Differential effects of mutations in NS4B on West Nile virus replication and inhibition of interferon signaling.
ABSTRACT West Nile virus (WNV) is a human pathogen that can cause symptomatic infections associated with meningitis and encephalitis. Previously, we demonstrated that replication of WNV inhibits the interferon (IFN) signal transduction pathway by preventing the accumulation of phosphorylated Janus kinase 1 (JAK1) and tyrosine kinase 2 (Tyk2) (J. T. Guo et al., J. Virol. 79:1343-1350, 2005). Through a genetic analysis, we have now identified a determinant on the nonstructural protein 4B (NS4B) that controls IFN resistance in HeLa cells expressing subgenomic WNV replicons lacking the structural genes. However, in the context of infectious genomes, the same determinant did not influence IFN signaling. Thus, our results indicate that NS4B may be sufficient to inhibit the IFN response in replicon cells and suggest a role for structural genes, or as yet unknown interactions, in the inhibition of the IFN signaling pathway during WNV infections.
- SourceAvailable from: PubMed Central[Show abstract] [Hide abstract]
ABSTRACT: West Nile virus (WNV) is an important emerging neurotropic virus, responsible for increasingly severe encephalitis outbreaks in humans and horses worldwide. However, the mechanism by which the virus gains entry to the brain (neuroinvasion) remains poorly understood. Hypotheses of hematogenous and transneural entry have been proposed for WNV neuroinvasion, which revolve mainly around the concepts of blood-brain barrier (BBB) disruption and retrograde axonal transport, respectively. However, an over‑representation of in vitro studies without adequate in vivo validation continues to obscure our understanding of the mechanism(s). Furthermore, WNV infection in the current rodent models does not generate a similar viremia and character of CNS infection, as seen in the common target hosts, humans and horses. These differences ultimately question the applicability of rodent models for pathogenesis investigations. Finally, the role of several barriers against CNS insults, such as the blood-cerebrospinal fluid (CSF), the CSF-brain and the blood-spinal cord barriers, remain largely unexplored, highlighting the infancy of this field. In this review, a systematic and critical appraisal of the current evidence relevant to the possible mechanism(s) of WNV neuroinvasion is conducted.Viruses 07/2014; 6(7):2796-2825. · 3.28 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Previous studies have shown that an attenuated West Nile virus (WNV) nonstructural (NS) 4B-P38G mutant induces stronger innate and adaptive immune responses than wild-type WNV in mice, which has important applications to vaccine development. To investigate the mechanism of immunogenicity, we characterized WNV NS4B-P38G mutant infection in two human cell lines-THP-1 cells and THP-1 macrophages. Although the NS4B-P38G mutant produced more viral RNA than the parental WNV NY99 in both cell types, there was no detectable infectious virus in the supernatant of either cell type. Nonetheless, the attenuated mutant boosted higher innate cytokine responses than virulent parental WNV NY99 in these cells. The NS4B-P38G mutant infection of THP-1 cells led to more diverse and robust innate cytokine responses than that seen in THP-1 macrophages, which were mediated by toll-like receptor (TLR)7 and retinoic acid-inducible gene 1(RIG-I) signaling pathways. Overall, these results suggest that a defective viral life cycle during NS4B-P38G mutant infection in human monocytic and macrophage cells leads to more potent cell intrinsic innate cytokine responses. Copyright © 2014. Published by Elsevier Ltd.Vaccine 01/2015; · 3.49 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: In nature, vector-borne flaviviruses are persistently cycled between either the tick or mosquito vector and small mammals such as rodents, skunks, and swine. These viruses account for considerable human morbidity and mortality worldwide. Increasing and substantial evidence of viral persistence in humans, which includes the isolation of RNA by RT-PCR and infectious virus by culture, continues to be reported. Viral persistence can also be established in vitro in various human, animal, arachnid and insect cell lines in culture. Although some research has focused on the potential roles of defective virus particles, evasion of the immune response through the manipulation of autophagy and/or apoptosis, the precise mechanism of flavivirus persistence is still not well understood. We propose additional research for further understanding of how viral persistence is established in different systems. Avenues for additional studies include determining if the multifunctional flavivirus protein NS5 has a role in viral persistence, the development of relevant animal models of viral persistence as well as investigating the host responses that allow vector borne flavivirus replication without detrimental effects on infected cells. Such studies might shed more light on the viral-host relationships, and could be used to unravel the mechanisms for establishment of persistence.This article is protected by copyright. All rights reserved.Pathogens and Disease. 04/2014;
JOURNAL OF VIROLOGY, Nov. 2007, p. 11809–11816
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Vol. 81, No. 21
Differential Effects of Mutations in NS4B on West Nile Virus
Replication and Inhibition of Interferon Signaling?
