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TLR2 Activation in Permissive Cells
Glycoproteins B and H Are Necessary for
Human Cytomegalovirus Envelope
Karl W. Boehme, Mario Guerrero and Teresa Compton
http://www.jimmunol.org/content/177/10/7094
2006; 177:7094-7102; ;J Immunol
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Print ISSN: 0022-1767 Online ISSN: 1550-6606.
Immunologists All rights reserved.
Copyright © 2006 by The American Association of
9650 Rockville Pike, Bethesda, MD 20814-3994.
The American Association of Immunologists, Inc.,
is published twice each month byThe Journal of Immunology
by guest on June 13, 2013http://www.jimmunol.org/Downloaded from
Human Cytomegalovirus Envelope Glycoproteins B and H
Are Necessary for TLR2 Activation in Permissive Cells
1
Karl W. Boehme,* Mario Guerrero,*
†
and Teresa Compton
2
*
†
Human CMV (HCMV) is a ubiquitous member of the Herpesviridae family and an opportunistic pathogen that poses significant
health risks for immunocompromised patients. HCMV pathogenesis is intimately tied to the immune status of the host, thus
characterization of the innate immune response to HCMV infection is critical for understanding disease progression. Previously,
we identified TLR2 as a host factor that detects and initiates inflammatory cytokine secretion in response to HCMV independent
of viral replication. In this study, we show that two entry-mediating envelope gp, gp B (gB) and gp H (gH), display determinants
recognized by TLR2. Neutralizing Abs against TLR2, gB and gH inhibit inflammatory cytokine responses to HCMV infection,
suggesting that inflammatory cytokine stimulation by HCMV is mediated by interactions between these envelope gp and TLR2.
Furthermore, both gB and gH coimmunoprecipitate with TLR2 and TLR1, indicating that these envelope gp directly interact with
TLR2 and that a TLR2/TLR1 heterodimer is a functional sensor for HCMV. Because our previous studies were conducted in
model cell lines, we also show that TLR2 is expressed by HCMV permissive human fibroblast cell strains, and that TLR2 is a
functional sensor in these cells. This study further elucidates the importance and potency of envelope gp as a class of molecules
displaying pathogen-associated molecular patterns that are recognized with immediate kinetics by TLRs in permissive cells. The
Journal of Immunology, 2006, 177: 7094 –7102.
H
uman CMV (HCMV)
3
is a ubiquitous member of the
Herpesviridae family that causes significant morbidity
and mortality in immune compromised patients (1).
Similar to other herpesviruses, HCMV establishes a lifelong rela-
tionship with its host as a latent infection, and disease can result
from either primary infection or reactivation from latency (2, 3).
HCMV has an extremely broad tissue tropism that allows it to
infect nearly every organ system in the body (4, 5). Consequently,
HCMV disease presents itself in a variety of clinical sequelae (1).
It is a major cause of postoperative disease in chemically immu-
nosuppressed transplant recipients and greatly increases the risk of
graft rejection (6 – 8). HCMV is also a leading cause of congenital
birth defects, and infection during the first trimester of pregnancy
often results in neurological and cognitive disorders in the devel-
oping child (9, 10). Furthermore, HCMV has been implicated as a
factor in coronary artery disease (11–14). There is currently no
vaccine for HCMV, and existing therapeutics exhibit toxicity pre-
cluding long-term use (15, 16). Thus, an understanding of the viral
and cellular determinants of the immune response to HCMV is
critical for the development of new vaccines and therapies.
We recently identified TLR2 as a host factor that activates in-
flammatory cytokine secretion in response to HCMV (17). The
TLRs are a family of pathogen-recognition receptors that initiate
innate immune responses to a myriad of invading microbes, in-
cluding viruses (18, 19). Eleven mammalian TLRs have been iden-
tified, and they are predominantly expressed on phagocytic cells
such as dendritic cells and macrophages; however, most cells ex-
press at least a subset of TLRs (19). The primary consequences of
TLR activation include NF-
B activation, inflammatory cytokine
secretion, dendritic cell maturation, up-regulation of immune co-
stimulatory molecules, and for a subset of TLRs, the production of
type I IFN (19 –22). TLRs detect microorganisms on the basis of
unique molecular structures termed pathogen-associated molecular
patterns (PAMPs). Analysis of the innate response to bacterial
PAMPs such as LPS, peptidoglycan, and unmethylated CpG DNA
are a cornerstone of TLR research, and great strides have been
made in our understanding of the relationship between bacteria and
the innate immune system (23–28). In contrast, the mechanisms by
which the TLR system recognizes and responds to viruses have
only begun to be explored. Viral genomic nucleic acids are one
major class of PAMP. TLR3 (dsRNA), TLR7 (ssRNA), TLR8
(ssRNA), and TLR9 (CpG DNA) (29 –33) signal from the endo-
some (34–38) where degradation of virus particles exposes the
viral genome for detection by this panel of TLRs (29, 31, 32).
Although significantly less well studied, envelope gps that deco-
rate the exterior of the virion are an emerging class of TLR activators
(18). To date, three envelope gp have been identified as TLR agonists.
The fusion protein from respiratory syncytial virus and the mouse
mammary tumor virus envelope protein activate TLR4, while the
hemagglutinin protein from measles virus activates TLR2 (39 – 42).
