JOURNAL OF VIROLOGY, Nov. 2008, p. 10735–10746
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Vol. 82, No. 21
Vaccinia Virus Subverts a Mitochondrial Antiviral Signaling
Protein-Dependent Innate Immune Response in
Keratinocytes through Its Double-Stranded
RNA Binding Protein, E3?
Liang Deng,1* Peihong Dai,2Tanvi Parikh,1,2Hua Cao,1,2Vijay Bhoj,4Qinmiao Sun,4
Zhijian Chen,4Taha Merghoub,3Alan Houghton,3and Stewart Shuman2*
Dermatology Service, Department of Medicine,1Molecular Biology Program,2and Immunology Program,3
Memorial Sloan-Kettering Cancer Center, New York, New York 10021, and Howard Hughes Medical Institute,
Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, Texas 753904
Received 23 June 2008/Accepted 7 August 2008
Skin keratinocytes provide a first line of defense against invading microorganisms in two ways: (i) by acting
as a physical barrier to pathogen entry and (ii) by initiating a vigorous innate immune response upon sensing
danger signals. How keratinocytes detect virus infections and generate antiviral immune responses is not well
understood. Orthopoxviruses are dermatotropic DNA viruses that cause lethal disease in humans. Virulence
in animal models depends on the virus-encoded bifunctional Z-DNA/double-stranded RNA (dsRNA)-binding
protein E3. Here, we report that infection of mouse primary keratinocytes with a vaccinia ?E3L mutant virus
triggers the production of beta interferon (IFN-?), interleukin-6 (IL-6), CCL4, and CCL5. None of these
immune mediators is produced by keratinocytes infected with wild-type vaccinia virus. The dsRNA-binding
domain of E3 suffices to prevent activation of the innate immune response. ?E3L induction of IFN-?, IL-6,
CCL4, and CCL5 secretion requires mitochondrial antiviral signaling protein (MAVS; an adaptor for the
cytoplasmic viral RNA sensors RIG-I and MDA5) and the transcription factor IRF3. IRF3 phosphorylation is
induced in keratinocytes infected with ?E3L, an event that depends on MAVS. The response of keratinocytes
to ?E3L is unaffected by genetic ablation of Toll-like receptor 3 (TLR3), TRIF, TLR9, and MyD88.
The interactions of viruses with the skin immune system are
not well understood. The interface of poxviruses with skin is
especially intriguing, given that (i) skin infection and disfigure-
ment are prominent features of smallpox disease; (ii) success-
ful smallpox immunization is achieved by intentional skin in-
fection with vaccinia virus; and (iii) eczema vaccinatum is a
major severe complication of vaccination in people with atopic
dermatitis (AD), a condition associated with defects in skin
barrier function and antiviral innate immunity (25, 26, 69).
Concerns about smallpox as a bioterrorism threat against an
unvaccinated population, the persistence of monkeypox in Af-
rica and episodes of its extracontinental spread, and the im-
perative to improve smallpox vaccination strategies all focus
attention on poxvirus-host dynamics, particularly with skin. A
recent case of life-threatening eczema vaccinatum in a child
with AD who became infected by mere household contact with
a smallpox vaccinee and then passed the infection on to a third
party (68) highlights just how delicate the balance is between
poxvirus virulence and skin immunity.
Keratinocytes comprise the predominant cell type in the
epidermis. Liu et al. (43) reported that vaccinia virus had
limited replicative capacity in human keratinocytes and that
infection induced keratinocytes to produce the Th2 cytokines
transforming growth factor ?, interleukin-10 (IL-10), and IL-
13, which suggested that vaccinia virus might downregulate
skin immune responses. Human keratinocytes express Toll-like
receptors (TLRs) that initiate innate immune signaling by
binding to ligands referred to as pathogen-associated molecu-
lar patterns (30, 40, 47, 49). Human keratinocytes produce a
repertoire of cytokines, chemokines, and antimicrobial pep-
tides in response to TLR stimulation (6, 40, 66). Such cytokines
and chemokines augment innate and acquired immunity me-
diated by dendritic cells, neutrophils, macrophages, NK cells,
and T cells, which reside in the skin or are recruited to the skin
in the setting of infection or inflammation (39).
