytoplasmic RNA was extracted from sBL cell
lines and the human pre-B-cell line Reh (American Type Cul-
ture Collection, Rockville, Md.) as previously described (4).
Poly(A)?RNA was isolated by using an oligo(dT)-cellulose
microcentrifuge pack (Collaborative Biomedical Products,
Bedford, Mass.) according to the manufacturer’s protocol.
RNA was electrophoresed in a 1.2% agarose-formaldehyde
gel, transferred to a nylon membrane (Micron Separations,
Westborough, Mass.), and hybridized to a32P-labeled probe
generated by random-primed labeling (11). Probe was derived
from an XhoI-HindIII digestion fragment of a cloned 6.6-kb
human RAG1 cDNA, H36 (gift of D. G. Schatz, Yale Univer-
Northern (RNA) blot analysis of poly(A)?RNA from un-
infected and EBV-converted sBL cell lines that had been
maintained in long-term culture revealed RAG1 RNA in four
of five EBV-infected sBL lines but not in their uninfected
counterpart (representative blot in Fig. 1; Table 1). To control
for RNA loading, blots were reprobed for the generally ex-
pressed glyceraldehyde 3-phosphate dehydrogenase gene, and
the integrated optical density of bands was quantitated with a
Visage 110 image analysis system (BioImage Products, Ann
Arbor, Mich.). RAG1 RNA was approximately 50-fold less
abundant in EBV-converted sBL lines than in the pre-B-cell
To confirm these findings and to detect less abundant RAG2
whose levels fluctuate throughout the cell cycle (23), we per-
formed reverse transcription (RT)-PCR to detect RAG RNAs
(Fig. 1B). For RT-PCR, total RNA was isolated by using RNA-
zol B (Cinna/BiotecX Laboratories International Inc., Friends-
wood, Tex.). RNA was incubated for 20 min at 37?C in DNase
I (RNase free; Promega, Madison, Wis.) to remove contami-
nating DNA. cDNA was reverse transcribed from 1 ?g of total
cellular RNA, using the 3? primers specified below with avian
myeloblastosis virus reverse transcriptase as specified by the
manufacturer (Life Sciences, St. Petersburg, Fla.). After unin-
corporated nucleotides were removed by centrifugation over a
Centricon-100 (Amicon, Beverly, Mass.), cDNA was amplified
for 35 cycles (1.5 min at 94?C, 2 min at 52?C, and 2 min at 70?C)
with Taq polymerase (Stratagene, La Jolla, Calif.). RAG1-
specific primers were 5?-CCATCATGCAGGGAAAGG-3? (5?
primer; bp 1479 to 1496) and 5?-GCTTCTCACTCACGTC
TC-3? (3? primer, bp 1928 to 1946) (38); RAG2 primers
were 5?-TGAGATGGAGACCCCAGATT-3? (5? primer, bp
2101 to 2120) and 5?-TCACAAGTAGGGCAGCATGT-3? (3?
primer, bp 2453 to 2472) (15). The PCR products were sepa-
rated on a 3% NuSieve–1% SeaKem agarose gel (FMC Bio-
products, Rockland, Maine), transferred onto a nylon mem-
brane, and hybridized to
probes specific to RAG sequences internal to the primers.
Results achieved by RT-PCR for RAG1 were identical to
those of Northern blot hybridizations (Fig. 1). In all infected
sBL cell lines except one (BL30/P3HR-1), RAG-1 and RAG-2
were coordinately transcribed (Table 1). These results concur
with recent findings derived independently in a second labo-
ratory by Kuhn-Hallek et al. (19) showing RAG expression in
EBV-infected BL cell lines. Detection of RAG2 in the absence
of RAG1, seen in BL30/P3HR-1 cells, has been described
previously in B-lineage acute lymphocytic leukemia cells (5)
* Corresponding author. Mailing address: Departments of Infec-
tious Diseases and Virology & Molecular Biology, St. Jude Children’s
Research Hospital, 332 N. Lauderdale, Memphis TN 38101-0318.
and chicken B cells undergoing immunoglobulin gene conver-
To relate RAG induction temporally to virus infection, one
sBL line (BL41) was acutely infected with the B95-8 strain of
EBV as described elsewhere (7). After exposure to virus, ap-
proximately 2% of the total cell population expressed EBV
nuclear antigen 2 (EBNA2), as determined by indirect immu-
nofluorescence staining with monoclonal antibody PE-2 (gift of
L. S. Young, University of Birmingham) (45). The bulk culture
was then cloned with a FACStar cell sorter (Becton Dickinson,
Braintree, Mass.) at one cell per well into 96-well plates con-
taining human foreskin fibroblast feeder layers. Of 300 clones
screened, one (5D3) was EBNA2 positive. Within the first
passage of this newly converted EBV-positive clone, we veri-
fied de novo induction of RAG1 and RAG2 (Fig. 1, clone
5D3). Five EBV-negative clones screened for RAG mRNA by
RT-PCR did not contain transcripts (data not shown).
