JOURNAL OF VIROLOGY, Sept. 2006, p. 9300–9309
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Vol. 80, No. 18
Poxvirus Tumor Necrosis Factor Receptor (TNFR)-Like T2 Proteins
Contain a Conserved Preligand Assembly Domain That Inhibits
Cellular TNFR1-Induced Cell Death
Lisa M. Sedger,1,2* Sarah R. Osvath,1Xiao-Ming Xu,3Grace Li,1Francis K.-M. Chan,4
John W. Barrett,3and Grant McFadden3
Institute for Immunology & Allergy Research and Centre for Virus Research, Westmead Millennium Institute, Department of Medicine,
University of Sydney, Sydney, Australia1; Department of Molecular Immunology, Immunex Corporation, Seattle, Washington2;
Department of Microbiology and Immunology, University of Western Ontario, and Robarts Research Institute, London,
Ontario, Canada3; and Department of Pathology, University of Massachusetts Medical School, Worcester, Massachusetts4
Received 22 November 2005/Accepted 23 June 2006
The poxvirus tumor necrosis factor receptor (TNFR) homologue T2 has immunomodulatory properties;
secreted myxoma virus T2 (M-T2) protein binds and inhibits rabbit TNF-?, while intracellular M-T2 blocks
virus-induced lymphocyte apoptosis. Here, we define the antiapoptotic function as inhibition of TNFR-medi-
ated death via a highly conserved viral preligand assembly domain (vPLAD). Jurkat cell lines constitutively
expressing M-T2 were generated and shown to be resistant to UV irradiation-, etoposide-, and cycloheximide-
induced death. These cells were also resistant to human TNF-?, but M-T2 expression did not alter surface
expression levels of TNFRs. Previous studies indicated that T2’s antiapoptotic function was conferred by the
N-terminal region of the protein, and further examination of this region revealed a highly conserved N-
terminal vPLAD, which is present in all poxvirus T2-like molecules. In cellular TNFRs and TNF-?-related
apoptosis-inducing ligand (TRAIL) receptors (TRAILRs), PLAD controls receptor signaling competency prior
to ligand binding. Here, we show that M-T2 potently inhibits TNFR1-induced death in a manner requiring the
M-T2 vPLAD. Furthermore, we demonstrate that M-T2 physically associates with and colocalizes with human
TNFRs but does not prevent human TNF-? binding to cellular receptors. Thus, M-T2 vPLAD is a species-
nonspecific dominant-negative inhibitor of cellular TNFR1 function. Given that the PLAD is conserved in all
known poxvirus T2-like molecules, we predict that it plays an important function in each of these proteins.
Moreover, that the vPLAD confers an important antiapoptotic function confirms this domain as a potential
target in the development of the next generation of TNF-?/TNFR therapeutics.
The leporipoxviruses myxoma virus and Shope fibroma virus
both encode a high-affinity tumor necrosis factor alpha (TNF-?)-
(S-T2) protein was reported to bind and neutralize both rabbit
and human TNF-? (49), but the myxoma virus T2 protein (M-T2)
exhibits strict species specificity and inhibits only rabbit TNF-?
(38). M-T2 is a genuine virulence factor, because rabbits infected
with the M-T2 open reading frame (ORF) knockout myxoma
virus vMyxT2G exhibit a markedly attenuated disease compared
to rabbits infected with the M-T2-expressing control virus vMyxlac
(54). On this basis, M-T2 has served as a model of poxvirus
subversion of host immune responses in vitro and in vivo,
emphasizing the importance of TNF-?/TNFR biology in the
immune response to poxvirus infection (41).
M-T2 also prevents apoptosis of myxoma virus-infected rab-
bit CD4?RL5 T cells (24). RL5 cells infected with the T2
knockout vMyxT2G virus die rapidly by apoptosis, thereby
precluding optimal virus replication. In contrast, RL5 cells
infected with the T2-encoding virus vMyxlac or the vMyxT2R
revertant virus do not undergo apoptosis and support produc-
tive virus replication (24). However, it is the intracellular ver-
sion of the M-T2 protein that is required for this antiapoptotic
activity because active purified M-T2 protein added to the
culture supernatants of vMyxT2G-infected RL5 cells fails to
rescue these cells from virus-induced apoptosis (24). Thus,
M-T2 has two distinct activities; extracellular or secreted M-T2
binds and inhibits rabbit TNF-?, whereas intracellular M-T2 acts
to block virus-infected lymphocyte apoptosis. That M-T2 serves
two distinct host evasion functions highlights the intricacies of
virus-host interactions (41, 58).
Here, we define the intracellular mechanism of T2’s anti-
apoptotic activity as inhibition of TNFR-mediated cell death.
Because myxoma virus and other poxviruses encode a number
of other antiapoptotic proteins, including T4 (4), T5 (29),
M11L (24), and Serp-2 (28, 33), M-T2 was expressed in mam-
malian cells in the absence of other poxvirus proteins. M-T2-
expressing human Jurkat T cells were found to be resistant to
TNF-?- and TNFR-induced cell death, thereby confirming that
M-T2 is a bona fide antiapoptotic protein. We demonstrate
that M-T2 inhibits human TNFR-induced cell death in a man-
ner that requires a preligand assembly domain (PLAD) located
in the N terminus and which is present and conserved in all
poxvirus T2-like proteins. We define a novel dominant-nega-
tive mechanism of viral subversion of TNF-?/TNFR biology.
MATERIALS AND METHODS
Plasmids. The full-length M-T2 ORF was PCR amplified and cloned into
pcDNA3.1myc/his (Invitrogen). pcDNA3-M-T2?PLADmyc was constructed by
* Corresponding author. Mailing address: Westmead Millennium In-
stitute, P.O. Box 412, Westmead, NSW 2145, Australia. Phone: 61-2-9845
7491. Fax: 61-2-9845 9100. E-mail: firstname.lastname@example.org.
PCR amplification of the 5? PLAD-adjacent cDNA spanning the first 54 nucle-
otides cloned into the BamHI and HindIII sites of pcDNA3.1myc/his and PCR
amplification of the 3? PLAD-adjacent T2 cDNA, beginning at the GGG codon
encoding glycine at nucleotide 166, cloned into the HindIII and XhoI sites of
pcDNA3.1myc/his. The 5? pre-PLAD BamHI-HindIII and 3?-post-PLAD
HindIII-XhoI M-T2 fragments were then ligated together into BamHI/XhoI-
digested pcDNA3.1myc/his. pcDNA3-humanTNFR1 and humanTNFR2 were
kindly provided by Chris Benedict (La Jolla Institute for Allergy and Immunol-
ogy, San Diego, Calif.), and pcDNA3-TNFR1-cyan fluorescent protein (CFP)
was generated by Francis Chan and is described elsewhere (8). Full-length
p16INK4a was subcloned into pCMV-myc (Clontech) and was kindly provided
by Helen Rizos (Westmead Millennium Institute, Westmead, Australia).
Viruses and cells. Control virus vMyxlac, T2 knockout virus vMyxT2G, and T2
revertant virus vMyxT2R were described previously (24, 54). Myxoma virus
stocks were grown in BGMK monkey kidney cells (obtained from S. Dales,
University of Western Ontario, London, Ontario, Canada) in Dulbecco’s mod-
ified Eagle’s medium (Gibco BRL) with 10% fetal bovine serum (FBS). Recom-
binant Autographa californica nucleopolyhedrosis virus encoding M-T2 (AcM-
T2) was constructed by insertion of the M-T2 ORF into the BamHI site of the
baculovirus vector, and recombinant baculovirus was propagated in Sf21 cells in
TNM medium with 10% FBS or in SF900-II serum-free medium (Gibco BRL).
The M-T2 ORF from plasmid pMTN-6 (54) was inserted into the XhoI site of
the BMG-neo plasmid and used to generate stable M-T2-expressing human
Jurkat T cells by electroporation with a Gene Pulser II instrument (Bio-Rad) at
250 V and 960 ?F capacitance. Multiple Jurkat lines constitutively expressing
M-T2, designated T2O-a, T2O-11, T2L-4, and T2L-3, were generated by limit-
ing-dilution cloning and expansion in 1 mg/ml G418 (Invitrogen). A BMG-neo
plasmid was used to generate the control G418-resistant line JNeo. Jurkat T cells
and M-T2-expressing Jurkat lines were cultured in RPMI 1640 medium with 10%
FBS; human embryonic kidney (HEK) 293T cells (kindly provided by Grant
Logan, Children’s Medical Research Institute, Westmead, Australia), Vero cells
(originally from the American Type Culture Collection), and U20S human os-
teosarcoma cells (gift from Helen Rizos, Westmead Millennium Institute, West-
mead Australia) were cultured in Dulbecco’s modified Eagle’s medium with 10%
fetal calf serum.
