Latent Membrane Protein 1 of Epstein-Barr Virus

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Latent Membrane Protein 1 of Epstein-Barr Virus
1
Latent Membrane Protein 1 of Epstein-Barr Virus coordinately regulates
proliferation with control of Apoptosis
Supplementary files
Ulrike Dirmeier1, Reinhard Hoffmann3, Ellen Kilger1, Ute Schultheiss1, Cinthia Briseno1, Olivier
Gires4, Arnd Kieser1, Dirk Eick2, Bill Sugden5, and Wolfgang Hammerschmidt1*
GSF-National Research Center for Environment and Health, Department of Gene Vectors1 and
Institute for Clinical Molecular Biology2, D–81377 Munich, Germany; Max-von-PettenkoferInstitute,
Department
Bacteriology,
D-80336
Munich,
Germany3;
Ludwig-Maximilian
University, Department of Otorhinolaryngology, D–81377 Munich, Germany4 and McArdle
Laboratory for Cancer Research, University of Wisconsin Medical School, Wisconsin 537065

Corresponding author. Phone: +49-89-7099-506. Fax: +49-89-7099-225.

E-mail: [email protected]
2
MATERIAL AND METHODS
Cells. The cell lines used in the study (293 cells, Raji, B95.8, 712 cells) as well as primary Blymphocytes were cultivated as described (Dirmeier et al., 2003; Feederle et al., 2000; Zeidler et
al., 1996). 293 cells stably transfected with maxi-EBV plasmid p2264 were selected and
cultivated in presence of 100µg/ml hygromycin (Calbiochem). B2264-19/3 cells were cultivated
on -irradiated WI38 human fibroblasts as feeder layer obtained from ATCC (Dirmeier et al.,
2003).
Recombinant maxi-EBV. Generation of recombinant EBVs. We generated a chimeric NGFR:LMP1 allele in the context of the maxi-EBV plasmid p2089, which encompasses the complete
genome of the B95.8 strain of EBV in E. coli (Delecluse et al., 1998). In order to exchange the
LMP1 wild-type locus for the NGF-R:LMP1 gene in p2089, several intermediate cloning steps
were necessary to establish a shuttle plasmid to be introduced into p2089 via homologous
recombination in E. coli. The chimeric NGF-R:LMP1 gene derived from p1755.1 (Gires et al.,
1997) was introduced into wild-type maxi-EBV p2089 by the aid of the shuttle plasmid p2167.1
(Dirmeier et al., 2003). The NGF-R:LMP1 molecule consists of aa 1-279 of the human low
affinity NGF-R fused to aa 192-386 of LMP1. The resulting p2264.19 maxi-EBV plasmid was
checked by restriction analyses, Southern blot hybridizations, and partial DNA sequencing (data
not shown). This maxi-EBV plasmid was then used to establish virus-producing 293 cell lines as
outlined in detail (Delecluse et al., 1998; Dirmeier et al., 2003; Humme et al., 2003; Neuhierl et
al., 2002). A 293 cell clone carrying the maxi-EBV plasmid p2264.19 was induced to provide
virus stocks as described (Dirmeier et al., 2003).
Infection of primary B cells. Primary human B-lymphocytes purified from adenoids or
umbilical cord blood were infected with the virus stock. In combination with -irradiated WI38
fibroblasts as feeder layer, 1x105 B cells were plated in a volume of 100µl per well of a 96 well
cluster plate and fed once a week. Proliferating cell clones were expanded in suspension in the
presence of irradiated WI38 feeder cells after 4 to 6 weeks (Dirmeier et al., 2003). One of the
many cell clones, termed B2264-19/3, was randomly picked, expanded and used in this study.
