Cell, Vol. 80, 389-399, February 10, 1995, Copyright © 1995 by Cell Press
The Epstein-Barr Virus Transforming Protein LMP1
Engages Signaling Proteins
for the Tumor Necrosis Factor Receptor Family
George Mosialos,* Mark Birkenbach,t
Ramana Yalamanchili,* Todd VanArsdale,t
Carl Ware,t and Elliott Kieff*
*Department of Medicine
Department of Microbiology and Molecular Genetics
Harvard Medical School
Boston, Massachusetts 02115
tDepartment of Pathology
Marjorie B. Kovler Viral Oncology Laboratories
University of Chicago
Chicago, Illinois 60637
SDivision of Biomedical Sciences
University of California
Riverside, California 92521
The cytoplasmic C-terminus of Epstein-Ban' virus (EBV)
latent infection membrane protein 1 (LMP1) is essen-
tial for B lymphocyte growth transformation and is now
shown to interact with a novel human protein (LMP1-
associated protein 1 [LAP1]). LAP1 is homologous to
a murine protein, tumor necrosis factor receptor-
associated factor 2 (TRAF2), implicated in growth sig-
naling from the p80 TNFR. A second novel protein (EBI6),
induced by EBV infection, is the human homolog of
a second murine TNFR-associated protein (TRAF1).
LMP1 expression causes LAP1 and EBI6 to localize to
LMP1 clusters in lymphoblast plasma membranes,
and LMP1 coimmunoprecipitates with these proteins.
LAP1 binds to the p80 TNFR, CD40, and the lympho-
toxin-p receptor, while EBI6 associates with the p80
TNFR. The interaction of LMP1 with these TNFR fam-
ily-associated proteins is further evidence for their
role in signaling and links LMPl-mediated transforma-
tion to signal transduction from the TNFR family.
Epstein-Barr virus (EBV) causes infectious mononucleosis
and lymphoproliferative diseases in immune-suppressed
patients. EBV is also an important etiologic factor in en-
demic Burkitt's lymphoma, AIDS-associated central ner-
vous system lymphomas, Hodgkin's disease, and naso-
pharyngeal carcinoma (Kieff and Liebowitz, 1990; Miller,
1990). EBV infection of B lymphocytes is primarily nonlytic,
resulting in the expression of EBV-encoded nuclear
(EBNAs) and integral membrane proteins (latent infection
membrane proteins [LMPs]) as well as perpetual cell prolif-
eration (Kieff and Liebowitz, 1990). EBV recombinant-
based molecular genetic analyses have demonstrated that
LMP1 and EBNA2, -LP, -3A, and -3C are critical for B
lymphocyte transformation (Cohen et al., 1989; Ham-
merschmidt and Sugden, 1989; Kieff and Liebowitz, 1990;
Mannick et al., 1991; Kaye et al., 1993; Tomkinson et al.,
1993), while LMP2, EBNA3B, the EBV-encoded small
RNAs and most of the rest of the genome are dispensable
(Marchini et al., 1991; Swaminathan et al., 1991; Long-
necker et al., 1993; Tomkinson and Kieff, 1992; Robertson
et al., 1994). EBNA1 is also important for transformation
since it enhances transcription and replication from EBV
episomes (Yates et al., 1984; Reisman and Sugden, 1986).
LMP1 is not only essential for primary B lymphocyte
growth transformation but is also the only EBV gene that
has transforming effects in nonlymphoid cells. In Rat1
cells, LMP1 induces growth in lower serum, loss of contact
inhibition, anchorage independence, and nude mouse tu-
morigenicity, while in BALB/c 3T3 cells, LMP1 induces
anchorage-independent growth (Wang et al., 1985, 1988a;
Baichwal and Sugden, 1988; Moorthy and Thorley-
Lawson, 1993). In B lymphoblasts, LMP1 induces most of
the phenotypic effects of EBV infection, including induced
expression of activation markers and adhesion molecules,
altered growth, and NF-~:B activation (Wang et al., 1988b,
1990; Hammarskjold and Simurda, 1992; Laherty et al.,
1992; Rowe et al., 1994). In epithelial cells, LMP1 blocks
differentiation (Dawson et al., 1990; Fahraeus et al., 1990;
Wilson et al., 1990). These activities are probably relevant
to tumorigenicity in humans since LMP1 is expressed not
only in EBV-induced lymphoproliferative disease but also
in EBV-associated Hodgkin's disease tumor cells and in
most anaplastic nasopharyngeal carcinoma cells (Herbst
et al., 1991; Pailesen et al., 1991; Brooks et al., 1992;
Busson et al., 1992).
LMP1 is an integral membrane protein that may trans-
form cells by constitutively activating a growth factor re-
ceptor pathway common to many cell types (Wang et al.,
1985). LMP1 consists of a 23 amino acid amino-terminal
cytoplasmic domain, 6 markedly hydrophobic transmem-
brane domains separated by short reverse turns, and a
200 amino acid carboxy-terminal cytoplasmic domain
(Fennewald et al., 1984; Hennessy et al., 1984). The trans-
membrane domains enable LMP1 to posttranslationally
insert into membranes and to accumulate in aggregates
in the plasma membrane (Hennessy et al., 1984; Liebowitz
et al., 1986).
Recent genetic analyses in primary B lymphocyte growth
transformation indicate that the amino terminus is im-
portant for tethering of the first transmembrane domain,
and the transmembrane domains are important for confer-
ring plasma membrane aggregation (Kaye et al., 1993,
1995; Izumi et al., 1994). EBV recombinants deleted for
the entire 200 or for the last 155 amino acids of the LMP1
carboxy-terminal cytoplasmic domain are incapable of
transforming primary B lymphocytes (Kaye et al., 1995).
While the nontransforming phenotype of EBV recombi-
nants deleted for the last 155 amino acids of LMP1 can
be transcomplemented by the growth of infected lympho-
cytes on fibroblast feeders, to date it has not been possible
to similarly rescue primary B lymphocytes infected with
EBV recombinants deleted for the entire 200 amino acid
carboxy-terminal cytoplasmic domain (Kaye et al., 1995).
HESS KKMDS PGALQTNPPLKLHTDRSAGT
MAAAS -VTSPGSLELLQP ..................
MASSS-GSSP ..... RP .................
PVFVPEQGGYKEKFVKT - VEDKYK~E 54
APDEN ..... EFPFGCP
APDEN ..... EFQFGCP
K _ _ CH LVLC S pKQT E~G HRFCE~M~IJ~
PTVCQD- - pKEPRALC - - - -~AGCLSZ~L~p.N[~EDQ ICPK~RG EDL - ~ u - Q S I SPG
PAPC QD - - p SZPRVLC - - - - CTA~LSENLRDDEDRI~pK~RADNL
* .... * * * .......
S S p K - CTACQ ....... ESIVKD,
I S I LES
62 - - -HPVSPG
.. *. * .
SRLRT~EKAHP EV'~AG I GC PFAGgGC SFKGSPQSVQEHZVTSQT S~LNLLL - - -
SpL - TQEKVXS DVA~AE IMC pFAGVGCSFKGSPQSMQEREATBQS
IYCRNESRGCA- - - EQLMLGHLLVHLKNDC
P - -NDGCTWKGT- - - LKEYESCHEGLCPFLLTEC
SHLYLLL - -
..... AVLKEWKS S pGSNLGSAPR~LERNL .....
HVDLE~HYEV-C PKF PLTC
..... SDLQ~QAJ~VE"V - AGDLEV~
157 TGD LEV~
- DGCGKKK I p RETFQDHVRAC SKCRVLC RF HTVGC ..... SEMVETEN- - - -LQD
....... -~ .............. YRAPC .................
....................... YRAPC .................
S FKRYGCVFQGTNQQ I EAHEASSAVQH 259
CES- - - -QEE
WLI~EWSNSSEKKVSLLQN~SW~S~S~'~C S ~I ~ J ~ S
HEL ..... QRLREHLALLLS SFLEAQASPGTLNQVGPELLQ LQI~CQ
284 K~ATF QELEQ TFEN
TRAFI LAL ..... QHLVE~ ...................... KL LAQ~-- ~%3tF-LEE A~ 192
TRAFI HLA- - -LAAS IH~LDREH ....... SLE g --- 233
I SAGQVAR1N TGLL E S Q~S RHDQM~ VHD I I~%DlqDL RF QVL ETAS YIqGVL IWI< I RD I
................... RS~/GLK DLA~DL ER LKD
DLEQ ~E~ELEVSTYDGVF IWKI SD
Q = ....... TN
F TRKRQEAVAGRT PAI F S PAFYT S RYGYKMC LRVYLNG DGTGRGT HL S LFFVVMK
DAFRPDVTS S S FQRPVSDI~IASGC
- E~IRE~AIDAFRPDLS S~F~RPQSETNVASGC
SFKKPTGEMN IASGC 534
PVFVAQTVLENG - - TYIKDDT IF I KVIVDTSDLPDP
PLFP'PLSI~.,QS p~F~FKDD'I'MFLKC IV- - - E - TST
F IKAIV- - - DLTGL
Figure I. AminoAcidAlignmentofHuman
TRAF1 and TRAF2
The four polypeptide sequences were aligned using the CLUSTAL
.program (PCGene [IntelliGenetics]). Identical (asterisks) and homolo-
gous (dots) amino acids are shown. Pairwise alignment of EBI6 and
TRAF1 is also Shown, witi~ identical amino acids designated by bold
faced characters. Amino acidsthat form the RING finger motif in LAP1
and TRAF2 and the zinc finger s~trdcture in EBI6 and TRAF1 are under-
lined. Amino acids that form putative coiled-coil structures are boxed.