Interestingly, a shared feature of these gp is that they play critical roles
in the entry of their respective viruses, and this shared feature suggests
that the molecular machinery used by viruses for entry is also targeted
by the innate immune system (43, 44).
Although we demonstrated previously that TLR2 is activated by
HCMV, the molecular trigger for TLR2 has not been determined
*McArdle Laboratory for Cancer Research, University of Wisconsin Medical School,
Madison, WI 53706; and
†
Department of Biomolecular Chemistry, University of
Wisconsin, Madison, WI 53706
Received for publication April 17, 2006. Accepted for publication August 29, 2006.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by National Institutes of Health Grants RO1AI34998
and R21A154915 (to T.C.) and National Institutes of Health Training Grant
T32GM07215 (to K.W.B. and M.G.).
2
Address correspondence and reprint requests to Dr. Teresa Compton, 100 Tech
-
nology Square, Novartis Institute for Biomedical Research, Cambridge, MA 02139.
E-mail address: teresa.compton@novartis.com
3
Abbreviations used in this paper: HCMV, human CMV; PAMP, pathogen-associ
-
ated molecular pattern; gB, gp B; gH, gp H; gL, gp L; gO, gp O; NHDF, normal
human dermal fibroblast; HEK, human embryonic kidney; MOI, multiplicity of in-
fection; eGFP, enhanced GFP; VSV-G, vesicular stomatitis virus G; HSV-1, herpes
simplex virus type 1; CHO, Chinese hamster ovary.
The Journal of Immunology
Copyright © 2006 by The American Association of Immunologists, Inc. 0022-1767/06/$02.00
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(17). In contrast with the RNA viruses listed above, HCMV dis-
plays as many as 12 envelope gp, four of which are required for
entry. gp B (gB) works in concert with a tripartite complex com-
prised of gp H (gH), gp L (gL), and gp O (gO) to mediate the
binding and entry of HCMV virons into host cells (45–48). In
addition to their roles in entry, there is a growing body of evidence
that gB and gH elicit responses from cells that are reminiscent of
TLR activation. Abs against gB and gH block the induction of
various innate markers, including NF-
B (49, 50), and cells ex-
posed to soluble forms of gB activate NF-
B and the type I IFN
(49 –53). Additionally, an anti-Id bearing the image of gH activates
NF-
B (50). Based on these observations, we hypothesized that gB
and gH are the target of innate sensing by the host cell. In this
study, we show that HCMV gB and gH activate TLR2 and asso-
ciate with TLR1 and TLR2. Abs against gB and gH, but not gL,
inhibit the inflammatory cytokine response to HCMV, and both gB
and gH coimmunoprecipitate with TLR2 and TLR1, indicating that
the functional sensor for HCMV is a TLR2/TLR1 heterodimer. We
also extend our initial studies to HCMV permissive human fibro-
blast cells and show that TLR2 mediates NF-
B activation and
inflammatory cytokine responses in cells that support productive
HCMV infection.
Materials and Methods
Cell lines, reagents, and virus
Human embryonic kidney (HEK) 293T cells (American Type Culture Col-
lection) and normal human dermal fibroblast (NHDF) (Cambrex) cells
were grown in 5% CO
2
in DMEM (Invitrogen Life Technologies) supple
-
mented with 10% FBS (HyClone) and 1% penicillin-streptomycin-ampho-
tericin B-fungizone (PSF; BioWhittaker). Monomac-6 cells were main-
tained in Ham’s F12 medium supplemented with 10% FBS and 1% PSF in
a5%CO
2
environment. LPS (from Escherichia coli 0111:B4) was ob
-
tained from Sigma-Aldrich and repurified by phenol extraction as de-
scribed previously (54). Recombinant human IL-1

was obtained from
R&D Systems, Pam
3
CSK
4
was obtained from EMC Microcollections, and
soluble CD14 (sCD14) was from Biometec. The AD169 strain of HCMV
was propagated in NHDF cells. Virion particles were purified from infected
supernatants by density-gradient centrifugation (55–57), and titers were
determined as described previously on NHDF cell monolayers (58).
RT-PCR
Total RNA was harvested from NHDF, HEK/CD14, or Monomac 6 cells
using RNA-STAT 60 (Tel-Test B) according to the manufacturer’s instruc-
tions. RNA was quantitated, and 1
g of RNA was used for RT-PCR
analysis with TLR2 and GAPDH-specific primers. RNA was quantitated,
and 1
g of RNA was used for RT-PCR analysis using the rTth DNA
polymerase (Applied Biosystems) with TLR2 (5⬘-GCC AAA GTC TTG
ATT GAT TGG-3⬘ and 5⬘-TTG AAG TTC TCC AGC TCC TG-3⬘) and
GAPDH (5⬘-GAG CCA AAA GGG TCA TC-3⬘ and 5⬘-GTG GTC ATG
AGT CCT TC-3⬘)-specific primers.
