JOURNAL OF VIROLOGY, Feb. 2005, p. 1343–1350
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Vol. 79, No. 3
West Nile Virus Inhibits the Signal Transduction Pathway of
Ju-Tao Guo, Junpei Hayashi, and Christoph Seeger*
Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, Pennsylvania
Received 4 August 2004/Accepted 3 September 2004
West Nile virus (WNV) is a human pathogen that can cause neurological disorders, including meningoen-
cephalitis. Experiments with mice and mammalian cell cultures revealed that WNV exhibited resistance to the
innate immune program induced by alpha interferon (IFN-?). We have investigated the nature of this
inhibition and have found that WNV replication inhibited the activation of many known IFN-inducible genes,
because it prevented the phosphorylation and activation of the Janus kinases JAK1 and Tyk2. As a conse-
quence, activation of the transcription factors STAT1 and STAT2 did not occur in WNV-infected cells.
Moreover, we demonstrated that the viral nonstructural proteins are responsible for this effect. Thus, our
results provided an explanation for the observed resistance of WNV to IFN-? in cells of vertebrate origin.
West Nile virus (WNV) is an enveloped positive-strand
RNA virus which, along with other mosquito-borne human
pathogenic viruses, including Yellow fever virus and dengue
viruses, belongs to the genus Flavivirus in the family Flaviviri-
dae (3). WNV infects migratory and other birds, which produce
high virus titers in their blood and thereby permit transmission
of the virus to mosquitoes and, eventually, to humans. Al-
though WNV was isolated from an individual in Uganda more
than 6 decades ago, it has been recognized as a major human
pathogen only recently, when outbreaks of human encephalitis
were reported in Romania, Russia, Israel, and, in particular, in
New York City in 1999 (1, 19, 31). Subsequently, the virus has
spread throughout the continental United States. Whether re-
cent disease outbreaks were a consequence of the emergence
of new, pathogenic WNV strains or reflected a lack of immu-
nity in the population is not yet known. Phylogenetic analyses
based on the nucleotide sequence of a segment of the envelope
gene led to the classification of WNV isolates into two lineages
(19). This analysis also revealed a close relationship among the
WNV isolates involved in the recent outbreaks in the western
Infectious cDNA clones were first reported for Kunjin virus,
a subtype of WNV belonging to lineage 1, and later for two
WNV isolates representing both lineages (16, 32, 36). The
genomes are approximately 11 kb long and contain a large
open reading frame that is flanked by noncoding regions con-
taining the promoters for RNA-dependent RNA synthesis (3,
35). The polyprotein is processed into 10 polypeptides by cel-
lular and viral proteases. Three of these products are structural
proteins required for capsid formation (capsid protein) and
assembly into enveloped viral particles (premembrane and en-
velope proteins). The nonstructural (NS) proteins comprise a
serine protease and ATP-dependent helicase (NS3), a RNA-
dependent RNA polymerase (NS5), and a cofactor of the NS3
protease (NS2B). The functions of the remaining four NS
proteins, NS1, NS2A, NS4A, and NS4B, are not yet known.
Khromykh and Westaway demonstrated that subgenomic rep-
licons of Kunjin virus expressing the NS proteins were compe-
tent for RNA replication (17). Their studies set the path for
the development of similar replicon systems with other mem-
bers of the Flaviviridae, including Hepatitis C virus (HCV) (23).
Like other arboviruses, WNV has the remarkable ability to
replicate and assemble virus particles in insect and mammalian
cells; hence, it can complete its life cycle under very different
environmental conditions. Both invertebrates and vertebrates
rely on cellular antiviral programs, the innate immune re-
sponse, to regulate amplification of viral genomes and to pro-
tect cells from infection. Darnell et al. discovered that WNV,
compared with other viruses, including vesicular stomatitis vi-
rus (VSV) and Sindbis virus, exhibited a marked resistance to
the innate immune response elicited by alpha interferon
(IFN-?) (4, 5). Moreover, additional studies with WNV and
Dengue virus revealed that IFN-? did not inhibit viral replica-
tion following the establishment of an infection, suggesting
that expression of one or several viral proteins may inhibit the
IFN response (7, 24, 27). These observations stand in marked
contrast with results reported with HCV, demonstrating that
this virus is very sensitive to the antiviral program induced by
IFN-? in tissue culture cells (2, 12, 15).