Cross-linking of NGF-R:LMP1. B2264-19/3 cells were removed from the WI38 cell feeder
3
layer and cultivated at a cellular density of 1x105/ml for 72 h at 37°C. Induction of NGF-R:LMP1
was achieved at a concentration of 5x105 cells/ml. For cross-linking, the culture medium was
supplemented with about 0.5µg/ml NGF-R antibody, derived from supernatant of HB8737
hybridoma cells. After 15 min. preincubation at 37°C, cross-linking was initiated by adding
5µg/ml of the secondary antibody (goat anti mouse, IgG+IgM (H+L), Dianova #115-005-068).
As negative controls, B2264-19/3 cells were processed identically except that the NGF-R
antibody was replaced by an irrelevant mouse monoclonal antibody of the same isotype class.
FACS analysis of LMP1, NGF-R:LMP1, CD40, CD69 and CD83 protein. For surface
detection of the NGF-R:LMP1 protein, cells were washed with PBS/3% FCS and incubated with
a concentration of 0.4 - 0.8 µg/ml NGF-R antibody/ml from the hybridoma cell line HB8737
(ATCC) for 20 min. on ice. For intracellular detection of LMP1’s C-terminus, cells were treated
with IntraPrepTM Permeabilization Reagent (Immunotech) and incubated with LMP1 (CS1-4,
DAKO) antibody mix (1:16) for 15 min. at room temperature (RT). After washing with PBS/3%
FCS, cells were incubated with PE-conjugated secondary antibody (PE-conjugated F(ab)
2
fragment -mouse antibody from donkey (DAKO) for 30 min. at RT. Control samples were
incubated with secondary antibody, only. Stained cells were washed, resuspended in PBS/3%
FCS and analysed by FACS. The expression of CD40, CD69 and CD83 was investigated 3h
before and after cross-linking of NGF-R:LMP1. Cells were washed with PBS/3% FCS and
incubated with CD40 (SantaCruz, LOB-11 #13528) antibody mix (1:50) for 20 min. on ice.
After washing cells were stained with PE-conjugated secondary antibody as described and
analysed by FACS. The staining and analysis of CD69 and CD83 surface molecules was done
with PE-conjugated CD83 antibody (Immunotech, # PNIM2218) or PE-conjugated CD69
antibody (exalpha #0694) according to the manufacturers’ recommendations.
Cell cycle analysis. For analysis of cell cycle profiles B2264-19/3 cells were removed from
feeder cell layer and 2x107 cells were induced as described above. At the indicated time points
3x106 cells were cultivated in the presence of BrdU (In Situ Cell Proliferation Kit, Roche) for 4 h
and fixated with 70% (vol/vol) ethanol/ 50mM glycine pH 2 for 30 min. at 4°C. For DNA
depurination, cells were incubated in 4M HCl for 20 min. at RT. After several washing steps in
PBS/0.5% BSA/0.1% Tween 20, cells were incubated with BrdU antibody (Amersham) for 45
4
min. at RT. Cells were washed in PBS/3% FCS and BrdU incorporation was detected by a Cy5conjugated secondary antibody (Cy5-conjugated goat anti mouse, Dianova #115-175-164).
Cellular RNA was degraded by 15 min. RNase (10 µg/ml) digestion and DNA was stained with
propidium iodide (50 µg/ml). Cells were analysed by FACS.
Electrophoretic mobility shift assays and Western blot analysis. Cells were treated as
described in legend of Fig.3. Nuclear extracts were prepared as described (Avvedimento et al.,
1991) and normalized for protein concentration. 6 µg of protein was incubated with
32
P labeled
probe containing a NF-B binding site (5’-TCG ACA GAG GGG GAT AAC CAA GAG GCC3’/ 5’-TCG AGG CCT CTT GGA AAT CCC CCT CTG-3’) as described (Kilger et al., 1998).