Detection of M-T2 expression in Jurkat cell lines. Jurkat cell RNA, DNA, and
protein extracts were prepared with TRIzol reagent (Invitrogen) according to the
manufacturer’s directions. M-T2-specific oligonucleotide primers were used to
confirm T2 DNA incorporation by PCR and M-T2 mRNA expression by reverse
transcription-PCR. TRIzol protein extracts were precipitated with isopropyl al-
cohol, washed with 0.3 M guanidine hydrochloride in ethanol, and pelleted by
centrifugation at 7,500 ? g at 4°C. Secreted M-T2 protein in culture medium was
precipitated with 100 ?g/ml sodium deoxycholate in 6% trichloroacetic acid for
1 h at 4°C. For controls, BGMK cells were infected with vMyxT2G or vMyxlac at
a multiplicity of infection of 10 and harvested 12 or 24 h postinfection.
M-T2-specific antibody B5. M-T2 protein expressed by the recombinant bac-
ulovirus AcM-T2 was purified from 20 mM Tris HCl (pH 7.5)-dialyzed culture
supernatants with a Hi-trap ion-exchange column (Pharmacia Biotech) equili-
brated with 20 mM Tris HCl (pH 7.5) and eluted with a 0 to 1 M linear NaCl
gradient. M-T2-containing fractions identified by Western immunoblotting were
purified on a Mono-Q column and eluted with a linear 0 to 300 mM NaCl
gradient in 20 mM bis-Tris (pH 6.4). Purified M-T2 protein was emulsified in
Freund’s complete adjuvant and injected intramuscularly into naive New Zeal-
and White rabbits (Riemens Co., St. Agatha, Ontario, Canada) housed at the
Robarts Animal Facility in accordance with approved ethics protocols. Rabbits
were boosted by a second injection of M-T2 in incomplete Freund’s adjuvant and
subsequently euthanized and exsanguinated. Serum containing M-T2-specific
antibody B5 was affinity purified with an M-T2-conjugated cyanogen bromide-
activated Sepharose 4B column (Pharmacia) by standard procedures (20).
Apoptosis assays. Rabbit peripheral blood lymphocytes were isolated from
peripheral blood from a healthy naive New Zealand White laboratory rabbit
(Riemens Co., St. Agatha, Ontario). Rabbit lymphocytes were isolated from
blood by centrifugation over Ficoll-Paque (Amersham Pharmacia Biotech) and
separated into nonadherent lymphocytes or adherent monocytes by adherence to
plastic petri dishes, exactly as described previously (13). The nonadherent cells,
which are primarily lymphocytes, were infected with vMyxlac, vMyxT2G, or
vMyxT2R at a multiplicity of infection of 10. Apoptosis of virus-infected cells was
detected by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick
end labeling (TUNEL) staining with fluorescein isothiocyanate-conjugated
dUTP (Boehringer Mannheim) according to the manufacturer’s instructions and
as described previously (24). Rabbit lymphocytes were judged to be infected by
detecting LacZ expression, staining with 1 mg/ml 5-bromo-4-chloro-3-indolyl-?-
D-galactopyranoside (X-Gal) in dimethylformamide, 5 mM potassium ferricya-
nide, 5 mM potassium ferrocyanide, and 2 mM MgCl2for 45 min at 37°C and
examining cells by light microscopy to detect blue cells. In this situation, LacZ
expression is an indication of virus infection and replication because it is ex-
pressed by these viruses; the LacZ cDNA has been incorporated into an inter-
genic region of vMyxlac and hence is also present in the vMyxT2G and vMyxT2R
Jurkat Jneo and M-T2-expressing cell lines were seeded in triplicate, exposed
to various inducers of apoptosis, and then incubated with Annexin-V-Fluos
(Boehringer Mannheim) and propidium iodide (Sigma) and analyzed by flow
cytometry. Specifically, cells were exposed to 10,000 J of UV irradiation, 20 ?M
etoposide (VP-16; Sigma), and 10 ?M cycloheximide (CHX; Sigma) and ana-
lyzed by flow cytometry. Additionally, cells were cultured in 30 ?M cisplatin or
120 ?M melphalan (both from Sigma), LZ-humanTRAIL (TNF-?-related apop-
tosis-inducing ligand), LZ-humanFasL, LZ-humanCD40L (all from Immunex),
or recombinant human TNF-? (R&D Systems), and cell death was assessed by
a chromium release assay with a Hewlett-Packard gamma counter. Percent
specific lysis was calculated as x ? [(experimental release ? minimum release)/
(maximum release ? minimum release)] ?100. In some assays, the blocking
agents human Fas-Fc, human TRAIL-R2-Fc, and human TNFR1-Fc (Immunex
Corporation) were added.
Flow cytometric measurement of TNFR expression and TNF-? binding. For
surface and intracellular staining, Jurkat cells were incubated for 1 h at 4°C with
5% normal human serum and then incubated with 10 ?g of the following
antibodies: mouse immunoglobulin G1 (IgG1) M180 anti-huTRAIL, mouse
IgG1 M271 anti-huTRAILR1, mouse IgG1 M413 anti-huTRAILR2, mouse
IgG1 M430 anti-huTRAILR3, mouse IgG1 M444 anti-huTRAILR4, mouse
IgG1 M38 anti-Fas, or isotype control antibody mouse IgG1 M330 anti-RANK
(receptor activator of NF?B) IgG1, which were described previously (12, 18, 43).
Binding of primary antibodies was detected by incubation with biotinylated
F(ab?)2 goat anti-murine IgG (Jackson Laboratories) and avidin-phycoerythrin
(Av-PE; Pharmingen). Jurkat cells and transfected 293T cells were also incu-
bated in PE-conjugated mouse IgG1 (clone 16803.1) anti-human TNFR1, PE-
conjugated mouse IgG2a anti-human TNFR2 (clone 22235), PE-conjugated
mouse IgG1 (clone 11711), or PE-conjugated mouse IgG2a (clone 20102) isotype
control antibodies, according to the manufacturer’s instructions (all from R&D
Systems). For intracellular staining, cells were fixed in 2% paraformaldehyde in
phosphate-buffered saline (PBS) for 10 min at 4°C and stained and washed in
0.1% saponin (Sigma).
For TNF-? binding, HEK 293T cells were transfected with pcDNA3-TNFR1
and either pcDNA3-M-T2myc or pcDNA3-M-T2?PLADmyc or with each plas-
mid alone, incubated 24 h later in recombinant human TNF-? (R&D Systems)
for 10 min at 4°C or room temperature, and washed three times in PBS with 5%
fetal calf serum to remove unbound TNF-?. Remaining bound TNF-? was
detected by flow cytometry with anti-human TNF-?–PE antibody (clone 6402.31;
R&D Systems) and compared to staining with isotype control PE-conjugated
mouse IgG1 (clone 11711; R&D Systems). The expression of ectopically ex-
pressed TNFRs in these experiments was also checked by flow cytometry with the
PE-conjugated TNFR-specific antibodies listed above. Cells were analyzed on a
FACScan or FACScalibur flow cytometer (Becton Dickenson), and 10,000 or
30,000 events were collected and analyzed with Cell Quest system software (BD
Transfection and immunoprecipitation studies. For viability studies, HEK 293T
cells seeded in six-well tissue culture plates were transfected with various combina-
tions of pcDNA3.1myc/his, pcDNA3-M-T2myc/his, pcDNA3-T2?PLADmyc/his,
and pcDNA3-TNFR1, together with pcDNA3-LacZ, by a calcium phosphate trans-
fection method described previously (17). ?-Galactosidase activity was quantitated
as follows. Lysates were prepared by three cycles of freezing-thawing on dry ice and
centrifugation at 13,000 rpm for 15 min to remove debris and then incubated with
o-nitrophenyl-?-D-galactopyranoside in 0.1 M MgCl2plus 5 mM ?-mercaptoethanol
in 0.1 M sodium phosphate buffer (pH 7.3), and LacZ expression was measured by
absorbance at 415 nm with a Titertek Multiskan plate reader.
For immunoprecipitation studies, HEK 293T cells seeded in 10-cm dishes were
transfected with pcDNA3-TNFR1 or pcDNA3-TNFR2 and either pcDNA3-M-
T2myc, pcDNA3-M-T2?PLADmyc, or pcDNA3 (empty vector) and 48 h later
harvested directly into RIPA buffer containing complete protease inhibitors
(Roche). Lysates were precleared with a 50/50 mixture of a protein A and protein
G-Sepharose (Sigma) slurry in PBS. Beads were pelleted by centrifugation, and
supernatants were incubated with protein A/G-Sepharose plus 1 ?l of rabbit
anti-human TNFR1 antibody (H5; Santa Cruz) or 1 ?l of goat anti-human
TNFR2 antibody (C20; Santa Cruz) and incubated overnight at 4°C with rota-
tion. Beads were washed three times, resuspended in sodium dodecyl sulfate-
polyacrylamide gel electrophoresis (SDS-PAGE) reducing sample buffer, and
examined by SDS-PAGE and immunoblotting. Briefly, samples were subjected
VOL. 80, 2006 POXVIRUS SUBVERSION OF TNFR1 BY vPLAD9301
to 12% SDS-PAGE, blotted onto polyvinylidene difluoride membrane (Bio-
Rad), incubated overnight in 10% skim milk in PBS plus 5% Tween 20 to block
nonspecific binding, then incubated with B5 anti-M-T2 rabbit serum and biotin-
ylated goat anti-rabbit antibody (Sigma), and developed with 5-bromo-4-chloro-
3-indolyl-?-D-galactopyranoside (BCIP)–nitroblue tetrazolium alkaline phos-
phatase substrate (Sigma). Alternatively, lysates were analyzed directly by
immunoblotting with B5 anti-M-T2, H5 anti-TNFR1, or C20 anti-TNFR2 anti-
body (Santa Cruz).