Jared D. Evans* and Christoph Seeger
Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111
Received 12 April 2007/Accepted 8 August 2007
West Nile virus (WNV) is a human pathogen that can cause symptomatic infections associated with
meningitis and encephalitis. Previously, we demonstrated that replication of WNV inhibits the interferon (IFN)
signal transduction pathway by preventing the accumulation of phosphorylated Janus kinase 1 (JAK1) and
tyrosine kinase 2 (Tyk2) (J. T. Guo et al., J. Virol. 79:1343–1350, 2005). Through a genetic analysis, we have
now identified a determinant on the nonstructural protein 4B (NS4B) that controls IFN resistance in HeLa
cells expressing subgenomic WNV replicons lacking the structural genes. However, in the context of infectious
genomes, the same determinant did not influence IFN signaling. Thus, our results indicate that NS4B may be
sufficient to inhibit the IFN response in replicon cells and suggest a role for structural genes, or as yet unknown
interactions, in the inhibition of the IFN signaling pathway during WNV infections.
West Nile virus (WNV) is a member of the flavivirus family,
which includes hepatitis C virus, Japanese encephalitis virus
(JEV), and dengue virus (30). After binding to cellular recep-
tors, these viruses enter the cell via active endocytosis and
release their genomes into the cytoplasm through a pH-depen-
dent mechanism, allowing translation to occur immediately.
Genome replication occurs at the membrane of the endoplas-
mic reticulum (ER) within a multiprotein complex (14, 34, 57).
The genome is packaged into a virion core composed of the
structural proteins in the ER. Immature virions move through
the trans-Golgi network to the plasma membrane. Infectious
virus is released through exocytosis at early times postinfection
and by virus-induced cell death later (14, 18).
Virions are small icosahedral particles that contain a single
positive-stranded RNA molecule of approximately 11 kilo-
bases. The viral genome encodes a single polyprotein, which is
then proteolytically processed into 10 individual proteins. The
cleavage of the precursor protein is facilitated by cellular pep-
tidase and furin (or furin-like enzyme) and the viral protease
NS2B/3 (13, 54, 55). The proteins include three structural
proteins (the capsid C, membrane M, and envelope E) and
seven nonstructural (NS) proteins (glycoprotein NS1, NS2A,
protease cofactor NS2B, protease and helicase NS3, NS4A,
NS4B, and the polymerase NS5). Although incompletely char-
acterized, the NS proteins all seem important to replication.
Thus, only a few deletion and complementation studies have
been possible as most mutations totally disable virus replica-
tion (21–23, 32). Enzymatic properties have been assigned to
only two NS proteins. NS5 is a large protein containing a
domain coding the RNA-dependent RNA polymerase respon-
sible for genome replication as well as a methyltransferase
required for the formation of the cap structure at the 5? end of
the viral genome (10, 13, 17).
WNV has emerged as a major cause of viral encephalitis.
Since its introduction into North America in 1999, WNV has
rapidly spread across the continental United States and has
recently been identified in Mexico and South America (26).
WNV isolates are generally separated into two lineages (I and
II) based on phylogenetic analysis of their envelope sequences
(2). To date, only lineage I viruses have been shown to cause
neurologic disease (26). The virus is generally maintained in an
enzootic cycle between mosquitoes and birds (4). Mosquitoes
act as the primary vectors of virus transfer. Mammals are
incidental hosts that cannot act as reservoirs due to low virus
levels in the blood. Upon infection, humans first develop a
febrile illness that is generally resolved quickly (5). In approx-
imately 10% of symptomatic cases, more serious sequelae oc-
cur, including meningoencephalitis. The immunocompro-
mised, elderly, and children are at highest risk for symptomatic
infections progressing to more severe central nervous system
disease and even death. Antiviral therapies or vaccines are not
yet available to treat or prevent WNV infections. In contrast to
hepatitis C virus infections, interferon (IFN)-based therapies
appear to be ineffective against WNV infections (6, 20, 36).
IFNs act as the earliest immune mediators against virus
infections. The alpha IFN (IFN-?) receptor (IFNAR) has two
major subunits, IFNAR1 and IFNAR2c, which dimerize upon
IFN-? binding (51). Associated with the cytoplasmic domains
of the subunits are members of the Janus kinase (JAK) family.
In the absence of ligand, tyrosine kinase 2 (Tyk2) is associated
with IFNAR1 while JAK1, STAT1, and STAT2 are associated
with IFNAR2 (27). Dimerization of the receptor components
leads to the activation of JAK1, which leads to a cross-phos-
phorylation cascade with Tyk2 (7, 8, 12). Activated JAK1 and
Tyk2 tyrosine phosphorylate the STAT proteins STAT1 and
STAT2, which then interact with IFN regulatory factor 9 to
form the heterotrimeric transcription factor IFN-stimulated
gene factor 3 (ISGF-3). ISGF-3 binds to specific DNA se-
quences known as IFN-specific response elements (ISRE) that
are present in the promoters of select genes (19).