DNA-protein complexes were resolved on a 7.5 % non-denaturating polyacrylamid gel. Dried
gels were exposed to X-ray film for autoradiograhy. Proteins for Western blot analysis were
prepared as described (Schultheiss et al., 2001). Antibodies directed against LMP1’s C-terminus
(CS1-4, DAKO), the human NGF-R (HB8737, ATCC), JNK (Santa Cruz Biotechnology),
phospho-JNK (NEB), phospho-p38 (NEB), STAT1 (p84/p91, Santa Cruz Biotechnology),
phosphotyrosine (PY20, Transduction Laboratories/ 4G10, Upstate Biotechnologies), p53 (DO-1
sc-126, Santa Cruz), PAC1 (N-19 sc-1621, Santa Cruz), and c-myc (GA10-2-1, 3H2-1, HH3-1-1
from E. Kremmer) were used for detection diluted 1:100 in RotiBlock (Roth). Immunoblots were
developed using peroxidase coupled secondary antibodies (Promega) and ECL reagent
(Amersham-Pharmacia).
Immunoprecipitation of STAT1. B2264-19/3 cells were induced as described above. 5x106
cells were lysed in 200 µl RIPA buffer (20 mM TrisCl pH 7.5, 150 mM NaCl, 1 % Triton X100,
0.5 % deoxycholate, 0.1% SDS, 2mM DTT, 1mM PMSF, 0.1 mM glycerol phosphate, 0.1mM
sodium vanadate, 0.1 mM sodium fluoride, 0.1 mM sodium molybdate). Protein lysates were precleared by centrifugation at 15,300 rpm. 50 µl protein A-sepharose beads (Roche) were
resuspended in 15 µl RIPA buffer and preincubated for 1 h at 4°C with 10 µl STAT1 antibody
(p84/p91, C-136 Santa Cruz). The coupled beads were combined with pre-cleared protein lysates
and incubated in an overhead tumbler over night. Samples lacking STAT1 antibody or protein
lysate served as negative controls. The beads were washed three times in RIPA buffer, protein
was resolved by boiling in 40 µl RIPA buffer/10 µl loading dye and monitored like described
above.
5
Isolation of lipid rafts. 2 x 107 HEK 293 cells stably transfected with the plasmid p1755.1 (Gires
et al., 1997) encoding NGFR:LMP1 were cross-linked in a total volume of 30ml with 2.5 ml
NGFR:LMP1 HB8737 for 1 hr and -mouse IgG+IgM (Dianova) for 45 min at 37°C to induce
recruitment of the receptor to membrane lipid rafts. After cross-linking, cells were washed once
with ice cold PBS and 4 x 106 cells were resuspended in 380 µl TXNE (1% Triton X-100, 50 mM
Tris-HCl [pH 7.4], 150 mM NaCl, 5 mM EDTA, protease inhibitors) for 30 min on ice. Cells
were homogenized with 20 strokes in a 200 µl pipet tip. Homogenates were mixed with 520 µl 60
% OptiPrep (Axis-Shield PoC AS, Oslo, Norway) and transferred to SW 60Ti ultracentrifuge
tubes. Overlaying sequentially 2.5 ml 30 % OptiPrep and 0.6 ml TXNE formed a discontinuous
gradient which was centrifuged at 160.000 g at 4°C for 4 hr. Eight 500-µl fractions were
harvested from top to bottom. Fractions were analyzed for the raft marker ganglioside GM1 by
dot blotting 1 µl of each fraction with cholera toxin B-conjugated HRP. Fraction 2 was identified
as the low-density lipid raft fraction. Fractions were also blotted with monoclonal LMP1 (CS14) antibody (Dako).