IFNs mediate their biological functions by binding to their
cognate receptors on target cells, which induces a signal trans-
duction pathway leading to the induction of tens or even hun-
dreds of genes (6, 21). Besides the IFN receptors, the principal
components include the Janus tyrosine kinases JAK1, JAK2,
and Tyk2 and the latent transcription factors STAT1, STAT2,
and IRF9 (p48). The IFN-induced signals are transduced
through sequential phosphorylations of tyrosine residues on
the Janus kinases, the IFN receptors, and the STAT proteins.
The induction of gene expression is mediated by the formation
of multimeric transcription factors that bind to their cognate
DNA sequence motifs located in the promoters of IFN-in-
duced genes (21, 33).
The purpose of this study was to investigate the basis for the
observed resistance of WNV to the antiviral program induced
by IFN-?. The results showed that the expression of one or
* Corresponding author. Mailing address: Institute for Cancer Re-
search, Fox Chase Cancer Center, 333 Cottman Ave., Philadelphia, PA
19111. Phone: (215) 728-4312. Fax: (215) 728-4329. E-mail: seeger
more of the WNV NS proteins interfered with the activation of
an early step in the JAK-STAT signal transduction pathway.
MATERIALS AND METHODS
Cells, viruses, and reagents. HeLa, Vero, and BHK cells were maintained in
Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine se-
rum, penicillin G, streptomycin, nonessential amino acids, and L-glutamine. SL1
is a HeLa cell line expressing an HCV subgenomic replicon (40). The KUNCD20
cells were derived from a pool of approximately 200 colonies of G418-resistant
HeLa cells obtained after transfection with the Kunjin virus subgenomic replicon
C20DxrepNeo RNA (14, 17). The cell lines were cultured in medium containing
500 ?g of G418/ml. WNV (lineage 2) was produced in BHK cells that were
electroporated with 1 ?g of synthetic RNA transcribed from plasmid
pSP6WNrep/Xba as described by Yamshchikov et al. (36) (accession number
Recombinant IFN-?2b (Intron A) and IFN-? were obtained from Schering-
Plough and Pierce, respectively. Antibodies against STAT1, STAT2, JAK1, Tyk2,
IFN-?/? receptor chain 1 (IFNAR1), and ?-actin were obtained from Santa Cruz
Biotechnology. Antibodies against p-STAT1, p-JAK1, and p-Tyk2 were obtained
from Cell Signaling Technologies. Antibodies against p-STAT2 and IFNAR2
were obtained from Upstate Biotechnology and Pierce, respectively.
RNA extraction and Northern blot hybridization. SL1 and KUNCD20 cells
were left untreated or were treated for 3 days with the indicated concentrations
of recombinant IFN-?2b (Intron A; Schering-Plough) and IFN-? (Pierce), re-
spectively. Total cellular RNA was extracted with TRIzol reagent (Invitrogen)
following the manufacturer’s directions. Five micrograms of total RNA was
fractionated on a 1% agarose gel containing 2.2 M formaldehyde and transferred
onto nylon membranes. Membranes were hybridized with a riboprobe specific for
the neomycin phosphotransferase II (NPT II) gene.
Western blot analysis. For the analysis of STAT1 and STAT2 phosphoryla-
tion, cells were left untreated or were treated for 30 min with 1,000 IU of IFN-?
and IFN-?/ml, respectively. For the analysis of IFN receptor and its associated
JAK family of protein tyrosine kinases, cells were cultured in Dulbecco’s mod-
ified Eagle’s medium containing 1% fetal bovine serum for 24 h and then mock
treated or treated for 15 min with 1,000 IU of IFN-? and IFN-?/ml, respectively.
Upon the completion of treatment, cells in 100-mm-diameter petri dishes were
washed once with ice-cold phosphate-buffered saline (PBS) and subsequently
lysed with 800 ?l of lysis buffer (20 mM Tris-HCl [pH 8.0], 100 mM NaCl, 1 mM
EDTA, 1% NP-40, 1% sodium deoxycholate, and 0.1% sodium dodecyl sulfate
[SDS]) supplemented with protease inhibitor cocktail (Roche) and 0.1 mM
sodium orthovanadate. Lysates were centrifuged for 5 min at 10,000 ? g at 4°C.
