The Journal of Experimental Medicine
JEM © The Rockefeller University Press
Vol. 201, No. 6, March 21, 2005 1007–1018
Vaccinia virus protein A46R targets multiple
Toll-like–interleukin-1 receptor adaptors and
contributes to virulence
Geoffrey L. Smith,
Ismar R. Haga,
Patrick C. Reading,
and Andrew G. Bowie
Katherine A. Fitzgerald,
Nathan W. Bartlett,
Department of Biochemistry, Trinity College, Dublin 2, Ireland
Department of Virology, Faculty of Medicine, Imperial College London, St. Mary’s Campus, London W2 1PG, England, UK
Department of Medicine, University of Massachusetts Medical School, Worcester, MA 01604
Viral immune evasion strategies target key aspects of the host antiviral response. Recently,
it has been recognized that Toll-like receptors (TLRs) have a role in innate defense against
viruses. Here, we define the function of the vaccinia virus (VV) protein A46R and show it
inhibits intracellular signalling by a range of TLRs. TLR signalling is triggered by homotypic
interactions between the Toll-like–interleukin-1 resistance (TIR) domains of the receptors
and adaptor molecules. A46R contains a TIR domain and is the only viral TIR domain–
containing protein identified to date. We demonstrate that A46R targets the host TIR
adaptors myeloid differentiation factor 88 (MyD88), MyD88 adaptor-like, TIR domain–
containing adaptor inducing IFN-
(TRIF), and the TRIF-related adaptor molecule and
thereby interferes with downstream activation of mitogen-activated protein kinases and
B. TRIF mediates activation of interferon (IFN) regulatory factor 3 (IRF3)
and induction of IFN-
by TLR3 and TLR4 and suppresses VV replication in macrophages.
Here, A46R disrupted TRIF-induced IRF3 activation and induction of the TRIF-dependent
gene regulated on activation, normal T cell expressed and secreted. Furthermore, we show
that A46R is functionally distinct from another described VV TLR inhibitor, A52R.
Importantly, VV lacking the A46R gene was attenuated in a murine intranasal model,
demonstrating the importance of A46R for VV virulence.
The discovery of the Toll-like receptor (TLR)
family of proteins has revolutionized the un-
derstanding of how the innate immune system
recognizes pathogens and initiates an effective
and appropriate response (1, 2). TLRs are part
of the larger IL-1R/TLR superfamily, which
includes IL-1Rs, IL-18Rs, and a group of or-
phan receptors (3). The family is defined by
the presence of a cytoplasmic Toll-like–IL-1
resistance (TIR) domain, which is responsible
for mediating downstream signaling. So far,
13 TLRs have been identified; TLRs 1–9
are common to mouse and human, whereas
TLR10 is only functional in humans, and
TLRs 11, 12, and 13 have been found only in
mice (2–5). Many but not all of these receptors
have been assigned a role in the initial detection
of, and response to, specific pathogen-associated
molecules (PAMs). In macrophages and neu-
trophils, this drives innate immune responses,
such as inflammation and induction of micro-
bicidal activity, whereas activation of TLRs
expressed on dendritic cells leads to the initia-
tion of adaptive immunity through induction
of IL-12 and costimulatory molecules (6, 7).
Although interest focused on the role of
TLRs in responding to bacteria initially, there
are now three strong lines of evidence that TLRs
are also involved in detecting viruses and initiat-
ing antiviral responses. First, some TLRs are re-
quired for cellular and whole animal responses to
certain viruses, viral proteins, and nucleic acids
(8, 9). Second, TLRs trigger antiviral signaling
pathways leading to the induction of IFN re-
sponses (8, 9). Third, viral immune strategies
used against TLRs have been identified (8, 9).
J. Stack and I.R. Haga contributed equally to this work.
I.R. Haga’s present address is Department of Biochemistry,
Trinity College, Dublin 2, Ireland.
P.C. Reading’s present address is Department of Microbiology
and Immunology, University of Melbourne, Victoria, 3010,
Abbreviations used: ERK, extra-
cellular signal–regulated kinase;
IRAK, IL-1 receptor–associated
kinase; IRF3, interferon regula-
tory factor 3; JNK, c-Jun NH
terminal protein kinase; Mal,
MyD88 adaptor-like; MOI,
multiplicity of infection; MyD88,
myeloid differentiation factor 88;
PAM, pathogen-associated mol-
ecule; RANTES, regulated on
activation, normal T cell ex-
pressed and secreted; SARM,
motifs-containing protein; TIR,
Toll–IL-1 resistance; TLR,
Toll-like receptors; TRAF,
TNF receptor–associated factor;
TRAM, TRIF-related adaptor
molecule; TRIF, TIR domain-
containing adaptor inducing
; VV, vaccinia virus.
A46R TARGETS TOLL-LIKE RECEPTOR ADAPTORS AND AFFECTS VACCINIA VIRULENCE | Stack et al.
Cell surface TLR2 and TLR4 may recognize viral glyco-
proteins on virions (8, 9). For example, measles virus hemag-
glutinin activates murine and human cells via TLR2, leading
to induction of proinflammatory cytokines and up-regula-
tion of surface expression of CD150, a receptor for measles
virus (10). In terms of viral nucleic acids, TLR3 is activated
in response to poly(I:C), a synthetic analogue of viral
dsRNA (11), whereas TLR7 and TLR8 recognize ssRNA
(from influenza, HIV, and vesicular stomatitis virus; refer-
ences 12–14) and TLR9 responds to dsDNA from herpes
simplex virus (15, 16).