Cloning and purification of recombinant gBs-GFP
gBs-GFP was constructed by fusing the ectodomain of gB (strain AD169)
ending at aa 750 to the enhanced GFP (eGFP). The ectodomain of gB was
amplified with the upstream primer (5⬘-CTC GAG CTC GAG ATG GAA
TCC AGG ATC-3⬘) incorporating a XhoI site and the downstream primer
(5⬘-TCT AGA TCT AGA GGG GTT TTT GAG GAA-3⬘) incorporating an
XbaI site. The eGFP sequence was amplified using the upstream primer
(5⬘-TCT AGA TCT AGA ATG GTG AGC AAG-3⬘) incorporating an XbaI
site and the downstream primer (5⬘-CGC GGC CGC GGC TCA CTT GTA
CAG CTC-3⬘) incorporating a NotI site. The gB fragment was inserted into
the pCI-Neo vector (Promega) using XhoI/XbaI. The eGFP fragment was
subsequently inserted using XbaI/NotI.A6⫻ histidine tag was added to the
3⬘ end of eGFP upstream of the stop codon using the downstream primer
(5⬘-TCA GTG GTG GTG GTG GTG GTG CTT GTA CAG CTC-3⬘).
Oligonucleotide primers were synthesized at the University of Wisconsin
Biotechnology Center. The construct was transfected into Chinese hamster
ovary (CHO) pgsD 677 cells for the generation of a stable gBs-GFP-ex-
pressing cell line. At 48 h posttransfection, GFP-positive cells were col-
lected by FACS (University of Wisconsin Flow Cytometry Facility) and
subjected to geneticin (Invitrogen Life Technologies) selection at a con-
centration of 1 mg/ml. The cells were subsequently adapted to suspension
in chemically defined CHO medium (Invitrogen Life Technologies) sup-
plemented with 1% PSF and geneticin at a concentration of 1 mg/ml. For
protein isolation, cells were pelleted and lysed by sonication in lysis buffer
(50 mM NaPO
4
, 300 mM NaCl, 0.5% Tween 20, 10 mM imidazole (pH
8.0)). Cell debris was removed by centrifugation at 27,000 ⫻ g for 30 min.
The supernatants were incubated for2hat4°Cunder rotation with Ni-
NTA-agarose beads (Qiagen). The beads were transferred to a chromatog-
raphy column and washed with 10-column volumes of lysis buffer, fol-
lowed by 10-column volumes of wash buffer (50 mM NaPO
4
, 300 mM
NaCl, 20 mM imidazole (pH 8.0)). gBs-GFP was eluted in 4 ml of elution
buffer (50 mM NaPO
4
, 300 mM NaCl, 300 mM imidazole (pH 8.0)) and
dialyzed overnight in PBS (50 mM NaPO
4
, 150 mM NaCl (pH 8.0)) at 4°C.
Dialyzed protein was separated from low m.w. contaminants by size-ex-
clusion chromatography. Samples were loaded onto a 50-ml column con-
taining Sephacryl S-200 substrate (Amersham Biosciences) in 1⫻ PBS
(Invitrogen Life Technologies) and run through by gravity flow at 4°C.
Collected fractions were stored at ⫺80°C.
Construction and generation of TLR2⌬C and TLR4⌬C-encoding
retroviruses
The mutants were constructed using full-length FLAG epitope-tagged
TLR2 and TLR4 provided by B. Williams (Cleveland Clinic Foundation,
Cleveland, OH). The TLR2 and TLR4 cytoplasmic tails were deleted by
PCR mutagenesis using a common upstream primer (5⬘-TAA TAT ACC
GGT GCC ACC ATG TCT GCA CTT CTG ATC C-3⬘) incorporating an
AgeI restriction site and TLR2-specific (5⬘-TTA AAT GCG GCC GCT
TAT GTA TTT CAT ATA CCA CAG GCC-3⬘) and TLR4-specific (5⬘-
TTA AAT GCG GCC GCT TAT GTA GCA GCC AGC AAG AAG C-3⬘)
downstream primers incorporating NotI restriction sites. The fragments
were digested and cloned into the retroviral transfer vector pCMMP.MCS.
IRES-GFP (a gift from B. Sugden, University of Wisconsin, Madison, WI).
The constructs were confirmed by sequencing (University of Wisconsin
Biotechnology Center), and recombinant retroviruses were generated as
described previously (59). NHDF cells were transduced with retroviruses
encoding TLR2⌬C, TLR4⌬C, or an empty vector control in a minimal
volume for1hinthepresence of 5
g/ml polybrene. At 96 h posttrans-
duction, GFP-positive cells were collected by FACS and used as indicated.
Cytokine ELISAs
Ninety-six-well plates were seeded with cells at a density of 5000 cells per
well. At 24 h postplating, the growth medium was removed and replaced
with serum-free DMEM. After 24 h serum starvation, the cells were chal-
lenged as indicated. At 18 h postchallenge, the supernatants were harvested
and IL-6 or IL-8 levels were determined by ELISA. OptEIA IL-6 or IL-8
dual Ab detection assay (BD Pharmingen) was used according to the man-
ufacturer’s instructions. For blocking Ab studies, virions were preincu-
bated for 15 min with isotype control (eBioscience), anti-gB 27-78 or 9-3
(60), or anti-gH 14-4b (61) mouse mAbs at 100
g/ml. For gL, a rabbit
polyclonal anti-gL 6394 (62) or rabbit IgG (Sigma-Aldrich) were used at
100
g/ml. For TLR2 blocking, Ab studies the cells were preincubated for
30 min with isotype control or anti-TLR2 Abs (eBioscience) at 1
g/ml.