M-T2myc and M-T2?PLADmyc binding to human and rabbit TNF-? was also
determined by immunoprecipitation. In these experiments, TNF-? binding was
determined by mixing 500 ?g of total protein from pcDNA3.1, pcDNA3.1-M-
T2myc, and pcDNA3.1-M-T2?PLADmyc transfected HEK 293T cells with ly-
sates and supernatants from Vero cells infected with vaccinia viruses (VV)
encoding human or rabbit TNF-?, which have been described previously (39). In
these assays, M-T2myc and M-T2?PLADmyc were mixed with anti-c-Myc anti-
body (clone 9E10; Santa Cruz) overnight at 4°C. Antibody-bound M-T2 com-
plexes containing TNF-? were then immunoprecipitated with protein G-Sepha-
rose beads (Upstate) for 1 h. Sepharose beads were washed four times in lysis
buffer containing 0.5% deoxycholate and proteinase inhibitor cocktail (Roche),
and immunoprecipitates were subjected to SDS-PAGE. TNF-? was detected by
Western immunoblotting with the TNF-?-specific antibodies rabbit anti-human
TNF-? (BioSource International) and goat anti-rabbit TNF-? (Fitzgerald Indus-
tries). These antibodies were detected with goat anti-rabbit immunoglobulin and
donkey anti-goat immunoglobulin secondary antibodies, respectively (both from
Jackson Immunoresearch). Immunoblots were visualized with Western Light-
ning chemiluminescence reagent plus (Perkin-Elmer).
Confocal microscopy. Confocal microscopy was performed on U2OS cells
seeded onto 13-mm round glass coverslips that were placed in six-well tissue
culture dishes. pcDNA3-TNFR1-CFP and pcDNA3-M-T2myc or pcDNA3-M-
T2?PLADmyc transfected U203 cells were fixed in 2% paraformaldehyde at 24 h
posttransfection. M-T2myc and M-T2?PLADmyc proteins were detected by
staining with rabbit anti-myc antibody (clone 9E10; Santa Cruz) and then goat
anti-rabbit immunoglobulin Cy3-labeled antibody (Amersham Pharmacia Bio-
tech) in 0.1% saponin–PBS. TNFR1-CFP and T2myc were visualized in U20S
cells with a Leica SP2 confocal microscope with helium neon and argon lasers.
Images were captured with Leica confocal software.
Statistical analysis. Where appropriate, data were analyzed statistically by a
two-tailed Student t test. A P value of ?0.05 was considered statistically signif-
M-T2 protects primary rabbit lymphocytes from virus-in-
duced apoptosis. M-T2 can protect rabbit RL5 CD4?T lym-
phocytes from virus-induced apoptosis (24). In order to deter-
mine whether this is a biologically relevant phenomenon or a
peculiarity of this rabbit cell line, primary rabbit lymphocytes
were examined. Rabbit peripheral blood leukocytes were col-
lected, lymphocytes were separated from monocytes by adher-
ence to plastic, and the nonadherent lymphocytes were in-
fected with the M-T2-deficient vMyxT2G virus or the parental
M-T2-expressing control virus and assessed by flow cytometry
by fluorescein isothiocyanate-dUTP (TUNEL) staining. Infec-
tion with vMyxT2G resulted in an increase in apoptotic cells in
primary blood lymphocytes, in that there were 14% TUNEL-
positive cells in vMyxLac-infected leukocytes, but this was in-
creased to 26% in vMyx-T2G-infected leukocytes at 24 h
postinfection (data not shown). Primary rabbit lymphocytes
were judged to be infected because approximately 70% of
rabbit lymphocytes were expressing LacZ, which is encoded by
these viruses (data not shown). Thus, M-T2 inhibits apoptosis
of primary rabbit blood lymphocytes (data not shown), not just
that of the transformed rabbit RL5 T-cell line (24).
Expression of M-T2 from stably transfected Jurkat clones.
Myxoma virus encodes a number of gene products that can
inhibit apoptosis (for a review, see reference 31). In order to
characterize the antiapoptotic properties of M-T2 in lympho-
cytes and to circumvent the limitations of secreted M-T2’s
ability to neutralize only rabbit TNF-? and not human TNF-?
(38), we expressed M-T2 in human Jurkat T cells in the ab-
sence of virus infection. Human CD4?Jurkat T-cell clones
T2O-a, T2O-11, T2L-4, and T2L-3 were generated and found
to express M-T2-specific mRNA (data not shown). M-T2 pro-
tein was judged to be properly synthesized in each of these
lines, as both lysates and culture supernatants contained the
55-kDa M-T2 protein (Fig. 1A and data not shown). No M-T2
protein was present in Jneo control cells (Fig. 1A). Since the
intracellular form of M-T2 was required for protection against
apoptosis (24), the relative amount of intracellular M-T2 ex-
pressed by the Jurkat clones was quantitated by immunoblot-
ting and densitometry. Jurkat clone T2O-a expressed the high-
est level of intracellular M-T2, followed by Jurkat clone
T2O-11 and then clones T2L-4 and T2L-3 (Fig. 1A). Thus,
M-T2 was efficiently expressed in human Jurkat lymphocytes in
the absence of virus infection and without DNA codon opti-
mization. Importantly, detection of M-T2 protein in both ly-
sates and supernatants of the Jurkat clones is completely anal-
ogous to the normal expression of M-T2 or S-T2 during virus
infection or when expressed via plasmid transfection in rabbit
RK13 fibroblasts (2, 53; L. Sedger, unpublished data).
M-T2 inhibits TNF-?-mediated apoptosis. Next, we investi-
gated whether Jurkat T cells expressing M-T2 are more resis-
tant to apoptosis induced by different stimuli. To determine
whether M-T2 blocks UV-induced apoptosis, control Jneo
Jurkat cells and M-T2-expressing Jurkat cells were exposed to
10,000 J of UV irradiation, which induced 32% of the Jneo
cells tested to undergo apoptosis (Fig. 1A). In contrast, only
9% of Jurkat T2O-a cells, 14% of Jurkat T2O-11 cells, 16% of
Jurkat T2L-4 cells, and 22% of Jurkat T2L-3 cells underwent
apoptosis after UV irradiation (Fig. 1A). In order to determine
whether M-T2 also protects Jurkat T cells against other induc-
ers of apoptosis, these cells were tested for sensitivity to eto-
poside (VP-16) and CHX. All M-T2-expressing Jurkat cells
were more resistant to VP-16- and CHX-induced apoptosis
compared to Jneo control cells, and the cells that had the
highest M-T2 expression (T2O-a) also exhibited the greatest
resistance (Fig. 1A). M-T2-expressing Jurkat T-cell clones
were also tested for sensitivity to the UV-mimetic agents cis-
platin and melphalan. Death induced by these agents is much
slower, and hence, 24-h and 48-h51Cr release assays were used
to measure apoptosis. However, Jurkat lines expressing M-T2
were not more resistant to these cytotoxic agents than Jneo
control cells (data not shown). Together, these data indicate
that M-T2 expressed in the absence of myxoma virus infection
induced resistance to UV irradiation-, VP-16-, and CHX-in-
duced cell death but not cisplatin- or melphalan-induced cell
UV irradiation is known to induce TNF-? production and
also increase the expression and aggregation of TNFR in ke-
ratinocytes and other cell lines (3, 23, 50), whereas the UV-
mimetic agents cisplatin and melphalan do not (46). Moreover,
caspase-8?/?cell lines are reportedly more resistant to apop-
tosis induced by both UV irradiation and etoposide (22).
Therefore, M-T2-expressing Jurkat clones were tested for the
ability to resist apoptosis induced by TNF-? and other caspase-
8-sensitive death receptor-binding cytokines TRAIL and FasL.
Recombinant human TNF-?, LZ-TRAIL, LZ-FasL, and LZ-
9302SEDGER ET AL.J. VIROL.
CD40 (control) were used because they induce ligand-medi-
ated receptor-specific signaling (56). In an 8- or 18-h
release assay, all M-T2-expressing Jurkat clones were signifi-
cantly more resistant to TNF-?-mediated death compared to
control Jneo cells (Fig. 1B and data not shown), but they were
not more resistant or susceptible to LZ-FasL or LZ-TRAIL,
and as expected, there was no proapoptotic effect by the con-
trol reagent LZ-CD40L (Fig. 1B). Thus, M-T2 expression con-
ferred specific protection from human TNF-?-mediated cell
death, which is consistent with resistance to UV, etoposide,
and CHX, which inhibit the synthesis of antiapoptotic proteins
such as inhibitors of apoptosis.
