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Early Innate Immune Responses to Sin
Nombre Hantavirus Occur Independently of
IFN Regulatory Factor 3, Characterized
Pattern Recognition Receptors, and Viral
Entry
Joseph B. Prescott, Pamela R. Hall, Virginie S.
Bondu-Hawkins, Chunyan Ye and Brian Hjelle
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The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2007 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2007; 179:1796-1802; ;
doi: 10.4049/jimmunol.179.3.1796
http://www.jimmunol.org/content/179/3/1796
The Journal of Immunology
Early Innate Immune Responses to Sin Nombre Hantavirus
Occur Independently of IFN Regulatory Factor 3,
Characterized Pattern Recognition Receptors,
and Viral Entry1
Joseph B. Prescott,* Pamela R. Hall,* Virginie S. Bondu-Hawkins,* Chunyan Ye,*
and Brian Hjelle2*†‡
M
icrobes are recognized by cells of the innate immune
system through their interactions with pattern recognition receptors (PRRs)3 expressed by many cell types
including endothelial and epithelial cells. Recognition of pathogen-associated molecular patterns (PAMPs) of viruses by cellular
PRRs results in the activation of signaling cascades and transcription factors that modulate the expression of type I IFNs and an
array of IFN-stimulated genes (ISGs) that possess diverse antiviral
functions. TLRs and the recently discovered caspase recruitment
domain-containing cytoplasmic RNA helicases, retinoic acid-inducible gene-I (RIG-I), and myeloid differentiation-associated factor 5 (MDA5), are the best characterized PRRs and their activity is
*Department of Pathology, †Department of Biology, and ‡Department of Molecular
Genetics and Microbiology, Center for Infectious Diseases and Immunity, School of
Medicine, University of New Mexico, Albuquerque, NM 87131
Received for publication January 19, 2007. Accepted for publication May 14, 2007.
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 U.S. Public Services Grants U01 AI56618 and U01
AI054779. J.B.P. was supported by National Institute of Allergy and Infectious Disease Grant 1 T32 AI07583.
2
Address correspondence and reprint requests to Dr. Brian L. Hjelle, Department of
Pathology, University of New Mexico, 329CRF (MSC08 4640), 1 University of New
Mexico, Albuquerque, NM 87131-0001. E-mail address: [email protected]
3
Abbreviations used in this paper: PRR, pattern recognition receptor; PAMP, pathogen-associated molecular pattern; ISG, IFN-stimulated gene; RIG-I, retinoic acidinducible gene I; MDA5, myeloid differentiation-associated factor 5; IRF, IFN regulatory factor; vRNA, viral RNA; SNV, Sin Nombre virus; HCPS, hantavirus
cardiopulmonary syndrome; TRIF, Toll/IL-1R domain-containing adaptor protein inducing IFN-␤; siRNA, small-interfering RNA; qRT-PCR, quantitative RT-PCR;
MxA, myxovirus resistance A; poly I:C, polyinosinic/polycytidylic acid; IPS-1, IFN
promoter stimulator-1; SeV, Sendai virus; CT, cycle threshold; SEAP, secreted alkaline phosphatase; CARD, caspase recruitment domain.
Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00
www.jimmunol.org
required for the recognition of several pathogenic RNA viruses
(1–5). In the majority of the described systems, viral RNA (vRNA)
serves as the PAMP and its binding to specific PRRs, such as
TLR3 or RIG-I, results in the activation of transcription factors
NF-␬B and IFN regulatory factor 3 (IRF3) via multiple signaling
pathways (6). These transcription factors are localized to the cytoplasm and translocate to the nucleus upon activation. Although
IRF3 is constitutively expressed, after phosphorylation it forms a
homodimer, or a heterodimer with IRF7, and binds IFN-stimulated
response elements in the promoter regions of many ISGs, including ISG56 (7–11). IRF3 also mediates the expression of IFN-␤,
although transcription of a subset of ISGs is independent of IFN-␤
expression early in viral infection (11–14). Several groups have
shown IRF3 to be indispensable for the expression of ISGs in
response to infection by a multitude of RNA viruses (14 –17).
Sin Nombre virus (SNV), an enveloped virus with a genome
comprised of three negative-sense RNA segments, is a New World
hantavirus (Bunyaviridae: Hantavirus) and is the primary etiologic
agent of hantavirus cardiopulmonary syndrome (HCPS) in North
America (18, 19). HCPS is characterized by pulmonary edema due
to capillary leak, followed by cardiogenic shock. Approximately
453 cases have been reported in the United States since 1993 with
a 35% case-fatality ratio (www.cdc.gov/ncidod/diseases/hanta/
hps/index.htm). SNV is carried by the deer mouse (Peromyscus
maniculatus) and transmitted to humans by inhalation of viruscontaminated urine, feces, and/or saliva (20). Interestingly, infection in the deer mouse is asymptomatic and detectable titers of
virus persist for at least many months after experimental inoculation, despite a substantial specific immune response (21). Vascular
endothelial cells are thought to be the primary sites of viral replication in humans and infected cells and/or adjacent cells secrete
high levels of chemokines and cytokines as a result (22–25).