Coimmunoprecipitations and immunoblotting
The gB, gH, and gL coding sequences from HCMV strain AD169 were
cloned previously into the pCAGGS expression vector (63). The vector
pCVSVG encoding vesicular stomatitis virus G (VSV-G) was a gift from
Y. Kawaoka (University of Wisconsin, Madison, WI). pFLAG-TLR1,
pFLAG-TLR2, and pFLAG-TLR6 plasmids were donated by B. Williams
(Cleveland Clinic Foundation). For coimmunoprecipitation experiments,
293T cells were cotransfected with plasmids encoding gB, gH, gL, VSV-G
pFLAG-TLR1, pFLAG-TLR2, or pFLAG-TLR6 as indicated. Transfec-
tions were performed using Lipofectamine 2000 (Invitrogen Life Technol-
ogies) according to the manufacturer’s directions. Dose-response precipi-
tations were performed in 6-well plates, with total DNA concentrations
ranging from 0 to 2
g/ml. For radiolabeled immunoprecipitation assays,
cells were incubated for 24 h in DMEM supplemented with 150
Ci/ml
35
S-express label (NEN-DuPont). Cells were harvested at 48 h posttrans
-
fection in lysis buffer (TBS plus 2% TX-100 (pH 8.8)). Lysates were clar-
ified twice by high-speed microcentrifugation (13,000 rpm, 5 min, 4°C),
diluted 2-fold in lysis buffer, and incubated with 30
l of anti-FLAG-
conjugated bead slurry (M2; Sigma-Aldrich) for 12 h with continuous
rocking at 4°C. The beads were pelleted under low-speed microcentrifu-
gation (2500 rpm, 2 min, 4°C) and washed five times in lysis buffer. Prod-
ucts were eluted by competition with 3⫻ FLAG peptide (Sigma-Aldrich)
and analyzed by 10% SDS-PAGE gel followed by immunoblotting using
7095The Journal of Immunology
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the following Abs: anti-gB 27-78 (60), anti-gH 6824 (62), anti-gL 6394
(62), anti-VSV-G 15F9 (Sigma-Aldrich), or anti-FLAG M2 (Sigma-
Aldrich). For the radiolabeled immunoprecipitation assay, the cells were
transfected with gB and FLAG-TLR2 expression constructs individually or
in combination. At 24 h posttransfection, the medium was replaced with
DMEM supplemented 10% FBS, 1% PSF, and 150
Ci/ml
35
S-express
label (NEN-DuPont). The cells were harvested at 48 h posttransfection and
processed as described above. The immunoprecipitation products were re-
solved by 10% SDS-PAGE, the gel was dried to Whatman paper, and
exposed to film. Images were collected using a Typhoon phosphor imager.
I
B
␣
degradation assay
Cells were serum starved for 24 h before infection and pretreated with
cycloheximide (100
g/ml) for 1 h before infection. The cells were treated
with IL-1

(100 pg/ml), Pam
3
CSK
4
(20
g/ml), LPS (1
g/ml) plus 4 (1
g/ml), or infected with UV-HCMV (multiplicity of infection (MOI) ⫽
10) as measured before UV treatment. At 3 h posttreatment,the cells were
harvested by scraping in Nonidet P-40 lysis buffer (50 mM Tris (pH 7.4),
150 mM NaCl, 30 mM NaF, 5 mM EDTA, 10% glycerol, 40 mM 2-glyc-
erophosphate; Sigma-Aldrich), 1 mM Na
3
VO
4
(Sigma-Aldrich), 0.1 mm of
PMSF, protease inhibitor mixture (56), and 1% Nonidet P-40). The cells
were subjected to two freeze-thaw cycles, and insoluble material was re-
moved by microcentrifugation (13,000 rpm, 5 min, 4°C). The total protein
content of each sample was quantitated using the Bio-Rad protein assay
reagent. Equivalent amounts of total protein for each sample were sepa-
rated by 10% SDS-PAGE. I
B
␣
and actin levels were analyzed by immu-
noblotting as described previously (56) using anti-I
B
␣
(sc-371; Santa
Cruz Biotechnologies) and anti-actin Abs. Densitometry was performed
using the ImageQuant software system (Amersham Biosciences).
Statistical analysis
The means of triplicate samples were compared using an unpaired Stu-
dent’s t test with GraphPad Prism software (version 4.00; GraphPad).
Results
gB and gH elicit inflammatory cytokine responses from cells
To assess the ability of envelope gp to elicit inflammatory cytokine
responses we used a panel of neutralizing Abs to block interactions
between gB and gH and receptors on the surface of the cell (Fig.
1A). Transcriptionally inert UV-inactivated HCMV virions (UV-
HCMV) were incubated with Abs for 15 min before infection, and
IL-6 levels were measured by ELISA at 18 h postinfection as a
marker of inflammatory cytokine activation. IL-6 levels were di-
minished by pretreatment of virions with gB (27-78 and 9-3) and
gH-specific (14-4b) Abs, whereas the isotype control Ab had a
modest effect on the IL-6 response. Furthermore, a rabbit poly-
clonal Ab against gL did not affect the IL-6 response (data not
shown). These data suggest that gB and gH, but not gL, are inter-
acting with cell surface receptors that elicit inflammatory cytokine
secretion.