Equal amounts of cell lysates were separated by SDS-polyacrylamide gel elec-
trophoresis, transferred on Immuno-P membrane (Millipore), probed with an-
tibodies for STAT1? p91 (sc-417; Santa Cruz Biotechnology) and STAT2 (sc-
1668; Santa Cruz Biotechnology), phosphotyrosine 701-STAT1 (9171; Cell
Signaling Technologies), phosphotyrosine 689-STAT2 (07-224; Upstate Tech-
nology), IFNAR1 (sc-7391; Santa Cruz Biotechnology), JAK1 (sc-277; Santa
Cruz Biotechnology), Tyk2 (sc-169; Santa Cruz Biotechnology), phosphotyrosine
1022/1023-JAK1 (3331; Cell Signaling Technologies), phosphotyrosine 1054/
1055-Tyk2 (9321; Cell Signaling Technologies), or ?-actin (sc-1616; Santa Cruz
FIG. 1. Antiviral activities of IFN-? and IFN-? against HCV and Kunjin replicons in HeLa cell lines. (A) Viral RNA levels present in SL1 and
KUNCD20 cells incubated with increasing concentrations of IFN-? (0 and 10?4to 100 IU/ml in increments of 10; lanes 1 to 8 and 10 to 17) and
IFN-? (lanes 18 to 25 and 27 to 34) for 72 h were determined by Northern blot analysis. rRNA (28S rRNA) served as a control for the amount
of RNA loaded per lane. (B) The amounts of HCV RNA were determined with a Fuji phosphoimager. The values plotted as e, ?, E, and ‚ are
the percentages of the values obtained with the cells in lanes 1, 18, 10, and 27, respectively, of panel A. KUN, KUNCD20 cells.
FIG. 2. Replication of Kunjin replicons in HeLa cells blocks the
antiviral response of IFN-? and IFN-? against VSV. Normal HeLa,
SL1, and KUNCD20 (KUN) cells were incubated for 24 h with the
indicated concentrations of IFN-? and IFN-? and then infected with
VSV at a multiplicity of infection of 1. Twenty-four hours later, cells
were stained with crystal violet to determine the cytopathic effects. All
cell lines were assayed in duplicate. The cells in columns C were not
infected with VSV.
1344 GUO ET AL.J. VIROL.
Biotechnology), and visualized with Super-Signal chemiluminescence reagents
DNA microarray analysis. Normal HeLa and KUNCD20 cells were mock
treated or treated with 100 IU of IFN-? or IFN-?/ml for 6 h, and total cellular
RNAs were extracted with TRIzol reagent (Invitrogen) and further purified with
a RNeasy kit (QIAGEN). A total of 25 ?g of each RNA sample was used as a
template to make double-stranded cDNA by reverse transcription with oli-
go(dT)12–18primer and deoxynucleoside triphosphate mixed with amino allyl-
dUTP by using a cDNA indirect labeling kit essentially as described previously
(13). The cDNA was then purified and labeled with N-hydroxysuccinimide ester
containing Cy3 or Cy5 dye in coupling reactions (Amersham Biosciences). The
labeled probes were purified by using a QIAquick PCR purification kit (QIA-
GEN) by following the manufacturer’s directions and hybridized to a microarray
containing 15,552 human oligonucleotide sets obtained from MWG Biotech
(High Point, N.C.) in 38 ?l of hybridization buffer (5? SSC [1? SSC is 0.15 M
NaCl plus 0.015 M sodium citrate], 0.1% SDS, and 50% formamide) at 42°C for
16 h. Arrays were then washed and analyzed with an Affymetrix 428 scanner. The
raw data were analyzed with Imagene 5.6 and Genesight 4 software (Biodiscov-
ery). The complete data set of the microarray experiments can be viewed at the
following URL: http://www.fccc.edu/research/labs/seeger/docs/.
CPE assay. Cells were seeded in 24-well tissue culture dishes with 5 ? 104cells
per well and were incubated for 24 h. The cells were treated with the indicated
concentrations of IFN-? or IFN-? for 24 h and then infected with VSV strain
Indiana at a multiplicity of infection of 1. Twenty-four hours postinfection, the
cells were washed once with PBS, fixed with 10% formaldehyde, and then stained
with crystal violet to examine the cytopathic effect (CPE).
Immunofluorescence. Cells cultured on glass coverslips in six-well plates were
mock-treated or treated with 1,000 IU of IFN-? or IFN-?/ml for 30 min and fixed
with 4% of paraformaldehyde in PBS for 10 min. After three washes with PBS,
cells were permeabilized with 0.5% Triton X-100 in a blocker solution (2%
bovine serum albumin and 10% fetal bovine serum in PBS) for 30 min. Samples
were then incubated with mouse monoclonal antibodies for STAT1 and STAT2
for 1 h. Fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse immu-
noglobulin G (IgG; Jackson Laboratories) was used to visualize STAT1 and
STAT2 proteins. For the double immunostaining of WNV-infected HeLa cells,
cells cultured on glass coverslips in six-well plates were infected with WNV.