Nuclear run-on analysis. Nuclear run-on RNA preparations were done as described
(Schuhmacher et al., 2001). Labeled RNAs were hybridized to ATLAS arrays human 1.2I, 1.2II,
and human cancer 1.2, each containing 1176 cDNA fragments (Clontech). After exposure to the
membranes, phosphoimager screens were scanned in a Fuji-BAS 1000 reader and signal
intensities were quantified with AtlasImage 1.5-software (Clontech). Normalization of different
arrays was done according to the intensity of control genes and the general background level,
which resulted in a normalization coefficient. Signal intensities were adjusted by subtraction of
the common background value and multiplication by the normalization coefficient. Comparison
of adjusted signal intensities resulted in ‘difference’ values (adjusted intensity signal 1 -adjusted
intensity signal 2) or ‘ratio’ values (adjusted intensity signal 2 / adjusted intensity signal 1). For
obvious reasons a ‘ratio’ could not be defined when the gene signal was at background level in
one of the two arrays. The threshold of differential gene expression was set at a ‘ratio’ value of
≥2.0 or a ‘difference’ value of ≥100.
RNA isolation, labeling and hybridization to oligonucleotide probe arrays. Sample
preparation was carried out essentially as described (Schuhmacher et al., 2001). In brief, total
6
RNA was isolated (RNeasy Midi Kit, Qiagen) and 20µg were used to synthesize cDNA with an
oligo-dT25-T7 primer (cDNA synthesis system, Roche). Double-stranded cDNA was treated with
RNaseA and proteinaseK/0.4% SDS, followed by phenol extraction. Purified cDNA was used for
an in vitro transcription reaction in the presence of biotinylated ribonucleotides (BioArray RNA
Transcript Labeling Kit, Affymetrix). After purification and fragmentation, 15µg labeled cRNA
was used in a 300µl hybridization mixture containing spiked IVT controls. The solution was
hybridized for 16 h at 45°C to the oligonucleotide arrays “Human Genome U95Av2” and
“U133A/B” (Affymetrix), which represent sequences of 12,625 and 44,928 human cDNAs
including control genes (http://www.netaffx.com). Chips were washed, stained, and scanned
according to the manufacturer’s recommendations, and images were analyzed with Affymetrix
Microarray Suite 5.0. As a measure of relative gene expression, signal values (the average of
weighted
fluorescence
intensities)
were
recorded
(http://www.affymetrix.com/products/statistical_algorithm_reference_guide_content.html).
Data analysis. To collect statistically relevant data, three to six independent replica experiments
for each time point were performed. Analysis of this data set consisted of three steps:
normalization of the individual arrays, identification of differentially expressed genes, and
detection of gene clusters with similar expression patterns. Normalization was performed by the
invariant set normalization of Stuart et al. (Stuart et al., 2001) to accommodate recent findings
(Hoffmann et al., 2002). Differentially expressed genes were defined by the permutation-based
method SAM (significance analysis of microarrays) by Tusher et al. (Tusher et al., 2001). The
‘false discovery rate’ (FDR) was calculated as the percentage of genes falsely detected as
differentially expressed among all differentially expressed genes (Tusher et al., 2001). Based on
this method, a FDR < 15% was accepted, because otherwise several target genes, which were
also confirmed with other methods would have been lost. A list of LMP1 target genes and genes
regulated by feeder layer at FDR < 3%, FDR < 6% and FDR < 15% is available upon request.
Genes with similar expression patters were clustered by Self Organizing Maps (Tamayo et al.,
1999).
Real-time PCR analysis of mRNAs. The mRNA transcripts of the p53 and c-myc genes were
reverse transcribed as described above and the resulting cDNAs were quantitatively amplified as
described in detail (Schepers et al., 2001). For detection of p53 cDNA molecules the primer pair
7
5’-GCTTTGAGGTGCGTGTTTGTG-3’ and 5’-TTCTTCTTTGGCTGGGGAGAGGAG-3’was
used; for detection of c-myc cDNA the primer pair 5’-GTCTTCCCCTACCCTCTCAACG-3’ and
5’-GGAGCCTGCCTCTTTTCCAG-3’ was used.
REFERENCES of Materials and Methods
Avvedimento, V.E., Musti, A.M., Ueffing, M., Obici, S., Gallo, A., Sanchez, M., DeBrasi, D. &
Gottesman, M.E. (1991). Genes Dev, 5, 22-8.