Additionally, the capacity of a soluble form of gB to activate
TLR2 was assessed. The ectodomain of gB (HCMV strain AD169)
was fused to the eGFP (gB-GFP), the resulting protein purified and
used to challenge HEK cells expressing CD14 alone (HEK/CD14)
or in combination with TLR2 (HEK/CD14/TLR2) or TLR4 (HEK/
CD14/TLR4) and IL-8 levels were measured by ELISA (Fig. 1B).
To date, we have been unable to generate a soluble form of the
gH/gL complex. The TLR2- and TLR4-specific controls, Pam
3
CSK
4
and LPS, elicited IL-8 responses from HEK/CD14/TLR2 and HEK/
CD14/TLR4, respectively; and, consistent with our previous study,
HCMV virions only activated cells expressing TLR2 (17). Simi-
larly, gB-GFP induced IL-8 secretion in a TLR2-dependent man-
ner. A soluble eGFP control did not elicit cytokine responses.
These data further support the hypothesis that HCMV envelope gp
serve as agonists for TLR2.
gB and gH physically associate with TLR2 and TLR1
TLR ectodomains are composed of varying numbers of leucine-
rich repeats, a motif that is commonly involved in protein–protein
interactions (64). Based on the preceding data, we hypothesized
that gB and gH directly interact with TLR2. To test this hypoth-
esis, we performed coimmunoprecipitation experiments from 293T
cells cotransfected with gB and FLAG-TLR2 expression con-
structs. FLAG-TLR2 was immunoprecipitated with anti-FLAG
Ab-conjugated agarose beads, the proteins resolved by SDS-
PAGE, and products detected by immunoblotting for FLAG-TLR2
or gB (Fig. 2A). A dose-dependent pulldown of gB was observed
from cells cotransfected with a constant amount of gB-expression
plasmid and increasing levels of FLAG-TLR2-expression plasmid.
The amount of gB precipitated increased in proportion to the level
of FLAG-TLR2 input. Immunoprecipitations from
35
S-labeled
cells confirmed that coexpression of both gB and FLAG-TLR2 is
required for this interaction (Fig. 2B). gH also coprecipitated with
TLR2 indicating that both gB and gH physically interact with
TLR2 (Fig. 2E). The envelope gp from VSV-G was included as a
specificity control and did not coprecipitate with any of the TLRs
tested (Fig. 2G).
In vivo, TLR2 functions as a heterodimer in combination with
either TLR1 or TLR6 (65, 66). To determine the interacting part-
ner for TLR2, gB and gH coimmunoprecipitation experiments
were performed with TLR1 and TLR6. Both gB and gH copre-
cipitated with TLR1, but not TLR6, suggesting that the operative
sensor for HCMV gB and gH is a TLR2/TLR1 heterodimer (Fig.
FIGURE 1. gB and gH elicit cytokine responses to HCMV. A, UV-
inactivated HCMV virions (MOI ⫽ 1) were incubated with the indicated
Abs (100
g/ml) for 15 min before infection of NHDF cells. At 18 h
postinfection, the supernatants were harvested and IL-6 levels were deter-
mined by ELISA. Error bars indicate SD. ⴱ, p ⬍ 0.05 in comparison to
untreated control. B, HEK cells expressing CD14 alone or in combination
with TLR2 or TLR4 were mock treated, treated with Pam
3
CSK
4
(100 ng/
ml) or LPS (20
g/ml), infected with HCMV (MOI ⫽ 0.01), treated with
gB-sGFP (250 nM), or treated with soluble eGFP alone (2
M). At 18 h
posttreatment, the supernatants were harvested and IL-8 levels were deter-
mined by ELISA. Error bars indicate SD.
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2, C–E). In contrast, gL did not coprecipitate with TLR2, TLR1, or
TLR6 (Fig. 2F). These results are consistent with inability of anti-
gL Abs to inhibit the inflammatory cytokine response to HCMV.
Together, these results indicate that gB and gH directly interact
with a TLR2/TLR1 heterodimer to activate inflammatory cytokine
responses from cells.
HCMV activates TLR2 in permissive fibroblasts
To appreciate the relationship between TLR2 and HCMV in a
context that is physiologically relevant to the life cycle of HCMV,
it is critical to use cells that are fully permissive for HCMV in-
fection. The initial studies that identified TLR2 as a host factor
mediating innate responses to HCMV used cell types that do not
support HCMV replication (17). Thus, we endeavored to translate
our findings into HCMV permissive human fibroblast cells, the
best-characterized cell culture system for the study of HCMV. The
TLR repertoire of NHDF cells has not been reported, and it is not
known whether these cells express TLR2. RT-PCR analysis of
total RNA from NHDF cells revealed the presence of TLR2 (Fig.
3), as well as TLR1, TLR6, and TLR4 (data not shown). RNA
harvested from Monomac-6 and 293T cells were included as pos-
itive and negative controls for TLR2 expression, respectively (Fig.