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Sin Nombre virus (SNV) is a highly pathogenic New World virus and etiologic agent of hantavirus cardiopulmonary syndrome.
We have previously shown that replication-defective virus particles are able to induce a strong IFN-stimulated gene (ISG) response
in human primary cells. RNA viruses often stimulate the innate immune response by interactions between viral nucleic acids,
acting as a pathogen-associated molecular pattern, and cellular pattern-recognition receptors (PRRs). Ligand binding to PRRs
activates transcription factors which regulate the expression of antiviral genes, and in all systems examined thus far, IFN regulatory factor 3 (IRF3) has been described as an essential intermediate for induction of ISG expression. However, we now describe
a model in which IRF3 is dispensable for the induction of ISG transcription in response to viral particles. IRF3-independent ISG
transcription in human hepatoma cell lines is initiated early after exposure to SNV virus particles in an entry- and replicationindependent fashion. Furthermore, using gene knockdown, we discovered that this activation is independent of the best-characterized RNA- and protein-sensing PRRs including the cytoplasmic caspase recruitment domain-containing RNA helicases and the
TLRs. SNV particles engage a heretofore unrecognized PRR, likely located at the cell surface, and engage a novel IRF3-independent pathway that activates the innate immune response. The Journal of Immunology, 2007, 179: 1796 –1802.
The Journal of Immunology
Materials and Methods
Cell culture and treatments with virus or
polyinosinic/polycytidylic acid (poly I:C)
We purchased human hepatoma cells (Huh7) and the RIG-I-defective clone
Huh7.5 from Apath and grew cells in DMEM (Invitrogen Life Technologies) containing 10% FCS, 1⫻ essential amino acids (Invitrogen Life
Technologies), penicillin/streptomycin, and gentamicin.
For SNV treatment, we seeded cells in 48- or 24-well plates at a density
to achieve 90 –95% confluent after overnight incubation at 37°C, 5% CO2.
We previously described the isolation of SNV strain SN77734 from a deer
mouse and its propagation and titration in Vero E6 cells under strict standard operating procedures using biosafety level 3 facilities and practices
(CDC registration number C20041018-0267; Centers for Disease Control)
(32). Treatment was done essentially as described previously (26). Briefly,
the cell culture medium was removed and we then added SNV at a multiplicity of infection equivalent to 1.0 (after 15 s inactivation by 254 nm
UV light, ⬃5 mW/cm2) to the cell cultures. SNV-treated cells were then
incubated for 1 h at 37°C in a 5% CO2 atmosphere. We then removed the
virus-containing medium and replaced it with fresh medium and incubated
the cells as above for the amount of time indicated.
For Sendai virus treatment, we exposed cells to Sendai virus (SeV)
(Cantell strain; Charles River Laboratories) at a final concentration of 150
hemagglutinin units/ml in serum-free DMEM. We incubated this mixture
at 37°C, 5% CO2 for 2 h, followed by the addition of an equal volume of
DMEM containing 20% FCS to the cells and incubated them for an additional 14 h. For poly I:C treatment, we seeded cells as above and at the time
of infection, and exposed cells to 10% DMEM containing 20 ␮g/ml poly
I:C (Sigma-Aldrich).
RNA silencing
We seeded Huh7 cells in 48-well plates at a density to achieve 50% confluency after overnight incubation. We then transfected these cells for 64 h
with the specified siRNA (siGENOME gene-specific pool or siCONTROL
nontargeting siRNA pool from Dharmacon) at a final concentration of 100
nM/well along with 0.6 ␮l/well of DharmaFect 1 transfection reagent as
per Dharmacon’s protocol. Control cells were treated in parallel with transfection reagent only. We then exposed cells to UV-SNV or poly I:C, or live
SeV as described above. After treatment, we extracted total RNA from
cells using the RNeasy kit (Qiagen) and used this RNA in quantitative
RT-PCR (qRT-PCR) assays.
Blocking by neutralizing antisera
We used heat-inactivated (56°C, 30 min) serum with a known focus-reduction neutralization titers of 1/1600/ml, to neutralize live or UV-killed
SNV (1.5 ⫻ 106 foci-forming units/ml) at a 1:40 ratio of serum to virus and
incubated this for 30 min at room temperature. We then treated Huh7 with
this mixture or with the virus alone for 1 h, removed the supernatant, and
added fresh medium. Six hours after treatment with UV-SNV, or 24 h after
incubation with live virus, we harvested total cellular RNA and quantitated
viral S-segment RNA and ISGs as above. As controls, we used the plasma
of two SNV-seronegative individuals as well as serum from a rabbit with
Abs raised against rSNV N Ag which is known to lack any neutralizing
activity.