That TLR signaling can induce an antiviral state was
shown clearly by Doyle et al. (17) in that pretreatment of
cells with poly(I:C) or lipid A (the moiety of LPS that is rec-
ognized by TLR4) inhibited the replication of a murine her-
pesvirus in macrophages. Even before the discovery of
TLRs, it was known that viral replication and viral patho-
genesis often involves NF-
B activation (18). TLRs, like IL-1,
mediate downstream signaling mainly through their cyto-
plasmic TIR domain. This domain mediates homotypic in-
teractions between TLRs, and also recruitment of TIR-con-
taining adaptor proteins, of which myeloid differentiation
factor 88 (MyD88) is a prototypical example. Recruitment
of MyD88 to TLR complexes leads to activation of IL-1 re-
ceptor–associated kinases (IRAKs), which engage with TNF
receptor–associated factor (TRAF) 6, leading ultimately to
the activation of mitogen-activated protein (MAP) kinases
and the transcription factor NF-
volved in NF-
B activation by every TLR tested thus far,
except for TLR3 (21, 22). MyD88 adaptor-like (Mal; refer-
ence 23), another TIR adaptor protein, is required specifi-
cally for TLR2 and TLR4 signaling (19, 20).
An important MyD88-independent signaling pathway is
the activation of the antiviral transcription factor IFN regula-
tory factor 3 (IRF3) by TLR3 and TLR4. IRF3 activation,
together with NF-
B activation, leads to IFN-
which initiates the IFN-based antiviral response. TIR do-
main-containing adaptor inducing IFN-
for MyD88-independent TLR3 and TLR4 signaling (21,
22, 24). TRIF activates IRF3 via TANK-binding kinase-1
(TBK1), leading to IFN-
expression (25, 26). TRIF proba-
bly associates directly with TLR3, but indirectly with
TLR4, via a bridging interaction with a fourth adaptor that
is unique to the TLR4 signaling pathway, TRIF-related
adaptor molecule (TRAM, 27, 28).
Further evidence for the role of TLRs in responding to
viruses came from the discovery of viral immune strategies
used against TLRs. Vaccinia virus (VV), the poxvirus used to
vaccinate against smallpox, encodes proteins that antagonize
important components of host antiviral defense. Previously,
we showed that VV protein A52R, which has no obvious
similarity to host proteins, can block the activation of NF-
by multiple TLRs, in particular TLR3 (29, 30). A52R associ-
ates with both IRAK2 and TRAF6, and disrupts signaling
complexes containing these proteins (30). Furthermore, dele-
tion of the
gene from VV reduced virus virulence (30).
B (19, 20). MyD88 is in-
(TRIF) is essential
In contrast with A52R, the VV protein investigated in
this work, A46R, has a TIR domain, and as such is the only
viral member of the IL-1R/TLR family identified to date
(29). Initial studies revealed that A46R could inhibit IL-1,
but not TNF-induced NF-
B activation (29). The effect on
IL-1, together with the presence of a TIR domain in the
protein suggested that A46R may have a role in immune
evasion. However, the mechanism of action of A46R, its
effect on TLR signaling pathways, and its potential role in
virulence were not determined. Here, we show that A46R
inhibits TLR-induced signaling, by associating with TIR-
domain containing adaptor molecules. This is the first dem-
onstration of direct viral targeting of TIR adaptors. We also
demonstrate a role for A46R in VV virulence, and show that
A46R and A52R are functionally distinct.
A46R inhibits multiple IL-1–induced signals
Previously, we showed that A46R was capable of suppress-
B activation induced by IL-1, whereas TNF-
B was unaffected (29). To further characterize
the effects of A46R on cell signaling, first we determined the
effect of A46R on other IL-1–dependent signals. The effect
of A46R on IL-1–, but not TNF-induced NF-
dent reporter gene activation was confirmed (Fig. 1 a). To
measure IL-1–induced transactivation of NF-
an assay was performed using an expression plasmid encod-
ing the transactivation domain of the p65 subunit of NF-
fused to the DNA-binding domain of Gal4, together with a
reporter plasmid under the control of a Gal4 upstream acti-
vation sequence (31). Reporter gene expression from this
plasmid requires p65 transactivation through phosphoryla-
tion. Ectopic expression of A46R inhibited IL-1–induced
p65 transactivation, but had no effect on basal levels of activ-
ity (Fig. 1 b). The effect of A46R on p65 transactivation
provided a rationale for the inhibition of the NF-
dent reporter gene. In a similar assay (23), A46R blocked
both c-Jun NH
-terminal protein kinase (JNK; Fig. 1 c) and
extracellular signal–regulated kinase (ERK; Fig. 1 d) activa-
tion induced by IL-1. Thus, A46R inhibited multiple dis-
tinct signals emanating from the IL-1 receptor.
A46R interacts with MyD88 and antagonizes MyD88-
The results from Fig. 1 suggest that A46R was acting close to
the IL-1 receptor complex. This was also likely given that
A46R has a TIR domain (29), as illustrated in Fig. 2 a, which
shows an alignment of VV and variola virus A46R with sev-
eral human TIR-containing proteins. Given that all IL-1
signals tested to date are dependent on the TIR domain-
containing adaptor MyD88 (32, 33), including p65 trans-
activation (31) and JNK activation (34), we reasoned
that A46R may target MyD88 via a TIR domain interac-
tion. To test this hypothesis, coimmunoprecipitation studies
were performed with A46R and MyD88 expressed ectopi-
cally. Fig. 2 b shows a clear interaction between A46R and
JEM VOL. 201, March 21, 2005
MyD88 (left). As a control, the ability of A46R to interact
with TRAF2, an adaptor used by TNF but not IL-1 (35),
was tested in parallel. In this case, no interaction was detected
(Fig. 2 a, right), consistent with the lack of effect of A46R on
TNF signaling (Fig. 1 a). These results were confirmed by
GST-pulldown experiments, whereby purified GST-A46R
interacted with ectopically expressed MyD88 in a cell lysate,
but not with TRAF2 (Fig. 2 c). A46R also failed to interact
with TAB1, a signaling molecule downstream of MyD88
(unpublished data). The interaction of A46R with MyD88
was also demonstrated in cells infected by VV and transfected
to express MyD88, to use the A46R protein at its physiological
concentration (Fig. 2 d). As a control for specificity, this was
compared with a VV deletion mutant lacking the A46R gene
A46R), whereupon no band for A46R was detected (Fig.