Recently, we along with others discovered that WNV and
various members of the genus Flavivirus in the Flaviviridae
* Corresponding author. Mailing address: Fox Chase Cancer Cen-
ter, Institute for Cancer Research, 333 Cottman Ave., Philadelphia,
PA 19111. Phone: (215) 728-4313. Fax: (215) 728-3574. E-mail: jared
?Published ahead of print on 22 August 2007.
family of viruses, including dengue virus and JEV, block the
IFN signal transduction pathway (3, 15, 29, 38). Replication of
these viruses inhibits the phosphorylation and activation of the
IFN receptor-associated kinases, JAK1 and Tyk2, thus block-
ing phosphorylation of the STAT proteins and the activation of
IFN-induced genes. These findings raised questions about the
nature of the viral determinants that play a role in the observed
inhibition of the IFN response. Although several reports ad-
dressing this question have been published, they have not yet
yielded firm conclusions. For example, Munoz-Jordan and col-
leagues reported that, as with dengue virus, NS4B of WNV is
the major determinant for inhibition (37, 38). In contrast, Liu
and colleagues reported that all NS proteins, save for NS1 and
NS5, could inhibit the IFN response (33). Alternatively, Lin et
al. concluded from their investigations that JEV, the closest
relative of WNV (?80% amino acid identity in NS5), inhibits
the IFN response through NS5 (28). The latter study was based
on more detailed work from Best and colleagues demonstrat-
ing that NS5 of Langat virus, a tick-borne encephalitis virus,
could antagonize the IFN response through direct binding to
the IFN-? and IFN-? receptor complexes (3).
While previous investigations relied on transient expression
assays, we used an alternative strategy based primarily on rep-
lication-competent subgenomic replicons and infectious WNV.
Our results revealed that the mechanism by which WNV in-
hibits the IFN response depends on multiple factors and is
much more complex than anticipated from previous studies.
MATERIALS AND METHODS
Plasmid construction and mutagenesis. For mutagenesis experiments, we used
plasmid C20DX-BlaM. This plasmid was derived from plasmid C20Dxrep/neo
expressing a subgenomic WNV lineage I ([WNI] Kunjin) replicon and neomycin
phosphotransferase (24). The BLaM (beta-lactamase) gene including the auto-
protease site 2A from foot and mouth disease virus was incorporated into the 5?
end of the C20DXrep/neo replicon as previously described (52, 53). The final
construct is described in Fig. 1.
To create NS4B-E22K24, we mutated NS4B residues E22K24in C20DX-BLaM
to alanines using a QuickChange mutagenesis kit (Stratagene), according to the
manufacturer’s instruction. The primers used for mutagenesis are available from
the authors on request. The mutations were verified by nucleotide sequence
analysis. To minimize the chances for the presence of unwanted mutations, a
1,216-nucleotide fragment was excised with BsiWI and AgeI and back-cloned
into naı ¨ve C20DX-BlaM. The NS4B-E22K24mutation was introduced into
C20DXrep/neo without the beta-lactamase gene; the BsiWI-AgeI fragment was
excised from the C20DX-BLaM plasmid and cloned into the corresponding
region of C20DXrep/neo to produce C20DX-EK (where EK indicates the mu-
tation of the E22K24residues to alanines).
To produce WNII-ER, NS4B residues E22R24were replaced with alanines in
the WNV lineage II (WNII) infectious clone pSP6WN/Xba. To create pWNII-
KUN4B and pWNII-KUN4B-EK infectious clones, we replaced nucleotides
6931 to 7000 of pSP6WN/Xba, encoding NS4B, with the corresponding nucleo-
tides from Kunjin replicon C20DXrep/neo and C20DX-BLaM-EK, respectively,
by a two-step PCR mutagenesis protocol (41). The sequences were verified by
restriction endonuclease digestion and DNA sequencing. The primers used for
plasmid construction are available on request from the authors.
To create pUNO-NS4B constructs, NS4B coding regions were cloned from
C20DX-BlaM and NS4B-E22K24replicons. Both constructs contained the 2-kDa
segment with the forward primer (restriction sites are highlighted in boldface)
5?-GCACCGGTCATCATGCAACGTTCGCAGACAGACAAC-3?. The reverse
primer was 5?-TACCTCTAGATCATCTTTTTAGTCCTGGTTTTTCC-3?. After
PCR amplification, PCR products were digested with AgeI and XbaI. These
fragments were cloned into pUNO using AgeI and NheI sites.