Twenty-four hours postinfection, cells were treated with IFNs, fixed with 4% of
paraformaldehyde, and permeabilized with 0.5% Triton X-100 in the blocker
solution as described above. After each subsequent antibody exposure, samples
were washed with PBS and blocked with blocker solution. Antibody staining was
performed sequentially, starting with STAT1 or STAT2 antibody at a 1:100
dilution, followed by FITC-conjugated goat anti-mouse IgG at a 1:200 dilution
and then West Nile hyperimmune ascitic fluid (V554-701-562; American Type
Culture Collection) at a dilution of 1:1,000 and rhodamine-conjugated goat
anti-mouse IgG (Jackson Laboratories) at a dilution of 1:1,000. Cell nuclei were
stained with DAPI (4?,6?-diamidino-2-phenylindole). The slides were examined
TABLE 1. IFN-?-inducible gene expression is inhibited in KUNCD20 cells
Gene identification no. and description
Mean induction level fora
HeLa cellsKUNCD20 cells
NM_001548_1; IFN-induced protein with tetratricopeptide repeats 1; ifit1
XM_038988_1; similar to unknown (protein for image: 3155889)
NM_005101_1; IFN-stimulated protein, 15 kDa; isg15
NM_006417_1; HCV-associated microtubular aggregate; mtap44
AL160008.3.1.85671.1; Ensembl Genscan prediction
NM_002053_1; guanylate binding protein 1, IFN inducible, 67 kDa; gbp1
NM_004223_1; ubiquitin-conjugating enzyme E2l6; ube2l6
AF086924_1; protein phosphatase 2al b gamma subunit; imypnol
NM_017654_1; hypothetical protein Flj20073; flj20073
NM_016323_1; cyclin-e binding protein 1; loc51191
BC005986_1; unknown (protein for image 3685398)
NM_002463_1; myxovirus (influenza) resistance 2; mx2
NM_032036_1; Tlh29 protein precursor; tlh29
NM_005531_1; IFN-?-inducible protein 16; ifi16
AB025254_1; tudor repeat associator with PCTAIRE 2
NM_001572_1; IFN regulatory factor 7 isoform a; irf7
NM_016817_1; 2?, 5?-oligoadenylate synthetase 2, isoform p71; oas2
NM_012420_1; retinoic acid- and IFN-inducible protein (58kDa), ri58
NM_002534_1; 2?, 5?-oligoadenylate synthetase 1, isoform e16, oas1
NM_006187_1; 2?, 5?-oligoadenylate synthetase 3; oas3
NM_007315_1; signal transducer and activator of transcription 1, 91 kDa; stat1
NM_015474_1; Dkfzp564a032 protein, samhd1
NM_022750_1; hypothetical protein Flj22693; flj22693
NM_006820_1; hypothetical protein, expressed in osteoblast; gs3686
NM_002675_1; promyelocytic leukemia protein, isoform 6; pml
NM_002462_1; myxovirus (influenza) resistance 1, mx1
AF159441_1; phospholipid scramblase 2
BC017212_1; unknown (protein for mgc 10254)
NM_014314_1; RNA helicase, rig-1
AF297093_5; UDP glucuronosyltransferase 1a6; ugt1a6
NM_024293; hypothetical protein Mgc3035, mgc3035
NM_007346_1; 7–60 protein; ogfr
NM_017414_1; ubiquitin specific protease 18; usp18
NM_003733_1; 2?, 5?-oligoadenylate synthetase-like; oas1
NM_001549_1; IFN-induced protein with tetratricopeptide repeats 4; ifit4
NM_006084_1; IFN stimulated transcription factor 3, gamma (48 kDa)
AF167472_1; double-stranded RNA-activated protein kinase; pkr
AF307338_1; b aggressive lymphoma long isoform; bal
NM_016582_1; peptide transporter 3; pht2
NM_003641_1; IFN-induced transmembrane protein 1 (9–27); ifitm1
aMean induction levels for two experiments are shown.