[he TRAF domain is shown by large boxes.
[::Bi6and LAPI andMurine
These results implicate the LM P1 carboxyl terminus in cell
growth transformation and suggest that the first 44 amino
acids ,may ,be stringently required for transformed cell
growth (Kaye et al., 1995). We have now discovered that
the first 44 amino acids of the LMP1 carboxy-terminal cyto-
plasmic domain interact with a human protein related to
the very recently described putative effectors of tumor ne-
crosis factor receptor (TNFR) signaling (Rothe et al.,
1994). The interaction of this human protein with LMP1
and with the cytoplasmic domains of the TNFR family
members is evidence for a central role of this protein as
an effector of cell growth or death signaling pathways.
A Yeast Two.Hybrid Screen Reveals Proteins That
Interact with the LMP1 Carboxy-Terminal
DNA encoding the200 amino acid LMP1 carboxy-terminal
Table 1. 13-Gal Assay of Protein-Protein Interactions in the Yeast
Transformant 13-Gal Units
G4DBDLMP1(187-386)-G4TAD LAP 1(346-568)
G4DBDLM P 1(187-231)-G4TAD LAP1(183-568)
G4DBD LM P 1(167-231)-G4TAD LAP 1 (346-568)
G4DBDLM P1 (187-231 )
The yeast strain Y190 was transformed with the indicated plasmids,
and transformants were selected on appropriate selective-defined me-
dia. Isolated colonies were grown to mid to late log density and assayed
for I~-gal activity as described in Experimental Procedures. Four indi-
vidual transformants were assayed in each case (except for the interac-
tion between G4DBDSNF1 and G4TADSNF4, for which two trans-
formants were tested), and the average values of ~,-Gal units are
shown. The interaction between G4DBDSNF1 and G4TADSNF4 was
used as a positive control (Harper et al., 1993) and scored 0.8 ~-gal
units or higher in different assays.
cytoplasmic domain was fused in frame to the GAL4 DNA-
binding domain for use as bait in a yeast two-hybrid screen
for cDNAs that encode interactive proteins. The GAL4-
activating domain was fused to cDNAs made from RNA
from EBV-transformed B lymphocytes (Durfee et al.,
1993). Of 5 x 10 s transformants that were tested for
growth in the absence of tryptophane, leucine, and histi-
dine and in the presence of 25 mM 3-aminotriazole, 147
colonies showed at least moderate growth and were ana-
lyzed for ~-galactosidase (15-gal) expression. Two clones
were strongly positive for 13-gal, scoring higher than 8 U
in a standard 13-gal assay, whereas the rest of the clones
had nearly background levels of 13-gal activity (less than
0.04 U). The GAL4-activating domain-fusion proteins
made from these two clones did not interact with GAL4
DNA-binding domain fusions to p53, pRB, lamin, or the
yeast protein kinase SNF1, indicating specificity for the
LMP1 cytoplasmic carboxyl terminus. The two clones
were 3' coterminal cDNAs from the same mRNA. The
clones have a single long open reading frame predicted
to encode part of a novel protein provisionally designated
LMPl-associated protein 1 (LAP1). To obtain the full-
length LAP1 open reading frame, the insert from the longer
clone was used to identify clones from a Xgtl0 library of
cDNAs (Birkenbach et ai., 1993) prepared from RNA from
an EBV-infected B lymphoblast cell line, BL41/B95-8. One
clone has the beginning and most of the 568 codon LAP1
open reading frame, whereas two other clones were de-
rived from an alternatively spliced mRNA that lacks 1061
bases, including the first 320 codons of LAP1.
From thesequence of the complete open reading frame,
full-length LAP1 is predicted to have an amino-terminal
RING finge.r metal-binding motif and a carboxy-terminal
domain that begins with an extended coiled-coil motif (Fig-
EBV LMP1 Transformation and TNFR Family Signaling
1.35- ii~i!i~!, ~,! ,,!
~, D ~,
Figure 2. LAP1 and EBI6 mRNA Abundance
Northern blot of RNA from human tissues (A
and B, both poly(A) + RNA) or cell lines ((3 and
D, poly(A) + RNA~ and total RNA, respecti~ely,~',
hybridized with LAP1 (A and C) or EBI6~(B;andJ
D) probes. The probe is shown belQw the,blot~,
and the origin of RNA is shown above each.
lane. Size markers are to the left of each blot,
and arrows indicate ~e position of specifically
and consistently detected mRNAs. The LAP1
probe detected 2.8 kb and 1.8 kb RNAs,
whereas the EBI6 probe~detected e 2.6 kb RNA.
The high molecular weight bands were not con-
sistently detected in other Northern blots with
these probes. LAP1 (C) a~d EBI6 (D) mRNAs
were also detected in RNA from EBV-infected
BL41 (BL41/B95-8), EBV-neg~tive SL41 (BL41),
and EBV-transformed (IS4) cells. An actin
probe (ACTIN) indicates the relative amounts
of RNA in (C) and (D).
Abbreviations: PA, pancreas, KI, kidney, SM,
skeletal muscle, LI, liver, LU, lu~ng, PL, pla-
centa, BR, brain, and HE, heart.
ure 1). The carboxy-terminal LAP1 domain (amino acids
302-568) has 45% colinear amino acid identity to the
TNFR-associated factor (TRAF) homology domain of the
recently identified murine TRAF1 or -2 (Rothe et al., 1994).
LAP1 is similar to TRAF2 in having an amino-terminal
RING finger motif, but is only 27% identical to TRAF2
overall. The longest open reading frame identified in the
alternatively spliced LAP1 mRNA encodes for a polypep-
tide that initiates at methionine codon 350 within the
coiled-coil motif of full-length LAP1 and includes the rest
of the TRAF domain. Since amino acids 345-568 of LAP1
interact strongly with the LMP1 carboxy-terminal cyto-
plasmic domain (Table 1), the protein encoded by the
spliced LAP1 m RNA is likely to interact also with the LM P1
carboxy-terminal cytoplasmic domain and could modulate
interactions of LAP1 with LMP1.
A Carboxy-Terminal LAP1 Domain Interacts with a
LMP1 Membrane Proximal Cytoplasmic Domain
The interaction of the full-length LMP1 cytoplasmic car-
boxyl terminus (amino acids 187-386) with the LAP1 car-
boxy-terminal 386 or 223 amino acid polypeptide scored
higher than the positive control in the yeast two-hybrid
dependent ~-gal assay (Table 1). The interaction of the
membrane proximal 44 amino acids of the LMP1 cyto-
plasmic carboxyl terminus (amino acids 187-231) with the
LAP1 carboxy-termina1386 or 223 amino acids also gener-
ated higher I~-gal activity than did the positive control (Ta-
ble 1). Thus, the membrane proximal 44 amino acids of
the LMP1 cytoplasmic carboxyl terminus and the LAP1
carboxy-terminal 223 amino acid-TRAF homology do-
main are sufficient for high level interaction in the yeast
two-hybrid assay. The EBV recombinant molecular ge-
netic evidence, which indicates that the LMP1 carbox~-
terminal cytoplasmic domain is essential for transforma-
tion, provides a linkage between this biochemical interaction
An EBV-Induced Cell Protein Is a
Human TRAF1 Homolog
LAP1 is 32% colinearly homologous to another novel hu-
man protein that had been identified because its encoding
mRNA is more abundant in the EBV-infected B lympho-
blast line BL41/B95-8 than in the uninfected control BL41
cells (Figure 1). This EBV-induced mRNA 6 (EBI6) is pre-
dicted to encode for a 416 amino acid protein that is 86%
colinearly identical to the murine TRAF1 (Rothe et al.,
1994) (Figure 1). EBI6 is probably the human homolog of
murine TRAF1 since these proteins are nearly identical
throughout their entire sequence (Figure 1). EBI6 and
TRAF1 have similar amino-terminal zinc finger motifs and
are 95% identical in their TRAF domains (Figure 1). When
fused to the GAL4-activating domain, EBI6 amino acids
53-416 do not interact in yeast with the LMP1 carboxy-
terminal cytoplasmic domain or with amino acids 12-568
of LAP1 fused to the GAL4 DNA-binding domain (Table
1). The failure of EBI6 to interact with LAP1 distinguishes
LAP1 from the murine RING finger protein TRAF2, which
,SGSLMm + +++++++
FLAGLAP1 -- "Jl- "1---
FLAGEBI6 -- -- "3 I- "t--
FLAOE2 -~ +
Figure 8. Intracellular Association of LMP1 with LAPI or EBI6 in
Transfected B JAB, Non-EBV-lnfected, B Lymphoma Cells
B JAB cells (10 x 106 cells per transfection) were electroporated with
plasmids expressing the proteins indicated by a plus sign at the bottom
of the figure. Approximately 20 hr posttransfection, 4 x 106 cells from
each transfection were lysed and subjected to immunoprecipitation
with 10 tlg of M2 anti-FLAG monoclonal antibody (Kodak). Equivalent
cell lysates obtained before immunoprecipitation (lanes 1-4) and im-
munoprecipitated material (lanes 5-8) were analyzed by SDS-PAGE
on a 7.5 % gel, transferred onto nitrocellulose, and subjected to West-
ern blot analysis using rabbit anti-LM P1 polyclonal antisera (Hennessy
et al., 1984) and ~2Sl-labeled protein A, followed by autoradiography.