2 d). A46R also interacted with MyD88 in a yeast two-hybrid
pairwise assay (unpublished data), thus demonstrating that the
association was direct.
Apart from its role in IL-1R signaling, MyD88 is also
used by murine TLRs 1, 2, 4, 5, 6, 7, and 9 (2). Therefore,
we tested the effect of A46R on signaling via these TLRs in
RAW264.7 cells, a murine macrophage cell line that ex-
presses most TLRs (36). Stimulation of RAW264.7 cells
with MALP-2 (TLR2 and 6), Pam
(TLR4), flagellin (TLR5), R848 (TLR7), or CpG DNA
(TLR9) led to induction of the NF-
gene (Fig. 2 e). In each case, the presence of A46R caused
strong inhibition of induction, whereas it had no suppressive
effect on control levels (Fig. 2 e). Thus, A46R inhibited
MyD88-dependent signaling by both IL-1R and TLRs, pre-
sumably by interacting with MyD88 via its TIR domain.
Cys (TLR2 and 1), LPS
A46R targets the TLR4 receptor complex
The effect of A46R on TLR4 signaling was examined in
greater detail. A chimeric form of TLR4, comprising the
murine CD4 extracellular domain fused to the cytoplasmic
domain of human TLR4, which renders TLR4 constitu-
tively active (37), was used. Overexpression of CD4-TLR4
B, and this was inhibited by coexpression of
A46R in a dose-dependent manner (Fig. 3 a, left). In fact,
the highest concentration of A46R-expressing plasmid al-
most completely prevented TLR4-induced NF-
tion, whereas it did not suppress basal levels of reporter gene
activity (Fig. 3 a, left, white bars). TLR4-induced activation
of the MAP kinases p38 and ERK was also inhibited by
A46R expression (Fig. 3, middle and right, respectively).
Given that TLR4-induced NF-
only partially MyD88 dependent (38), it was difficult to ac-
count for the potent effects of A46R on these signals by an
interaction with MyD88 alone. Therefore, we assessed the
ability of A46R to target other TIR domain-containing pro-
teins involved in the TLR4 receptor complex. Fig. 3 b
shows that A46R could be immunoprecipitated with TLR4
itself, suggesting that A46R can also interact with the TLR4
TIR domain. Next, we tested the ability of A46R to target
two important TLR4 TIR adaptors, Mal and TRAM. Mal
and TRAM are thought to interact directly with TLR4, and
subsequently recruit MyD88 and TRIF, respectively (23, 27,
39). Both Mal and TRAM are essential for robust TLR4-
B activation (19, 23, 27, 28). Consistent
with this, A46R immunoprecipitated with both Mal (Fig. 3 c)
and TRAM (Fig. 3 d). The interactions with TLR4, Mal,
and TRAM were confirmed by GST-pulldown experiments
(Fig. 3 e). Furthermore, Mal was shown to coimmunopre-
cipitate with A46R in infected cells, associate directly with
A46R in the yeast two-hybrid pairwise assay, and rMal in-
teracted with GST-A46R in vitro (unpublished data).
Next, we demonstrated that the inhibitory effects of
A46R on TLR4 signaling pathways also resulted in a suppres-
sion of gene induction. Fig. 3 f shows that LPS stimulation of
HEK293 cells expressing TLR4 led to the release of IL-8,
which is NF-
B and p38 dependent (not depicted). Transient
transfection of these cells with A46R suppressed IL-8 release
50%. Thus, the viral TIR domain of A46R can target
B, p38, and ERK are
cells were transfected with 100 ng A46R or pcDNA3.1 (EV) and the NF-?B
(a), p65 (b), JNK (c), or ERK (d) reporter plasmids as described in Materials
and methods. 6 h before harvesting, the cells were stimulated with either
100 ng/ml IL-1 or 100 ng/ml TNF as indicated, and luciferase reporter gene
activity was measured.
A46R inhibits multiple IL-1–dependent signals. HEK 293
A46R TARGETS TOLL-LIKE RECEPTOR ADAPTORS AND AFFECTS VACCINIA VIRULENCE | Stack et al.
signaling. (a) Alignment of A46R with human TIR domains. The conserved
motifs Box 1, Box 2, and Box 3 are indicated by a solid line. The eight dif-
ferences between VV and variola virus A46R sequences are indicated by an
asterisk. For A46R, amino acids 35–238 are shown. (b) HEK 293T cells were
transfected with A46R and AU1-MyD88 (left) or Flag-TRAF2 (right) as
indicated. After 24 h, lysates were subject to immunoprecipitation, SDS-
PAGE, and immunoblotting with the indicated antibodies. (c) HEK 293T
cells were transfected with 8 ?g AU1-MyD88 (top) or Flag-TRAF2 (bottom).
After 24 h, lysates were incubated with GST alone (lane 2) or GST-A46R
(lane 3), and together with whole cell lysates (lane 1) were analyzed by
SDS-PAGE and immunoblotting with the indicated antibodies. (d) HEK 293
A46R associates with MyD88 and blocks MyD88-dependent
cells were transfected with 4 ?g of myc-MyD88. After 24 h, cells were
infected with viruses either containing (vWT-A46R) or not (v?A46R) the
A46R gene (MOI ? 1) and harvested 24 h after infection. Lysates were
subjected to immunoprecipitation, SDS-PAGE, and immunoblotting with
the indicated antibodies. Whole cell lysates were analyzed for expression
of A46R. (e) Murine macrophage RAW 264.7 cells were transfected with
the phRL-TK reporter gene and the NF-?B luciferase construct as described
in Materials and methods, together with pcDNA3.1 or 100 ng A46R. Cells
were stimulated for 6 h with 10 nM MALP-2 (MALP), 5 ?g/ml Pam3Cys
(Pam), 1 ?g/ml LPS, 250 ng/ml Flagellin (Flag), 1 ?M R-848, or 5 ?g/ml
CpG DNA. Cells were harvested 24 h after transfection and the reporter
gene activity was measured.