Cell culture and virus infections. HeLa, Vero, and BHK-21 cells were main-
tained at 37°C in a humidified atmosphere with 5% CO2in Dulbecco’s modified
Eagle medium (DMEM) (Gibco) supplemented with 10% fetal calf serum and
100 ?g/ml penicillin, 100 ?g/ml streptomycin, 1% nonessential amino acids
(Gibco), and 50 ?g/ml glutamine. Mouse embryo fibroblasts (MEFs) were main-
tained under similar conditions as above except in DMEM supplemented with
10% heat-inactivated fetal calf serum and 100 ?g/ml glutamine, as described
previously (45, 47).
Prior to infection, HeLa or Vero cells were seeded at a density of 1 ? 106cells
in 100-mm plates. Cells were infected with virus in phosphate-buffered saline
containing 2% fetal bovine serum for 1 h. Unadsorbed virus was removed by
washing, and DMEM supplemented with 2% fetal bovine serum was added to
the cells. After 24 h, cells were treated with IFN-?2a (Schering-Plough) or left
untreated, as described below. For growth curves, Vero cells or MEFs were
seeded at 5 ? 104cells/well in a 24-well plate. After 16 h, cells were infected.
Medium was collected at indicated times (see Fig. 7 and 9). Virus titers were
determined on BHK-21 cells as previously described (59).
In vitro transcription and transfection of RNA. To produce replicon-bearing
cells, the C20DX-BLaM plasmid (10 ?g) was linearized with XhoI. Linearized
plasmid (1 ?g) was in vitro transcribed using the SP6 in vitro transcription kit
(Promega). HeLa cells were electroporated with 5 ?g of C20DX-BLaM RNA.
Forty-eight hours posttransfection, cells were selected with 500 ?g/ml G418.
After 3 weeks of selection, colonies were harvested and expanded as pools. The
same procedure was followed to produce replicon-bearing cells without the
To produce infectious virus, WNII was produced from the plasmid construct
pSP6WN/Xba or mutants as previously described (59). BHK-21 cells were elec-
troporated with 2 ?g of in vitro transcribed RNA. Medium was collected 3 days
posttransfection, cleared of cell debris, and stored at ?70°C. Titers were deter-
mined by plaque assay on BHK-21 cells as previously described (59).
Beta-lactamase assay. HeLa or replicon-bearing cells were seeded into 24-well
tissue culture dishes at 5 ? 104cells/well for 24 h. The cells were then assayed for
beta-lactamase activity using a beta-lactamase loading solutions kit (Invitrogen)
according to the manufacturer’s instructions. Briefly, the cells were washed two
times in Opti-Mem (Gibco), incubated for 1.5 h at room temperature in Opti-
MEM containing the fluorescent substrate CCF2A, and then viewed on a Nikon
Antibodies. Rabbit polyclonal antibodies to WNV NS protein NS4B were
produced by Pacific Immunology (San Diego, CA) against a peptide spanning
positions 2 to 12 (EMGWLDKTKSD) of WNV. Goat polyclonal antibodies
against actin (sc1616; Santa Cruz Biotechnology), mouse monoclonal antibodies
against STAT1? p91 (sc417; Santa Cruz Biotechnology), and rabbit polyclonal
antibodies against phosphotyrosine 701-STAT1 (9171; Cell Signaling Technolo-
gies) were used in Western blotting experiments.
VSV cytopathic effect (CPE) assay. Parental HeLa and replicon cells were
seeded in 24-well tissue culture dishes at 5 ? 104cells/well. After 24 h, the cells
were treated with indicated concentration of IFN for 24 h and then infected with
vesicular stomatitis virus ([VSV] Indiana strain) at a multiplicity of infection
(MOI) of 1. Twenty-four hours postinfection, the cells were washed with phos-
phate-buffered saline, fixed with 10% formaldehyde for 30 min, and stained with
2% crystal violet for 20 min. Excess stain was removed with multiple rounds of
washes with water.