The position of LMP1 is shown by the arrow, and molecular weight
markers are shown on the right side of the panel. LMP1 was readily
coimmunoprecipitated with FLAGLAP1 or FLAGEBI6 (lanes 6 and 7).
No detectable LMP1 was seen in anti-FLAG immunoprecipitations
from cells cotransfected with pSG5LMP1 and either vector control
(pSG5) or a construct expressing a FLAG-tagged EBNA2 (FLAGE2,
lanes 5 and 8).
scored positive in the yeast two-hybrid system with TRAF1
(Rothe et al., 1994). While the failure could be due to differ-
ences between the human RING finger (LAP1) or zinc
finger (EBI6) TRAFs and murine TRAF1 and -2, the lower
level of homology of LAP1 to TRAF2 is more likely to ac-
count for this discrepancy given the extensive identity be-
tween EBI6 and LAP1.
Expression of LAP1 and EBI6 in Human Tissues
and Cell Lines
The full-length 2.8 kb and alternatively spliced 1.8 kb LAP1
mRNAs are expressed in all tissues examined (Figure 2A).
In contrast, the 2.6 kb EBI6 mRNA is readily detected in
lung, spleen, and tonsil, is barely detected in placenta,
and is not detectable in pancreas, kidney, smooth muscle,
liver, brain, or heart (Figure 2B; data not shown). To evalu-
ate the effects of EBV infection on LAP1 and EBI6 mRNA
abundance, LAP1 or EBI6 probes were hybridized to RNAs
from EBV-negative BL41 B lymphoblasts, from EBV-infected
BL41 B lymphoblasts (BL41/B95-8), or from an EBV-trans-
formed B lymphoblast cell line (IB4) (Figures 2C and 2D).
LAP1 mRNAs were similarly abundant in EBV-positive or
-negative cells, whereas the EBI6 message was at least
8-fold more abundant in the EBV-infected BL41/B95-8 or
IB4 cells than in noninfected BL41 cells.
LMP1 Associates with LAP1 and EBI6
in B Lymphoblasts
LMP1 and FLAG epitope-tagged LAP1 (FLAGLAP1) or
FLAG-tagged EBI6 (FLAGEBI6) were expressed in an effi-
ciently transfectable non-EBV-infected human B lympho-
blast cell line (BJAB). As controls, LMP1 and FLAG epi-
tope-tagged EBNA2 (FLAGEBNA2) were expressed in
B JAB cells. Approximately 18 hr posttransfection, the cells
were lysed in nonionic detergent; FLAG fusion proteins
were immunoprecipitated with an anti-FLAG monoclonal
antibody; and LMP1 was detected by Western blotting
(Hennessy et al., 1984). LMP1 was expressed at similar
levels in all transfections (Figure 3, lanes 1-4). A substan-
tial fraction of LMP1 immunoprecipitated along with FLAG-
LAP1 or FLAGEBI6 and did not immunoprecipitate along
with FLAGEBNA2 (Figure 3, lanes 4-8). Thus, LMP1 spe-
cifically associates with LAP1 or EBI6 in B lymphoblasts.
LMP1 Redirects the Localization of LAP1 and EBI6
to LMP1 Plasma Membrane Patches
FLAG-tagged LAP1 or EBI6 expressed in BJAB cells local-
izes throughout the cytoplasm to punctate structures that
resemble cytoplasmic vesicles (Figures 4A and 4C). When
LMP1 and FLAGLAP1 or FLAGEBI6 were expressed in the
same cells, LM P1 localized to its characteristic patches or
caps in the plasma membrane (Figures 4F, 41, 4L, 40),
and FLAGLAP1 or FLAGEBI6 now colocalized with LMP1
in plasma membrane patches or caps (Figures 4E and 4H
or Figures 4K and 4N, respectively). FLAGEBI6 did not
localize as precisely with LMP1 as did FLAGLAP1, sug-
gesting a weaker, less direct, or more transient interaction
with LMP1. Thus, LMP1 expression redirects LAP1 and
(to a lesser extent) EBI6 from scattered cytoplasmic struc-
tures to LMP1 plasma membrane patches.
LAP1 and EBI6 Associate with TNFR Family
The homology of LAP1 and EBI6 to routine TRAF2 and
TRAF1 prompted us to examine whether LAP1 and EBI6
can interact with members of the TNFR family, including
CD40, lymphotoxin-I~ receptor (LTI~R), and Fas (Bancher-
eau et al., 1994; Nagata, 1994; Smith et al., 1994; Ware
et al., 1995), using receptor cytoplasmic domains con-
structed as fusion proteins with glutathione S-transferase
(GST). In vitro translated LMP1 did not interact with GST
or GST fusions to TNFRs (Figure 5A, lanes 1-3). However,
in vitro translated LAP1 bound specifically (112-fold over
GST background) to the p80 cytoplasmic domain and less
well (14-fold over GST background) to the p60 cytoplasmic
domain (Figure 5A, lanes 4-6; Figure 5B, lanes 3 and 4;
data not shown). LAP1 also showed nearly quantitative
binding to the cytoplasmic domains of TNFR-related pro-
teins CD40 and LTI~R and less efficient binding to Fas
(Figure 5B). EBI6 bound less efficiently than did LAP1 to
p80 and showed weak binding to p60 (Figure 5A, lanes
LAP1 and EBI6 also associate with the p80 receptor in
vivo. The p80 TNFR was coexpressed with FLAGLAP1,
FLAGEBI6, or FLAGEBNA2 in B JAB cells that do not ex-
press any surface pS0 receptor (data not shown). FLAG-
tagged proteins were immunoprecipitated from cell ly-
sates using M2 anti-FLAG antibody, and the immune
complexes were analyzed for the presence of p80 TNFR
by Western blotting (Figure 5C). The M2 anti-FLAG anti-
body coimmunoprecipitated the p80 receptor from cells
EBV LMP1 Transformation and TNFR Family Signaling
ii !I~Z~ ~ .......
Figure 4. Subcellular Localization of LAP1 and EBI6 in the Presence
or Absence of LMP1
The intracellular distribution of FLAG-tagged LAP1 and EBI6 was de-
termined by indirect immunofluorescence using M2 anti-FLAG mono-
clonal antibody and rabbit anti-LMP1 polyclonal antisera. B JAB cells
were transfected with FLAGLAP1 (A, B, and E-J) or FLAGEBI6 (C,
D, and K-P) expressing constructs in the presence of vector pSG5
(A-D) or pSG5LMP1 (E-P). M2 anti-FLAG reactivity was visualized
with a fluorescein isothiocyanate-conjugated goat anti-mouse second-
ary antibody (A, C, E, H, K, N). LMP1 was detected with a Texas Red-
conjugated goat anti-rabbit secondary antibody (F, I, L, O). Phase-
contrast pictures are shown in (B), (D), (G), (J), (M), and (P). M2 and
anti-LMP1 antibodies did not show any reactivity in untransfected cells.
No cross-reactivity was observed between M2 and the goat anti-rabbit
secondary antibody or between the rabbit anti-LMP1 and goat anti-
mouse secondary antibody (data not shown).
expressing FLAGLAP1 or FLAGESI6 (Figure 5C, lanes 1
and 2), but not from cells expressing FLAGEBNA2 (Figure
5C, lane 3). FLAGLAP1 and FLAGEBI6 associated with
both the -70 kDa precursor and the mature p80 TNFR
(Ware et al., 1991), indicating that the association with
LAP-1 occurs prior to receptor expression at the cell sur-
face. These studies indicate the potential of LAP1 and
EBI6 to participate in TNFR signaling and also provide
evidence that LAP1 may be a component common to the
CD40, LTI3R, and TNFR signal transduction pathways.
]r IF I
2 3 4 5 6 7 8 9
8 o~ A
C 1 2 3 4 5 6 7
+ - -
-- + --
+ + +
-- + _ _
FLAGE2 -- -- + -- -- +--
Figure 5. Association of LAP1 and EBI6 with TNFR-Related Proteins
(A) Cytoplasmic domains of the p60 or p80 TNFR constructed as fusion
proteins with GST and bound to glutathione beads were incubated
with [3~S]methionine-labeled LMP1, LAP1, or EBI6 translated in vitro
(5 I11 reaction mix), and the fraction bound to glutathione beads was
analyzed on a 8.5% SDS-PAGE and processed by a phosphorimager.