JEM VOL. 201, March 21, 2005
multiple human TIR domain-containing proteins important
for TLR4 signaling, leading to antagonism of signaling path-
ways and subsequent suppression of gene induction.
A46R antagonizes TRIF-dependent pathways
TRIF was identified as a TIR adaptor molecule capable of
directing both TLR4- and TLR3-induced IRF3 activation,
leading to IFN-
induction, which is independent of
MyD88 (21, 22, 24). The MyD88-independent late activa-
tion of NF-
B by TLR4 was also explained by the discovery
of TRIF (21). Given the central role of TRIF in the activa-
tion of the antiviral transcription factor IRF3, we wondered
whether A46R would also target this TIR adaptor directly.
Co-expression of A46R and TRIF, with subsequent coim-
munoprecipitation, demonstrated that these two proteins
could indeed form a complex (Fig. 4 a). An interaction be-
tween A46R and TRIF was confirmed by a GST-pulldown
experiment (Fig. 4 b) and also by a yeast two-hybrid pair-
wise assay (not depicted).
To determine the functional relevance of this interac-
tion, the effect of A46R on TRIF-dependent signals was ex-
amined. For this, IRF3 activation by TLR4 and TLR3 was
assessed by using a Gal4–IRF3 fusion protein together with a
Gal4-dependent reporter plasmid, which requires IRF3
Mal, and TRAM. (a) HEK 293 cells were transfected with 50 ng CD4-TLR4,
25–100 ng A46R, or pcDNA3.1 (EV) and the NF-?B (left), p38 (middle), or
ERK (right) reporter plasmids as indicated. Cells were harvested 24 h after
transfection, and luciferase reporter gene activity was measured. (b–d)
HEK 293T cells were transfected with A46R and Flag-TLR4 (b), Flag-Mal (c),
or Flag-TRAM (d) as indicated. After 24 h, lysates were subject to immuno-
precipitation, SDS-PAGE, and immunoblotting with the indicated antibodies.
A46R inhibits TLR4 signaling and interacts with TLR4,
(e) HEK 293T cells were transfected with 8 ?g of Flag-TLR4 (top), Flag-Mal
(middle), or Flag-TRAM (bottom). After 24 h, lysates were incubated with
GST-A46R (lane 3) or GST alone (lane 2), and together with whole cell
lysate (lane 1), were analyzed by SDS-PAGE and immunoblotting with the
indicated antibodies. (f) HEK-TLR4 cells were transfected with the indi-
cated amounts (ng) of A46R 24 h before stimulation with 1 ?g/ml LPS,
and 24 h after stimulation, supernatants were harvested and assayed for
IL-8 by ELISA.
A46R TARGETS TOLL-LIKE RECEPTOR ADAPTORS AND AFFECTS VACCINIA VIRULENCE | Stack et al.
transactivation to express the reporter gene (27). Ectopic ex-
pression of CD4-TLR4 resulted in a threefold induction of
this IRF3-dependent reporter, and this was completely
blocked by coexpression of A46R (Fig. 4 c, top). Although
the interaction of A46R with TLR4 and TRAM probably
contributes to this inhibitory effect, induction of an IFN-
promoter reporter gene by ectopic expression of TRIF was
also blocked by A46R (unpublished data), suggesting a direct
effect on TRIF, which is downstream of TLR4 and TRAM.
Further TLR3-dependent activation of IRF3 by poly(I:C),
which is entirely TRIF dependent, was also completely
blocked by A46R (Fig. 4 c, bottom). A46R also inhibited
LPS and poly(I:C)-mediated induction of an ISRE-dependent
reporter (unpublished data). In addition, no interaction be-
tween A46R and TLR3 was detected (unpublished data).
Furthermore, poly(I:C)-induced regulated on activation,
normal T cell expressed and secreted (RANTES) release
from HEK293 cells stably expressing TLR3 was inhibited by
A46R (Fig. 4 d). This represents inhibition of gene induc-
tion that is entirely independent of MyD88 (27). Therefore,
A46R blocks TLR-induced IRF3 activation and subsequent
gene induction by directly targeting TRIF.
Thus, A46R was capable of interacting with four TIR
adaptors known to have a key role in IL-1R and TLR-induced
signaling. In contrast, A46R did not interact with the fifth hu-
man intracellular TIR domain-containing protein, sterile
HEAT/Armadillo motifs–containing protein (SARM) in a
coimmunoprecipitation (Fig. 4 e) or GST-pulldown (Fig. 2 f)
assay. This is consistent with the fact that SARM does not lead
B or IRF3 activation (40), and provided an important
specificity control for A46R interactions.
A46R is expressed early during infection and contributes to
To determine when A46R was expressed during the virus
life cycle, cells were infected for different lengths of time in
the presence or absence of cytosine
(AraC) (an inhibitor of virus DNA replication and, there-
fore, of intermediate and late genes) and analyzed by immu-
noblotting. Fig. 5 a (top) shows that A46R expression was
detected in the presence of AraC, whereas a late protein
signaling and gene induction.
A46R and Flag-TRIF as indicated. After 24 h, lysates were subject to immuno-
precipitation, SDS-PAGE, and immunoblotting with the indicated antibodies.