Ectopic expression of M-T2 does not alter the expression of
TNFR, Fas, or TRAIL receptors. The finding that M-T2-ex-
pressing Jurkat clones were more resistant to human TNF-?
was surprising because secreted M-T2 binds to and inhibits
only rabbit TNF-? and not human TNF-? (38). Thus, we rea-
soned that forced expression of M-T2 might have influenced
TNFR expression on Jurkat cell lines, potentially explaining
these results. Therefore, death receptor-specific monoclonal
antibodies and flow cytometry were used to assess TNFR fam-
ily molecule expression levels on control Jneo cells and M-T2-
expressing Jurkat lines T2-Oa, T2O-11, T2-L3, and T2-L3.
First, Jneo cells were assessed and found to express detectable
surface levels of Fas but only low levels of surface TRAIL-R2
and TNFR1 and no surface TRAIL, TRAIL-R1, TRAIL-R3,
TRAIL-R4, or TNFR2 relative to staining with isotype control
antibodies. All M-T2-expressing Jurkat cells expressed essen-
tially similar levels of these molecules at the cell surface, with
only a very minimal reduction in surface TNFR1 (Fig. 1C). The
intracellular levels of death receptors found in each of these
cell lines were also assessed with permeabilized cells; however,
none of these molecules, including TNFR1 and TNFR2, were
significantly altered in M-T2-expressing Jurkat lines (Fig. 1C).
Thus, M-T2 expression did not significantly alter surface or
intracellular TNFR molecule expression levels.
Poxvirus T2 ORFs contain a highly conserved PLAD. The
N-terminal cysteine-rich repeat domain (CRD)-containing re-
gion of the myxoma virus and S-T2 ORFs are highly similar to
human TNFR (41), and previous mutational studies had indi-
cated that the N-terminal region of M-T2 contains distinct
domains responsible for TNF-? binding and inhibition of virus-
induced apoptosis (40). Further analysis of the first 200 N-
terminal amino acids of the M-T2 and S-T2 proteins and hu-
man TNFR1 and TNFR2 identified a highly conserved region
in M-T2 and S-T2 which resides within the first CRD (8) and
has clear homology to the PLAD of human TNFRs (Fig. 2).
The human TNFR PLAD is important in conferring a confor-
mation change in the structure of TNFRs and allows TNFR
self-association prior to ligand binding and signaling (8). As
such, the PLAD is required for signaling competency. This
vPLAD is present in all poxvirus T2-like molecules: myxoma
virus M-T2 (Lausanne strain; accession no. NC_001132) (7),
Shope fibroma virus S-T2 (Kasza strain; accession no.
FIG. 1. Expression of M-T2 in stably transfected Jurkat T-cell lines and susceptibility to apoptosis. (A) M-T2 expression in stably transfected
Jurkat T-cell clones T2O-a, T2O-11, T2L-4, T2L-3, and Jneo analyzed by Western immunoblotting and densitometry. Percent apoptosis induced
in M-T2-expressing and control Jurkat T cells after exposure to 10,000 J of UV irradiation, 20 ?M etoposide (VP-16), and 10 ?M CHX for 8 h.
Data shown are means ? standard deviations and are representative of replicate experiments. (B) Susceptibility of Jneo (unfilled squares) and
M-T2-expressing Jurkat T cells (filled symbols) after 8 h of culture in LZ-TRAIL, LZ-FasL, LZ-CD40L (control), or recombinant human TNF-?.
Data are means ? standard errors of the means, which were ?5%, and error bars have been omitted for clarity. (C) Flow cytometry analysis of
death receptor expression in Jurkat T-cell clones. Histograms show death receptor expression on the control Jneo Jurkat line (solid lines), both
at the cell surface and intracellularly, relative to staining with isotype control antibodies (filled histograms). Death receptors on M-T2-expressing
Jurkat lines T2O-a (dots), T2O-11 (spaced dots), T2L-4 (small dashes), and T2L-3 (large dashes) are overlaid with control Jneo cells (solid lines).
VOL. 80, 2006POXVIRUS SUBVERSION OF TNFR1 BY vPLAD 9303
NC_001266) (57), camelpoxvirus CMLV002 and CMLV210
(Kazakhstan M-96 strain; accession no. NC_003391) (1), var-
iola minor virus G2R (strain Garcia; accession no. Y16780)
(45), variola major virus G2R (strain Bangladesh-1975; acces-
sion no. L22579) (26), monkeypox virus J2L (strain Zaire 96-
1-16; accession no. NC_003310) (44), ectromelia virus CrmD
(strain Moscow; accession no. NC_004105) (9), and even the
fragmented T2-like ORFs in VV (strains Lister; accession no.
U86871 [V. N. Loparev, J. M. Parsons, and J. J. Esposito, unpub-
lished data], and Copenhagen; accession no. NC_001559) (16)
(Fig. 2). In fact, there is 22 to 37% amino acid identity and 27 to
47% amino acid similarity between human TNFR1 and TNFR2
PLAD/CDR1 and the vPLAD present in poxvirus T2-like ORFs
(Fig. 2 and data not shown). Hence, all known viral T2 family
ORFs contain a highly conserved vPLAD that overlaps the first
CRD in viral and cellular TNFR molecules.
M-T2 inhibits TNFR1-mediated apoptosis acting via its con-
served vPLAD. Because M-T2 specifically inhibited Jurkat T
cells from human TNF-?-mediated apoptosis (Fig. 1B) and
since secreted M-T2 does not bind or inhibit human TNF-?
(38) nor influence endogenous human TNFR expression levels
(Fig. 1C), we hypothesized that M-T2 functions as a dominant-
negative TNFR inhibitor molecule (31, 41). To determine
whether M-T2 specifically inhibits TNFR1-induced death and
the relative importance of this vPLAD, a series of cotransfec-
tion experiments were performed with full-length M-T2 or a
T2 mutant in which the vPLAD was deleted (M-T2?PLAD).
Plasmid pcDNA3-M-T2myc, pcDNA3-M-T2?PLADmyc, or
pcDNA3-LacZ, and TNFR-induced cell death was assayed by
measuring ?-galactosidase activity in surviving HEK 293T
cells. Overexpression of TNFR1 alone (or with an empty con-
trol plasmid) induced significant cell death (P ? 0.001) com-
pared to pcDNA3, and most cells appeared rounded and be-
came detached from the culture dish. In contrast, expression of
full-length M-T2myc significantly inhibited TNFR1-induced
HEK 293T cell death (P ? 0.001) (Fig. 3 and data not shown).
In all T2-cotransfected cultures, 293T cells appeared healthy,
as judged by phase-contrast microscopy (data not shown). In
contrast, M-T2?PLADmyc did not protect 293T cells from
TNFR1-induced death, and in fact, M-T2?PLADmyc con-
ferred increased sensitivity to TNFR1-induced death (P ?
0.01) for reasons that are unclear (Fig. 3). As expected, both
M-T2myc and M-T2?PLADmyc proteins were detected in
transfected 293T cells (Fig. 3, insert), and interestingly,
M-T2myc also inhibited transfection-induced HEK 293T cell
death (Fig. 3), perhaps implying that the transfection proce-
dure itself induces TNF-? and/or TNFR-mediated cell death.
Moreover, S-T2 similarly inhibited human TNFR1-induced
cell death even in the presence of saturating amounts of puri-
fied human TNFR1-Fc, which was added to the culture super-
natants to sequester any possible production of transfection-
induced TNF-? (data not shown). Thus, leporipoxvirus T2
proteins inhibit human TNFR1-induced death in vitro, and for
M-T2, this activity requires an intact vPLAD.
M-T2 physically associates with TNFR1 and TNFR2. Due to
the highly conserved N-terminal region of leporipoxvirus T2
with human cellular TNFRs (41), we hypothesized that M-T2
inhibited TNFR signaling by directly interacting with TNFRs
themselves, forming an inhibitory heterocomplex (31, 41). To
determine whether M-T2 specifically interacts with human TN-
FRs, cotransfections were again performed and TNFR1 or
FIG. 2. Poxvirus T2-like molecules contain a conserved PLAD. Sequence similarity analysis of the N-terminal region of the myxoma virus
(MV), Shope fibroma virus (SFV), VV strain Lister and Copenhagen, camelpox virus (CMLV), variola major virus strain Bangladesh (VAR bsh
major), variola minor virus strain Garcia (VAR gar minor), monkeypox virus (MPV), and ectromelia virus (EV) T2 ORFs and human TNFR1 and
TNFR2 reveals a highly conserved PLAD that overlaps the first CRD. Identical amino acids (bold) and conserved amino acid differences (gray)
FIG. 3. T2 protects against TNFR1-induced cell death. Survival of
HEK 293T cells 48 h posttransfection with pcDNA3-TNFR1 and
pcDNA3-M-T2myc or pcDNA3-M-T2?PLADmyc together with LacZ,
and other control plasmids, as indicated. Viability was determined by
measuring ?-galactosidase activity in surviving cells. Data shown are
means ? the standard errors of the means of triplicate transfections, each
measured in quadruplicate, and are representative of repeated assays. An
asterisk indicates a statistically significant difference (P ? 0.05). (Insert)
Western immunoblot detection of M-T2myc and M-T2?PLADmyc in
transfected HEK 293T cells.