Plasmid transfections
We seeded Huh7.5 cells in 48-well plates at a density to achieve 50%
confluency after an overnight incubation. We then transfected these cells
for 64 h with the indicated amounts of pNiFTy-secreted alkaline phosphatase (SEAP) (Invivogen), pEGFP, pTLR3 (a gift from G. Sen, Lerner Research Institute, Cleveland, OH (33)), or pRIG-I (a gift from M. Gale,
University of Texas Southwestern, Dallas, TX (34)) using 4 ␮l/well effectene (Qiagen) as per the Qiagen transfection protocol. We then removed
the medium and treated cells as described above for the amount of time
indicated in the experiments.
NF-␬B-SEAP assay
We cotransfected Huh7.5 cells seeded in 48-well plates with 100 ng of
pNiFTy and 100 ng of either pEGFP or pTLR3 for 48 h using Effectene as
above. After transfection, we exposed cells to ligands followed by the
collection of cell supernatants, and heat inactivated the endogenous alkaline phosphatases for 30 min in a 65°C water bath before performing the
assay. To perform the phosphatase assay, we added 40 ␮l of each supernatant to 20 ␮l of 0.05% CHAPS/PBS (Sigma-Aldrich) and added this to
100 ␮l of equal parts BluePhos A and B solutions (KPL) in 96-well plates.
After a 20-min incubation, we read the 96-well plate using an ELISA plate
reader at 630 nm. We calculated the fold change of SEAP by subtracting
the OD of control wells and comparing the sample ODs to a standard curve
generated using serial dilutions of a sample known to have a high concentration of SEAP.
Real-time SYBR Green and TaqMan RT-PCR
For SYBR Green RT-PCR, we performed reverse transcription using 2 ␮g
of total RNA extracted from cell cultures using the RNeasy kit (Qiagen)
and random hexamer primers in 50-␮l reactions using the Applied Biosystems TaqMan Reverse Transcription Reagents kit. For PCR, we used
the Applied Biosystems Power SYBR Green Reagents kit to perform reactions in triplicate using 3 ␮l of cDNA and a 0.4 ␮M final concentration
of each primer in a 25 ␮l total reaction volume. The primers we used were:
␤-actin, sense (5⬘-ccatcatgaagtgtgacgtgg-3⬘) and antisense (5⬘-gtccgccta
gaagcatttgcg-3⬘); IFN promoter stimulator-1 (IPS-1), sense (5⬘-agcaa
gagaccaggatcgactg-3⬘) and antisense (5⬘-cgcaatgaagtactccaccca-3⬘); IRF7,
sense (5⬘-taccatctacctgggcttcg-3⬘) and antisense (5⬘-agggttccagcttcacca3⬘); ISG56, sense (5⬘-tctcagaggagcctggctaag-3⬘) and antisense (5⬘-ccacact
gtatttggtgtctagg-3⬘); MDA5, sense (5⬘-cagaaggaagtgtcagctgcttag-3⬘) and
antisense (5⬘-tgctgccacattctcttcatct-3⬘); myxovirus resistance A (MxA),
sense (5⬘-tgatccagctgctgcatccc-3⬘) and antisense (5⬘-ggcgcaccttctcctcatac3⬘); MyD88, sense (5⬘-agcattgaggaggattgcca-3⬘) and antisense (5⬘-gtccgt
gggacactgctgt-3⬘); RIG-I, sense (5⬘-gactggacgtggcaaaacaa-3⬘) and antisense (5⬘-ttgaatgcatccaatatacacttctg-3⬘); Toll/IL-1R domain-containing
adaptor protein inducing IFN-␤ (TRIF), sense (5⬘-ccagatgcaacctccactgg3⬘) and antisense (5⬘-ctgttccgatgatgattcc-3⬘). We incubated the reactions at
50°C for 2 min, then 95°C for 10 min followed by 40 cycles of 95°C for
15 s and 60°C for 1 min using an Applied Biosystems Prism 7000 Sequence Detection System. We subjected the reactions to dissociation curve
analysis to exclude the possibility of nonspecific amplification and then
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Despite dysregulation of the vasculature and a strong adaptive immune response, no cytopathic effect is observed in infected tissues.
These observations suggest that the inflammatory response in local
tissues contributes to the pathogenic process.