Western blotting. Cells were lysed in 1% Triton lysis buffer (1% Triton X-100,
150 mM sodium chloride, 50 mM Tris, pH 8.0), and protein amounts were
quantified by Bradford assay (Bio-Rad). Proteins (15 ?g) were separated by
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and
FIG. 1. Schematic of C20DX-BLaM. WNV subgenomic replicon
containing the beta-lactamase (BLaM) transgene. The coding regions
for NS4A (gray box) and NS4B are magnified. The transmembrane
domains of NS4B are represented by dashed boxes. The double-ala-
nine site-directed substitutions are highlighted. UTR, untranslated
region; IRES, internal ribosome entry cite; EMCV, encephalomyocar-
11810 EVANS AND SEEGER J. VIROL.
transferred to Immobilon-P (Millipore) membranes. Membranes were cut ac-
cording to molecular weight markers, and membrane strips were probed with the
indicated antibodies at the following dilutions: actin, 1:600; STAT1? p91, 1:500;
P-STAT1, 1:750. Horseradish peroxidase-conjugated secondary antibodies
against goat, mouse, and rabbit immunoglobulin G (Amersham) were used at a
dilution of 1:2,500. The bands were visualized using SuperSignal West Pico
RNA analysis. Total RNA was extracted with TRIzol reagent (Invitrogen)
according to the manufacturer’s instructions. For Northern blotting, 5 ?g of total
RNA was fractionated on a 1% agarose gel containing 2.2 M formaldehyde and
transferred to nitrocellulose. Membranes were hybridized with a riboprobe spe-
cific for the neomycin phosphotransferase II gene. For PCR, total RNA (1 ?g)
was used for cDNA synthesis with random primers. Real-time PCR was per-
formed in triplicate using 200 pg of amplified cDNA and primer-probe sets
specific to ?-actin, IFIT1, IFI27, and MxA (ABI Biosystems) on an ABI7000
sequence detection system. Results were normalized to ?-actin prior to deter-
mination of relative induction.
Reporter gene assays. HeLa cells were transfected using Lipofectamine 2000
(Invitrogen), according to the manufacturer’s instructions. As a positive control,
pEF-HA-HPIV2 expressing the V protein of human parainfluenza virus type 2
(HPIV2) was used (16, 39). Each transfection of 7.5 ? 104cells contained 0.8 ?g
of the plasmid of interest (HPIV2 V protein or pUNO-NS4B constructs), 0.2 ?g
of pISRE-Luc (Clontech), and 0.02 ?g of pCMV-RL (Promega). Transfections
were performed in triplicate. At 24 h posttransfection, cells were mock treated or
treated with 100 U of IFN-?2a. Cells were maintained for 24 h and then har-
vested and lysed. Luciferase assays were performed using a dual luciferase assay
system (Promega). Firefly luciferase expression was normalized to Renilla lucif-
Nucleotide sequence accession number. The nucleotide sequence of the
C20DX-BlaM construct has been deposited in the GenBank database under
accession number EF536932.
Mutations in the NS4 region. To analyze the role of WNV
NS proteins in the inhibition of IFN signaling, a panel of
mutants was created in C20DX-BLaM, a WNV subgenomic
replicon expressing the beta-lactamase transgene. We targeted
the NS4 coding regions to more clearly define their functions
in viral replication and IFN signaling. Unlike other NS pro-
teins, the NS4 proteins do not encode specific enzymatic ac-
tivities. Because both NS4A and NS4B are required in cis for
viral replication, we created mutants with only two amino acid
changes to maximize the chances for obtaining mutants sensi-
tive to IFN but competent for viral replication. We selected
regions containing clusters of five charged amino acids and
mutated two charged residues to alanine (1) (Fig. 1). Because
clusters of charged residues are generally located on the sur-
face of proteins, altering them increases the chances of dis-
rupting protein-protein interactions or functions while leaving
the overall structures intact. In vitro transcribed RNA derived
from the wild-type and the mutants was electroporated into
HeLa cells. Twenty-four hours after electroporation, more
than 25% of the cells expressed beta-lactamase, indicating that
all the genomes used in these experiments were translated with
similar efficiency (data not shown).
To identify genomes that were competent for RNA replica-
tion, we maintained the transfected HeLa cells in medium
containing G418. Only one of the eight mutants produced
colonies after 3 weeks of selection. This replicon had a muta-
tion in amino acids E22K24in the NS4B protein (NS4B-
E22K24). The colonies were pooled and expanded for all sub-
sequent analyses. Analyzing the cells for beta-lactamase
activity revealed that ?95% of the cells were positive and,
hence, replicated the mutant genomes (Fig. 2B and C). North-
ern blot analysis confirmed this result and demonstrated that
the mutant replicated in HeLa cells with an efficiency that was
comparable to wild-type replicons (Fig. 2D). Similarly, West-
ern blot analysis revealed that cells expressing wild-type and
mutant replicons expressed equivalent levels of NS4B proteins,
indicating proper genome replication and translation (see Fig.
4). Finally, transfection of BHK-21 cells with wild-type and
NS4B-E22K24replicons produced stable colonies, as observed
with HeLa cells, demonstrating that the results were not influ-
enced by cell-type-specific factors (data not shown).