9304 SEDGER ET AL.J. VIROL.
TNFR2 was immunoprecipitated and examined for the pres-
ence of associated M-T2myc or M-T2?PLADmyc protein. An-
tibodies specific to either TNFR1 or TNFR2 bound to a TNFR
complex that contained M-T2myc (Fig. 4). In contrast, the T2
mutein T2?PLADmyc interacted considerably less well and
not at all with TNFR1 and TNFR2, respectively (Fig. 4). Anal-
ogous experiments also demonstrated that S-T2 physically as-
sociates with human TNFRs (data not shown). Taken together,
these data indicate that leporipoxvirus T2 physically associates
with human TNFR1 and TNFR2 and that the interaction
largely requires the presence of the vPLAD.
Intracellular localization of the M-T2-TNFR1 complex. To
determine where M-T2 physically associates with TNFRs, we
examined TNFR and T2 expression by confocal microscopy.
For this, the TNFR1-CPF and M-T2myc or M-T2?PLADmyc
proteins were coexpressed by transfection into U20S human
osteosarcoma cells and examined for CFP (TNFR1) expres-
sion and M-T2myc expression with a Cy3-tagged anti-myc an-
tibody. TNFR1-CFP was clearly detectable intracellularly in a
compartment resembling the Golgi apparatus, which is consis-
tent with the fact that most TNFR1 protein is found in intra-
cellular compartments within cells and specifically in the trans-
Golgi (21). Although M-T2 was historically described as a
secreted protein (54), it was clearly detectable within lysates
from virus-infected cells or cDNA-transfected 293T cells (Fig.
3 and data not shown), and confocal-microscopy examination
demonstrated that M-T2myc and M-T2?PLADmyc are
abundantly present intracellularly in transfected U20S cells
(Fig. 5 and data not shown). Furthermore, M-T2myc and
M-T2?PLADmyc clearly colocalized with TNFR1-CFP within
intracellular compartments (Fig. 5, merge panels). For control
purposes, U20S cells were also cotransfected with pcDNA3-
TNFR1-CPF and pCMV-p16INK4a. p16INK is found as a
diffuse cytoplasmic protein (34), and although both p16INK
and TNFR1-CFP are clearly expressed, p16INK does not
colocalize with TNFR1-CFP (Fig. 5, merge panel). Hence,
M-T2’s inhibition of TNFR1-induced cell death occurs by vir-
tue of its association with TNFR1, and these proteins are
clearly found localized together within cells.
Cell-associated M-T2 and TNF-? binding. Finally, we tested
whether the presence of cell-associated M-T2 impeded the
binding of human TNF-?. For this, HEK 293T cells were
transfected with TNFR1 and M-T2myc or M-T2?PLADmyc or
with TNFR2 and M-T2myc or M-T2?PLADmyc and 24 h
later, incubated with recombinant human TNF-? for 10 min at
4°C. Bound TNF-? was then detected by flow cytometry. Cell
surface-bound TNF-? was detectable at equal levels in cells
expressing TNFR1, TNFR1 and M-T2myc, or TNFR1 and
M-T2?PLADmyc (Fig. 6A). Surface TNF-? binding was also
unaltered in 293T cells expressing TNFR2, TNFR2 and
M-T2myc, or TNFR2 and M-T2?PLADmyc (Fig. 6A). Impor-
tantly, TNFR1 is only present at low levels at the cell surface
compared to TNFR2, which is abundantly present after trans-
fection (Fig. 6A). Therefore, at least for TNFR1-expressing
cells, TNF-? binding is likely to have reached saturation, and
hence coexpression of M-T2 does not impinge on human
TNF-? binding to this receptor. However, in order to accu-
rately interpret these data we investigated whether M-T2myc is
detectable at the cell surface. Confocal-microscopic examina-
tion of U20S cells transfected with TNFR1-CPF and M-T2myc
clearly detected M-T2myc at the cell surface, that is, on un-
permeabilized cells, but it is uncertain whether M-T2myc and
TNFR1-CFP colocalize at the cell surface, because all intra-
FIG. 4. Immunoprecipitation analysis of M-T2 and TNFR. HEK
293T cells were cotransfected with pcDNA3-TNFR1 or pcDNA3-
TNFR2 and either pcDNA3-M-T2myc, pcDNA3-T2?PLADmyc, or
pcDNA3-LacZ. TNFR1 was immunoprecipitated with anti-TNFR1-
specific H5 antibody, TNFR2 was immunoprecipitated with TNFR2-
specific C20 antibody, and TNFR-associated M-T2 was detected with
anti-M-T2 B5 antibody. IP, immunoprecipitation; WB, Western im-
FIG. 5. Intracellular localization of the M-T2-TNFR1 complex.
Confocal-microscope detection of human TNFR1-CFP, M-T2myc
(Cy3), or p16INKmyc (Cy3) in cotransfected U20S cells. Cells were
fixed with 2% paraformaldehyde, permeabilized in 0.1% saponin,
stained, and examined at 24 h posttransfection with a Leica SP2 con-
focal microscope. Shown are CFP fluorescence, Cy3 fluorescence, and
merged images of both. Results are representative of repeated
VOL. 80, 2006 POXVIRUS SUBVERSION OF TNFR1 BY vPLAD9305
cellular CFP-labeled TNFR1 protein is detected in this assay
(Fig. 6B). Hence, M-T2 is detectable at the cell surface but
does not interfere with human TNF-? binding to human
TNFR1 or human TNFR2 (Fig. 6A and B). Moreover, that
TNF-? binding is unaltered in HEK 293T cells expressing
M-T2myc alone (data not shown) or M-T2 plus TNFR1 or
M-T2 plus TNFR2 (Fig. 6A) implies that cell-associated
M-T2myc does not itself bind human TNF-?. To confirm that
cell-associated M-T2 only binds rabbit TNF-?, we performed
a mixing experiment in which lysates from pcDNA3-M-
T2myc and pcDNA3-M-T2?PLADmyc transfected 293T cells
were combined with lysates from Vero cells infected with re-
combinant VV encoding VV-rabbit TNF-? or VV-human
TNF-?. In these experiments, immunoprecipitated M-T2myc
and M-T2?PLADmyc bound rabbit TNF-?, as expected (Fig.
6C), but human TNF-? was not detected in complex with immu-
noprecipitated M-T2myc or M-T2?PLADmyc (data not shown).
This is consistent with our previous reports that secreted M-T2
binds TNF-? in a species-specific manner; that is, it binds rabbit
TNF-? but not mouse or human TNF-? (38, 39). Furthermore,
these results confirm our previous findings that the TNF-?-bind-
ing and antiapoptotic functions of M-T2 are conferred by differ-
ent domains, both within the N-terminal TNFR-homologous re-
M-T2 is a bone fide antiapoptotic protein that acts by virtue of its
ability to physically associate with cellular TNFRs in intracellular
compartments and through its highly conserved vPLAD. Thus,
cell-associated M-T2 inhibits TNF-? and TNFR1 signaling in a
species nonspecific manner by forming a dominant-negative in-
tracellular receptor complex, and this is independent of secreted
M-T2’s documented ability to bind and inhibit soluble TNF-?,
which is species specific.
We have demonstrated that leporipoxvirus M-T2, a viral
TNFR homologue, is a genuine antiapoptotic factor that can
inhibit human TNFR1-mediated apoptosis independently of its
known ability to bind rabbit TNF-?, and in the absence of any
other myxoma virus protein. Moreover, we demonstrated that the
ability of M-T2 to inhibit TNFR1-induced cell death is dependent
on an N-terminal PLAD-homologous domain within T2 (vP-
LAD) and involves the formation of an intracellular TNFR–
M-T2 heterocomplex. This complex appears to work by prevent-
ing TNFR signaling rather than by impeding TNF-? binding
to cellular TNFRs at the cell surface.
Although it is clear that poxviruses have evolved multiple
strategies to prevent apoptosis, including the specific ability to
inhibit secreted TNF-? (39, 54), we have described a mecha-
nism of viral inhibition of TNFR-mediated apoptosis that ap-
pears to be unique (42). It is different mechanistically from the
actions of the myxoma virus ubiquitin ligase M153R gene
product that down-regulates Fas from the surface of infected
cells (19, 25), and unlike the adenovirus receptor internaliza-
tion domain (RID) proteins that specifically interact with Fas,
TNFRs, and TRAIL-Rs, forcing their internalization and deg-
radation (15, 47, 51, 52). Moreover, there are no similarities to
other previously described antiapoptotic strategies utilized by
lymphotropic viruses (27). Although the papillomavirus E6
protein can specifically inhibit TNFR signaling without de-
creasing surface TNFR expression (14), there are no func-
tional or amino acid sequence similarities between T2 and E6.