We have previously demonstrated that SNV can induce a strong
innate immune response in HUVEC even after rendering the virus
replication-defective and markedly reducing the titers of vRNA
through UV irradiation, suggesting that viral particles themselves
contain a PAMP that can induce ISGs independently of the described RNA-recognizing PRRs (26). This, along with the observation that human primary cells infected in vitro mount a robust
inflammatory response, indicates that pathology may be due to
maladaptive immune responses. One important difference between
the hantavirus-human disease model, compared with many other
viral disease models, is that humans are an incidental and terminal
host for hantaviruses and that neither the virus nor the human
immune system have evolved under selective pressures from this
host-pathogen interaction. Therefore, we hypothesize that human
cells may recognize and signal the activation of innate responses to
hantaviruses, such as SNV, differently that those described for
other common human pathogenic viruses. Several groups have reported on the innate immune response to hantaviruses, but it has
yet to be determined how the human innate immune system recognizes these viruses and how this recognition results in the transcription of innate immunity genes (27–31). To address these questions, we used well-characterized human hepatoma cell lines to
investigate the requirements for viral entry, PRR expression, and
the downstream transcription factors in the activation of the innate
response to SNV. We found that neither IRF3 nor IRF7 are required for the induction of ISGs in response to replication-defective SNV particles. Furthermore, viral entry and the well-described
virus-sensing PRRs are not involved in the initial innate immune
response to SNV. These results confirm that some human cells
possess an RNA-independent mechanism to detect viruses, distinct
from the well-described IRF3-dependent PRR-signaling cascades,
which leads to the transcription of ISGs and activation of the innate immune system.
1797
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IRF3-INDEPENDENT INNATE IMMUNE RESPONSE TO VIRUS
calculated the fold-change for each gene using the mean of the change in
cycle threshold (CT) values (⌬CT) normalized to the CT values of GAPDH
for each sample (2⫺⌬⌬CT).
We performed TaqMan RT-PCR for viral S-segment RNA as previously
described (21). Briefly, we extracted vRNA from cells using the RNeasy
kit as above. We then reverse-transcribed 3 ␮l of RNA using an S-segmentspecific sense primer (5⬘-agcacattacagagcagacgggc-3⬘) and subjected 5 ␮l
of the resulting cDNA to PCR with S-segment-specific primers, sense (5⬘gcagacgggcagctgtg-3⬘) and antisense (5⬘-agatcagccagttcccgct-3⬘) and a
fluorescently labeled TaqMan probe 5⬘-(FAM)-tgcattggagaccaaactcg
gagaactt-(TAMRA)-3⬘ in triplicate PCRs using an Applied Biosystems
Prism 7000 Sequence Detection System. We quantitated the vRNA using a
standard curve generated using S-segment templates of known copy number.
Western blotting
Density gradient purification of SNV
We added 10 ml of UV-SNV to a 2-ml cushion of a 50% solution of
Optiprep (iodixanol, 5,5⬘-[(2-hydroxy-1–3 propanediyl)-bis(acetylamino)]
bis [N,N⬘-bis (2,3dihydroxypropyl-2,4,6-triiodo-1,3-benzenecarboxamide]) (Sigma-Aldrich) contained in a 14 ⫻ 89 polyallomer centrifuge tube
FIGURE 2. Blocking SNV entry does not inhibit ISG responses. A–C, Huh7 were transfected with the indicated siRNAs for 64 h. A, Equal amounts of
total cell lysates, or soluble CD61 (lane 3), were used for Western blots which were probed with either anti-CD61 (top panel) or anti-actin (bottom panel).
B, Cells were infected with live SNV and at the indicated time points, total cellular RNA was extracted and used for quantitation of viral S-segment by
TaqMan assays. Results are reported as the mean ⫾ SEM from triplicate experiments. C, Gene-silenced cells were exposed to UV-SNV and 6 h
posttreatment total RNA was extracted and used to determine the expression of ISG56 and MxA by real-time qRT-PCR. Results are reported as the mean ⫾
SEM from duplicate experiments. D and E, SNV (D) or UV-SNV (E) was incubated with serum samples from two convalescent HCPS patients ⫹(a) and
⫹(b) or two control sera -(c) and -(d), or anti-N Abs for 1 h. Huh7 were then exposed to virus samples as above and S-segment vRNA (D) was quantitated
24 h postinfection or ISG56 and MxA mRNA abundances (E) were determined 6 h postexposure.
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FIGURE 1. Huh7 cells respond to UV-SNV and purified UV-SNV by
expression of ISGs. A, Confluent Huh7 were treated with medium (Con),
uninfected Vero-conditioned cell culture supernatants (SN), or UV-SNV
grown on Vero E6 cells. At the indicated time points posttreatment, ISG56
and MxA expression was analyzed by real-time qRT-PCR. B, UV-SNV
was density-purified in Optiprep and equal volumes of UV-SNV stock or
purified UV-SNV, or the indicated amounts of recombinant N were used in
an anti-N Western blot. C, Huh7 were treated as in (A) with UV-SNV or
the indicated ratio of purified UV-SNV relative to N concentration and 6 h
posttreatment ISG56 and MxA mRNA levels were determined as above.
Results are representative of several individual experiments. Real-time
qRT-PCR results are reported as the mean ⫾ SEM from duplicate
experiments.
We cultured Huh7 cells in 24-well plates and transfected them with the
indicated small-interfering RNA (siRNA) or transfection reagent only. At
64 h posttreatment, we washed the cells once with ice-cold PBS and made
whole cell extracts using 100 ␮l of a buffer containing 1% Triton X-100,
10 mM Na2HPO4, 150 mM NaCl, 2 mM EDTA, 1% sodium deoxycholate,
0.25% SDS, 1 mM PMSF, and complete mini protease inhibitor (Roche).