Lanes 1,4, and 7 are pull downs with GST-p80; lanes 2, 5, and 8 are
those with GST-p60; and lanes 3, 6, and 9 are those with GST-control.
Coomassie blue staining of the gel demonstrated the presence of ap-
proximately equivalent amounts of GST or GST-fusion proteins.
(B) Glutathione beads containing the cytoplasmic domains of p60 (lane
3), p80 (lane 4), Fas (lane 5), CD40 (lane 6), and LTJ3R (lane 7) ex-
pressed as GST-fusion proteins or GST (lane 2) were incubated with
[3SS]methionine-labeled LAP1 (2 p.I of in vitro translation reaction mix
were used per reaction) as in (A) and were analyzed by SDS-PAGE
and autoradiography. In vitro translated LAP1 (2 p.I) was analyzed in
(C) Coimmunoprecipitation of LAP1 and EBI6 with p80 TNFR in
cotransfected cells. BJAB cells were cotransfected with plasmids ex-
pressing the FLAG-tagged proteins indicated by a plus sign at the
bottom of the figure or were left untransfected (lane 7). Approximately
20 hr posttransfection, the cells were lysed, and lysates from 10 x
106 cells were subjected to immunoprecipitation with M2 anti-FLAG
monoclonal antibody. Equivalent cell lysates obtained before immuno-
precipitation (lanes 4-7) and immunoprecipitated complexes (lanes
1-3) were analyzed by Western blotting using an anti-p80 TNFR anti-
body (VanArsdale and Ware, 1994). The positions of mature p80 TNFR
and the immunoglobulin heavy chain (Ig) are shown by arrows. The
asterisk shows the position of a precursor form of the p80 TNFR. The
p80 receptor was readily coimmunoprecipitated with FLAGLAP1 or
FLAGEBI6 (lanes 1 and 2). No detectable p80 receptor was immuno-
precipitated with anti-FLAG antibody from cells cotransfected with
plasmids expressing p80TNFR and FLAGEBNA2 (FLAGE2, lane 3).
We have identified two human TRAFs and confirmed their
association with the human p80 TNFR. The TRAF domain
was initially defined because of more than 50% colinear
primary sequence identity through the carboxy-terminal
230 amino acids of murine TRAF1 and -2 (Rothe et al.,
1994). Genetic and biochemical data link TRAF1 and -2
to the growth and NF-•B-transducing effects of a domain
near the carboxyt terminus of the p80 TNFR cytoplasmic
tail (Rothe et al., 1994). EBI6 is almost certainly the human
TRAF1, based on the ability of both proteins to associate
with the p80 TNFR, similar tissue restricted expression,
similar amino-terminal zinc finger motifs, 95% primary se-
quence identity in the TRAF domains, and an overall 86%
colinear primary sequence identity. LAP1 is probably not
the human TRAF2, but rather a human RING finger ana-
log. If so, there may be one or more human RING finger
TRAFs as yet unidentified. Although LAP1 and TRAF2
are similar in size, have an amino-terminal RING finger
domain, and are constitutively expressed in most tissues,
LAP1 is 45% identical to either TRAF2 or TRAF1 in the
TRAF domain and is quite divergent from TRAF2 outside
of the TRAF domain. Overall, LAP1 and TRAF2 are only
27% identical. LAP1 also appears to be functionally dis-
tinct from TRAF2 in not directly interacting with EBI6, the
LAP1 and EBI6 interact with the cytoplasmic domain of
not only p80 TNFR but also with p60 TNFR, albeit less
well than with p80. This is the first evidence of interaction
beyond p80 between these putative effectors and the
TNFR family. Indeed, LAP1 also interacts strongly with
the LTI3R and CD40 cytoplasmic domains. These findings
provide a new molecular basis for understanding the com-
mon effects of activation of these members of the TNFR
family. Heterogeneity among TRAFs or other cell proteins
may provide components to the receptor-TRAF com-
plexes that determine the specific phenotypic outcome(s)
of receptor activation.
The identification of these human TRAFs is in the con-
text of an investigation into the mechanisms by which
LMP1 transforms cells. These biochemical and genetic
experiments and previous EBV recombinantmolecular
genetic analysis establish a connection between the role
of LMP1 in B lymphocyte growth transformation and TNFR
signaling pathways. As briefly reviewed in the introduction,
LMP1 is a dominant oncogene that has multiple down-
stream effects on cell growth and gene expression, at least
some of which are NF-KB mediated. LMP1 interacts
strongly and directly with LAP1 and also associates with
EBI6 in human lymphoblasts. Our genetic, biochemical,
and intracellular localization data on the association of
~LMP1 with LAP1 and EBI6, taken together with the previ-
ous genetic and biochemical linkage of TRAF1 and -2 to
p80 TNFR signaling, reinforce a role for the TRAFs as
mediators of cell growth or death and NF-~B responses
(for schematic model, see Figure 6).
The six markedly hydrophobic transmembrane domains
of LMP1 enable it to aggregate in the plasma membrane
and to present aggregated cytoplasmic domains to the
Figure 6. Schematic Representation of p80 TNFR Activation and
LMP1 Complexes at the Plasma Membrane
(A) Model forthe activation of the p80 TNFR is shown. The extracellular
region of the TNFR is composed of four domains with characteristic
cysteine patterns. The cytoplasmic domain of the receptor is known
to associate with complexes of TRAF molecules (TRAF), including
TRAF1, TRAF2, and LAP1. Upon binding of TNF (shown here as a
trimer), the extracellular domains of several receptor molecules are
believed to be cross-linked, causing aggregation of intracellular do-
mains and their associated TRAF molecules. Clustering of receptor
molecules and their intracellular domains results in signal transduction
as manifested by a number of phenotypic alterations, including the
activation of transcription factor NF-KB (NFkB) and cell growth.
(B) Three LMP1 molecules are shown to form a constitutive complex
at the plasma membrane (depicted by the stippled area between the
two solid horizontal lines). The amino-terminal (N) and carboxy-
terminal (C) cytoplasmic regions of LMP1 are shown by short and long
lines, respectively. The transmembrane domains of LMP1 are depicted
by vertical cylinders, which are joined by short reverse turns (short
curved lines). Aggregation of LMP1 molecules at the plasma mem-
brane brings together LMPl-associated TRAF molecules ('rRAF) in a
complex, thus, generating a constitutive signal that results in pleiotro-
pic effects, including activation of NF-KB (NFkB) and cell growth.
TRAFs (Figure 6B). In presenting aggregated TRAF-
interacting domains, LMP1 mimics TNFR aggregation,
which appears to be essential for signal transduction (En-
gelmann et al., 1990; Loetscher et al., 1991; Pennica et
al., 1992; Tartaglia and Goeddel, 1992). Receptor cross-
linking probably locally aggregates TRAFs and associated
molecules, creating a second messenger signal perhaps
mediated by a receptor-associated serine/threonine ki-
nase (Figure 6A) (Darnay et al., 1994a, 1994b; VanArsdale
and Ware, 1994). Since LMP1 constitutively aggregates
EBV LMP1 Transformation and TNFR Family Signaling
LAP1 and EBI6 in oligomeric complexes at plasma mem-
brane patches, these complexes could constitutively
activate growth signals and NF-KB in the absence of
extracellular stimuli (Figure 6B). LM P1 signaling via TRAF
molecules may thus proceed independently of TNFR mol-
ecules. Alternatively, LMP1-TRAF aggregates may nucle-
ate larger, more diverse, or more stable TNFR family-
TRAF complexes through interactions among the extended
coiled-coils of the TRAF domains. In fact, some evidence
favors the latter alternative in that lymphotoxin-~ (LT(~) is
an autocrine growth factor for EBV-transformed lympho-
blastoid cell lines (Estrov et al., 1993; Gibbons et al., 1994).