Latent infection by prototype EBV is associated with con-
stitutive expression of six EBNAs and three latent membrane
proteins. Whereas both B95-8 and P3HR-1 virus strains in-
duced RAG expression in sBL cells (Fig. 1), infection with
P3HR-1 results in a more restricted pattern of viral gene ex-
pression (28) due to deletions in that virus of DNA encoding
EBNA-LP and the transactivating protein EBNA2 (6, 18, 33).
On this basis, we selected three gene constructs for transfec-
tion into sBL to determine what viral genes might be respon-
sible for RAG induction: EBNA1 (required for EBV plasmid
maintenance in infected cells) (34, 36, 44), EBNA3C (a tran-
scription factor essential for EBV transformation) (27, 42),
and EBNA2 (deleted in P3HR-1 and not anticipated to stim-
ulate RAG expression).
Fifteen micrograms of vector DNA (pSG5; Stratagene),
pSG-EBNA1, pSG-EBNA2 (type A), or pSG-EBNA3C (pro-
vided by C. Sample, St. Jude) was transfected into 8 ? 106
BL41 cells, using an electroporator (Gene Pulser; Bio-Rad,
Hercules, Calif.) at 250 V and capacitance of 960 ?F. After
resuspension in 10 ml of RPMI 1640, cells were cultured for 48
h and then harvested. Comparable expression of introduced
genes was confirmed by protein immunoblotting and immuno-
fluorescence staining with monoclonal antibody PE-2 (anti-
EBNA2) and monospecific human sera AM (anti-EBNA1)
and JT (anti-EBNA3C) (from C. Sample). Total RNA was
analyzed for RAG1 and RAG2 transcripts by RT-PCR. At 48
h, EBNA1-transfected sBL cells contained both RAG1 and
RAG2 RNA by RT-PCR analysis. Cells mock transfected or
electroporated with EBNA2, EBNA3C, or the pSG5 vector
alone did not express RAG genes (Fig. 2).
Although characteristically episomal, EBV regularly inte-
grates in converted sBL lines (14). To determine if integration
events coincided with recombinase expression, total cellular
DNA was digested with restriction endonuclease BamHI, and
the molecular configuration of EBV DNA was determined by
analysis of terminal repeat sequences as described previously
(32). After electrophoretic separation on 0.8% agarose gels,
DNA was transfered to nylon membranes for hybridization
with riboprobes to unique DNA at either side of EBV terminal
FIG. 1. RAG1 and RAG2 expression in EBV-infected sBL cells. (A) North-
ern blot analysis for the approximate 6.6-kb RAG1 mRNA in BL41 cells infected
with two EBV strains. BL41 lanes contained 10 ?g of poly(A)?RNA; the REH
lane contained 0.2 ?g of mRNA from control pre-B-cell line Reh, which ex-
presses abundant RAG. The glyceraldehyde 3-phosphate dehydrogenase
(GAPDH) probe controlled for RNA loading. (B) Ethidium bromide gel of
RT-PCR amplification products for RAG1 and RAG2 in infected BL41 cells.
Reaction mixtures contained 50 ng of positive control Reh RNA; 1 ?g of RNA
was used for all other assays. Amplifications without reverse transcriptase (RT
?) excluded contaminating DNA as a reaction template. Data are representative
of four separate assays.
FIG. 2. EBNA1 induction of RAG1 and RAG2 in sBL cells. Shown is a
Southern blot of RT-PCR products after BL41 cells were transiently transfected
with EBV gene EBNA1, EBNA2 (type A), or EBNA3C or with pSG5 vector
alone. BL41, mock-transfected cells. RT, reverse transcriptase.
TABLE 1. EBV-associated RAG1 and RAG2 transcription in
aP3HR-1 and B95-8 are nontransforming and transforming laboratory strains
bDetermined by Northern blot analysis and RT-PCR.
cDetermined by RT-PCR analysis only.