A46R associates with TRIF and inhibits TRIF-dependent
(a) HEK 293T cells were transfected with
(b) HEK 293T cells were transfected with 8
were incubated with GST-A46R (lane 3) or GST alone (lane 2), and together
with whole cell lysates (lane 1), were analyzed by SDS-PAGE and immuno-
blotting with anti-Flag Ab. (c) HEK 293 cells were transfected with the
IRF3 reporter plasmids (as described in Materials and methods) with either
50 ng CD4-TLR4 (top) or 0.5 ng TLR3 (bottom) and 50–150 ng A46R or
pcDNA3.1 (EV) as indicated. (bottom) Cells were stimulated with 25
poly(I:C) 6 h before harvesting. Luciferase activity was measured after 24 h.
(d) HEK-TLR3 cells were transfected with the indicated amounts (ng) of
A46R 24 h before stimulation with 25
stimulation supernatants were harvested and assayed for RANTES by
ELISA. (e and f) As in a and b, except Flag-SARM was transfected instead
g Flag-TRIF. After 24 h, lysates
g/ml poly(I:C), and 24 h after
JEM VOL. 201, March 21, 2005
(D8L) was not (Fig. 5 a, bottom), showing early expression.
A46R remained in infected cells up to 24 h after infection.
To explore the role of
letion mutant lacking the
A46R). As a control, a revertant virus was also con-
structed in which the A46R gene was reinserted into the
A46R mutant (vA46R-REV). Western blot analysis con-
firmed expression of A46R in wild-type virus (vWT-A46R)
and vA46R-REV, but not in v
picted). Mice were infected intranasally with v
vA46R-REV, or vWT-A46R and were weighed and as-
sessed for signs of illness daily. Fig. 5 b shows that v
was attenuated relative to both vWT-A46R and vA46R-
REV in terms of reduced weight loss (Fig. 5 b, top graph)
and milder signs of illness (bottom graph). Assessment of the
total number of cells in lungs after infection revealed a differ-
ence in the kinetics of the host response to v
pared with vA46R-REV or vWT-A46R, in that when the
, the number of cells present on day two
was increased, whereas on days 5 and 8, it was reduced. The
difference in cell recruitment between the vWT-A46R and
A46R on day 5 was statistically significant (P
The p-value for the difference between vWT-A46R and
A46R on day 8 was 0.09, whereas the value for the dif-
ference between vA46R-REV and v
in VV virulence, a VV de-
gene was constructed
A46R (Fig. 2 d and not de-
A46R was 0.04.
A46R and A52R are not functionally redundant
Previously, we showed that the poxviral protein A52R
could also inhibit TLR signaling and contribute to virulence
(30). A52R does not resemble any host proteins, but does
have some similarity to A46R (29, 30). A52R blocked
B activation by targeting TRAF6 and
IRAK2, which act downstream of TIR adaptors (30).
Therefore, we looked in more detail at the effects of both
proteins on signaling by a single TLR, to ascertain whether
or not they were functionally redundant. We chose TLR3,
given its proposed role in the antiviral response, and also be-
cause it signals via a single TIR adaptor, TRIF, thus making
the interpretation of the results more definitive. Analysis of
B and IRF3 activation by TLR3 demonstrated a clear
difference between A46R and A52R. Fig. 6 a shows that
A52R was a potent inhibitor of poly(I:C)/TLR3-induced
B activation, whereas A46R had little effect. In con-
trast, Fig. 6 b shows that IRF3 activation induced by poly(I:C)/
TLR3 was sensitive to A46R, but not A52R. Furthermore,
A52R was not capable of inhibiting IL-1 or TLR4-induced
MAP kinase activation (not depicted), which were clearly
blocked by A46R (Fig. 1, c and d, and Fig. 3, a and b). Hence,
A46R and A52R are not functionally redundant in that both
are required to effectively shut down TLR3 signaling, whereas
only A46R is capable of inhibiting TLR-induced MAP ki-
The results demonstrate that A46R has a viral TIR do-
main with which it targets TIR adaptor molecules, resulting
in inhibition of both MyD88-dependent and TRIF-depen-
tributes to virus virulence. (a) BSC-1 cells were mock infected (M) or
infected with VV Western reserve (MOI ? 5) in the absence or presence of
40 ?g/ml cytosine ?-D-arabinofuranoside (AraC). Cells were harvested at
the indicated times (h) after infection, and lysates were subjected to SDS-
PAGE and immunoblotting, using either anti-A46R (top) or anti-D8L
(bottom) Ab. (b) A46R contributes to virus virulence. Groups of 15 female,
6-wk-old Balb/c mice were infected intranasally with 5 ? 103 PFU of vWT-
A46R, v?A46R, or vA46R-REV. Each day, animals were weighed and the
signs of illness were scored. Data are presented as the mean weight of
each group of animals compared with the mean weight of the same group
on day 0 (top graph), and the mean signs of illness score (bottom graph).
Error bars are SEM. Asterisks represent days on which there was a statisti-
cally significant difference (P ? 0.05; Student’s t test) between the vWT-
A46R and both control groups. (c) Number of cells recruited to the lungs
of VV-infected mice. On days 2, 5, and 8 after infection, mice were killed,
lungs were harvested, and cell suspensions were prepared. The total number
of viable lung cells per animal was determined by Trypan blue exclusion.
Data are means ? SEM.
A46R is expressed early during virus infection and con-
A46R TARGETS TOLL-LIKE RECEPTOR ADAPTORS AND AFFECTS VACCINIA VIRULENCE | Stack et al.
dent signaling and gene induction. Furthermore, A46R is
functionally distinct from A52R and contributes to VV viru-
lence, most likely due to its inhibitory effects on TIR-depen-
The identification of viral immune evasive strategies and the
analysis of the molecular aspects of host–pathogen interac-
tions are crucial to enhancing understanding of microbial
pathogenesis and immunity to infection. Given the emerg-
ing importance of the TLR system in the antiviral response,
understanding how viruses target this receptor family is of
particular interest. During a database search for novel TIR
domain–containing proteins, A46R from VV was identified
(29). This was potentially interesting because many poxviral
immunomodulatory proteins, such as cytokine-binding pro-
teins, bear sequence similarity to host factors (41). To date,
A46R is the only identified viral TIR domain–containing
protein. In this paper, we show that A46R is an intracellular
inhibitor of multiple TLR-dependent signaling pathways,
define host signaling molecules that it targets, and demon-
strate that the protein contributes to VV virulence in vivo.