Furthermore, expression of the full range of EBV latent
infection-associated proteins in Burkitt lymphoma cell
lines induces LTa and the p80 receptor (Gibbons et al.,
1994). Moreover, antagonistic antibodies to the p60 TNFR
have a negative growth effect in such cells (Gibbons et
al., 1994). The LMP1 cytoplasmic carboxy-terminal do-
main and TNFR family members could even interact with
different domains of the same LAP1 molecule since there
is no obvious homology between the LMP1 cytoplasmic
carboxyl terminus and the cytoplasmic domains of TNFR
The induction of EBI6 by latent EBV infection and the
association of EBI6 with I_MP1 in B lymphoblasts are also
evidence of an important role for EBI6 in EBV-mediated
B lymphocyte growth transformation. The interaction ap-
pears to be less direct than with LAP1 and may be medi-
ated by another as yet unidentified human RING finger
TNF and CD40 ligand are well known mediators of
growth of B lymphocytes and of other cell types that are
targets for LMPl-transforming effects (Noelle et al., 1992;
Boussiotis et al., 1994). In fact, CD40 ligation and interleu-
kin-4 treatment are sufficient to sustain the proliferation
of primary B lymphocytes in vitro for several months, and
the cells are phenotypically similar to EBV-transformed
lymphocytes (Saeland et al., 1993; Banchereau et al.,
1994; Galibert et al., 1994). The LTI3R is expressed on
epithelial cells (C. W. and J. Browning, unpublished data),
while basal epithelial cells and anaplastic nasopharyngeal
carcinoma cells also express high levels of CD40 (Busson
et al., 1988; Young et al., 1989). LMP1, through constitu-
tive direct interaction with LAP1, may amplify or usurp
LTI~R and CD40 signal transduction and constitutively pro-
mote cell growth. Nasopharyngeal carcinoma is tightly as-
sociated with EBV, and LMP1 is frequently expressed in
the tumor cells (Brooks et al., 1992). Hodgkin's disease
is another EBV-associated malignancy in which LMP1 is
expressed (Herbst et al., 1991). CD40, TNFRs, and the
related receptor CD30 are up-regulated in Hodgkin's dis-
ease cells (Froese et al., 1987; Pfreundschuh et al., 1989;
Carde et al., 1990; O'Grady et al., 1994; Trumper et al.,
1994). A potentially important consequence of the demon-
strated interaction between LAP1 and LMP1 is that inhibi-
tors of that interaction may affect the growth or development
of these LMPl-associated malignancies.
In interacting with components of receptor signaling,
LMP1 is reminiscent of bovine papilloma virus E5. Bovine
papilloma virus E5 dimerizes in the plasma membrane, pre-
sumably through hydrophobic interactions, and activates re-
ceptors for epidermal growth factor, platelet-derived growth
factor, or colony-stimulating factor 1 (Martin et al., 1989;
Petti et al., 1991; Petti and DiMaio, 1992). E5 binds a
component of vacuolar H+-ATPases, and this may affect
receptor recycling (Goldstein et al., 1991).
A curious aspect of our data is the finding that LAP1 or
EBI6 localized to vesicle-like structures in the cytoplasm
of B lymphoblasts. Furthermore, LAP1 is constitutively ex-
pressed in cells, and there may be another constitutively
expressed RING finger protein that intermediates between
LMP1 and EBI6. These Constitutively expressed proteins
may also have a role in vesicle biology.
The interaction of LMP1 with TNFR signaling pathways
may also be important in enabling EBV-infected cells to
evade cellular host defense mechanisms in latent or lytic
EBV infection. LMP1 is one of the few EBV genes ex-
pressed in both phases of the virus life cycle (Mann et al.,
1985; Rowe et al., 1992). Several virus families appear to
specifically target the TNF/lymphotoxin pathways, pre-
sumably to avoid these immune cell mediators of cytotox-
icity. Pox viruses produce soluble versions of the p80
TNFR (Smith et al., 1991; Massung et al., 1993); proteins
encoded by the adenovirus E3 region block the apoptotic
function of TNF (Gooding, 1992); and HIV utilizes NF-KB-
activating signals induced by TNF signaling to enhance
transcription (Poli et al., 1990). The binding of EBV LMP1
to LAP1 may effectively compete with normal LAP1 bind-
ing to the p60 TNFR, blocking the induction of cell death
mediated by that receptor (Tartaglia et al., 1993b) or
blocking other functions critical to host defense (Pfeffer
et al., 1993; Rothe et al., 1993), while simultaneously usurp-
ing the growth-promoting signals of the ~)80 TNFR (Tartag-
lia et al., 1993a).
The GAL4 DNA-binding domain (G4DBD) fusions were constructed
in vector pAS2 (Harper et al., 1993). G4DBDLMPI(187-386) was con-
structed by polymerase chain reaction-mediated (PCR-mediated) am-
plification of the LMP1 cDNA fragment, encoding amino acids 187-
386, using oligos L1-5PCR (5'-CGCGGATCCATGGACAACGACA-
CAGTG-3') and L1-4PCR (5'-CGCGGATCCTTAGTCATAGTAGCT-
TAG-39, followed by cloning into the BamHI site of pAS2. G4D-
BDLMP1(187-231) was constructed by PCR amplification of the LMP1
cDNA fragment, encoding amino acids 187-231, using oligos L1-
5PCR and LCA231 (5"CGCGGATCCTTAGGCTCCACTCACGAG-
CAG-3~, followed by cloning into the BamHI site of pAS2. G4DBDL-
AP1(12-568) was constructed by isolating the BssHII-BamHI
fragment of LAP1 cDNA from pSG5LAP1, blunt-ending it using T4
DNA polymerase, and subcloning it into the Smal site of pASI. GAL4
transactivating domain (G4TAD) fusions were as follows. G4TA-
DEBI6(53-416) was constructed by subcloning the Bglll fragment of
EBI6 cDNA into the BamHI site of pACTII (a gift of S. EUedge). G4TA-
DEBI6(53-416) encodes for an in-frame fusion of EBI6 amino acids
53-416 to the acidic transactivating domain of GAL4. Plasmids
G4TADLAP1 (183-568) and G4TAD LAP1(345-568) were isolated from
the yeast two-hybrid screening. Plasmids expressing SNF1 fused to
the DNA-binding domain of GAL4 (G4DBDSNF1) or SNF4 fused to
the activation domain of GAL4 (G4TADSNF4) were gifts of S. Elledge.
SNF1 and SNF4 are two yeast proteins that are known to interact with
each other, and they were used as a positive control in the yeast
two-hybrid-dependent l}-gal assay (Harper et al., 1993). LAP1 cDNAs
were subcloned into the EcoRI site of plasmid pSG5 (Stratagene) for
sequencing analysis, pSG5 subclones of the longest LAP1 cDNAs
were spliced at the Nrul site to generate the full-length LAP1-
expressing construct pSG5LAP1. The EcoRI insert of Zgtl0 clone EBI6
was subcloned into the plasmid pBluescript for sequencing analysis.
pSG5FLAGLAP1 and pSG5FLAGEBI6 were constructed in vector
pSG5 by placing through PCR a FLAG-encoding DNA fragment just
after the initiator AUG codon. Expression plasmids for TNFRs were
previously described (Ware et al., 1991).
Construction of the ~.gtl 0 cDNA library from the EBV-positive cell line
BL41/B95-8 was previously described (Birkenbach et al., 1993). Sub-
tractive hybridization and homology screening of the Xgtl0 library was
done as described before (Birkenbach et al., 1993).
Yeast Two-Hybrid Screening
Yeast transformation was performed according to the method of
Schiestl and Geitz (1989). The yeast strain Y190 (Du rfee et al., 1993)
was transformed with plasmid construct G4DBDLMP 1(187-386), and
transformants were plated on Trp selective-defined media. A single
colony was picked, and the expression of the LMP1 fusion protein
was verified by Western blotting using the $12 anti-LMP1 monoclonal
antibody. The G4DBDLM P 1 (187-386) transformant was subsequently
transformed with a cDNA library constructed previously from an EBV-
transformed lymphoblastoid cell line (Durfee et al., 1993), and selec-
tion was done on Trp Leu- His- selective-defined media in the pres-
ence of 25 mM 3-aminotriazole (Sigma) as previously described
(Durfee et al., 1993). Colonies that showed moderate to intense growth
were streaked on Trp- Leu- Hi~ selective-defined media containing
50 mM 3-aminotriazole and tested for 13-gal expression by a filter lift
assay (Breeden and Nasmyth, 1985). For quantitation of ~-gal expres-
sion, yeast clones were grown in appropriate selective media to ODsoo
of 0.5-1.2 and were assayed for ~-gal activity using ONPG and stan-
dard conditions as previously described (Breeden and Nasmyth,
1985). I~-Gal units were expressed as (1000/hls)/(assay time in mi-
nutes)(ceU culture volume in milliliters)(OD~). The interactions of
G4DBD with G4TADLAPl(183-568), G4TADLAPl(346-568), or
G4TADEBI6(53-416) and of G4TAD with G4DBDLMP1(187-386),
G4DBDLMPl(187-231), or G4DBDLAP1(12-568) scored ~<0.1 U of
~-gal. Library-derived piasmids were recovered by transformation of
competent bacteria with total yeast DNA preps, followed by selection
for ampicillin resistance as previously described (Ausubel et al., 1987).
Northern blots containing poly(A) ÷ RNA (2 p.g per lane) from eight
human tissues were purchased from CLONTECH. RNA was prepared
from EBV-positive (BL41/B95-8) or EBV-negative (BL41) Burkitt's
lymphoma cell lines and a lymphoblastoid cell line (IB4) as previously
described (Birkenbach et al., 1993). cDNA probes were labeled by
random hexanucleotide priming (Stratagene) using [32P]dCTP. The
RNA blots were hybridized to 32P-labeled cDNA probes under high
stringency conditions as described (Mosialos et al., 1994). Northern
blot filters were exposed to autoradiography film or were processed
by phosphorimager analysis.