Mutation in NS4B relieves inhibition of IFN signaling. In
the next step, we investigated whether expression of the NS4B-
E22K24mutant in HeLa cells inhibited the IFN response, as
observed in cells expressing the wild-type replicon. For this
purpose, we took advantage of the known antiviral activity of
IFN against VSV. We incubated cells with IFN prior to VSV
infection and determined the protective effects by observing
virus-induced cell death (Fig. 3). The results showed that IFN
protected HeLa cells from VSV-induced CPE at 10 U; in
contrast, wild-type replicon cells were not protected at any
concentration. The NS4B-E22K24mutant replicon-containing
cells were protected against lysis by 100 U of IFN, a slightly
higher amount than needed to prevent lysis in parental HeLa
cells. The results indicated that the IFN signaling pathway
remained functional in the presence of the NS4B-E22K24rep-
FIG. 2. C20DX-BLaM replicons are stable in HeLa cells. HeLa
cells were electroporated with wild type or mutant RNA. The trans-
fected cells were selected with G418. Colonies were pooled and passed
as cell lines. Parental HeLa (A), C20DX-BLaM (B), or NS4B-E22K24
(C) cells were analyzed for beta-lactamase activity. (D) Northern blot
analysis of cell lines for the amount of WNV RNA present. Actin
served as the loading control. WT, wild type; E22K24,NS4B-E22K24.
FIG. 3. The NS4B-E22K24mutation relieves WNV repression of
IFN antiviral response. Parental HeLa, C20DX-BLaM, or NS4B-
E22K24(C20DX-EK) cells were incubated for 24 h with the indicated
concentrations of IFN-? and then infected with VSV at an MOI of 1.
Twenty-four hours postinfection, cells were stained with crystal violet
to determine CPE. The cells in the control column were not infected
or treated with IFN. WT, wild type.
VOL. 81, 2007INHIBITION OF IFN RESPONSE BY WNV11811
licon and was able to induce downstream effects resulting in
protection from VSV lysis.
To validate the results obtained with VSV infection and
correlate these findings with the molecular components of the
IFN activation cascade, we incubated cells with IFN and de-
termined the levels of phosphorylated STAT1. As previously
seen with KUNCD20 cells, wild-type C20DX-BLaM replicon
cells significantly inhibited the levels of phosphorylated STAT1
(Fig. 4) (15). In contrast, HeLa cells harboring the NS4B-
E22K24replicon still exhibited stimulation of the IFN pathway,
as seen by induced phosphorylation of the STAT1 protein (Fig.
4). The level of STAT1 phosphorylation was increased in com-
parison to cells with the wild-type replicon but slightly reduced
in relation to the parental HeLa cells (Fig. 4). Surprisingly, the
overall level of STAT1 protein was decreased in the mutant
replicon cells. Although the basis for reduced STAT1 expres-
sion in cells harboring the NS4B-E22K24replicon is currently
unclear, the ratio of phosphorylated to total protein is compa-
rable to that found in parental HeLa cells.
To better quantitate the effects of wild-type and mutant
replicons on IFN signaling, we examined the induction of ISGs
in parental HeLa cells and replicon cells treated with IFN.
Using TaqMan quantitative PCR, we analyzed the expression
of three genes known to be up-regulated by IFN treatment, the
IFIT1, IFI27, and MxA genes (Fig. 5) (9). The wild-type rep-
licon inhibited ISG induction to almost the background level.
Induction of ISGs in the NS4B-E22K24replicon cells was less
than in HeLa cells but much greater than in wild-type replicon
cells. For instance, the MxA gene, which has been shown to
play an important role in antiviral activities against a number
of viruses, is induced fivefold less in NS4B-E22K24replicon
cells than in parental HeLa cells, but expression is eightfold
higher than in wild-type replicon cells. These results agree with
those above demonstrating that the mutation of two amino
acids in the NS4B coding region of the WNV replicon does not
suppress genome replication but relieves the inhibition of IFN
signaling. To validate our results, we produced a second pool
of HeLa cells expressing the NS4B-E22K24mutant in the con-
text of a C20DXrep/neo vector lacking the beta-lactamase
transgene (Fig. 1). As before, the cells expressing the mutant
were sensitive to IFN, demonstrating that the outcomes of our
experiments were not influenced by the selection of the vector.
Moreover, these cells expressed normal levels of STAT1 (Fig.
To determine the effect individually expressed NS4B pro-
teins had on IFN signaling, wild-type and mutant NS4B-
E22K24proteins were exogenously expressed in HeLa cells
treated with IFN. As a reporter for the activation of the IFN
signal transduction pathway, we used the luciferase gene ex-
pressed from the IFN-responsive ISG54 promoter. Cells trans-
fected with a plasmid expressing the V protein of HPIV, lead-
ing to the degradation of STAT2, served as a positive control
for the inhibition of IFN signaling (39). As reported previously,
NS4B inhibited activation of the IFN-responsive promoter, but
the inhibition was weaker than observed with the V protein
(Fig. 6) (37, 38). Consistent with the results described above,
wild-type NS4B blocked IFN signaling more efficiently than the
mutant (Fig. 6).