VOL. 79, 2005 INHIBITION OF IFN RESPONSE BY WNV 1345
with a Nikon fluorescence microscope and photographed with a charge-coupled
Attenuated IFN responses in cells expressing Kunjin but
not HCV subgenomes. Our investigations were prompted by
the observation that the replication of subgenomic replicons
expressing the NS proteins of Kunjin virus was much more
resistant than the replication of similar replicons derived from
HCV to the antiviral response induced by both IFN-? and
IFN-? (Fig. 1) (14). The 50% inhibitory concentration of
IFN-? against Kunjin virus was approximately 100 times that
observed with HCV replicons; with IFN-?, the 50% inhibitory
concentration differed about 1,000-fold. Although Kunjin virus
and WNV belong to the same lineage of known WNV isolates,
sharing more than 95% amino acid homology, we will refer to
the subgenomic replicon as Kunjin, which is consistent with the
original nomenclature of this isolate (17).
To determine whether the replication of Kunjin replicons
led to a general inhibition of the IFN response or whether the
replicons were otherwise protected from the antiviral program
induced by the cytokines, we compared the protective effects of
IFN-? and IFN-? on VSV infection in HeLa cells, in HeLa
cells expressing HCV (termed SL1), and in HeLa cells express-
ing Kunjin virus subgenomes (termed KUNCD20). The results
showed that IFN-? did not protect KUNCD20 cells from the
cytopathic effects caused by VSV and that IFN-? had only a
modest effect (Fig. 2). In contrast, the cytokines were effective
in HeLa and HCV-replicating SL1 cells. As reported previ-
ously, a fraction of SL1 cells underwent apoptosis in the pres-
ence of the cytokine (14). Hence, these results indicated that
the resistance of Kunjin replicons to IFN was caused by a more
general inhibition of the IFN response in KUNCD20 cells
rather than by the absence of a viral target for the IFN-induced
innate immune response.
Replication of Kunjin subgenomic replicons in HeLa cells
inhibited IFN-induced gene expression. To gain a better un-
derstanding of the mechanism responsible for the observed
inhibition of the IFN response in KUNCD20 cells, we per-
formed DNA microarray analyses to compare the induction of
IFN-stimulated genes (ISG) in parental HeLa and KUNCD20
cells treated with IFN-?. The results revealed that after IFN-?
treatment, many of the known ISG were expressed at reduced
levels in KUNCD20 cells compared with HeLa cells (Table 1).
Among 41 genes that were induced more than 2-fold in HeLa
cells, the levels of inhibition in KUNCD20 cells varied from
more than 10-fold (ifit1, p56) to 2- to 3-fold depending on the
gene. Notably, the induction of at least one gene [NM_003641_
1; interferon-induced transmembrane protein 1 (9-27); ifitm1]
was not inhibited by Kunjin replication, and five other genes
differed less than twofold, suggesting that the observed inhibi-
tion was not absolute. Nevertheless, these results invoked the
hypothesis that one or several NS proteins interfered with the
activation of the IFN-induced antiviral program. Consistent
with the CPE results (Fig. 2), IFN-? treatment of SL1 cells
resulted in the induction of ISG essentially as observed with
HeLa cells (J. P. Hayashi, J.-T. Guo, and C. Seeger, unpub-
WNV infection prevented phosphorylation of STAT1 and
STAT2. To test our hypothesis, we investigated the levels of
tyrosine phosphorylation and nuclear translocation of STAT1
and STAT2 in IFN-treated cells. Both events are required for
the induction of IFN-induced genes. Analyses of total STAT1
and STAT2 proteins in cell extracts prepared from IFN-?- and
IFN-?-treated cells showed that they were present at similar
levels in all three cell lines analyzed (Fig. 3A). In contrast, the
levels of tyrosine-phosphorylated STAT1 and STAT2 were
reduced in IFN-?-treated KUNCD20 cells compared with
HeLa or SL1 cells. In IFN-?-treated KUNCD20 cells, some
phosphorylated STAT1 could still be detected, which could
FIG. 3. WNV infection prevents the activation of STAT1 and STAT2. (A) Normal HeLa, SL1, and KUNCD20 cells were left untreated or were
treated with 1,000 IU of IFN-? or IFN-?/ml for 30 min., and equal amounts of cell lysates were separated by SDS-polyacrylamide gel
electrophoresis and transferred onto Immobilon-P membranes. The expression of STAT and tyrosine-phosphorylated STAT proteins were
detected by Western blot analysis with the corresponding specific antibodies. ?-Actin levels served as a loading control. (B) Vero cells were infected
with WNV, and at 24 h postinfection, the cells were left untreated or treated for 30 min. with 1,000 IU of IFN-? or IFN-?/ml. Western blot analysis
was performed as described above. The figure shows results from one of three experiments that yielded consistent results.