Delecluse, H.J., Hilsendegen, T., Pich, D., Zeidler, R. & Hammerschmidt, W. (1998). Proc Natl
Acad Sci U S A, 95, 8245-50.
Dirmeier, U., Neuhierl, B., Kilger, E., Reisbach, G., Sandberg, M.L. & Hammerschmidt, W.
(2003). Cancer Res, 63, 2982-9.
Feederle, R., Kost, M., Baumann, M., Janz, A., Drouet, E., Hammerschmidt, W. & Delecluse,
H.J. (2000). EMBO J, 19, 3080-9.
Gires, O., Zimber-Strobl, U., Gonnella, R., Ueffing, M., Marschall, G., Zeidler, R., Pich, D. &
Hammerschmidt, W. (1997). EMBO J, 16, 6131-40.
Hoffmann, R., Seidl, T. & Dugas, M. (2002). Genome Biol, 3, RESEARCH0033.
Humme, S., Reisbach, G., Feederle, R., Delecluse, H.J., Bousset, K., Hammerschmidt, W. &
Schepers, A. (2003). Proc Natl Acad Sci U S A, 100, 10989-94.
Kilger, E., Kieser, A., Baumann, M. & Hammerschmidt, W. (1998). EMBO J, 17, 1700-9.
Neuhierl, B., Feederle, R., Hammerschmidt, W. & Delecluse, H.J. (2002). Proc Natl Acad Sci U
S A, 99, 15036-41.
Schepers, A., Ritzi, M., Bousset, K., Kremmer, E., Yates, J.L., Harwood, J., Diffley, J.F. &
Hammerschmidt, W. (2001). EMBO J, 20, 4588-602.
Schuhmacher, M., Kohlhuber, F., Holzel, M., Kaiser, C., Burtscher, H., Jarsch, M., Bornkamm,
G.W., Laux, G., Polack, A., Weidle, U.H. & Eick, D. (2001). Nucleic Acids Res, 29, 397406.
Schultheiss, U., Puschner, S., Kremmer, E., Mak, T.W., Engelmann, H., Hammerschmidt, W. &
Kieser, A. (2001). EMBO J, 20, 5678-91.
Stuart, R.O., Bush, K.T. & Nigam, S.K. (2001). Proc Natl Acad Sci U S A, 98, 5649-54.
Tamayo, P., Slonim, D., Mesirov, J., Zhu, Q., Kitareewan, S., Dmitrovsky, E., Lander, E.S. &
Golub, T.R. (1999). Proc Natl Acad Sci U S A, 96, 2907-12.
Tusher, V.G., Tibshirani, R. & Chu, G. (2001). Proc Natl Acad Sci U S A, 98, 5116-21.
Zeidler, R., Meissner, P., Eissner, G., Lazis, S. & Hammerschmidt, W. (1996). Cancer Res, 56,
5610-4.
8
Tab.1. Functional groups of LMP1 target genes in B2264-19/3 cells as identified by selforganizing maps and run-on hybridizations.