3). Efforts to detect TLR2 protein expression in NHDF cells have
been unsuccessful; however, the presence of the TLR2 transcript,
coupled with the ability of NHDF cells to respond to the synthetic
TLR2 ligand Pam
3
CSK
4
(Fig. 4
), indicates that these cells express
TLR2. Additionally, TLR1, TLR6, and TLR4 transcripts were de-
tected in NHDF cells by RT-PCR (data not shown). NHDF cells
secrete IL-6 in response to zymosan (data not shown).
To determine whether TLR2 mediates innate responses to
HCMV in NHDF cells, we tested the effect of an anti-TLR2 Ab on
the cytokine response to HCMV (Fig. 4). Compared with medium
alone (No Ab), an isotype control Ab had no effect on the IL-6
response to UV-HCMV infection, the TLR-independent control
FIGURE 2. HCMV envelope glycoproteins
coimmunoprecipitate with TLR2 and TLR1.
293T cells were cotransfected with a constant
amount of gB expression plasmid and increasing
amounts of plasmids encoding FLAG-TLR2 (A),
FLAG-TLR1 (C), or FLAG-TLR6 (D). At 48 h
posttransfection, immunoprecipitations were per-
formed with anti-FLAG agarose beads. The pre-
cipitation products were separated by SDS-PAGE
and immunoblotted with anti-gB and anti-FLAG
Abs as indicated. B, 293T cells were transfected
with gB or FLAG-TLR2 expression plasmids as
indicated. At 24 h posttransfection, the cells were
metabolically labeled with [
35
S]methionine. At
48 h posttransfection, immunoprecipitations were
performed as described above, the products were
separated by SDS-PAGE, and labeled proteins
were detected by autoradiography. Bands corre-
sponding to gB and TLR2 are indicated. 293T cells
were transfected with gH (E), gL (F), or VSV-G
(G) expression plasmids, along with FLAG-TLR1,
FLAG-TLR2, or FLAG-TLR6 as indicated. At
48 h posttransfection, immunoprecipitations were
performed as described above. The precipitation
products were analyzed with anti-gH, anti-gL, anti-
VSV-G, or anti-FLAG Abs as indicated.
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IL-1

, or the synthetic TLR2 ligand Pam
3
CSK
4
. In contrast, the
levels of IL-6 secreted in response to UV-HCMV and the TLR2
control Pam
3
CSK
4
were dramatically reduced by pretreatment
with the TLR2 blocking Ab, but no effect was observed on the
response to IL-1

. These results indicate that inflammatory cyto-
kine responses to HCMV in permissive NHDF cells are mediated
by TLR2.
To further address the role of TLR2 in NHDF cells, we con-
structed dominant-negative versions of TLR2 and TLR4 by re-
moving their cytoplasmic tails (TLR2⌬C and TLR4⌬C, respec-
tively) (Fig. 5A). These signaling-defective constructs lack the
TLR1 domain common to all TLRs and cannot recruit the cyto-
plasmic adaptor molecules that propagate downstream signaling
events (67). NHDF cells were transduced with recombinant retro-
viruses that coexpress FLAG epitope-tagged TLR2⌬C or TLR4⌬C
in combination with eGFP (59). A control population expressing a
GFP vector was also generated. GFP-positive cells were collected
by FACS and expression of the dominant-negative constructs was
confirmed by immunoprecipitation and immunoblotting (Fig. 5B).
IL-6 secretion was used as a marker of inflammatory cytokine
activation after challenge of the dominant-negative cell panel with
UV-HCMV (Fig. 6). The GFP vector control cells responded nor-
mally to all stimuli, including UV-HCMV. TLR2⌬C-expressing
cells responded normally to IL-1

and LPS ⫹ sCD14, the TLR4
ligand. However, these cells displayed a reduced response to UV-
HCMV and the TLR2 control ligand Pam
3
CSK
4
, confirming that
TLR2 mediates inflammatory cytokine responses to HCMV in per-
missive NHDF cells. The IL-6 response from TLR4⌬C-expressing
cells to IL-1

and Pam
3
CSK
4
were unaffected, and as predicted,
the response to LPS ⫹ sCD14 was completely eliminated. Inter-
estingly, the response to UV-HCMV was partially diminished in
these cells. No role for TLR4 was found in previous studies (17);
however, these experiments were performed in nonpermissive
FIGURE 4. Anti-TLR2 Abs block inflammatory cytokine responses to
HCMV. NHDF cells were incubated without Ab (No Ab) or with 1
g/ml
isotype control Ab or anti-TLR2 Ab for 30 min before HCMV challenge.
The cells were treated with IL-1

(1 pg/ml), Pam
3
CSK
4
(20
g/ml), or
infected with UV-inactivated HCMV at a MOI of 1. The supernatants were
harvested at 18 h postinfection, and IL-6 levels were determined by
ELISA. Error bars indicate SD. ⴱ, p ⬍ 0.001 in comparison to No Ab
control.
FIGURE 5. Dominant-negative TLR constructs. A, Dominant-negative
TLR2 and TLR4 constructs were generated that lack the cytoplasmic TLR1
domain (TLR2⌬C and TLR4⌬C, respectively). The dominant-negative
constructs contain an aminoterminal FLAG epitope tag for detection pur-
poses. B, Expression of the constructs was confirmed by immunoprecipi-
tation-immunoblot analysis.