Mutations in NS4B do not affect IFN inhibition in infectious
virus. Although the replicon system faithfully mimicked cer-
FIG. 4. The NS4B-E22K24mutation cannot inhibit IFN-induced
STAT phosphorylation in replicon cells. (A) Parental HeLa, C20DX-
BLaM (wild type [WT]) and NS4B-E22K24(E22K24) cells were left
untreated or treated with 1,000 IU of IFN-? for 20 min. (B) A second
pool of replicon-bearing HeLa cells was generated with the
C20DXrep/neo replicon harboring the NS4B-E22K24mutation without
the beta-lactamase transgene. Cells were treated with IFN-?. Equal
amounts of cell lysates were separated by SDS-PAGE and transferred
to Immobilon-P membranes. The expression of STAT1, phosphory-
lated STAT (P-STAT), and NS4B proteins was detected by Western
blotting with the corresponding specific antibodies. Actin served as the
lane loading control. One representative experiment of three is shown.
FIG. 5. IFN-stimulated gene expression in replicon cells. Parental
HeLa, C20DX-BLaM (C20DX-WT), and NS4B-E22K24(C20DX-
E22K24) cells were left untreated or treated with 100 IU of IFN-? for
6 h. Total RNA was isolated by TRIzol reagent extraction. Equivalent
amounts of total RNA were reverse transcribed with gene-specific
primers to create cDNA pools. Real-time PCR with ISG-specific
TaqMan primer-probe sets was performed. Results shown are the
average of two independent experiments.
FIG. 6. Individually expressed NS4B proteins reflect activity in rep-
licons. HeLa cells were transfected with each of indicated plasmids. At
24 h posttransfection, cells were treated with 100 U of IFN-?. The
firefly luciferase activities were normalized to Renilla luciferase to
determine ISRE-specific gene induction. Firefly luciferase activities
were determined as mean values from three independent experiments
performed in triplicate (P values of 0.005 to 0.01). WT, wild type;
11812EVANS AND SEEGER J. VIROL.
tain aspects of WNV replication, it still had limitations with
respect to virus infection. For instance, replicon-bearing cells
are selected, and this leads to propagation of variants that are
less cytopathic (31, 44). Further, replicons do not express the
viral structural proteins, which play an active role in the virus
life cycle, thus eliminating their effects as well as possible
interactions between NS and structural proteins.
Therefore, to analyze the effects of the NS4B-E22K24muta-
tion in a more natural context, we introduced the correspond-
ing mutations into a WNII infectious clone. The WNII-derived
clone was used because attempts to introduce the mutations
into an infectious clone derived from lineage I (NY99) were
unsuccessful due to instability of the plasmid during mutagen-
esis (48). Although WNII and Kunjin virus (lineage I) exhibit
very high overall amino acid homology (?90%), they differ
between residues 11 and 32 in NS4B. Moreover, NS4B in
WNII carries an arginine (R) residue in lieu of lysine (K) at
position 24. To minimize the chance for any lineage- or con-
text-specific effects, we also produced chimeric WNII infec-
tious clones with Kunjin sequences spanning amino acids 10 to
33 in NS4B and introduced the E22K24mutations, yielding
mutant WNII-KUN4B-EK. The resulting RNAs were trans-
fected into BHK-21 cells, and virus was collected 3 days later.
The presence of the mutations was verified by sequence anal-
ysis (data not shown). The titer of the WNII-ER mutant virus
was approximately fivefold less than the wild type. The wild-
type and mutant chimeric viruses (WNII-KUN4B and WNII-
KUN4B-EK) replicated to levels equivalent to parent WNII.
Further, in single-step growth curves in Vero cells, both mu-
tants replicated to identical levels compared to their respective
wild types (Fig. 7).
HeLa cells were incubated with wild-type or mutant virus at
an MOI of 1 to ensure that a majority of cells were infected,
and then cells were treated with IFN to analyze signaling.
Western blotting for phosphorylated STAT1 showed that wild-
type and mutant viruses all completely inhibited IFN stimula-
tion (Fig. 8). The duration of infection did not influence these
results. Moreover, the viruses behaved identically in Vero cells,
suggesting that the effects were not cell type specific (data not
shown). Further, the chimeric viruses WNII-KUN4B and
WNII-KUN4B-EK were analyzed for their ability to block IFN
signaling. Both chimeric viruses inhibited STAT1 phosphory-
lation in both HeLa and Vero cells (data not shown). These
observations were surprising since they did not reflect the
results seen with the replicon-bearing cells.