We then separated equal volumes of lysates on 12.5% SDS-PAGE gels and
electrophoretically transferred the proteins to nitrocellulose membranes for
1 h at 100 v. We blocked the membranes in 1% BSA-PBS for 1 h and then
probed them with a primary rabbit anti-CD61 (AB1932; Chemicon International) or anti-IRF3 (SC-9082; Santa Cruz Biotechnology) or anti-actin
Ab (Cruz SC-1616-R; Santa Cruz Biotechnology) overnight at a dilution of
1/750, 1/200 and 1/2000, respectively. We then washed the membranes
three times for 5 min each in Western wash, and incubated them with a
phosphatase-labeled anti-rabbit IgG secondary Ab for 1 h. To visualize the
proteins, we washed the membranes again and developed using the colorimetric substrate NBT-5-bromo-4-chloro-3-indolyl phosphate.
For N protein detection, we heated supernatants containing virus particles with 5⫻ sample loading buffer containing SDS to 95°C for 5 min
before electrophoresis and transfer as above. We then blocked the membranes for 1 h with 5% milk-PBS and then incubated them with a polyclonal rabbit-anti SNV-N Ab (1/1000) for 1 h in 5% milk-PBS. After the
primary incubation, we washed the membranes three times for 5 min each
in Western wash, and incubated them with a peroxidase-labeled anti-rabbit
IgG secondary Ab (1/1000) for 1 h. After an additional washing step, we
visualized the proteins using ECL and Kodak BioMax MR film.
The Journal of Immunology
1799
(Beckman). This was placed in a TH641 swing bucket rotor (Sorvall) and
centrifuged at 40,000 rpm for 3 h at 4°C. After centrifugation, we collected 4
ml of solution from the bottom of the tube and mixed it with 1 ml of a 50%
Optiprep solution. We added this to a 5-ml Optiseal (Bechman) tube and centrifuged at 65,000 rpm for 5 h at 4°C in a NVT90 rotor. We then collected
100-␮l fractions from the bottom of the tube and determined the location and
concentration of the virus proteins by Western blot (data not shown). Fractions
that contained the viral particles were pooled and used in experiments.
Results
Replication-defective SNV particles induce ISG transcription in
human hepatoma cells
We previously demonstrated that live SNV, or SNV treated with a
minimal dose of UV irradiation (UV-SNV) that renders the virus
unable to replicate and express N protein, can nonetheless induce
ISG expression in primary endothelial cells (26). In an effort to
investigate ISG transcription in a tractable and well-studied cell
line, we exposed the human hepatoma line Huh7 to UV-inactivated
UV-SNV obtained from Vero E6 cell cultures or, as a control,
supernatants from uninfected Vero cells. We then measured ISG
expression at early time points after such treatment (Fig. 1A). We
found that ISG56 and MxA were both up-regulated by 1 h postexposure to supernatants from SNV-infected Vero cell cultures,
while Vero-conditioned medium did not elicit a response.
SNV is propagated on Vero E6 cells which lack the ability to
express type I IFNs (35, 36). Despite this, we sought to confirm
that the observed ISG responses are due to factors associated with
the virus and not soluble mediators released by these cells into the
culture supernatants. We compared responses elicited by UV-SNV
obtained directly from Vero cultures as above to those elicited by
Optiprep-purified UV-SNV. Purification of virus stocks resulted in
an ⬃6- to 8-fold concentration of the virus as determined by Western blotting of N protein isolated from those fractions that contained high concentrations of viral proteins (Fig. 1B). Treatment of
Huh7 with either UV-SNV or purified UV-SNV preparations at
similar concentrations of N protein resulted in comparable ISG
inductions and stimulation with purified virus was dose dependent,
FIGURE 4. ISG responses to UV-SNV do not require intact TLR3 or
RIG-I signaling pathways. A, Confluent Huh7 and Huh7.5 were treated
with either medium, UV-SNV, SeV, or poly I:C as described in Materials
and Methods. Six hours posttreatment with UV-SNV or poly I:C, and 16 h
after SeV infection, total cellular RNA was used to quantitate ISG56 and
MxA mRNA expression by real-time qRT-PCR. Results are representative
of several individual experiments. B, Huh7.5 were cotransfected with
pNiFTy and either pTLR3 or pEGFP. Sixty-four hours posttransfection,
cells were treated with medium, UV-SNV, or poly I:C. At the indicated
time points, duplicate samples of cell supernatants were collected and used
in phosphatase assays as described in Materials and Methods. C, Huh7.5
were transfected with either pEGFP or pRIG-I for 64 h. Cells were then
treated with either medium, UV-SNV, or SeV. Six hours postinfection with
UV-SNV and 16 h postinfection with SeV, total cellular RNA was used to
determine ISG56 and MxA mRNA abundance by qRT-PCR. Results are
reported as the mean ⫾ SEM from triplicate experiments.
further supporting the hypothesis that the PAMP activity was associated with the viral particle (Fig. 1C).