The type I interferons (IFN-? and IFN-?) are key mediators
of antiviral innate immunity (65). Double-stranded RNA
(dsRNA) introduced during virus infection is a potent inducer
of the type I IFN response. dsRNA can trigger distinct signaling
pathways by engaging either (i) the endosomal membrane-bound
receptor TLR3 (42), (ii) the soluble cytoplasmic receptors RIG-I
and MDA5 (28, 29, 31, 73), or (iii) the dsRNA-dependent
protein kinase PKR (16). Signaling through TLR3 leads to
activation of the transcription factors IFN regulatory factor 3
(IRF3) and NF-?B via the adaptor molecule TRIF. As a con-
sequence, IRF3 is phosphorylated and then moves to the nu-
cleus to activate IFN-? expression (59). In contrast, cytoplas-
mic dsRNA produced during replication of RNA viruses binds
to RIG-I or MDA5 and triggers activation of IRF3 and NF-?B
through the mitochondrial antiviral signaling protein, MAVS
* Corresponding author. Mailing address for S. Shuman: Molecular
Biology Program, Memorial Sloan-Kettering Cancer Center, 1275
York Ave., New York, NY 10021. Phone: (212) 639-7145. Fax: (212)
772-8410. E-mail: firstname.lastname@example.org. Mailing address for L.
Deng: Dermatology Service, Department of Medicine, Memorial
Sloan-Kettering Cancer Center, 1275 York Ave., New York, NY 10021.
Phone: (212) 610-0785. Fax: (212) 308-0739. E-mail: email@example.com.
?Published ahead of print on 20 August 2008.
(58), also known as IPS-1 (32), VISA (71), or Cardif (48).
Cytoplasmic 5?-triphosphate-terminated single-stranded RNA
is also able to activate the RIG-I and PKR pathways (24, 52).
Relatively little is known about these pathways in skin cells.
Dai et al. (13) reported that treatment of human keratinocytes
with exogenous poly(I:C), a TLR3 agonist, induced phosphor-
ylation and nuclear translocation of IRF3. To our knowledge,
there is no report of the status of cytoplasmic viral RNA
sensing in keratinocytes or during a poxvirus infection.
Poxviruses are extraordinarily adept at evading and antago-
nizing multiple innate immune signaling pathways by encoding
proteins that interdict the extracellular and intracellular com-
ponents of those pathways (57). Chief among the poxvirus
antagonists of intracellular innate immune signaling is the vac-
cinia virus dsRNA-binding protein E3, which can inhibit the
PKR and NF-?B pathways (10, 14, 38) that would otherwise be
activated by vaccinia virus infection. A mutant vaccinia virus
lacking the E3L gene (?E3L) has a restricted host range, is
highly sensitive to IFN, and has greatly reduced virulence in
animal models of lethal poxvirus infection (5, 7, 8). Recent
studies have shown that infection of cultured cell lines with
?E3L virus elicits proinflammatory responses that are masked
during infection with wild-type vaccinia virus (14, 38). We
reported that infection of a mouse epidermal dendritic cell line
with wild-type vaccinia virus attenuated proinflammatory re-
sponses to the TLR agonists lipopolysaccharide (LPS) and
poly(I:C), an effect that was diminished by deletion of E3L.
Moreover, infection of the dendritic cells with ?E3L virus
triggered NF-?B activation in the absence of exogenous ago-
nists (14). These results suggested that E3L might play a role
in actively suppressing skin innate immunity during poxvirus
Here, we explore this theme by studying the responsiveness
of primary murine keratinocytes to TLR agonists and to infec-
tion by wild-type and ?E3L vaccinia viruses. The instructive
finding is that sensing of poxvirus infection by keratinocytes is
blocked by E3L, deletion of which unmasks a proinflammatory
response entailing secretion of IFN-?, IL-6, CCL4, and CCL5.
Using primary keratinocytes from knockout mice, we deter-
mined that the innate immune response to ?E3L infection is
independent of the TLR pathway components TLR3, TRIF,
TLR9, and MyD88. Rather, the ?E3L response is completely
dependent on MAVS and IRF3. This is, to our knowledge, the
first report of the activation of the MAVS-driven cytoplasmic
RNA-sensing pathway during infection by a poxvirus.
MATERIALS AND METHODS
Cell lines. BSC40 cells (African green monkey kidney cells) were maintained
in Dulbecco’s modified Eagle’s medium supplemented with 5% fetal bovine
serum (FBS). BHK-21 (baby hamster kidney) and RK13 (rabbit kidney) cells
were cultured in Dulbecco’s modified Eagle’s medium containing 10% FBS, 0.1
mM nonessential amino acids, and 50 ?g/ml gentamicin. All cells were grown at
37°C in a 5% CO2incubator.
Viruses. The WR strain of vaccinia virus was propagated, and virus titers were
determined in BSC40 monolayers at 37°C. The ?E3L and E3L?83N viruses were
kindly provided by B. L. Jacobs (Arizona State University). ?E3L was propa-
gated in BHK-21 cells, and virus titers were determined on RK13 cells. E3L?83N
was propagated, and virus titers were determined BSC40 cells. UV-inactivation
of ?E3L was performed with a UV Stratalinker 2400 by exposing the virus stock
to three cycles of radiation with 0.36 J of energy in the auto-cross-link mode.