1346GUO ET AL. J. VIROL.
explain the attenuated antiviral response observed with VSV-
infected cells (Fig. 2). However, the amounts of phosphory-
lated STAT proteins in HeLa and SL1 cells were relatively low;
therefore, we could not determine whether the levels of phos-
phorylated STAT1 in KUNCD20 cells were reduced more than
two- to threefold compared with the other HeLa cells.
To validate our observations made with HeLa cells replicat-
ing subgenomic Kunjin replicons, we performed additional
experiments with Vero cells that were infected with WNV
released from BHK cells transfected with an infectious full-
length cDNA clone (see Materials and Methods). The Vero
cells were harvested 24 h after infection, when almost all of the
cells were infected as determined by immunofluorescence but
before the appearance of CPE (results not shown). While the
accumulation of total STAT1 and STAT2 proteins was ele-
vated in WNV-infected Vero cells, IFN-?-induced phosphor-
ylation of both STAT proteins was significantly inhibited com-
pared with control cells (Fig. 3B). As with KUNCD20 cells,
IFN-?-induced STAT1 phosphorylation could be detected in
WNV-infected Vero cells, but at a significantly reduced level
compared with uninfected cells.
To further confirm our results, we determined the cellular
distribution of STAT1 and STAT2 in IFN-treated HeLa cells.
As expected, IFN-? induced the nuclear translocation of both
proteins in HeLa and SL1 cells. In contrast, in IFN-?-treated
KUNCD20 cells both STAT proteins remained in the cyto-
plasm (Fig. 4A and B). STAT1 could be detected in the nuclei
of IFN-?-treated KUNCD20 cells, which was consistent with
the observed accumulation of tyrosine-phosphorylated STAT1
in these cells (Fig. 3A and 4A). Similar observations were
made with WNV-infected HeLa cells that were used in lieu of
Vero cells for these experiments. Under our experimental con-
ditions, approximately 40% of HeLa cells were infected by
WNV. IFN-?-induced nuclear translocation of both STAT
proteins was completely blocked only in cells that expressed
WNV proteins (Fig. 5A and B). In contrast, in IFN-?-treated
cells STAT1 appeared to accumulate in both the cytoplasmic
and nuclear compartments.
Taken together, our results showed that WNV and Kunjin
subgenomes did not inhibit the accumulation of total STAT
proteins, but either reduced the steady-state levels of tyrosine
phosphorylated STAT proteins, inhibited their phosphoryla-
FIG. 4. Nuclear accumulation of STAT1 and STAT2 in the presence of IFN-? and IFN-?. Normal HeLa, SL1, and KUNCD20 cells were
seeded on glass coverslips and left untreated (control) or were treated with 1,000 IU of IFN-? or IFN-?/ml for 30 min. The cells were fixed with
4% paraformaldehyde, permeabilized with 0.5% Triton X-100, and incubated first with STAT1 (A) and STAT2 (B) monoclonal antibodies and
then with FITC-conjugated goat anti-mouse IgG. The cell nuclei were stained with DAPI. The slides were examined with a Nikon fluorescence
microscope and photographed with a charge-coupled device camera.
VOL. 79, 2005INHIBITION OF IFN RESPONSE BY WNV 1347
tion, or both. Hence, our results were consistent with the hy-
pothesis predicting that replication of WNV blocked the signal
transduction pathways responsible for the activation of IFN-
?-and IFN-?-induced genes.
WNV reduces the tyrosine phosphorylation of JAK1 and
Tyk2. A first step in the activation of the IFN signal transduc-
tion cascade is the autophosphorylation of the tyrosine kinases
JAK1 and Tyk2 in response to a ligand-induced conforma-
tional change of the IFN-?/? receptors. Expression of Kunjin
replicons in HeLa cells did not significantly alter the steady-
state levels of JAK1 and Tyk2, whereas WNV infection of
Vero cells slightly down-regulated the expression of both ki-
nases (Fig. 6). However, IFN-?-induced phosphorylation of
both JAK1 and Tyk2 was efficiently blocked in KUNCD20 and
in WNV-infected Vero cells (Fig. 6). Moreover, reduced levels
of JAK1 phosphorylation were also observed with IFN-?-
treated KUNCD20 and WNV-infected cells. As expected,
IFN-? treatment did not induce Tyk2 phosphorylation in any
of the cell lines examined during this study.