Viral entry is not required for UV-SNV-mediated ISG inductions
ISG expression is initiated by 1 h following exposure to UV-SNV
(Fig. 1A) and this activation is more rapid than that elicited by poly
I:C, a synthetic dsRNA that activates the innate response (our unpublished observations), suggesting that early events, perhaps preceding viral entry, may signal the transcription of ISGs. Therefore, we
sought to examine viral entry requirements in the context of ISG
inductions by silencing CD61, the ␤3 integrin receptor for pathogenic
hantaviruses, including SNV (37). A panel of siRNAs directed against
CD61 mRNA specifically decreased ␤3 integrin protein levels in
Huh7 as demonstrated by Western blotting (Fig. 2A). To determine
whether receptor knockdown can effectively inhibit viral entry, we
exposed CD61-silenced Huh7 to live SNV and measured the
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FIGURE 3. IRF3 and IRF7 are not required for ISG responses to UVSNV. A–C, Huh7 were transfected with the indicated siRNAs for 64 h. A,
Equal amounts of total cell lysates were used for anti-IRF3 (top panel) or
anti-actin (bottom panel) Western blots. B, Total cellular RNA was extracted from cells and used to determine IRF7 mRNA levels by real-time
qRT-PCR. C, Total cellular RNA from cells treated with medium, UVSNV, poly I:C, or SeV, as in Materials and Methods was used to determine
ISG56 and MxA mRNA expression levels by real-time qRT-PCR. Results
are reported as the mean ⫾ SEM from triplicate experiments.
1800
IRF3-INDEPENDENT INNATE IMMUNE RESPONSE TO VIRUS
FIGURE 5. TLRs and CARDRNA helicases are not necessary for
ISG responses to UV-SNV. A–C,
Huh7 were transfected with the indicated siRNAs for 64 h. A, Total cellular RNA was extracted and used in
qRT-PCR with ␤-actin and the specified gene-specific primers. B and C,
siRNA-transfected Huh7 were treated
with medium, UV-SNV, SeV or poly
I:C as in Materials and Methods and
total RNA used to determine ISG56
and MxA mRNA expression levels by
qRT-PCR. PCR was performed in
triplicate for duplicate biological experiments. All results are reported as
the mean ⫾ SEM from triplicate
experiments.
IRF3 and IRF7 are not required for ISG responses to SNV
viral particles
Transcription of ISGs is mediated by binding of the IRF family of
transcription factors to the promoters of these genes. Signals generated from virus binding to specific, identified PRRs, most of
which require viral entry, results in the activation of IRF3, and at
later time points, IRF7. IRF3 is activated early and controls the
expression of a subset of ISGs, including ISG56 and the initial
transcription of IFN-␤. IRF7 is subsequently activated and mediates the expression of IFN-␣ (39). In this system, we observe ISG
responses that occur independently of viral entry and we have
previously reported that SNV particles do not induce IFN-␣/IFN-␤
expression in primary endothelial cells. Other investigators have
shown the lack of early induction of IFN expression by hantaviruses (28). Based on these observations, we hypothesized that
SNV activates a novel entry-independent pathway that results in
the transcription of ISGs and that this mechanism is likely to be
IRF3 and 7 independent. To determine whether either molecule is
required for ISG transcription, we used siRNAs to knock down
their expression in Huh7 and measured ISG inductions mediated
by UV-SNV or control ligands. Western blotting of IRF3 protein
and real-time qRT-PCR of IRF7 mRNAs confirmed effective
knockdown of these genes (Fig. 3, A and B). As expected, silencing of IRF3 almost completely inhibited ISG56 and MxA transcriptional responses to poly I:C and SeV, which activate ISGs in
an IRF3-dependent fashion (40, 41), but induction of ISGs following exposure to UV-SNV was not attenuated (Fig. 3C). Silencing
of IRF7 partially inhibited poly I:C-mediated ISG responses, but
again, responses to UV-SNV were not inhibited (Fig. 3C).
ISG responses to UV-SNV in human hepatoma cells does not
require engagement of TLR3 and RIG-I
The lack of involvement of IRF3 and IRF7 in induction of ISGs by
SNV suggested to us the well-described viral nucleic acid-sensing
PRRs, TLR3 and RIG-I, might not be the molecules that detect the
SNV PAMP. To confirm or refute this hypothesis, we exposed
Huh7 or Huh7.5 cells, which do not express TLR3 and possess a
defective form of RIG-I (34), to UV-SNV and measured the induction of ISG mRNAs. Exposure to UV-SNV induced ISG56 and
MxA in both cell lines at 6 h posttreatment, although ISGs were
induced slightly better in Huh7 than in Huh7.5 (Fig. 4A). As positive controls to assess the function of TLR3 and RIG-I pathways
in our cultures, we used poly I:C presented in the medium and
replication-competent SeV, respectively. As expected, Huh7.5
cells did not respond to either treatment, confirming the expected
deficiencies, whereas Huh7 cells responded well to both treatments. Both SeV and poly I:C induced ISG56 transcription much
better than MxA transcription in responsive Huh7, results opposite
that seen when UV-SNV was used as the stimulus.