The observation that A46R blocked all IL-1 signals
tested (NF-?B, JNK, and ERK activation) suggested that it
was acting close to the IL-1R complex. Furthermore, the
presence of a TIR domain within A46R, the knowledge
that TIR domains participate in homotypic interactions, and
the fact that all the signals blocked by A46R were MyD88
dependent (31–34), suggested that MyD88 may be seques-
tered by A46R. Coimmunoprecipitation and GST-pull-
down experiments demonstrated an interaction between
A46R and MyD88. This also suggested that A46R would
antagonize TLR signaling, given the central role of MyD88
in many of these pathways. In fact, A46R inhibited every
murine TLR pathway to NF-?B activation known to in-
volve MyD88 in a mouse macrophage cell line, together
with TLR4-mediated NF-?B, p38, and ERK activation,
and IL-8 induction in human 293 cells.
Inhibition of TLR4-dependent NF-?B signaling in hu-
man cells was particularly potent, which led us to test the ef-
fect of A46R on other TIR domain–containing proteins
with a role in this pathway. Altogether, five such proteins
are known to be involved in TLR4-mediated NF-?B acti-
vation, namely the receptor itself, MyD88, Mal, TRAM,
and TRIF (19–23, 28). A46R was found to be capable of as-
sociating with all five of these proteins, and hence it proba-
bly prevents the formation of the TLR4 receptor complex,
thus accounting for its potent inhibition of NF-?B activa-
tion. Although these results were based on overexpression,
several lines of evidence suggest that the interactions de-
tected are specific, direct, and likely to occur in vivo. First,
normally VV-expressed A46R interacted with MyD88 and
Mal. Second, the interaction of A46R with MyD88, Mal,
and TRIF were confirmed in yeast two-hybrid. Third, rMal
interacted directly with GST-A46R in vitro. Finally, A46R
displayed specificity for certain TIR domain–containing
proteins and did not interact with TLR3 or SARM.
A46R is the first viral protein identified that can target
host TIR domain–containing proteins. Given its ability to
interact with different and diverse TIR domains (TRIF and
TRAM are quite distinct in sequence from MyD88 and Mal;
Fig. 1 a and reference 20), elucidation of the crystal structure
of A46R should provide important information of general
relevance as to how TIR domains interact. Because Box 2 of
the TIR domain is particularly important in signaling (42),
the extra amino acids surrounding the A46R Box 2 (Fig. 1 a)
may represent inhibitory loops that account for the ability of
A46R to prevent TIR-dependent signaling. This hypothesis
is being explored using mutagenesis studies.
The fact that A46R associated with all four TLR4 adap-
tors may suggest that TLR4 is a particularly important target
for VV immune evasion. Indeed, TLR4 has been proposed
to have a role in responding to fusion (F) protein of respira-
tory syncytial virus (43), although the functional significance
of this remains to be clarified (44). TLR4 is also activated by
envelope proteins from both mouse mammary tumor virus
and Moloney murine leukemia virus, which could also be
coimmunoprecipitated with TLR4 (45). VV might interact
with other or multiple TLRs that also use these adaptors.
Possible VV PAMs detected by TLRs could be proteins on
the surface of the intracellular mature virus or extracellular
enveloped virus particles (potentially detected by TLR2 or
TLR4; references 8, 9), intracellular dsRNA produced from
the bidirection transcription of the VV genome (potentially
detected by TLR3; reference 11), or the dsDNA genome it-
pathways. (a and b) HEK 293 cells were transfected with 0.5 ng TLR3 and
the indicated amounts (ng) of either A52R or A46R, together with the
NF-?B (a) or IRF3 (b) reporter plasmids as described in Materials and
methods. Cells were stimulated with 25 ?g/ml poly(I:C) 6 h before har-
vesting where indicated. Luciferase activity was measured after 24 h.
Relative stimulation is shown on the y axis.
A46R and A52R target different TLR3-mediated signaling
JEM VOL. 201, March 21, 2005
self (potentially detected by TLR9, which responds to the
dsDNA genome of herpes simplex virus; reference 15, 16).
The role of TLRs in responding to VV PAMs is currently
Although Mal and TRAM are yet to be directly impli-
cated in responding to viruses, MyD88 (for TLR7; refer-
ences 8, 9) and TRIF (for TLR3) have been shown to have
a role, particularly in relation to type I IFN production (15,
16, 21, 22). TRIF is essential for both TLR4- and TLR3-
mediated IRF3 activation and IFN-? production (21, 22)
and probably has a key role in the initiation of the IFN-
based antiviral response, leading to the inhibition of viral
replication and spread. Although the relevance of TLR3 in
responding to viruses in vivo has recently been questioned
(46, 47), there is evidence that TRIF is important in con-
trolling VV replication. Hoebe et al. (22) showed that mac-
rophages from mice in which trif was disrupted supported
the replication of VV to a higher titre than did macrophages
from normal mice. Thus, the ability of A46R to inhibit
TRIF-mediated IRF3 activation may be of primary impor-
tance to VV infection.
Consistent with its proposed role in antagonizing early
innate TLR responses, A46R was expressed early during in-
fection. Furthermore, deletion of A46R from VV caused at-
tenuation in a murine intranasal model, and also led to en-
hanced levels of cells on day 2 in lungs of infected animals.
The use of a revertant virus in which the A46R gene was re-
inserted into the deletion virus confirmed that the attenua-
tion observed in the deletion virus, and the difference in
lung cell numbers, were due solely to the absence of A46R.