Thus, these proteins appear to act via distinctly different mech-
anisms: papillomavirus E6 inhibits apoptosis by binding to the
intracellular region of human TNFR, preventing the associa-
tion of TRADD and FADD (14), while M-T2 acts via its
N-terminal vPLAD to form a dominant-inhibitory T2-TNFR
complex (Fig. 3 and 4). Furthermore, the ability of M-T2 to
inhibit human TNF-? function by preventing cellular TNFR
signaling while not impeding TNF-? binding to cellular TNFRs
(Fig. 6) highlights how this mechanism of subverting TNF-?
prevents any potential exacerbation of immunopathological
effects of TNF-?. Indeed, because the cellular TNFRs can still
bind human TNF-? there is not likely to be an increase in
FIG. 6. Cell-associated M-T2 and TNF-?-binding. (A) Flow cytom-
etry analysis of human TNF-? binding to the surface of HEK 293T
cells transfected with TNFR1, TNFR1 plus M-T2myc, or TNFR1 plus
M-T2?PLADmyc (top row) or with TNFR2, TNFR2 plus M-T2myc,
or TNFR2 plus M-T2?PLADmyc (second row) at 24 h posttransfec-
tion. Shown is the surface fluorescence of bound TNF-?, detected with
anti-human TNF-?–PE antibody, after 10 min of culture with (red,
unfilled histograms) or without (black, unfilled histograms) recombi-
nant human TNF-?. Isotype control staining (filled histograms) and
surface expression levels of TNFR1 and TNFR2 24 h after transfection
are also shown, as are overlays of TNF-? staining in each transfec-
tion (orange, TNFR; green, TNFR plus M-T2; blue, TNFR plus
M-T2?PLAD). Data shown are representative of independently re-
peated experiments. (B) Surface detection of TNFR1-CFP and
M-T2myc protein at 24 h posttransfection on unpermeabilized U20S
cells (TNFR1-CFP, M-T2myc [Cy3], and merged images of both).
(C) Cell-associated M-T2myc and M-T2?PLADmyc binding to rabbit
TNF-?. M-T2myc- and M-T2?PLADmyc-containing HEK 293T trans-
fected-cell lysates were mixed with Vero cell lysates containing rabbit
TNF-? expressed by recombinant VV. M-T2myc-bound rabbit TNF-?
was detected by immunoprecipitation (IP) with anti-myc antibody and
Western immunoblotting (WB) with rabbit TNF-?-specific antibody
(as indicated). Expression of M-T2, M-T2?PLAD, and rabbit TNF-?
was confirmed by Western immunoblotting.
9306SEDGER ET AL.J. VIROL.
bioavailable TNF-? which might otherwise affect neighboring
uninfected cells or act systemically. Thus, the ability of M-T2 to
specifically inhibit human TNFR function indicates its ability
to indirectly limit human TNF-?. In this sense, M-T2 vPLAD
confers intracellular M-T2 with a potent species nonspecific
inhibitory activity against both TNF-? and TNFR, while se-
creted M-T2 inhibits only soluble TNF-? and acts in a strictly
species-specific manner (38). Therefore, M-T2 is a “dominant-
negative” inhibitor that appears to be acting in a manner some-
what similar to that recently ascribed to TRAIL-R4 PLAD in
inhibiting TRAIL-R2-mediated cell death (10).
Modeling of a predicted secondary structure of M-T2 to-
gether with the known secondary structure of TNFRs (30)
illustrates how M-T2 might exert its TNFR-inhibitory activity
(Fig. 7). These modeling predictions suggest that the N-termi-
nal TNFR-homologous region of M-T2 is structurally similar
to the N-terminal regions of human TNFRs (Fig. 7). This is
consistent with the predicted secondary structures of the N-
terminal regions of other TNFR superfamily molecules which
are also remarkably similar (32). Because the extracellular
regions of TNFRs have been found as parallel and antiparallel
dimers (30), it is possible that T2 can form heterocomplexes in
either of these conformations. The antiparallel conformation is
found under low-pH conditions, which are thought to mimic
the environment in intracellular compartments. The modeling
predictions indicate that antiparallel T2-TNFR heterocom-
plexes would share considerably more surface interface than a
parallel T2-TNFR heterocomplex (Fig. 7) and thus predicts
that M-T2 occurs in an antiparallel conformation with cellular
TNFR within intracellular vesicles, where we find these mole-
cules colocalized (Fig. 5). However, this model also potentially
explains how even the most severely truncated M-T2 protein,
T2?L113 (which we described previously [40, 41]), retains its
antiapoptotic function, as it likely retains a strong propensity to
stably associate with TNFR in an antiparallel, apoptotic sig-
naling-incompetent conformation (Fig. 7). These modeling
predictions are consistent with our experimental data, but fur-
ther experiments, such as with FRET technology and C- and
N-tagged proteins, are needed to confirm these hypotheses.
Nevertheless, our data clearly suggest that when bound to
M-T2, TNFRs are in a conformation that is nonconducive to
death signaling. TNFR1 transduces a number of other signal-
ing pathways in addition to caspase-mediated apoptotic signal-
ing; TNFR1 can transduce activation of NF?B, Jun, and MAP
kinase, and TNFR1 induces neutral and acid sphingomyelin-
ase, resulting in the production of ceramide (55). Recent evi-
dence suggests that these pathways appear to be tightly regu-
lated by TNFR internalization (36). Our data indicate that
M-T2 prevents TNFR1-induced apoptotic signaling from in-
tracellular locations, and in this regard it will be interesting to
determine whether M-T2 affects the recruitment of particular
TNFR1-association proteins and hence other TNFR1 signaling
It is clear that apoptosis is an innate response to virus in-
fection, and in many situations this necessitates that viruses
express multiple antiapoptotic proteins in order to maintain
cellular viability for long enough periods to sustain productive
virus replication in different cell types (31). Apoptosis of virus-
infected parenchymal cells can dramatically influence the virus
burden, and apoptosis of virus-infected lymphocytes is thought
to be a key event controlling the dissemination of lymphotropic
virus in vivo. However, in many cases the trigger(s) that ini-
tiates virus-induced apoptosis has not been defined. We have
demonstrated that M-T2 protects against virus-induced lym-
phocyte apoptosis (24) and specifically inhibits TNFR1-medi-
ated cell death. The fact that viruses encode proteins that act
to subvert nearly all aspects of TNFR signaling (5, 6, 42)
emphasizes the importance of the TNF-?/TNFR axis in anti-
viral immunity and virus-host interactions. Indeed, it is par-
ticularly noteworthy that there is virtually no detectable
alteration in gene expression of TNFR or TNFR signaling
molecules in variola virus (smallpox virus)-infected cells, which
strongly implies that variola virus employs a nontranscriptional
and nontranslational strategy to inhibit these pathways (35).
We have identified a vPLAD within all poxvirus T2-like
vTNFR ORFs and demonstrated that it is required for M-T2’s
inhibition of TNFR-induced death without altering TNFR ex-
pression levels. Given that the vPLAD is also present within
variola virus G2R, these T2-like proteins are prime candidates
for mediating variola virus’s subversion of the TNFR axis. It is
unknown whether other vTNFR molecules, such as the poxvi-
rus CrmB, -C, -D, and -E proteins, vCD30, and human cyto-
megalovirus UL144, act in an analogous manner to M-T2 by
forming inhibitory complexes with their cellular homologues
TNFR, LT?R, CD30, and HVEM, but this can easily be tested.
Finally, the demonstrated role of N-terminal vPLAD as critical
for the inhibition of TNFR signaling confirms that this is a
functional region within the ectodomain of TNFRs that regu-
lates TNFR biology, and notably, it is distinct from the ligand-
binding domain which resides within CRD2 and CRD3 (8, 41,
42). The M-T2 vPLAD spans amino acids 18 to 52, which lies
within CRD1, and this is entirely analogous to the situation
FIG. 7. Secondary structural predictions of M-T2 and TNFR.
Structural data from human TNFR1 (green) were used to generate a
predicted secondary structure for the N-terminal region of M-T2
(blue). Identical residues were given the same structural constraints as
in TNFR1, but nonidentical residues were weighted less stringently.
Panels: i, TNF-? bound to a parallel TNFR1:M-T2 dimer; ii, TNFR1
and M-T2?L113 mutein in parallel dimer conformation; iii, M-T2
dimer and M-T2:TNFR1 heterodimer in antiparallel conformation; iv,
M-T2?L113 dimer in antiparallel conformation.