FIGURE 3. TLR2 is expressed in NHDF cells. RT-PCR analysis of to-
tal RNA harvested from NHDF, HEK, and Monomac-6 cells using TLR2
(left) and GAPDH-specific primers (right). The resulting PCR products
were analyzed by 1% agarose gel electrophoresis and visualized by
ethidium bromide staining.
FIGURE 6. Dominant-negative TLR2 inhibits inflammatory cytokine
responses to HCMV in permissive fibroblasts. NHDF cells expressing a
GFP control vector, TLR2⌬C, or TLR4⌬C were mock infected, treated
with IL-1

(1 pg/ml), Pam
3
CSK
4
(40
g/ml), LPS (1
g/ml) ⫹ sCD14 (1
g/ml), or infected with UV-inactivated HCMV at a MOI of 10. The
supernatants were harvested at 18 h postinfection, and IL-6 levels were
determined by ELISA. Error bars indicate SD. ⴱ, p ⬍ 0.05, compared with
GFP control.
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cells. It is possible that TLR4 is involved in the innate response to
HCMV in permissive cells and this possibility is currently under
consideration.
TLR2 mediates NF-
B activation in response to HCMV infection
Another signature TLR response is activation of the pleiotropic
transcription factor NF-
B (19). Previous studies have shown that
HCMV activates NF-
B within minutes after infection, kinetics
that are suggestive of receptor-induced signaling (50, 68, 69). To
determine whether TLR2 mediates NF-
B activation upon HCMV
infection, we used our dominant-negative TLR cell panel to assess
the degradation of I
B
␣
as a marker of NF-
B activation (Fig. 7).
I
B
␣
binds and sequesters NF-
B in the cytoplasm as part of a
transcriptionally inactive complex (70). Many stimuli, including
TLRs, induce I
B
␣
degradation thereby releasing NF-
B to trans-
locate to the nucleus where it complexes with numerous other
factors to modulate transcription. Thus, the loss of I
B
␣
though
degradation correlates with the activation of NF-
B. In GFP con-
trol cells IL-1

caused complete I
B
␣
degradation, whereas
Pam
3
CSK
4
and LPS ⫹ sCD14 induced a lesser degree of degra
-
dation. Furthermore, I
B
␣
degradation is blocked in response to
Pam
3
CSK
4
and LPS ⫹ sCD14 in TLR2⌬C and TLR4⌬C-express
-
ing cells, respectively. Similar results were observed in a second
experiment. In response to UV-HCMV near-complete degradation
of I
B
␣
is observed in GFP vector control and TLR4⌬C-express-
ing cells. However, in cells expressing TLR2⌬C, the level of I
B
␣
degradation is reduced. Densitometric analysis of the blots indi-
cates that, although dominant-negative TLR2 completely prevents
I
B
␣
degradation in response to the TLR2 control ligand
Pam
3
CSK
4
,I
B
␣
degradation in response to HCMV infection is
not completely blocked (Fig. 7, lower panel). This observation
suggests that TLR2 is not the only mechanism by which HCMV
can activate NF-
B. Together, these observations indicate that
TLR2 mediates a portion of NF-
B activation in response to
HCMV infection and further support the hypothesis that TLR2 is
a key cellular factor for the innate immune response to HCMV.
FIGURE 7. HCMV activates NF-
B via TLR2 in permissive fibroblasts. NHDF cells expressing a GFP control vector (A), TLR2⌬C(B), or TLR4⌬C
(C) were treated with cycloheximide for 30 min before challenge. The cells were mock infected, treated with IL-1

(1 pg/ml), Pam
3
CSK
4
(40
g/ml), LPS
(1
g/ml) ⫹ sCD14 (1
g/ml), or infected with UV-inactivated HCMV at a MOI of 10. At 3 h postchallenge, whole-cell lysates were prepared. I
B
␣
and
actin levels were determined by SDS-PAGE analysis followed by immunoblotting (left panel). Right panel, The immunoblot shown in the upper panel was
subjected to densitometric analysis using ImageQuant software. The intensity of the I
B
␣
bands for each sample was normalized to the intensity of the
corresponding actin band. The resulting I
B
␣
intensity was plotted as a percentage of the I
B
␣
signal in the mock-infected cells for each transductant. The
results are indicative of two independent experiments.
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Discussion
The goal of this study is to further elucidate the relationship be-
tween viruses and the host innate immune response. We previously
identified TLR2 as a cellular factor that mediates innate immune
responses to HCMV infection (17). However, many questions re-
main with respect to the mechanism by which HCMV activates
TLR2, as well as the effect of TLR2 activation on the virus. In this
study, we show that two HCMV envelope gp, gB and gH, activate
TLR2. mAbs against both gB and gH inhibit cytokine responses to
HCMV, and both gB and gH physically associate with TLR2 in
coimmunoprecipitation experiments. gB and gH also coprecipitate
with TLR1, but not TLR6, indicating that the functional sensor for
HCMV is a TLR2/TLR1 heteromeric complex. Our previous stud-
ies used cell types that do not support HCMV infection, such as
HEK and CHO indicator lines and murine fibroblasts. In this study,
we extend our studies into HCMV permissive human fibroblasts,
which allows for a greater appreciation for the role that TLR2
plays in HCMV biology. Although human fibroblasts have been
shown previously to respond to TLR2 ligands, we demonstrate in
this study that these cells express TLR2 mRNA (71, 72). Using
TLR2 function-blocking Abs and dominant-negative TLR con-
structs, we show that TLR2 mediates NF-
B activation and in-
flammatory cytokine responses to HCMV in these cells. Together,
these data add to the growing body of evidence suggesting that
HCMV can activate innate immunity during binding and entry into
host cells. We propose that envelope gp gB and gH, already well
appreciated for their roles as mediators of virus entry, also interact
directly with TLR2 and TLR1 during entry to initiate a signaling
cascade that results in the activation of NF-
B and secretion of
inflammatory cytokines.