functional
group
transcription
factors
Gene ID
M69043
X5668
X51345
M62831
X63741
M92843
AT225
X61498
U44975
J04111
V00568
J04076
U73036
U91616
X66079
S76638
M58603
Z35278
D10522
U73477
AF016898
U51127
AF054589
M55643.1
U13991
AL021154
signal mediators L11329
U15932
NM004418.2
U16996
U33284
X83368
Z18859
L08246
X80200
X61587
U43185
M79321
X07743
U60060
M86752
U71364
X69549
description
Homo sapiens MAD-3+
Human junD mRNA+
Human jun-B mRNA+
Human ETR101 mRNA+
H.sapiens pilot mRNA
Human NUP475 mRNA+
transcription factor ETR103*
nuclear factor NF-kappa-B p100 subunit*
DNA binding protein CPBP*
c-jun proto-oncogene; AP-1*
c-myc oncogene*
early growth response protein 2*
interferon regulatory factor 7 (IRF-7)*
Human I kappa B epsilon (IkBe)*
H.sapiens Spi-B mRNA+
p50-NF-kappa B homolog mRNA
Human NF-kappa-B DNA binding subunit
H.sapiens PEBP2aC1 mRNA
H. sapiens mRNA for 80K-L protein
Human nuclear phosphoprotein pp32 mRNA
H. sapiens B-ATF gene+
Human interferon regulatory factor 5 mRNA
H. sapiens HIC protein mRNA
Human factor KBF1 mRNA
Human tafII30 mRNA
HEIR-1 mRNA
PAC1+
Dual-spec. protein phosphat. mRNA
Dual-spec. protein phosphat. 2 mRNA
dual-specificity protein phosphatase 5+
focal adhesion kinase 2 (FADK2; FAK2)*
PI3 kinase P110 subunit*
transducin alpha-2 chain(GNAT2)*
Human MCL1 mRNA*
H.sapiens MLN62 mRNA
H.sapiens rhoG mRNA for GTPase+
Stat5A mRNA
Human Lyn B protein mRNA
Human mRNA for pleckstrin (P47)
Human FEZ1 mRNA
Human protein IEF SSP 3521 mRNA
Human serine protease inhibitor (P19) mRNA
H.sapiens rho GDP-dissociation Inhibitor 2
cluster
1
2
3
4
1
2
3
4
9
apoptosis/cell
cycle
receptors/
ligands
cytokines
EBV induced
metabolism/
structure
U12707
U66464
X61123
U72649
U83981
AF078077
U45878
U27467
M59465
AF005775
AF058696
AJ000414
X02910
M16441
U03398
Z22576
U33017
M64749
D12614
BF664114
L31584
Z11697
M24283
NM000595.2
L05424
X60592
X94630
Y00636
X52425
L20859
M32315
U46751
NM004240.1
NM005658.1
M63928
X62744
D90144
NM002984.1
NM002983.1
J04130
U83171
L08177
U19261
NM004922.1
AI200589
U60266
M2341
M22300
X00351
X66435
Human WASP mRNA
Human HPK mRNA
B-cell translocation gene 1 protein (BTG1)*
Human BTG2 (BTG2) mRNA+
GADD34 mRNA
H. sapiens GADD45 beta mRNA
Human inhib. of apoptosis protein 1 mRNA
Human Bcl-2A1/Bfl-1 mRNA+
Human A20 mRNA+
H. sapiens clarp mRNA
H. sapiens p95/NBS1 mRNA
H. sapiens mRNA for CIP4
TNF-alpha
HUMTNFAB
Human receptor 4-1BB ligand mRNA
H.sapiens CD69 gene+
signaling lymphocytic activation molecule*
G-protein-coupled receptor rdc1 homolog*
Human mRNA for TNF-beta
TNFR superfamily, member 5
Human G protein-coupled receptor EBI1
H. sapiens mRNA for CD83+
Human ICAM1 (CD54) mRNA
lymphotoxin  precursor
Human hyaluronate receptor (CD44) gene
Human CDw40 mRNA+
H.sapiens CD97 gene
Human mRNA for LFA-3
Human IL-4-R mRNA
Human GLVR1 mRNA
Human TNF-R mRNA
Hum. ligand p62 for the Lck SH2 domain mRNA
H. sapiens TRIP10mRNA
H. sapiens TRAF1 mRNA
H. sapiens CD27 mRNA
Human RING6 mRNA
H. sapiens gene for LD78  precursor+
H.sapiens cytokine A4 mRNA
H.sapiens cytokine A3 mRNA
Human activation (Act-2) mRNA
Human MDC mRNA
EBV-induced receptor 2 (EBI2)+