VOL. 80, 2006 POXVIRUS SUBVERSION OF TNFR1 BY vPLAD9307
already demonstrated for PLAD in human TNFR and Fas
receptors (11, 48). Furthermore, for Fas, dominant interfering
mutations are only effective when this N-terminal PLAD of
CRD1 is intact (48). Finally, targeting cellular PLAD function
by recombinant protein mimotopes or humanized monoclonal
antibodies is likely to be efficacious clinically in any disease in
which anti-TNF-?-based therapies are currently used, and in
fact, cellular “PLAD-only” proteins have recently been dem-
onstrated to have clinical efficacy in murine models of exper-
imental inflammatory arthritis (11). Therefore, this study not
only defines a novel mechanism of viral subversion of TNF-?/
TNFR biology, but it also substantiates the targeting the
TNFR PLAD in the development of the next generation of
We thank Bruce Seet and Colin Macauley (Robarts Research Insti-
tute) for help in generating T2-specific antibody; Kathryn Bateman
(University of Alberta) for T2 predicted structures; Sabine Piller,
Helen Rizos, and Monica Miranda-Saksena (Westmead Millennium
Institute) for antibody reagents; Jacqui Mills (Westmead Millennium
Institute) for technical assistance with confocal microscopy; and Peter
Kerr (CSIRO, Australia) for thoughtful comments on the biology of
myxoma virus infection.
F.K.-M.C. is supported by NIH grant AI065877 and a Cancer Re-
search Institute investigator award. This work was supported by CIHR
and NCIC grants to G.M. and a University of Sydney U2000 fellowship
and NH&MRC (Australia) project grant 211128 to L.S.
1. Afonso, C. L., E. R. Tulman, Z. Lu, L. Zsak, N. T. Sandybaev, U. Z.
Kerembekova, V. L. Zaitsev, G. F. Kutish, and D. L. Rock. 2002. The genome
of camelpox virus. Virology 295:1–9.
2. Alcami, A., A. Khanna, N. L. Paul, and G. L. Smith. 1999. Vaccinia virus
strains Lister, USSR and Evans express soluble and cell-surface tumour
necrosis factor receptors. J. Gen. Virol. 80:949–959.
3. Aragane, Y., D. Kulms, D. Metze, G. Wilkes, B. Poppelmann, and T. A.
Luger. 1998. Ultraviolet light induces apoptosis via direct activation of CD95
(Fas/APO-1) independently of its ligand CD95L. J. Cell Biol. 140:171–182.
4. Barry, M., S. Hnatiuk, K. Mossman, S. F. Lee, L. Boshkov, and G. McFadden.
1997. The myxoma virus M-T4 gene encodes a novel RDEL-containing protein
that is retained within the endoplasmic reticulum and is important for the
productive infection of lymphocytes. Virology 239:360–377.
5. Benedict, C. A., T. A. Banks, and C. F. Ware. 2003. Death and survival: viral
regulation of TNF signaling pathways. Curr. Opin. Immunol. 15:59–65.
6. Benedict, C. A., P. S. Norris, and C. F. Ware. 2002. To kill or be killed: viral
evasion of apoptosis. Nat. Immunol. 3:1013–1018.
7. Cameron, C., S. Hota-Mitchell, L. Chen, J. Barrett, J.-X. Cao, C. Macaulay,
D. Willer, D. Evans, and G. McFadden. 1999. The complete DNA sequence
of myxoma virus. Virology 246:298–318.
8. Chan, F. K., H. J. Chun, L. Zheng, R. M. Siegel, K. L. Bui, and M. J.
Lenardo. 2000. A domain in TNF receptors that mediates ligand-indepen-
dent receptor assembly and signaling. Science 288:2351–2354.
9. Chen, N., M. I. Danila, Z. Feng, R. M. Buller, C. Wang, X. Han, E. J.
Lefkowitz, and C. Upton. 2003. The genomic sequence of ectromelia virus,
the causative agent of mousepox. Virology 317:165–186.
10. Clancy, L., K. Mruk, K. Archer, M. Woelfel, J. Mongkolsapaya, G. Screaton,
M. J. Lenardo, and F. K.-M. Chan. 2005. Preligand assembly domain-me-
diated ligand-independent association between TRAIL receptor 4 (TR4)
and TR2 regulates TRAIL-induced apoptosis. Proc. Natl. Acad. Sci. USA
11. Deng, G.-M., L. Zheng, F. K.-M. Chan, and M. Lenardo. 2005. Amelioration
of inflammatory arthritis by targetting the pre-ligand assembly domain of
tumour necrosis factor receptors. Nat. Med. 11:1304.
12. Dougall, W. C., M. Glaccum, K. Charrier, K. Rohrbach, K. Brasel, T. De
Smedt, E. Daro, J. Smith, M. E. Tometsko, C. R. Maliszewski, A. Armstrong,
V. Shen, S. Bain, D. Cosman, D. Anderson, P. J. Morrissey, J. J. Peschon,
and J. C. Schuh. 1999. RANK is essential for osteoclast and lymph node
development. Genes Dev. 13:2412–2424.
13. Everett, H., M. Barry, S. F. Lee, X. Sun, K. Graham, J. Stone, R. C.
Bleackley, and G. McFadden. 2000. M11L: a novel mitochondria-localized
protein of myxoma virus that blocks apoptosis of infected leukocytes. J. Exp.
14. Filippova, M., H. Song, J. L. Connolly, T. S. Dermody, and P. J. Duerksen-
Hughes. 2002. The human papillomavirus 16 E6 protein binds to tumor
necrosis factor (TNF) R1 and protects cells from TNF-induced apoptosis.
J. Biol. Chem. 277:21730–21739.
15. Friedman, J. M., and M. S. Horwitz. 2002. Inhibition of tumor necrosis
factor alpha-induced NF-?B activation by the adenovirus E3-10.4/14.5K
complex. J. Virol. 76:5515–5521.
16. Goebel, S. J., G. P. Johnson, M. E. Perkus, S. W. Davis, J. P. Winslow, and
E. Paoletti. 1990. The complete DNA sequence of vaccinia virus. Virology
17. Gorman, H. 1985. High efficiency gene transfer into mammalian cells,
p. 143–165. In D. M. Glover (ed.), DNA cloning. IRL Press, Oxford, United
18. Griffith, T. S., C. T. Rauch, P. J. Smolak, J. Y. Waugh, N. Boiani, D. H.
Lynch, C. A. Smith, R. G. Goodwin, and M. Z. Kubin. 1999. Functional
analysis of TRAIL receptors using monoclonal antibodies. J. Immunol. 162:
19. Guerin, J. L., J. Gelfi, S. Boullier, M. Delverdier, F. A. Bellanger, S. Bertagnoli,
I. Drexler, G. Sutter, and F. Messud-Petit. 2002. Myxoma virus leukemia-
associated protein is responsible for major histocompatibility complex class I
and Fas-CD95 down-regulation and defines scrapins, a new group of surface
cellular receptor abductor proteins. J. Virol. 76:2912–2923.
20. Harlow, E., and D. Lane. 1988. Antibodies: a laboratory manual. Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y.
21. Jones, S. J., E. C. Ledgerwood, J. B. Prins, J. Galbraith, D. R. Johnson, J. S.
Pober, and J. R. Bradley. 1999. TNF recruits TRADD to the plasma mem-
brane but not the trans Golgi network, the principal subcellular location of
TNF-R1. J. Immunol. 162:1042–1048.
22. Juo, P., C. J. Kuo, J. Yuan, and J. Blenis. 1998. Essential requirement for
caspase-8/FLICE in the initiation of the Fas-induced apoptotic cascade.
Curr. Biol. 10:1001–1008.
23. Kothny-Wilkes, G., D. Kulms, T. A. Luger, M. Kubin, and T. Schwarz. 1999.
Interleukin-1 protects transformed keratinocytes from tumor necrosis factor-
related apoptosis-inducing ligand- and CD95-induced apoptosis but not from
ultraviolet radiation-induced apoptosis. J. Biol. Chem. 274:28916–28921.
24. Macen, J. L., K. A. Graham, S. F. Lee, M. Schreiber, L. K. Boshkov, and G.
McFadden. 1996. Expression of the myxoma virus tumor necrosis factor
receptor homologue and M11L genes is required to prevent virus-induced
apoptosis in infected rabbit T lymphocytes. Virology 218:232–237.
25. Mansouri, M., E. Bartee, K. Gouveia, B. T. Hovey Nerenberg, J. Barrett, L.
Thomas, G. Thomas, G. McFadden, and K. Fruh. 2003. The PHD/LAP-
domain protein M153R of myxomavirus is a ubiquitin ligase that induces the
rapid internalization and lysosomal destruction of CD4. J. Virol. 77:1427–
26. Massung, R. F., L. I. Liu, J. Qi, J. C. Knight, T. E. Yuran, A. R. Kerlavage,
J. M. Parsons, J. C. Venter, and J. J. Esposito. 1994. Analysis of the com-
plete genome of smallpox variola major virus strain Bangladesh—1975. Vi-
27. Mein, E., H. Fickenscher, M. Thome, J. Tschopp, and B. Fleckenstein. 1998.
Anti-apoptotic strategies of lymphotropic viruses. Immunol. Today 19:474–
28. Messud-Petit, F., J. Gelfi, M. Delverdier, M. F. Amardeilh, R. Py, G. Sutter,
and S. Bertagnoli. 1998. Serp2, an inhibitor of the interleukin-1?-converting
enzyme, is critical in the pathobiology of myxoma virus. J. Virol. 72:7830–
29. Mossman, K., S. F. Lee, M. Barry, L. Boshkov, and G. McFadden. 1996.
Disruption of M-T5, a novel myxoma virus gene member of poxvirus host
range superfamily, results in dramatic attenuation of myxomatosis in infected
European rabbits. J. Virol. 70:4394–4410.