HCMV gB and gH brings the number of viral envelope gp that
are detected by TLRs to five (39–42). Notably, a shared feature of
all of these gp is that they play critical roles in the binding and
entry of their respective viruses (43– 47, 60, 73–79). Viral enve-
lope proteins are a compelling target for the TLR system, as they
are the first component of the virus to come into contact with the
cell. Consequently, detection of viral envelope gp would allow the
cell to set the innate response in motion at the earliest stages of
infection, perhaps even before the virus entering the cell. The rapid
recognition and response could provide a temporal advantage for
the host immune response, which would be extremely beneficial
for combating a viral infection.
Activation of TLRs by envelope gp also suggests that the pro-
cesses of virus entry and innate immune activation are coordinated.
The viral envelope is studded with numerous copies of each gp,
and each copy is able to interact with one or more cellular recep-
tors. Multiple interactions between viral envelope proteins and dif-
ferent types of cellular receptors may induce the formation of an
organized structure reminiscent of the immunological synapse
(80). This type of receptor clustering would allow the cell to syn-
chronize innate immune activation with the process of viral entry.
Cellular integrins have been identified as receptors for HCMV gB
and gH (46, 47) and have also been linked to TLRs (81, 82). It is
possible that HCMV binding to integrins could facilitate interac-
tion with TLR2/TLR1 heterodimers. Furthermore, receptor clus-
tering may provide a mechanism by with integrin and TLR sig-
naling can be coordinated. Fig. 7 indicates that HCMV-mediated
NF-
B activation is only partially attributable to TLR2. As NF-
B
activation is also a downstream consequence of integrin usage, it
is possible that TLR2 and integrins both contribute to the activa-
tion of NF-
B upon HCMV infection (83). However, it remains to
be determined whether NF-
B activation by TLR2 and integrins is
coordinated or coincidental.
In addition to HCMV, several other members of the Herpesviri-
dae activate innate immunity through TLRs. Herpes simplex virus
type 1 (HSV-1), HSV-2 and mouse CMV harbor CpG-rich ge-
nomes that activate TLR9 (32, 33, 84, 85), and HSV-1 and vari-
cella-zoster virus activate TLR2 (86, 87). An emerging possibility
is that herpesviruses are subject to innate detection by multiple
TLRs, with each TLR providing a distinct contribution to the over-
all response. For instance, TLR2 is associated with inflammatory
cytokine responses, whereas TLR9 elicits the secretion of type I
IFNs. Using multiple TLRs would allow the host to tailor its re-
sponse to fit the pathogen through the combined actions of each
TLR. In addition, HCMV infects a variety of cell types in vivo,
including fibroblasts, endothelial cells, epithelial cells, monocytes/
macrophages, smooth muscle cells, stromal cells, neuronal cells,
and hepatocytes (4, 5), and each of these cell types may express a
unique subset of TLRs and respond differently to HCMV infection.
Thus, it is possible that the cell type infected and the different
combinations of TLRs activated may have a profound influence on
the outcome of infection. Experiments addressing the role of mul-
tiple TLRs simultaneously will provide valuable insights into how
each TLR influences the global immune response to herpesviruses.
HCMV, like all herpesviruses, establishes a lifelong association
with the host as a latent infection. To accomplish this goal, HCMV
maintains a particularly close relationship with the host immune
system and employs multiple immune modulation strategies that
allow it to avoid detection by the host and persist in the face of a
potent immune response (3). Because of this close relationship, it
is tempting to speculate that HCMV may have adapted to use TLR
responses to its advantage. HCMV disseminates in neutrophils and
monocytes, and CD14-positive cells are hypothesized as a reser-
voir for latent virus (88). Each of these cell types are either acti-
vated by TLRs or are subject to recruitment by the mixture of
cytokines and chemokines that result from TLR activation. Thus,
HCMV may have evolved to use TLR responses as a means of
recruit its dissemination and latency vehicles to the site of infec-
tion, where these cells could then become infected. Further exam-
ination of the role that TLRs play at both the cellular and organ-
ismal levels may provide further clues toward understanding the
complex relationship between herpesviruses and their hosts.
Acknowledgments
We thank Bryan Williams (Cleveland Clinic Foundation) for the TLR1,
TLR2, TLR4, and TLR6 plasmids; and Yoshi Kawaoka and Bill Sugden
(both from the University of Wisconsin, Madison, WI) for the VSV-G
construct and retroviral vectors, respectively. We also thank members of
the Compton laboratory for critical review of the manuscript.
Disclosures
The authors have no financial conflict of interest.
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