Mice. Female BALB/c and C57B/6 mice between 8 and 12 weeks of age were
purchased from the Jackson Laboratory and were used for the preparation of
epidermal cells and primary keratinocytes. These mice were maintained in the
animal facility at the Sloan-Kettering Cancer Institute. All procedures were
performed with the consent of the Institutional Animal Use and Care Commit-
tee. IRF3?/?, TLR3?/?, MyD88?/?, TLR9?/?, and TRIF?/?(TRIFLPS2/LPS2)
mice were generated in the laboratories of Tadatsugu Taniguchi (University of
Tokyo), Richard Flavell (Yale University), Shizuro Akira (Osaka University),
and Bruce Beutler (Scripps Research Institute). Mice deficient for IFN ?/?
receptor (IFNAR?/?) were provided by Eric Pamer (Sloan-Kettering Institute);
the mice were purchased from B & K Universal and were backcrossed with
C57BL/6 mice for five generations. The MAVS?/?mice were maintained at the
animal facilities at the University of Texas Southwestern Medical Center.
Keratinocyte preparation. Female BALB/c and C57BL/6 mice at 8 to 12 weeks
of age were used. Mice were shaved and chemically depilated. Truncal skins were
removed and depleted of subcutaneous fat. The skin samples were floated,
dermis side down, on a solution containing 0.5 U/ml of dispase (Boehringer
Mannheim) and 0.38% trypsin (Sigma) in phosphate-buffered saline (PBS) for 40
min at 37°C. Epidermal sheets were collected and dissociated in Hanks balanced
salt solution with 2% heat-inactivated FBS for 20 min at room temperature
under gentle agitation. The cells were then filtered through a 40-?m-pore-size
cell strainer (BD Biosciences) and washed in Hanks balanced salt solution–2%
FBS. To remove Langerhans cells, the epidermal cells were incubated with
anti-Ia antibody (BD PharMingen), followed by washing and incubation with
Dynabeads M-450 coated with goat anti-mouse immunoglobulin (Dynal AS)
Materials. LPS and poly(I:C) were purchased from Sigma. An LPS stock
solution (1 mg/ml) in PBS was stored at ?80°C. A poly(I:C) stock solution (1
mg/ml) in PBS was stored at ?20°C. CpG oligodeoxynucleotide ODN2395 was
purchased from Alexis Biochemicals. Its stock solution (1 mg/ml) was stored at
?20°C. Cytosine arabinoside (araC) was from Sigma. Concentrations of cyto-
kines and chemokines were determined by enzyme-linked immunosorbent assay
(ELISA) with assay kits purchased from PBL Biomedical Laboratories and R &
D Systems. The assays were performed as specified by the kit vendor; the ELISA
data were converted to cytokine/chemokine concentrations by interpolation to
standard curves generated with cytokine/chemokine standards provided with
Responsiveness of mouse keratinocytes to TLR agonists and
vaccinia virus infection. Epidermal keratinocytes isolated
freshly from mouse skin were exposed to the TLR agonists
LPS (specific for TLR4), poly(I:C) (which acts via TLR3), and
CpG (which stimulates TLR9). Cell supernatants were col-
lected after 18 h and tested by ELISA for secreted cytokines
and chemokines. Treatment of keratinocytes with 1 ?g/ml LPS
or CpG failed to induce production of TNF-?, IL-6, IL-1?,
IFN-?, IFN-?, IFN-?, CCL3, CCL4, CCL5, CXCL9, or
CXCL10. Incubation of keratinocytes with 10 ?g/ml poly(I:C)
uniquely induced production of CCL5 (Fig. 1A) but not of
IFN-?, IL-6, or CCL4 (Fig. 1A) or any of the other aforemen-
tioned cytokines and chemokines (data not shown). Thus,
mouse keratinocytes display a narrow response to exogenous
TLR agonists. The positive response to poly(I:C) by secretion
of CCL5 attests to the presence of a TLR3-driven signaling
pathway. CCL5 is a proinflammatory chemokine that has an-
tiviral activities (51). CCL5 expression is controlled by both
NF-?B and IRFs (18).
Infection of keratinocytes with wild-type vaccinia virus
(strain WR) at a multiplicity of 3 was nonproductive; i.e., only
a twofold increase in viral titer was observed at 2 days postin-
fection (not shown). Pulse labeling of vaccinia virus-infected
keratinocytes with [35S]methionine highlighted the appearance
of new, labeled polypeptides at 4 to 12 h postinfection that
were not evident in uninfected control cells; a subset of virus-
specific labeled species appeared at 8 to 12 h that was not
evident at 4 h, consistent with a transition from early to inter-
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