The ability of A46R to target intracellular TIR-dependent
signaling most likely accounts for its role in virulence, prob-
ably by blocking the induction of immune response genes
downstream of TLRs, as has been shown here for the chemo-
kines IL-8 and RANTES. Possibly, inhibition of chemokine
induction by A46R might account for the early enhanced
levels of cells in the absence of A46R expression.
Previously, we identified another VV TLR antagonist,
A52R, which could inhibit TLR-dependent NF-?B activa-
tion. There are several lines of evidence that A46R and
A52R are not functionally redundant. First, they target dis-
tinct TLR signaling molecules (30). Second, although they
do have some overlapping effects (such as the inhibition of
MyD88-dependent NF-?B activation), their overall effects
on TLR signaling are quite distinct. Although A52R is a
good NF-?B inhibitor, it has no inhibitory effect on MAP
kinase activation (not depicted), nor on TLR3-mediated
IRF3 activation (Fig. 6 b). In contrast, A46R blocks both
MAP kinase activation and, importantly, TLR3- and TRIF-
mediated IRF3 activation. Furthermore, A46R has little ef-
fect on TLR3-mediated NF-?B activation, which A52R
blocks potently (Fig. 6 a). This was surprising because A46R
interacts with TRIF. But, presumably, the interaction of
A46R with TRIF has a greater effect on downstream IRF3
activation compared with NF-?B because these two path-
ways bifurcating from TRIF have been shown to be quite
distinct (48, 49). Finally and crucially, the deletion of either
A46R or A52R from VV causes attenuation (Fig. 5 b and
reference 30) and, thus, both contribute to virulence and are
N1L is another intracellular VV protein that contributes
to virulence (50), which has also been shown recently to
function by antagonizing TLR signaling, but at the level of
I?B kinases and related kinases involved in IRF3 activation
(51). Thus, the importance of blocking TLR signaling is
demonstrated by the retention by VV of at least three dis-
tinct mechanisms of disrupting these pathways. Furthermore,
the attenuated phenotypes seen in the absence of A46R,
A52R, or N1L provide evidence for a role for TLRs in con-
taining VV infections. The action of these intracellular TLR
inhibitory proteins would be expected to be restricted to in-
fected cells, whereas immunomodulatory proteins secreted
from VV-infected cells, such as cytokine, chemokine, and
IFN-binding proteins, can act on ligands produced from
both infected and uninfected cells.
Finally, A46R is also found in variola virus, the causative
agent of smallpox. Concern about the threat of the use of
variola as a bioweapon has led to a renewed desire to under-
stand this human pathogen. However, little is known about
the role of human TLRs in sensing variola virus. Given that
VV A46R targets human adaptors, the knowledge that
A52R is truncated in variola virus, together with the fact
that the VV and variola virus A46R amino acid sequences
differ by only eight residues (Fig. 2 a), it is likely that variola
virus A46R would have an important role in interactions
with the human TLR system.
MATERIALS AND METHODS
Expression plasmids. Sources of expression plasmids were as follows:
AU1-MyD88 and Flag-TLR4 (M. Muzio, Mario Negri Institute, Milan, It-
aly; references 52, 53), flag-TRAF2 (Tularik Inc.), chimeric receptor CD4-
TLR4 (R. Medzhitov, Yale University, New Haven, CT), TLR3 (D. Go-
lenbock, University of Massachusetts Medical School, Worcester, MA),
Flag-TRIF (S. Akira, Osaka University, Osaka, Japan), and Myc-MyD88
(L. O’Neill, Trinity College, Dublin, Ireland). Construction of A46R,
Flag-A46R, HA-Mal, Flag-TRAM, and Flag-SARM were described previ-
ously (23, 27, 29, 40). The glutathione S-transferase (GST) fusion of A46R
was synthesized by inserting full-length A46R in the bacterial expression
Antibodies and reagents. Anti-A46R polyclonal Ab was raised against a
purified, bacterial-expressed A46R–GST fusion protein. Other antibodies
used were anti-Flag M2 mAb, anti-Flag M2–conjugated agarose, anti-myc
mAb clone 9E10 (all obtained from Sigma-Aldrich), anti-AU1 mAb
(BabCO), anti-HA polyclonal Ab (Y-11), anti-TRAF2 Ab (both obtained
from Santa Cruz Biotechnology, Inc.), and anti-D8L mAb (54).
Human rIL-1? was a gift from the National Cancer Institute and human
rTNF-? was a gift from S. Foster (Zeneca Pharmaceuticals, Macclesfield,
England). TLR agonists used were poly(I:C) (Amersham Biosciences), LPS
(Sigma-Aldrich), R-848 (a gift from D. Golenbock, University of Massachu-
setts Medical School, Worcester, MA), flagellin (a gift from A. Gewirtz, Em-
ory University, Atlanta, GA), phosphothioate CpG DNA (Sigma-Aldrich),
synthetic tripalmitoyl lipopeptide Pam3Cys-Ser-(Lys)4 (Pam3Cys; Invivo-
gen), and macrophage-activating lipopeptide 2 kD (MALP-2; Qbiogene).
A46R TARGETS TOLL-LIKE RECEPTOR ADAPTORS AND AFFECTS VACCINIA VIRULENCE | Stack et al.
Reporter gene assays. HEK 293 cells (2 ? 104 cells per well) or
RAW264.7 cells (4 ? 104 cells per well) were seeded into 96-well plates and
transfected 24 h later with expression vectors and luciferase reporter genes us-
ing GeneJuice (Novagen). In all cases, 20 ng/well of phRL-TK reporter gene
(Promega) was cotransfected to normalize data for transfection efficiency. The
total amount of DNA per transfection was kept constant at 220 ng (HEK293)
or 200 ng (RAW264.7) by addition of pcDNA3.1 (Stratagene). After 24 h,
reporter gene activity was measured (30). Data are expressed as the mean fold
induction ? SD relative to control levels, for a representative experiment
from a minimum of three separate experiments, each performed in triplicate.