Because JAK1 and Tyk2 phosphorylation is the first event in
the IFN-induced phosphorylation cascade, our results sug-
gested that the observed inhibition could occur at the level of
the IFN-?/? receptor. Therefore, we sought to compare the
levels of IFN-?/? receptor in normal and WNV-expressing
cells. The results showed that the total levels of IFNAR1 as
well as the surface expression of IFNAR2 did not vary in a
significant way among normal HeLa, SL1, and KUNCD20 cells
and between normal and WNV-infected Vero cells (Fig. 6 and
results not shown). We concluded that WNV likely interferes
with signaling between the IFN receptor and JAK1/Tyk2.
Our results demonstrated that WNV inhibits one of the first
steps of the IFN signal transduction pathway and, hence, pro-
vided an explanation for previous observations made with
WNV-infected mice and tissue culture cells (5, 27). Interac-
tions between viral proteins and components of the innate
immune response are major determinants in viral pathogene-
sis. This fact is best exemplified with genetically modified mice
that are deficient in their response to IFN-? due to the lack of
functional IFN-?/? receptors or STAT proteins and, as a con-
sequence, rapidly succumb to viral infections (9, 26, 28). On
the other hand, most, if not all, viruses depend on mechanisms
to attenuate the IFN response for their survival (22). For
example, certain parainfluenza viruses can inhibit the IFN
pathway in human cells with the help of the accessory protein
V that induces the proteasome-dependent degradation of
STAT proteins (8, 30).
The exact mechanism by which WNV inhibits the IFN re-
sponse is not yet known. Our results showed that the structural
proteins are not required and, hence, by inference, have in-
voked a role for one or several NS proteins in blocking the
activation of the innate immune response. However, it is also
possible that the mechanism of inhibition was indirect. For
example, it could have been caused by changes in the cyto-
plasm of infected cells that are known to occur as a conse-
quence of viral replication. These changes include the induc-
tion and rearrangement of intracellular membranes (25, 34).
Because HCV replication induces similar changes (10) and yet
is very sensitive to IFNs (Fig. 1), we do not favor this possibil-
ity. Instead, we propose the following scenarios to explain our
results. First, NS proteins may directly bind to the IFN recep-
tor and block the activation of the Janus kinases. This model
would imply that the NS viral proteins bind to both IFN re-
ceptors, because both IFN responses were inhibited albeit with
slightly different efficiencies (Fig. 2, 3, and 6). Alternatively, NS
proteins could directly interact with JAK1 and Tyk2 and inhibit
their tyrosine kinase activities, perhaps similar to the V pro-
teins of human parainfluenza viruses as described above. In
fact, we observed a more profound inhibition of Tyk2 than of
JAK1, which could be caused by differences in the affinity of
one or more NS proteins for the two kinases. Second, WNV
replication may lead to the activation of negative feedback
loops that could inhibit or reverse JAK1 and Tyk2 phosphor-
ylation. The suppressor of cytokine signaling-3 (SOCS3) or the
protein tyrosine phophatases, such as the Src homology 2
(SH2) domain-containing protein tyrosine phophatases (SHP-
2), are known members of such pathways (18, 39). For exam-
ple, herpes simplex virus type I (HSV1) was recently shown to
FIG. 5. Localization of STAT1 and STAT2 in WNV-infected HeLa
cells. HeLa cells were seeded on glass coverslips and infected with
WNV for 24 h. The cells were then left untreated (control) or were
treated with 1,000 IU of IFN-? or IFN-?/ml for 30 min, fixed with 4%
paraformaldehyde, and permeabilized with 0.5% Triton X-100.
STAT1, STAT2, and WNV proteins were visualized by sequential
staining of the samples with STAT1 (A) or STAT2 (B) antibodies and
WNV-specific antibodies. The cell nuclei were stained with DAPI. The
slides were examined with a Nikon fluorescence microscope and pho-
tographed with a charge-coupled device camera. Arrows mark unin-
fected cells with nuclear accumulation of STAT1.