To further examine any possible involvement by TLR3 or
RIG-I, we expressed TLR3 and RIG-I in Huh7.5 and measured the
responses elicited by UV-SNV or control ligands. Ligand binding
to TLR3 results in the activation of NF-␬B, as well as IRF3 (42).
We cotransfected Huh7.5 with a NF-␬B reporter construct
(pNiFTy-SEAP) and either pTLR3, or a control plasmid (pEGFP)
before addition of each ligand. As expected, TLR3 expression conferred NF-␬B-mediated responsiveness to poly I:C, but did not
enhance UV-SNV-mediated responses (Fig. 4B). Likewise, transfection of a RIG-I expression plasmid into Huh7.5 conferred responsiveness to SeV, but inductions of MxA and ISG56 mRNAs
following UV-SNV exposure were unaffected (Fig. 4C).
SNV-mediated ISG transcription does not require known
candidate PRRs
TLR family members other than TLR3, and another described
caspase recruitment domain (CARD)-RNA helicase, MDA5, have
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abundance of viral S-segment genomic RNA 24 and 72 h
postinfection. As expected, CD61 knockdown resulted in a 60%
decrease in accumulation of vRNA at both time points, showing
that viral entry was indeed inhibited by silencing this receptor
(Fig. 2B). Despite this inhibition, Huh7 in which CD61 had
been silenced responded by up-regulation of ISG mRNAs 6 h
after exposure to UV-SNV, and ISG expression was higher,
rather than reduced, in those cells than in cells transfected with
control siRNAs (Fig. 2C). To assess the effects of neutralizing
Abs, we exposed Huh7 to SNV that had been pretreated with
human convalescent sera known to contain high titers of neutralizing Abs (38) and measured the abundance of S-segment
vRNA 24 h postinfection with live SNV, and ISG expression
6 h posttreatment with UV-SNV. Virus neutralization resulted
in a 56 – 66% decrease in vRNA titers (Fig. 2D). This neutralization did not inhibit ISG responses to UV-SNV (Fig. 2E).
The Journal of Immunology
been shown to recognize viral PAMPs. Therefore, we used gene silencing to inhibit members of these signaling pathways in responsive
Huh7 and measured ISG transcription elicited by UV-SNV or control
ligands. We transfected Huh7 with panels of siRNAs directed against
RIG-I or MDA5, or the downstream adaptor molecule for both helicases, IPS-1, and demonstrated diminished steady-state levels of these
mRNAs as determined by real-time qRT-PCR studies (Fig. 5A). As
expected, responses to SeV were almost completely inhibited by
knockdown of both RIG-I and IPS-1. However, ISG induction by
UV-SNV was unaffected by silencing of RIG-I and only slightly inhibited by IPS-1 silencing (Fig. 5B). Knockdown of MDA5 had no
effect on UV-SNV-mediated ISG responses (Fig. 5C). To examine
TLR-dependent pathways, we successfully silenced both TRIF and
MyD88, molecules used by all identified TLRs. Silencing of MyD88
had no effect on UV-SNV-mediated ISG inductions despite decreased
MyD88 mRNA levels (Fig. 5, A and C). Silencing of TRIF, which can
be used by TLR4, but is the sole adaptor molecule for TLR3, inhibited
ISG inductions by poly I:C as expected, but only slightly inhibited
UV-SNV-mediated ISG inductions (Fig. 5, A and C).
In this study, using a pathogenic hantavirus, we provide the first description of a pathway that is independent of the transcription factors
IRF3 and IRF7, that nonetheless leads to the activation of ISG transcription. IRF3 is activated early upon infection by several viruses and
is responsible for the transcription of a subset of ISGs, including
ISG56, in a type I IFN-independent fashion (10). IRF3 also directs the
transcription of IFN-␤, which subsequently acts to amplify the ISG
response and stimulates the expression of additional ISGs. Here, we
demonstrate that this normally critical transcription factor is dispensable for the innate immune response to SNV in human cell lines and
that ISG56 is nonetheless transcribed quickly following exposure to
hantavirus particles. Cells of the innate immune system generally detect RNA viruses through recognition of nucleic acids, although there
are a limited number of reports where viral proteins are detected by
TLRs expressed on the cell surface (43– 46). In this system, we show
that vRNA is unlikely to be the PAMP and the protein-sensing TLRs
are not responsible for ISG responses to SNV.