Immunoprecipitations, Western Blotting,
BJAB cells were eiectroporated at 220 V and 960 p~F in 400 pl of
RPMI-1640 medium containing 10% fetal calf serum. Approximately
20 hr posttranfection, cells were lysed for 30 rain on ice in 0.5% NP-40
lysis buffer containing 50 mM HEPES (pH 7.4), 250 mM NaCI, 10%
glycerol, 2 mM EDTA, 1 mM PMSF, 2 i~g/ml aprotinin, 2 i~g/ml pepstatin
A, and 2 ~g/ml leupeptin. Cell debris were removed by centrifugation
at 10,000 x g for 10 rain at 4°C. The cell lysates were precieared
with protein G-Sepharose beads for I hr at 4°C. The primary antibody
was then added for 1 hr at 4°C, and immunoglobulin complexes were
collected on protein G-Sepharose beads for 1 hr at 4°C. The beads
were then washed six times with 1 ml of lysis buffer each time, and
protein complexes were recovered by boiling in SDS sample buffer
and were analyzed by SDS-polyacrylamide gel electrophoresis (SDS-
PAGE). Western blotting was done using standard techniques as pre-
viously described (Mosiales et al., 1994).
Indirect immunofluorescence analysis on transfected cells was
done approximately 18-20 hr posttransfection as previously described
(Mosialos et al., 1994).
Production and Purification of GeT-Fusion Proteins
The cytoplasmic domains Of the p80 and p60 TNFRs were amplified
from the corresponding cDNAs by PCR and were cloned in frame into
the pGEX-4T-1 expression vector (Pharmacia) using the EcoRI and
Xhol restriction sites for the p60 TNFR and the EcoRI and Xhol sites
for the p80 TNFR. A similar strategy was employed for the construction
of a GST fusion to the LT~R cytoplasmic domain. GST-fusion proteins
of the cytoplasmic domains of CD40 and Fas were a gift of J. Reed
(La Jolla Cancer Research Institute). Expression and purification of
GST-fusion proteins were performed essentially as described pre-
viously (Smith and Johnson, 1988). Fusion protein concentrations of
3-5 mg/ml of glutathione-agarose beads (Pharmacia) were routinely
obtained. In vitro translations were done using the rabbit reticulocyte-
coupled in vitro transcription translation system (TNT, Promega) ac-
cording to the protocol of the manufacturer. In vitro translated proteins
were diluted with binding buffer (PBS containing 0.1% NP-40, 0.5
mM DTT, 10% glycerol, 1 mM PMSF, 2 i~g/mi aprotinin) and were
preclearad with glutathione beads for 45 rain at 4°C. GST or GST-
fusion proteins bound to glutathiene beads were then incubated with in
vitro translated proteins for 1 hr at 4°C. The beads were subsequently
washed five times with 0.5 ml of binding buffer each time, and bound
proteins were recovered by boiling in SDS sample buffer and were
analyzed by SDS-PAGE.
The corresponding author for this work is E. K. We are grateful to
S. Elledge for the GAL4-transactivating domain cDNA library and for
reagents necessary for the two-hybrid screening and to J. Reed for
GST constructs. We thank K. Kaye, E. Hatzivassiliou, E. Robertson,
K. Izumi, J. Minton, D. Tzamarias, W. Lesslauer, S. Fields, and J.
Pietenpol for reagents and helpful discussions. L. Vara provided excel-
lent technical assistance. This work was supported by grant CA47006
from the National Cancer Institute (to E. K.), grant IM663 from the
American Cancer Society (to C. W.), and grant RT0261 from the Ciga-
rette and Tobacco Surtax Fund of the State of California through the
Tobacco-Related Diseases Research Program (to C. W). G. M. was
supported by a postdoctoral fellowship of the Leukemia Society of
America, and M. B. was supported by a Physician Scientist Award
(grant 5K11CA01341) of the National Cancer Institute of the United
States Public Health Service, by a Scholarship of the James S. McDon-
nell Foundation, and by a Junior Faculty Research Award of the Ameri-
can Cancer Society. R. Y. was supported by a National Research
Service Award of the U. S. Public Health Service (AI08548-02).
Received November 28, 1994; revised December 19, 1994.
Ausubel, F. M., Brent, R., Kingston, R. E., Moere, D. D., Seidman,
J. G., Smith, J. A., and Struhl, K. (1987). Current Protocols in Molecular
Biology (New York: John Wiley and Sons).
Baichwal, V. R., and Sugden, B. (1988). Transformation of Balb/3T3
cells by the BNLF-1 gene of Epstein-Barr virus. Oncogene 4, 67-74.
Banchereau, J., Bazan, F., Blanchard, D., Briere, F., Galizzi, J. P.,
van Kooten, C., Liu, Y. J., Rousset, F., and Saelar~d, S. (1994). The
CD40 antigen and its ligand. Annu. Rev. Immunol. 12, 881-922.
Birkenbach, M., Josefsen, K., Yalamanchili, R., Lenoir, G., and Kieff,
E. (1993)~ Epstein-Barr virus-induced genes: first lymphocyte-specific
G protein-coupled peptide receptors. J. Virol. 67, 2209-2220.
Bouesiotis, V. A., Nadler, L. M., Strominger, J. L., and Goldfeld, A. E.
(1994). Tumor necrosis factor alpha is an autocrine growth factor for
normal human B cells. Proc. Natl. Acad. Sci. USA 91, 7007-7011.
Breeden, L., and N asmyth, K. (1985). Regulation of the yeast HO gene.
Cold Spring Harbor Symp. Quant. Biol. 50, 643-650.
Brooks, L., Yao, Q. Y., Rickinson, A. B., and Young, L. S. (1992).
Epetein-Barr virus latent gene transcription in nasopharyngeal carci-
EBV LMP1 Transformation and TNFR Family Signaling
noma cells: coexpression of EBNA 1, LMP1, and LMP2 transcripts. J.
Virol. 66, 2689-2697.
Busson, P., Ganem, G., Flores, P., Mugneret, F., Clausse, B., Caillou,
B., Braham, K., Wakasugi, H., Lipinski, M., and Tursz, T. (1988). Estab-
lishment and characterization of three transplantable EBV-containing
nasopharyngeal carcinomas. Int. J. Cancer 42, 599-606.
Busson, P., McCoy, R., Sadler, R., Gilligan, K., Tursz, T., and Raab-
Traub, N. (1992). Consistent transcription of the Epstein-Barr virus
LMP2 gene in nasopharyngeal carcinoma. J. Virol. 66, 3257-3262.
Carde, P., Da Costa, L., Manil, L., Pfreundschuh, M., Lumbroso, J.,
Saccavini, J., Caillou, B., Ricard, M., Boudet, F., Haya, M., Diehl, V.,
and Parmentier, P. (1990). Immunoscintiography of Hodgkin's disease:
in vivo use of radiolabeled monoclonal antibodies derived from Hodg-
kin cell lines. Eur. J. Cancer 26, 474.
Cohen, J. I., Wang, F., Mannick, J., and Kieff, E. (1989). Epstein-Barr
virus nuclear protein 2 is a key determinant of lymphocyte transforma-
tion. Proc. Natl. Acad. Sci. USA 86, 9558-9562.
Darnay, B. G., Reddy, A. G., and Aggarwal, B. B. (1994a). Identification
of a protein kinase associated with the cytoplasmic domain of the p60
tumor necrosis factor receptor. J. Biol. Chem. 269, 20299-20304.
Darnay, B. G., Reddy, S. A. G., and Aggarwal, B. B. (1994b). Physical
and functional association of a serine-threonine protein kinase to the
cytoplasmic domain of the p80 form of the human tumor necrosis factor
receptor in human histiocytic lymphoma U-937 cells. J. Biol. Chem.
Dawson, C. W., Rickinson, A. B., and Young, L. S. (1990). Epstein-Barr
virus latent membrane protein inhibits human epithelial cell differentia-
tion. Nature 344, 777-780.
Durfee, T., Becherer, K., Chen, P.-L., Yeh, S.-H., Yang, Y., Kilburn,
A. E., Lee, W.-H., and Elledge, S. J. (1993). The retinoblastoma protein
associates with the protein phosphatase type 1 catalytic subunit.
Genes Dev. 7, 555-569.
Engelmann, H., Holtmann, H., Brakebusch, C., Avni, Y. S., Sarov, I.,
Nophar, Y., Hadas, E., Leitner, O., and Wailach, D. (1990). Antibodies
to a soluble form of a tumor necrosis factor (TNF) receptor have TNF-
like activity. J. Biol. Chem. 265, 14497-14504.
Estrov, Z., Kurzrock, R., Poscik, E., Pathak, S., Kantarjian, H, M., Zipf,
T. F., Harris, D., Talpaz, M., and Aggarwal, B. B. (1993). Lymphotoxin
is an autocrine growth factor for Epstein-Barr virus-infected B cell
lines. J. Exp. Med. 177, 763-774.
Fahraeus, R., Rymo, L., Rhim, J. S., and K~ein, G. (1990). Morphologi-
cal transformation of human keratinocytes expressing the LMP gene
of Epstein-Barr virus. Nature 345, 447-449.