1348GUO ET AL.J. VIROL.
induce SOCS3 expression leading to the inhibition of the IFN
response (37). Finally, inhibition may occur through a reduc-
tion in the surface expression of IFN receptors. Although a
comparison of IFNAR2 present on the surface of parental
HeLa and KUNCD20 cells by flow cytometry analysis revealed
a slightly lower expression in KUNCD20 cells, similar compar-
ison with normal and WNV-infected Vero cells did not reveal
any substantial differences (results not shown). Moreover, a
Western blot analysis of IFNRA1 expression did not reveal any
significant alteration in KUNCD20 and WNV-infected Vero
cells compared with normal HeLa and Vero cells (Fig. 6 and
results not shown). Hence, based on these results, we consider
this possibility unlikely.
While this study was in progress, Mun ˜oz-Jorda ´n et al. (29)
reported that the Dengue virus replication could inhibit the
phosphorylation of STAT1 and, hence, explain the previously
observed inhibition of the IFN response. Moreover, these au-
thors provided evidence for a role of the NS protein 4B in the
modulation of Stat1 phosphorylation. However, this activity
was relatively weak compared with that of other known inhib-
itors of the IFN response and was amplified when NS4B was
coexpressed with other NS proteins. To identify the WNV
protein(s) responsible for the observed inhibition of the IFN
signaling pathway, we used a similar approach and transfected
HeLa cells with plasmids encoding individual NS proteins.
Unfortunately, our efforts have so far not yielded any positive
results (results not shown). It is possible that under our se-
lected conditions, the expression levels of individual NS pro-
teins were lower than those obtained with Dengue NS proteins
and those present in KUNCD20 or WNV-infected cells. Al-
ternatively, it is conceivable that during natural replication
viral proteins are sequestered in a manner that was not repro-
duced under our experimental conditions.
The physiological relevance of the well-documented resis-
tance of WNV to IFN treatment can so far only be explained
by inference based on other systems, because viral mutants that
have lost the ability to block the JAK-STAT pathway are not
yet available. One of the best examples is herpes simplex virus,
where a functional relationship between the viral infected cell
protein 34.5 (ICP34.5) and the IFN-inducible double-stranded
RNA-dependent protein kinase R exists that controls viral
replication (20). Interestingly, Gale and colleagues have just
reported that WNV could delay the expression of IFN-? (11).
The expression of this gene is controlled by a pathway that
depends on its activation by proteins that can sense virus par-
ticles or products of replication, such as certain viral RNA
structures or viral proteins. An example of a sensor would be
the recently identified retinoic acid inducible gene 1 (RIG-1)
(38). Hence, under physiological conditions, WNV might in-
hibit more than one cellular pathway of the innate immune
In summary, our study revealed the mechanism by which
WNV inhibits the cellular innate immune response elicited by
IFN-? and IFN-? and provided an explanation for the previ-
ously observed resistance of this virus to IFN treatment in
animals and cell cultures. These findings set the stage for the
identification of the viral proteins and cellular factors that
control the innate immune reaction and, hence, will provide
opportunities to better understand novel virus-host interac-
We thank Bill Mason and Rich Katz for their helpful comments on
the manuscript, and we acknowledge the services provided by the
FCCC tissue culture and cell imaging facilities and DNA microarray
facilities. Alex Khromykh (SASVRZ, Brisbane, Australia), Vladimir
Yamshchikov (University of Kansas), and Ron Harty (University of
Pennsylvania) provided reagents that were essential to conduct this
study. We acknowledge Margo Brinton for valuable information about
FIG. 6. WNV inhibits the tyrosine phosphorylation of JAK1 and Tyk2. (A) Normal HeLa, SL1, and KUNCD20 cells were left untreated or were
treated with 1,000 IU of IFN-? or IFN-?/ml for 15 min. Proteins from total cellular extracts were analyzed by Western blot analysis. The blots were
probed with antibodies against IFNAR1, JAK1, phosphotyrosine 1022/1023-JAK1, Tyk2, phosphotyrosine 1054/1055-Tyk2, and ?-actin. (B) Vero
cells were infected with WNV, and at 24 h postinfection, the cells were left untreated or were treated with 1,000 IU of IFN-? or IFN-?/ml for 15
min. Western blot analysis was performed as described above. Results shown are representative of those obtained from three independent
VOL. 79, 2005INHIBITION OF IFN RESPONSE BY WNV1349
WNV biology and Qing Zhu and Frank Puig for their help with flow
cytometry and the construction of plasmids.
This work was supported by grants from the National Institutes of
Health and by an appropriation from the Commonwealth of Pennsyl-
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