Others groups have demonstrated that viruses are able to induce
ISG expression following UV treatment and recently it has been
discovered that enveloped viruses can induce ISGs following interactions with the cell surface independent of the commonly implicated virus-sensing PRRs (14, 16). IRF3 activity is required in
these systems for the transcription of ISGs. The results we report
here support the findings that viral entry and these PRRs are not
essential for the initial response to virus; however, the new finding
that the innate immune system is able to detect and respond to
SNV independent of IRF3 suggests the existence of an alternate
signaling pathway that nonetheless leads to ISG transcription. It is
not surprising that the innate immune system has evolved redundant transcriptional pathways to detect viral infection. Several recent studies have focused on the discovery of virally encoded proteins that modulate the antiviral program and a number of
molecules within the recently described PRR-IRF signaling pathways, including IRF3 itself, are targeted for antagonism by viral
proteins (47). The existence of these viral defense pathways confirms the importance of the PRR-IRF pathway, but also provides
selective pressures that favor the evolution of alternative recognition mechanisms to sense invading microbes. It is possible that our
use of a pathogen that is non-native to human cells as a probe
facilitated our discovery of this novel antiviral sensing system.
Individual viruses interact with multiple cell surface receptors
and use a wide array of receptor classes to gain entry into cells
including cellular adhesion molecules, integrins, extracellular ma-
trix molecules, growth factor receptors, and complement cascade
proteins, and many of these receptors modulate cellular functions
with possible innate immunity implications (48). Although CD61
has been described as an entry receptor for hantaviruses, and possibly is an important mediator of vascular dysregulation in the
context of the pathogenic process, it is likely that other cellular
receptors participate in virus-cell interactions (29, 37, 49). SNV
may interact with additional plasma membrane receptors, such as
coreceptors for tethering or entry, and interaction with one or more
of these molecules may initiate the observed ISG transcriptional
response. We show here that CD61 is not required for the activation of ISGs, although its role in entry was confirmed.
It remains unclear whether the antiviral response described herein
contributes to SNV pathogenesis. Of potential interest in this regard is
our inability to elicit an ISG response to UV-SNV using murine embryonic fibroblasts as targets (our unpublished observations), in light
of the inability of SNV to cause disease in murine models (32). Furthermore, several differences in the response to SNV are observed
between our previous experiments in primary endothelial cells, and
the cell lines used here, that indicate that some mechanistic heterogeneity may be present in detection of SNV by human cells. Although
both cell types can be infected by SNV, neutralizing Abs block ISG
responses in endothelial cells (26), but not in Huh7 cells, and ISG
expression is activated more rapidly in Huh7. These data indicate that
either a divergent PAMP is detected by these cells, or differences in
PRR expression, context or localization, allow unique requirements
for engagement and signaling by the same PAMP. We have recently
investigated SNV-mediated ISG transcription in a number of diverse
cell lines that support or do no support SNV replication, including
those that lack expression of CD61 and that nonetheless respond by
increasing the expression of ISG mRNAs. The PRRs used by these
cell lines generate responses similar to that of Huh cells (our unpublished observations), indicating that these responses are not unique to
the widely used Huh cell lines. These results differ from what we have
previously reported in HUVEC, although additional primary cells
have not been examined. In support of the findings described here is
our previous observation that HUVEC exposed to either UV-SNV or
live SNV do not transcribe IFN-␣/IFN-␤, even at late time points
posttreatment. Because IRF3 controls the initial expression of IFN-␤,
these data suggest that IRF3 may not be essential for the immunologic
recognition of SNV by endothelial cells. Although others have shown
that IRF3 and IRF7 are activated in endothelial cells upon infection
with hantaviruses at later time points than those examined here, it was
not determined whether these transcription factors were required for
the observed transcription of ISGs in those systems (30, 50).
In summary, SNV particles can induce the expression of ISGs rapidly without a requirement for IRF3 or IRF7. To our knowledge, this
is the first demonstration of the activation of the innate immune response that does not require these transcription factors to respond to
a viral infection. Because viral entry is not required for the initiation
of ISG transcriptional activation, the cellular responses to the virus
particle are transduced directly from the plasma membrane. Furthermore, activation is independent of the TLR and CARD-containing
RNA helicase families of receptors commonly identified as important
for these responses. Considered together, these results suggest that a
heretofore-undefined PAMP-PRR pathway is engaged by SNV that
can induce expression of ISGs without the need for IRF3 or IRF7. The
SNV PAMP offers a useful instrument through which the identity of
this alternative PAMP-PRR axis can be dissected.
Acknowledgments
We thank Ganes Sen from the Lerner Research Institute for providing the
TLR3 expression clone and for helpful discussion, and Michael Gale from the
University of Texas Southwestern for providing the RIG-I expression clone.
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Discussion
1801
1802
Disclosures
The authors have no financial conflict of interest.
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IRF3-INDEPENDENT INNATE IMMUNE RESPONSE TO VIRUS