H. sapiens EBV-induced protein mRNA
H.sapiens SEC24C mRNA
H.sapiens ribosomal protein S16
lysosomal alpha-mannosidase*
desmoplakin III (DP3)*
L-plastin; lymphocyte cytosolic protein 1*
cytoplasmic beta-actin (ACTB)*
hydroxymethylglutaryl-CoA synthase*
1
2
3
1
2
3
4
1
2
3
1
2
1
10
others/
unknown
M97164
U14971
U29344
X16396
AF034544
U29171
Z82244
L06845
U09510
U03057
M80244
X79535
M26880
X65923
M57567
J03077
U70063
U14970
X06617
AL031228
M81757
M17885
U32576
NM000992. 1
M33197.1
AA587372
AA320764
W67644
NM025079.1
NM017648.1
AB037925.1
AA831661
gAI459194
gb:AI459194
D00174
M27364
X51466
M25296
M16660
U76248
U96876
AB002344
AF055001
AJ011896
Z24724
D42043
U71364
Z97054
AI865431
ferritin heavy chain (FTH1); FTHL6*
40S ribosomal protein S9*
Human BCA-fatty acid synthase mRNA
Hum. methylene tetrahydrofolat DH cyclohydroase
H. sapiens delta7-sterol reductase mRNA
Human casein kinase I delta mRNA
bK286B10.2 Heme Oxygenase 1
Human cysteinyl-tRNA synthetase mRNA
Human glycyl-tRNA synthetase mRNA
Human actin bundling protein (HSN) mRNA
Human E16 mRNA
H.sapiens mRNA for beta tubulin
Human ubiquitin mRNA
H.sapiens fau mRNA
Human hARF5 mRNA
Human co-beta glucosidasemRNA
Human acid ceramidase mRNA
Human ribosomal protein S5 mRNA
Human mRNA for ribosomal protein S11
Human 40S ribosomal protein S18
H.sapiens S19 ribosomal protein mRNA
Human ribos. phosphoprotein P0 mRNA
Human APOC4 gene
H.sapiens ribosomal protein L29 mRNA
Human GAPDH mRNA
nn82f03.s1 Homo sapiens cDNA
EST23183
Est:zd42a07.s1
H. sapiens protein FLJ23231 mRNA
H. sapiens protein FLJ20063 mRNA
Homo sapiens MAIL mRNA
EST /DB_XREF=gi:2904760
EST /DB_XREF=gi:4311773
EST /DB_XREF=gi:4311773
alpha-2-antiplasmin*
elongation factor 1 alpha (EF1 alpha)*
elongation factor 2 (EF2)*
natriuretic peptide precursor B*
Human 90-kDa heat-shock protein gene
Human hSIAH2 mRNA
H. sapiens insulin induced protein 1 mRNA
Human mRNA for KIAA0346 gene
H. sapiens clone 24560 unknown mRNA
H. sapiens mRNA for HIV-1, Naf1 beta
H.sapiens polyA site DNA
Human mRNA for KIAA0084 gene
Human serine proteinase inhibitor (P19) mRNA
Human DNA sequence from PAC 339A18 on
chromosome Xp11.2
wk11h09.x1 Homo sapiens cDNA
3
4
1
2
3
11
D87071
AL120815
W25986
AL096723
R87876
AB028969
AB014540
X81625
AI017574
D87076
D13630
AL050021
AL080109
U79259
J04164
X57809
U15131
U58515
AI525665
AA808961
S73591
H12458
AF070616
AJ245416
NM005101.1
Human mRNA for KIAA0233 gene
DKFZp762F172_r1 Homo sapiens cDNA
17e7 Homo sapiens cDNA
Homo sapiens mRNA; cDNA DKFZp564H2023
yo45h01.r1 Homo sapiens cDNA
Homo sapiens mRNA for KIAA1046 protein
Homo sapiens mRNA for KIAA0640 protein
H.sapiens mRNA for Cl1 protein
ou23f10.x1 Homo sapiens cDNA
Human mRNA for KIAA0239 gene
Human mRNA for KIAA0005 gene
Homo sapiens mRNA; cDNA DKFZp564D016
Homo sapiens mRNA; cDNA DKFZp586G1822
Human clone 23945 mRNA
Human INF-inducible protein 9-27 mRNA
Human rearranged Iglight chain mRNA
Human p126 (ST5) mRNA
Human chitinase (HUMTCHIT) mRNA
PT1.3_04_D06.r Homo sapiens cDNA
nw16h03.s1 Homo sapiens cDNA
Human HL-60 mRNA
Nb2HP Homo sapiens cDNA clone
BDP-1 protein mRNA
Homo sapiens mRNA for G7b protein