30. Naismith, J. H., T. Q. Devine, T. Kohno, and S. R. Sprang. 1996. Structures
of the extracellular domain of the type I tumor necrosis factor receptor.
31. Nash, P., J. Barrett, J. X. Cao, S. Hota-Mitchel, A. S. Lalani, H. Everett, X. M.
Xu, J. Robichaud, S. Hnatiuk, C. Ainslie, B. T. Seet, and G. McFadden. 1999.
Immunomodulation by viruses: the myxoma virus story. Immunol. Rev. 168:
32. Peitsch, M. C., and J. Tschopp. 1995. Comparative molecular modelling of
the Fas-ligand and other members of the TNF family. Mol. Immunol. 32:
33. Petit, F., S. Bertagnoli, J. Gelfi, F. Fassy, C. Boucraut-Baralon, and A.
Milon. 1996. Characterization of a myxoma virus-encoded serpin-like pro-
tein with activity against interleukin-1?-converting enzyme. J. Virol. 70:
34. Rizos, H., A. P. Darmanian, E. A. Holland, G. J. Mann, and R. F. Kefford.
2001. Mutations in the INK4a/ARF melanoma susceptibility locus function-
ally impair p14ARF. J. Biol. Chem. 276:44.
35. Rubins, K. H., L. E. Hensley, P. B. Jahrling, A. R. Whitney, T. W. Geisbert,
J. W. Huggins, A. Owen, J. W. Leduc, P. O. Brown, and D. A. Relman. 2004.
The host response to smallpox: analysis of the gene expression program in
peripheral blood cells in a nonhuman primate model. Proc. Natl. Acad. Sci.
36. Schneider-Brachert, W., V. Tchikov, J. Neumeyer, M. Jakob, S. Winoto-
Morbach, J. Feindt, M. Henrich, O. Merkel, M. Ehrenschwender, D. Adam,
9308 SEDGER ET AL.J. VIROL.
R. Mentlein, D. Kabelitz, and S. Schutze. 2004. Compartmentalization of
TNF receptor 1 signalling: internalized TNF receptorsomes as death signal-
ing vesicles. Immunity 21:415–428.
37. Schreiber, M., and G. McFadden. 1996. Mutational analysis of the ligand-
binding domain of M-T2 protein, the tumor necrosis factor receptor homo-
logue of myxoma virus. J. Immunol. 157:4486–4495.
38. Schreiber, M., and G. McFadden. 1994. The myxoma virus TNF-receptor
homologue (T2) inhibits tumor necrosis factor-alpha in a species-specific
fashion. Virology 204:692–705.
39. Schreiber, M., K. Rajarathnam, and G. McFadden. 1996. Myxoma virus T2
protein, a tumor necrosis factor (TNF) receptor homolog, is secreted as a
monomer and dimer that each bind rabbit TNF?, but the dimer is a more
potent TNF inhibitor. J. Biol. Chem. 271:13333–13341.
40. Schreiber, M., L. Sedger, and G. McFadden. 1997. Distinct domains of
M-T2, the myxoma virus tumor necrosis factor (TNF) receptor homolog,
mediate extracellular TNF binding and intracellular apoptosis inhibition.
J. Virol. 71:2171–2181.
41. Sedger, L., and G. McFadden. 1996. M-T2: a poxvirus TNF receptor homo-
logue with dual activities. Immunol. Cell Biol. 74:538–545.
42. Sedger, L. M. 2005. Viral inhibition of tumour necrosis factor-? (TNF?) and
TNF-receptor induced apoptosis and inflammation. Curr. Med. Chem.-Anti-
Inflamm. Anti-Allergy Agents 4:597–615.
43. Sedger, L. M., D. M. Shows, R. A. Blanton, J. J. Peschon, R. G. Goodwin, D.
Cosman, and S. R. Wiley. 1999. IFN-? mediates a novel antiviral activity
through dynamic modulation of TRAIL and TRAIL receptor expression.
J. Immunol. 163:920–926.
44. Shchelkunov, S. N., A. V. Totmenin, I. V. Babkin, P. F. Safronov, O. I.
Ryazankina, N. A. Petrov, V. V. Gutorov, E. A. Uvarova, M. V. Mikheev, J. R.
Sisler, J. J. Esposito, P. B. Jahrling, B. Moss, and L. S. Sandakhchiev. 2001.
Human monkeypox and smallpox viruses: genomic comparison. FEBS Lett.
45. Shchelkunov, S. N., A. V. Totmenin, V. N. Loparev, P. F. Safronov, V. V.
Gutorov, V. E. Chizhikov, J. C. Knight, J. M. Parsons, R. F. Massung, and
J. J. Esposito. 2000. Alastrim smallpox variola minor virus genome DNA
sequence. Virology 266:361–386.
46. Sheikh, M. S., M. J. Antinore, Y. Huang, and A. J. J. Fornace. 1998. Ultra-
violet irradiation-induced apoptosis is mediated via ligand independent ac-
tivation of tumor necrosis factor receptor 1. Oncogene 17:2555–2563.
47. Shisler, J., C. Yang, B. Walter, C. F. Ware, and L. R. Gooding. 1997. The
adenovirus E3-10.4K/14.5K complex mediates loss of cell surface Fas
(CD95) and resistance to Fas-induced apoptosis. J. Virol. 71:8299–8306.
48. Siegel, R., J. K. Frederiksen, D. A. Zacharias, F. K.-M. Chan, M. Johnson,
D. Lynch, R. Y. Tsien, and M. J. Lenardo. 2000. Fas preassociation required
for apoptosis signalling and dominant inhibition by pathogenic mutations.
49. Smith, C. A., T. Davis, J. M. Wignall, W. S. Din, T. Farrah, C. Upton, G.
McFadden, and R. G. Goodwin. 1991. T2 open reading frame from the
Shope fibroma virus encodes a soluble form of the TNF receptor. Biochem.
Biophys. Res. Commun. 176:335–342.
50. Tobin, D., M. van Hogerlinden, and R. Toftgard. 1998. UVB-induced asso-
ciation of tumor necrosis factor (TNF) receptor 1/TNF receptor-associated
factor-2 mediates activation of Rel proteins. Proc. Natl. Acad. Sci. USA
51. Tollefson, A. E., T. W. Hermiston, D. L. Lichtenstein, C. F. Colle, R. A.
Tripp, T. Dimitrov, K. Toth, C. E. Wells, P. C. Doherty, and W. S. Wold.
1998. Forced degradation of Fas inhibits apoptosis in adenovirus-infected
cells. Nature 392:726–730.
52. Tollefson, A. E., K. Toth, K. Doronin, M. Kuppuswamy, O. A. Doronina,
D. L. Lichtenstein, T. W. Hermiston, C. A. Smith, and W. S. Wold. 2001.
Inhibition of TRAIL-induced apoptosis and forced internalization of TRAIL
receptor 1 by adenovirus proteins. J. Virol. 75:8875–8887.
53. Upton, C., A. M. DeLange, and G. McFadden. 1987. Tumorigenic poxviruses:
genomic organization and DNA sequence of the telomeric region of the
Shope fibroma virus genome. Virology 160:20–30.
54. Upton, C., J. L. Macen, M. Schreiber, and G. McFadden. 1991. Myxoma
virus expresses a secreted protein with homology to the tumor necrosis factor
receptor gene family that contributes to viral virulence. Virology 184:370–
55. Wajant, H., K. Pfizenmaier, and P. Scheurich. 2003. Tumor necrosis factor
signaling. Cell Death Differ. 10:45–65.
56. Walczak, H., R. E. Miller, K. Ariail, B. Gliniak, T. S. Griffith, M. Kubin, W.
Chin, J. Jones, A. Woodward, T. Le, C. Smith, P. Smolak, R. G. Goodwin,
C. T. Rauch, J. C. Schuh, and D. H. Lynch. 1999. Tumoricidal activity of
tumor necrosis factor-related apoptosis-inducing ligand in vivo. Nat. Med.
57. Willer, D. O., G. McFadden, and D. H. Evans. 1999. The complete sequence
of Shope (rabbit) fibroma virus. Virology 264:319–343.
58. Xu, X., P. Nash, and G. McFadden. 2000. Myxoma virus expresses a TNF
receptor homolog with two distinct functions. Virus Genes 21:97–109.
VOL. 80, 2006 POXVIRUS SUBVERSION OF TNFR1 BY vPLAD9309