Fennewaid, S., van Santen, V., and Kieff, E. (1984). The nucleotide
sequence of a messenger RNA transcribed in latent growth trans-
forming virus infection indicates that it may encode a membrane pro-
tein. J. Virol. 51,411-419.
Froese, P., Lemke, H., Gerdes, J., Havensteen, B., Schwarting, R.,
Hansen, H., and Stein, H. (1987). Biochemical characterization and
biosynthesis of the Ki-1 antigen in Hodgkin-derived and virus-
transformed human B and T lymphoid cells. J. Immunol. 139, 2081-
Galibert, L., Durand, I., Banchereau, J., and Rousset, F. (1994). CD40-
activated surface IgD-positive lymphocytes constitute the long term
IL-4-dependent proliferating B cell pool. J. Immunol. 152, 22-29.
Gibbons, D. L., Rowe, M., Cope, A. P., Feldmann, M., and Brennan,
F. M. (1994). Lymphotoxin acts as an autocrine growth factor for Epstein-
Barr virus-transformed B cells and differentiated Burkitt lymphoma cell
lines. Eur. J. Immunol. 24, 1879-1885.
Goldstein, D. J., Finbow, M. E., Andresson, T., McLean, P., Smith,
K., Bubb, V., and Schlegel, R. (1991). Bovine papiilomavirus E5 onco-
protein binds to the 16K component of vacuolar H÷-ATPases. Nature
Gooding, L R. (1992). Virus proteins that counteract host immune
defenses. Cell 71, 5-7.
Hammarskjold, M. L, and Simurda, M. C. (1992). Epstein-Barr virus
latent membrane protein transactivates the human immunodeficiency
virus type 1 long terminal repeat through induction of NF-kappa B
activity. J. Virol. 66, 6496-6501.
Hammerschmidt, W., and Sugden, B. (1989). Genetic analysis of im-
mortalizing functions of Epstein-Barr virus in human B lymphocytes.
Nature 340, 393-397.
Harper, J. W., Adami, G. R., Wei, N., Keyomarsi, K., and Elledge,
S. J. (1993). The p21 CDK-interacting protein Cipl is a potent inhibitor
of G1 cyclin-dependent kinases. Ceil 75, 805-816.
Hennessy, K., Fennewald, S., Hummel, M., Cole, T., and Kieff, E.
(1984). A membrane protein encoded by Epstein-Barr virus in latent
growth-transforming infection. Proc. Natl. Acad. Sci. USA 81, 7201-
Herbst, H., Dallenbach, F., Hummel, M., Niedobitek, G., Piled, S.,
Muller-Lantzsch, N., and Stein, H. (1991). Epstein-Barr virus latent
membrane protein expression in Hodgkin and Reed-Sternberg cells.
Proc. Natl. Acad. Sci. USA 88, 4766-4770.
Izumi, K., Kaye, K., and Kieff, E. (1994). Epstein-Barr virus recombinant
molecular genetic analysis of the LMP1 amino-terminal cytoplasmic
domain reveals a probable structural role, with no component essential
for primary B-lymphocyte growth transformation. J. ViroL 68, 4369-
Kaye, K. M., Izumi, K. M, and Kieff, E. (1993). Epstein-Barr virus latent
membrane protein 1 is essential for B-lymphocyte growth transforma-
tion. Proc. Natl. Acad. Sci. USA 90, 9150-9154.
Kaye, K. M., Izumi, K. M., Mosialos, G., and Kieff, E. (1995). The
Epstein-Barr virus LMP1 cytoplasmic carboxyl terminus is essential for
B lymphocyte transformation; fibroblast co-cultivation complements a
critical function within the terminal 155 residues. J. Virol. 69, 675-
Kieff, E., and Liebowitz, D. (1990). Epstein-Barr virus and its replica-
tion. In Virology, B. N. Fields and D. M. Knipe, eds. (New York: Raven
Press), pp. 1889-1920.
Laherty, C., Hu, H., Opipari, A., Wang, F., and Dixit, V. (1992). The
Epstein-Barr virus LMP1 gene product induces A20 zinc finger protein
expression by activating nuclear factor, KB. J. Biol. Chem. 267, 24157-
Liebowitz, D., Wang, D., and Kieff, E. (1986). Orientation and patching
of the latent infection membrane protein encoded by Epstein-Barr vi-
rus. J. Virol. 58, 233-237.
Loetscher, H., Gentz, R., Zulauf, M., Lustig, A., Tabuchi, H.,
Schlaeger, E.-J., Brockhaus, M., Gallati, H~, Manneberg, M., and Les-
slauer, W. (1991). Recombinant 55-kDa tumor necrosis factor (TNF)
receptor. J. Biol. Chem. 266, 18324-18329.
Longnecker, R., Miller, C. L., Tomkinson, B., Miao, X. Q., and Kieff,
E. (1993). Deletion of DNA encoding the first five transmembrane do-
mains of Epstein-Barr virus latent membrane proteins 2A and 2B. J.
Virol. 67, 5068-5074.
Mann, K. P., Staunton, D., and Thorley-Lawson, D. A. (1985). Epstein-
Barr virus-encoded protein found in plasma membranes of trans-
formed cells. J. Virol. 55, 710-720.
Mannick, J. B., Cohen, J. I., Birkenbach, M., Marchini, A., and Kieff,
E. (1991). The Epstein-Barr virus n u clear protein encoded by the leader
of the EBNA RNAs is important in B-lymphocyte transformation. J.
Virol. 65, 6826-6837.
Marchini, A., Tomkinson, B., Cohen, J. I., and Kieff, E. (1991). BHRF1,
the Epstein-Barr virus gene with homology to Bcl2, is dispensable for
B-lymphocyte transformation and virus replication. J. Virol. 65, 5991-
Martin, P., Vass, W. C., Schiller, J. T., Lowy, D. R., and Velu, T. J.
(1989)~ The bovine papillomavirus E5 transforming protein can stimu-
late the transforming activity of EGF and CSF-1 receptors. Cell 59,
Massung, R. F., Esposito, J. J., Liu, L., Qi, J., Utterback, T. R., Knight,
J. C., Aubin, L., Yuran, T. E., Parsons, J. M., Loparev, V. N., Selivanov,
N. A, Cavallaro, K. F., Kerlavage, A. R., Mahy, B. W. J., and Venter,
J. C. (1993). Potential virulence determinants in terminal regions of
variola smallpox virus genome. Nature 366, 748-751.
Miller, G. (1990). Epstein-Barr virus. In Virology, B. N. Fields and
D. M. Knipe, eds.(New York: Raven Press), pp. 1921-1958.
Moorthy, R. K., and Thorley-Lawson, D. A. (1993). All three domains
of the Epstein-Barr virus-encoded latent membrane protein LMP-1
are required for transformation of Rat-1 fibroblasts. J. Virol. 67, 1638-
Mosialos, G., Yamashiro, S., Baughman, R. W., Matsudaira, P., Vara,
L., Matsumura, F., Kieff, E., and Birkenbach, M. (1994). Epstein-Barr
virus infection induces expression in B lymphocytes of a novel gone
encoding an evolutionarily conserved 55-kilodalton actin-bundling pro-
tein. J. Virol. 68, 7320-7328.
Nagata, S. (1994). Fas and fas ligand: a death factor and its receptor.
Adv. Immunol. 57, 129-144.
Noelle, R. J., Ledbetter, J. A., and Aruffo, A. (1992). CD40 and its
ligand, an essential ligand-receptor pair for thymus-dependent B-cell
activation. Immunol. Today 13, 431-433.
O'Grady, J. T., Stewart, S., Lowrey, J., Howie, S. E., and Krajewski,
A. S. (1994). CD40 expression in Hodgkin's disease. Am. J. Pathol.
Pallesen, G., Hamilton-Dutoit, S. J., Rowe, M., and Young, L. S. (1991).
Expression of Epstein-Barr virus latent gone products in turnout cells
of Hodgkin's disease. Lancet 337, 320-322.
Pennica, D., Kohr, W. J., Fendly, B. M., Shire, S. J., Raab, H. E.,
Borchardt, P. E., Lewis, M., and Goeddel, D. V. (1992). Characteriza-
tion of a recombinant extracellular domain of the type I tumor necrosis
factor receptor: evidence for tumor necrosis factor-alpha induced re-
ceptor aggregation. Biochemistry 31, 1134-1141.
Petti, L., and DiMaio, D. (1992). Stable association between the bovine
papillomavirus E5 transforming protein and activated platelet-derived
growth factor receptor in transformed mouse cells. Proc. Natl. Acad.
Sci. USA 89, 6736-6740.
Petti, L., Nilson, L. A., and DiMaio, D. (1991). Activation of the platelet-
derived growth factor receptor by the bovine papillomavirus E5 trans-
forming gene. EMBO J. 10, 845-855.