For NF-?B assays, 60 ng of a ?B-luciferase reporter gene was used (30).
For MAP kinase reporter assays, the Pathdetect System (Stratagene) was used,
whereby 0.25 ng c-jun–, 2 ng Elk1–, or 0.25 ng CHOP–Gal4 fusion vectors
were used in combination with 60 ng pFR-luciferase reporter to measure
JNK, ERK1/2, and p38 activation, respectively. For the p65 transactivation
assay, 1 ng of a p65 Gal4 fusion vector was used in combination with 60 ng
pFR-luciferase reporter (31). For the IRF3 assay, an IRF3-Gal4 fusion vector
(3 ng) was used in combination with 60 ng pFR luciferase reporter (27).
Immunoprecipitation and immunoblotting. HEK293 cells were
seeded into 10-cm dishes (1.5 ? 106 cells) 24 h before transfection with
GeneJuice. For coimmunoprecipitations, 4 ?g of each construct was trans-
fected. Cells were harvested after 24 h in 850 ?l of lysis buffer (50 mM
Hepes, pH 7.5, 100 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40
containing 0.01% aprotinin, 1 mM sodium orthovanadate, and 1 mM
PMSF). For assessment of the A46R–TLR4 interaction, the cells were har-
vested after 48 h and lysed for 1 h in a different lysis buffer (50 mM Tris/
HCl, pH 7.5, 1 mM EDTA, 10% glycerol, 0.05% CHAPS. 0.5% Triton
X-100, 250 mM NaCl containing 0.01% aprotinin, 1 mM sodium ortho-
vanadate, and 1 mM PMSF). For assessment of interactions involving VV-
expressed A46R, cells were infected (multiplicity of infection [MOI] ? 1)
with VV Western Reserve 24 h after transfection for 90 min at 37?C. The
virus inoculum was aspirated and cell monolayers were overlaid with 2.5%
FBS DMEM and harvested 24 h after infection in lysis buffer. For all immu-
noprecipitations, the appropriate antibodies were precoupled to either pro-
tein A or protein G–Sepharose for 1 h at 4?C before incubation with the
cell lysates overnight at 4?C. The immune complexes were precipitated,
washed, and analyzed by SDS-PAGE and immunoblotting (30).
For analysis of the kinetics of A46R expression, confluent monolayers
of BSC-1 cells were infected (MOI ? 5) for 90 min at 37?C. After removal
of inoculum, cell monolayers were overlaid with 2.5% FBS DMEM in the
presence or absence of 40 ?g/ml cytosine ?-D-arabinofuranoside (Sigma-
Aldrich). Cell lysates were analyzed by immunoblotting using anti-A46R or
GST pulldown assays. Plasmid GEX.4T2-A46R or GEX.4T2 was trans-
formed into Escherichia coli BL21 (DE3) and grown in Terrific Broth. Pro-
tein expression was induced with 0.7 mM IPTG. Cells were lysed in
NETN and proteins were purified from the insoluble fraction by glu-
tathione sepharose 4B affinity chromatography (Amersham Biosciences).
For GST pulldown experiments, HEK 293T cells were transfected and
harvested as described for coimmunoprecipitation. 800 ?l of cell lysate was
added to purified GST-fusion protein coupled to glutathione-sepharose and
incubated for 2 h at 4?C. The immune complexes were precipitated, sub-
jected to SDS-PAGE, and were analyzed by immunoblotting.
Determination of cytokine concentrations. HEK293 clonal cell lines
expressing either TLR3 (HEK-TLR3) or TLR4 and MD-2 (HEK-TLR4;
reference 27) and were used for determination of cytokine production.
Cells (2 ? 104 cells per well) transfected with the A46R expression plasmid
for 24 h were stimulated with 1 ?g/ml LPS or 25 ?g/ml poly(I:C) 24 h
later. Supernatants were harvested 24 h later and IL-8 and RANTES con-
centrations were determined by ELISA (R&D Systems). Experiments were
performed four times in triplicate and data are expressed as the mean ? SD
from one representative experiment.
Recombinant VV viruses. A VV mutant (strain WR) lacking 93.5% of
the A46R gene (v?A46R) was constructed by transient dominant selection
(55). A plaque-purified wild-type virus (vWT-A46R) and a revertant virus
(vA46R-REV) in which the A46R gene was reinserted at its natural locus
were also isolated. The virulence of the viruses was investigated in a mouse in-
tranasal model. Female, 6-wk-old BALB/c mice were anesthetized and inocu-
lated with 5 ? 103 plaque-forming units of VV in 20 ?l of phosphate-buffered
saline. A control group was mock-infected with phosphate-buffered saline.
Each day, the weights of the animals and signs of illness were measured as de-
scribed previously (56). On days 2, 5, and 8, single cell suspensions of lung
cells were prepared by sieving lungs through a 100-?m nylon mesh followed
by hypotonic lysis of erythrocytes. Cell viability was assessed using trypan blue
exclusion. Statistical significance was assessed using Student’s t test. The animal
experiments were conducted under the appropriate licence and regulations
stipulated by the Animals (Scientific Procedures) Act 1986, UK government.
Alignment of TIR domains. TIR domains from human proteins with
assigned functions were aligned with VV and variola virus A46R using
Clustal W. The alignment was viewed and adjusted using GeneDoc (57).
This work was supported by the Irish Higher Education Authority, Enterprise Ireland,
The Irish Health Research Board, and Science Foundation Ireland. G.L. Smith is a
Wellcome Trust Principal Research Fellow. I.R. Haga was a CNPq (Brazil) scholar. K.A.
Fitzgerald is supported by a fellowship from the Wellcome Trust, London.
The authors have no conflicting financial interests.
Submitted: 16 July 2004
Accepted: 14 January 2005
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