H.sapiens ISG 15 mRNA
4
Target genes of LMP1 after induction of B2264-19/3 cells for 0, 30, 60, and 180 min were
identified by the permutation based method (Tusher et al., 2001) with 15% false detection rate
cut-off. Genes with similar expression patterns were sorted in 4 clusters by self-organizing maps
(Tamayo et al., 1999). The cluster numbers are indicated in the rightmost column: genes in
cluster 1 are most rapidly upregulated in response to LMP1’s activation, genes in cluster 2 and 3
are upregulated within 60 min and 180 min, respectively. Genes in cluster 4 are repressed by
LMP1 signaling. Numbers of genes in each cluster are 30, 19, 62, and 31 in cluster 1, 2, 3 and 4,
respectively. Asterisks (*) indicate exclusive detection of the transcripts in run-on hybridizations,
only, which were grouped together with cluster 1 candidates. Cellular genes which scored
positive in both run-on hybridizations and high density oligonucleotide arrays are indicated by a
plus (+) sign. The assignment to different functional groups was decided according to published
data. No information was available for 29 LMP1 regulated genes.
12
Figure legends
Fig.S1. FACS analyses of EBV-infected B-cell lines expressing wild-type LMP1 (721) or
NGF-R:LMP1 chimeric protein (B2264-19/3). Expression level and surface presentation of the
NGF-R:LMP1 chimera was analyzed by FACS analyses with antibodies directed against the
extracellular NGF-R domain as well as LMP1’s intracellular C-terminus. (A) extracellular
detection using NGF-R antibody; (B) intracellular detection of LMP1’s C-terminus via LMP1
antibody (CS1-4). The surface expression of NGF-R on B2264-19/3 cells was clearly detectable,
wildtype EBV immortalized 721 cells express no NGF-R (A). The intracellular detection of
LMP1’s C-terminus demonstrated an about three fold higher expression level of NGF-R:LMP1 in
B2264-19/3 LCLs compared to wild-type LMP1 in 721 cells (B).
Fig.S2. Antibody-mediated cross-linking of NGF-R:LMP1 induced the p53 regulated
cellular gene PAC1but not p53. (A) To confirm the regulation of one LMP1 target gene (Fig.4
and Tab.1), Western blot immunodetection of the PAC1/DUSP2 (phosphatase of activated
cells/dual specificity phosphatase 2) gene product at different time points after LMP1 activation
was performed. B2264-19/3 cells were separated for 72 h from feeder cells prior to induction of
LMP1 signaling by antibody-mediated cross-linking. (B) Quantitative real-time PCR analysis of
reverse transcribed p53 mRNA indicated its detectable expression but no transcriptional
regulation upon antibody-mediated cross-linking of NGF-R:LMP1. The inset is a reconstruction
and calibration experiment (Schepers et al., 2001) to demonstrate the quantitative detection of
p53 reverse transcribed cDNA. (C) By immunodetection of p53 and EBNA2 (as a control) of
biochemically fractionated cytoplasmic and nuclear extracts of B2264-19/3 neither a change of
steady-state p53 protein levels nor a change of its subcellular localization could be detected
before or after NGF-R:LMP1 cross-linking.

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