Pfeffer, K., Matsuyama, T., KLindig, T. M., Wakeham, A., Kishihara,
K., Shahinian, A., Wiegmann, K., Ohashi, P. S., Kr6nke, M., and Mak,
T. W. (1993). Mice deficient for the 55 kd tumor necrosis factor receptor
are resistant to endotoxic shock, yet succumb to L. monocytogenes
infection. Cell 73, 457-467.
Pfreundschuh, M., Carde, P., Da Costa, L., Manil, L., Lumbroso, J.-D.,
Caillou, B., Boudeta, F., Ricard, M., Parmentier, C., and Saccavini,
J.-C. (1989). In vivo imaging of Hodgkin's lymphomas with monoclonal
antibodies. Onkologie 12 (Suppl. 1), 30-42.
Poll, G., Kinter, A., Justement, J. S., Kehrl, J. H., Bressler, P., Stanley,
S., and Fauci, A. S. (1990). Tumor necrosis factor a functions in an
autocrine manner in the induction of human immunodeficiency virus
expression. Proc. Natl. Acad. Sci. USA 87, 782-785.
Reisman, D., and Sugden, B. (1986). Transactivation of an Epstein-
Barr viral transcriptional enhancer by the Epstein-Barr viral nuclear
antigen 1. Mol. Cell. Biol. 6, 3838-3846.
Robertson, E. S., Tomkinson, B., and Kieff, E. (1994). An Epstein-Barr
virus with a 58-kilobase-pair deletion that includes BARF0 transforms
B lymphocytes in vitro. J. Virol. 68, 1449-1458.
Rothe, J., Lesslauer, W., Lotscher, H., Lang, Y., Koebel, P., Kontgen,
F., Althage, A., Zingernagel, R., Steinmetz, M., and Bluethmann, H.
(1993). Mice lacking the tumor necrosis factor receptor 1 are resistant
to TNF-mediated toxicity but highly susceptible to infection by Listeria
rnonocytogenes. Nature 364, 798-802.
Rothe, M., Wong, S. C., Henzel, W. J., and Goeddel, D. V. (1994). A
novel family of putative signal transducers associated with the cyto-
plasmic domain of the 75 kDa tumor necrosis factor receptor. Cell 78,
Rowe, M., Lear, A. L., Croom-Carter, D., Davies, A. H., and Rickinson,
A. B. (1992). Three pathways of Epstein-Barr virus gene activation
from EBNAl-positive latency in B lymphocytes. J. Virol. 66, 122-131.
Rowe, M., Peng-Pilon, M., Huen, D. S., Hardy, R., Croom-Carter, D.,
Lundgren, E., and Rickinson, A. B. (1994). Upregulation of bcl-2 by
the Epstein-Barr virus latent membrane protein LMP1: a B-cell specific
response that is delayed relative to NF-KB activation and to induction
of cell surface markers. J. Virol. 68, 5602-5612.
Saeland, S., Duvert, V., Moreau, I., and Banchereau, J. (1993). Human
B cell precursors proliferate and express CD23 after CD40 ligation.
J. Exp. Med. 178, 113-120.
Schiestl, R. H., and Gietz, R. D. (1989). High efficiency transformation
of intact yeast cells using single-stranded nucleic acids as a carrier.
Curr. Genet. 16, 339-346.
Smith, D. B., and Johnson, K. S. (1988). Single-step purification of
polypeptides expressed in Escherichia coil as fusions with glutathione
S-transferase. Gene 67, 31-40.
Smith, C. A., Davis, T., Wignall, J. M., Din, W. S., Farrah, T., Upton,
C., McFadden, G., and Goodwin, R. G. (1991). T2 open reading frame
from the Shope fibroma virus encodes a soluble form of the TNF recep-
tor. Biochem. Biophys. Res. Commun. 176, 335-342.
Smith, C. A., Farrah, T., and Goodwin, R. G. (1994). The TNF receptor
superfamily of cellular and viral proteins: activation, costimulation, and
death. Cell 76, 959-962.
Swaminathan, S., Tomkinson, B., and Kieff, E. (1991). Recombinant
Epstein-Barr virus with small RNA (EBER) genes deleted transforms
lymphocytes and replicates in vitro. Proc. Natl. Acad. Sci. USA 88,
Tartaglia, L. A., and Goeddel, D. V. (1992). Tumor necrosis factor
receptor signalling. A dominant negative mutation suppresses the acti-
vation of the 55-kDa tumor necrosis factor receptor. J. Biol. Chem.
Tartaglia, L. A., Goeddel, D. V., Reynolds, C., Figari, I. S., Weber,
R. F., Fendly, B. M., and Palladino, M. A. J. (1993a). Stimulation of
human T-cell proliferation by specific activation of the 75-kDa tumor
necrosis factor receptor. J. Immunol. 151, 4637-4641.
Tartaglia, L. A., Rothe, M., Hu, Y.-F., and Goeddel, D. V. (1993b).
Tumor necrosis factor's cytotoxic activity is signaled by the p55 TNF
receptor. Cell 73, 213-216.
Tomkinson, B., and Kieff, E. (1992). Use of second-site homologous
recombination to demonstrate that Epstein-Barr virus nuclear protein
3B is not important for lymphocyte infection or growth transformation
in vitro. J. Virol. 66, 2893-2903.
Tomkinson, B., Robertson, E., and Kieff, E. (1993). Epstein-Barr virus
nuclear proteins (EBNA) 3A and 3C are essential for B lymphocyte
growth transformation. J. Virol. 67, 2014-2025.
Trumper, L., Jung, W., Dahl, G., Diehl, V., Gause, A., and Pfeund-
schuh, M (1994). Interleukin-7, interleukin-8, soluble TNF receptor,
and p53 protein levels are elevated in the serum of patients with Hodg-
kin's disease. Ann. Oncol. 5, 93-96.
VanArsdale, T. L., and Ware, C. F. (1994). TNF receptor signal trans-
duction. Ligand-dependent stimulation of a serine protein kinase activ-
ity associated with (CD120a) TNFR60. J. Immunol. 153, 3043-3050.
Wang, D., Liebowitz, D., and Kieff, E. (1985). An EBV membrane pro-
tein expressed in immortalized lymphocytes transforms established
rodent cells. Cell 43, 831-840.
Wang, D., Liebowitz, D., and Kieff, E. (1988a). The truncated form of
the Epstein-Barr virus latent-infection membrane protein expressed
in virus replication does not transform rodent fibroblasts. J. Virol. 62,
Wang, D., Liebowitz, D., Wang, F., Gregory, C., Rickinson, A., Larson,
R., Springer, T., and Kieff, E. (1988b). Epstein-Barr virus latent infec-
tion membrane protein alters the human B-lymphocyte phenotype:
deletion of the amino terminus abolishes activity. J. Virol. 62, 4173-
Wang, F., Gregory, C., Sample, C., Rowe, M., Liebowitz, D., Murray,
R., Rickinson, A., and Kieff, E. (1990). Epstein-Barr virus latent mem-
brane protein (LMP1) and nuclear proteins 2 and 3C are effectors of
phenotypic changes in B lymphocytes: EBNA-2 and LMP1 coopera-
tively induce CD23. J. Virol. 64, 2309-2318.
Ware, C. F., Crowe, P. D., VanArsdale, T. L., Andrews, J. L., Grayson,
M. H., Jerzy, R., Smith, C. A., and Goodwin, R. G. (1991). Tumor
necrosis factor (TNF) receptor expression in T lymphocytes. Differen-
tial regulation of the type I TNF receptor during activation of resting
and effector T cells. J. Immunol. 147, 4229-4238.
Ware, C. F., VanArsdale, T. L., Crowe, P. D., and Browning, J. L.
(1995). The ligands and receptors of the lymphotoxin system. In Cur-
rent Topics in Microbiology and Immunology, G. Griffiths and
J. Tschoop, eds. (Berlin: Springer-Verlag) 198, in press.
Wilson, J. B., Weinberg, W., Johnson, R., Yuspa, S., and Levine,
EBV LMP1 Transformation and TNFR Family Signaling
A. J. (1990). Expression of the BNLF-1 oncogene of Epstein-Barr virus
in the skin oftransgenic mice induces hyperplasia and aberrant expres-
sion of keratin 6. Cell 61, 1315-1327.
Yates, J., Warren, N., Reisman, D., and Sugden, B. (1984). Acis-acting
element from the Epstein-Barr virus genome that permits stable repli-
cation of recombinant plasmids in latently infected cells. Proc. Natl,
Acad. Sci. USA 81, 3806-3810.
Young, L. S., Dawson, C. W., Brown, K. W., and Rickinson, A. B.
(1989). Identification of a human epithelial cell surface protein sharing
an epitope with the C3d/Epstein-Barr virus receptor molecule of B
lymphocytes. Int. J. Cancer 43, 786-794.
GenBank Accession Numbers
The accession numbers for the LAP1 and EBI6 sequences reported
in this study ere U19260 and U19261, respectively.
Note Added in Proof
In the December 2, 1994 issue of the Journal of Biological Chemistry,
Hu et al. reported the sequence of a protein that is nearly identical to
that of LAP1. This protein became bound to wild type, but not to a
signaling-defective mutant CD40 cytoplasmic domain.