8156NOTES J. VIROL.
repeats (1.9-kb XhoIa and BamHI J portion of EcoRI-I in
pGEM2; gift of N. Raab-Traub, University of North Carolina).
By analysis of EBV terminal sequences, we found complete
concordance between expression of RAG genes and EBV in-
tegration. The infected cell line (BL30/P3HR-1) that expressed
only RAG2 maintained EBV in its episomal form (Fig. 3).
Virus in newly infected BL41 cell clone 5D3 also integrated,
indicating that recombination detected in sBL cells is not a
consequence of long-term cell culture.
To strengthen the correlation between integration and RAG
expression, five additional EBV-converted sBL cell lines
(BL30/B95-8, BL31/B95-8, BL31/P3HR-1, BL40/B95-8, and
BL41/72; provided by D. Thorley-Lawson, Tufts University)
previously described as containing viral episomes (14) were
analyzed by RT-PCR for RAG1 expression. Ubiquitously ex-
pressed c-abl mRNA was amplified as a control to ensure RNA
integrity. RAG1 mRNA was not detected in these sBL lines
(data not shown). Also of note is the distinct correlation in two
B95-8-converted clones of BL30 from separate laboratories:
one contained a single copy of integrated EBV and expressed
RAG (Fig. 3), whereas the second was RAG negative with
multiple copies of episomal EBV (14). Although insufficient to
implicate V(D)J recombinase specifically, viral integration
does indicate the presence of recombinogenic activity in RAG-
positive cells. Whether increased RAG transcription correlates
with a functional V(D)J recombinase or represents a more
general stimulation of cellular recombination mechanisms (3,
41) is the focus of ongoing studies.
Our findings indicate inappropriate RAG expression after
B-cell infection by EBV and identify EBNA1 as sufficient for
induction of both RAG1 and RAG2 in some cells. Whereas all
EBV-converted sBL lines express EBNA1, RAG induction was
sporadic and presumably reflects cell context. For example, we
detected only transient RAG expression on infection of pe-
ripheral blood B cells (40). Despite a mature B-cell phenotype,
sBL may represent a subset of B lymphocytes, such as those in
the pre-B-to-B-cell transition (26), capable of simultaneously
expressing surface immunoglobulin as well as genes normally
restricted to the pre-B developmental stage. Because DNA
viruses induce a variety of cellular genes required for viral
replication, it is likely that cellular recombinogenic machinery
is activated for cleavage and packaging of EBV DNA con-
catameric structures or, alternatively, for circularization of in-
coming linear DNA (3, 10, 17, 41, 46). These findings may be
useful in delineating potential regulatory factors affecting
What makes activation of this host enzymatic machinery by
EBNA1 of particular note is the well-documented role that
aberrant V(D)J recombination plays in human tumorigenesis.
At least one-fifth of childhood malignancies appear to involve
translocations derived from site-directed lymphoid recombina-
tion (21). The one constant feature of EBV-positive African
BL is chromosomal translocation of the c-myc proto-oncogene
into an immunoglobulin locus (13). In this tumor, only
EBNA1, a protein which activates the EBV plasmid origin of
replication (34, 36, 44) but has no growth-transforming activity
per se, is expressed (37). Rather than a mistake in V(D)J
joining at the pre-B-cell stage as the developmentally regulated
expression of V(D)J recombinase has presupposed, our find-
ings raise the possibility that such translocations also occur at
later maturational states as a result of EBV-initiated V(D)J
recombinase activity. Given the widespread nature of EBV
infection, such a mechanism would have far-reaching implica-
tions and could contribute to V(D)J recombinase-generated
cytogenetic alterations reported in lymphoid tissue and periph-
eral blood lymphocytes of a large percentage of the healthy
human population (2, 22, 24, 25).
This work was supported by the American Lebanese Syrian Associ-
ated Charities and by grants from the National Institutes of Health
(CA21765, CA52258, CA38877, and CA67372).
We thank R. Goorha, and G. Neale for thoughtful discussions; D. G.
Schatz, N. Raab-Traub, J. Sample, C. Sample, C. Rooney, L. S. Young,
C. Naeve, and D. Thorley-Lawson for reagents; V. Holder for techni-
cal assistance; and J. Gilbert for review of the manuscript.
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8158 NOTES J. VIROL.