The infection experiments in HeLa cells could have masked
a subtle effect of the NS4B mutations, since they were per-
formed with a relatively high infectious dose. To further char-
acterize the NS4B virus mutant, we analyzed the impact of IFN
production and response on mutant virus replication and
spread. We infected MEFs derived from wild-type B6 mice,
which are competent for IFN production and activation. To
control for any effects not related to the IFN response, we
infected MEFs from congenic IFN-?/? receptor knockout an-
imals. Low-passage MEFs were infected with WNII and mu-
tant viruses at low MOIs of 0.2 and 0.002 and assayed for virus
production. The mutant viruses exhibited slightly lower virus
titers at early times (12 h postinfection) but produced levels of
virus equivalent to wild-type later (24 to 72 h postinfection).
Since the mutants exhibited a similar delay in MEFs lacking
the IFN-?/? receptor, this effect was not caused by activity of
the IFN system but most likely reflected reduced replication
following the infection. Although the starting inocula did not
appear to make a significant difference in the outcome of the
experiments, IFN-?/? receptor knockout MEFs produced
higher amounts of virus than wild-type cells. This observation
is consistent with the proposed activation of IFN-? following
FIG. 7. Mutant viruses exhibit growth kinetics similar to wild type. Vero cells were infected at an MOI of 0.5 with WNII, WNII-ER,
WNII-KUN4B, or WNII-KUN4B-EK for 12, 24, 48, and 72 h. Virus titers in medium from infected cells were determined by plaque assay on
FIG. 8. Mutant viruses inhibit STAT1 phosphorylation. HeLa cells
were infected at an MOI of 1 with WNII or WNII-ER for 24 h. The
infected (lanes 3 and 4) or uninfected (Uninf; lane 2) cells were treated
with 1,000 U of IFN-? (?IFN) for 20 min. As a control, cells were not
infected (Uninf; lane 1) and left untreated (?IFN). Equal amounts (15
?g) of cell lysates were separated by SDS-PAGE and transferred to
Immobilon-P membranes. The expression of phosphorylated STAT1
(P-STAT1), STAT1, and NS4B proteins was detected by Western
blotting with the corresponding specific antibodies. Actin served as the
lane loading control. One representative experiment of three is shown.
VOL. 81, 2007INHIBITION OF IFN RESPONSE BY WNV11813
infection of cells with WNV (Fig. 9), (11). Interestingly, the
WNII-ER mutant viruses caused less cell death than observed
with the wild type, suggesting that the mutations in NS4B
reduced the cytopathic potential of this virus (data not shown).
In this report, we established a biologically relevant system
in which to examine the effects of WNV NS proteins on the
IFN signaling pathway. Our findings demonstrate that alanine
substitution mutations at residues E22K24in the NS4B coding
region resulted in a WNV replicon that was replication com-
petent yet unable to completely inhibit the IFN phosphoryla-
tion cascade. Further, homologous mutations in infectious
WNV do not mirror these results, suggesting a complex inter-
play between the structural and NS proteins. Although it has
been hypothesized that NS4B is the protein responsible for
WNV-mediated IFN inhibition, these data are the first to de-
fine the importance of NS4B for the inhibition of IFN signaling
in the biological context of replicating genomes. In fact, our
results are surprising in view of previous studies with ectopi-
cally expressed proteins of WNV and other flaviviruses that
suggest roles for essentially all other NS proteins in the inhi-
bition of the IFN response (3, 28, 33). One major difference
between our experimental approach and that of other investi-
gators was that we relied primarily on cells replicating viral
genomes rather than transient transfection assays. Neverthe-
less, we also observed that wild-type NS4B blocks IFN signal-
ing and that the NS4B-E22K24mutant partially relieves the
inhibition. Our results show, similar to others, that wild-type
NS4B can block the majority of IFN signaling, whereas the
NS4B-E22K24mutant partially relieves the inhibition. Al-
though our in vitro reporter assays confirm previously pub-
lished data concerning the role of NS4B in the inhibition of
IFN signaling as well as support our own work with replicon
cells, we must interpret these overexpression studies with cau-
tion. We cannot exclude the possibility that transfection and
overexpression disrupt other mechanisms that lead to alter-
ation of the cell. Moreover, as our studies with full-length
infectious virus show, there may be more than one player
important in blocking IFN signaling pathways.
NS4B is a small, predominantly helical, membrane-associ-
ated protein. Hydrophobicity plots based on primary amino
acid sequence indicate that the protein consists of four or five
transmembrane domains (42, 58). Recently, Miller and col-
leagues showed that the NS4B protein of the closely related
dengue virus has three transmembrane domains (35). Al-
though neither the crystal structure nor topology of WNV
NS4B has been elucidated, the targets we selected for mu-
tagenesis are located in predicted exposed regions of the pro-
tein, based on the above criteria (Fig. 1). Further, deletion
mutagenesis of overexpressed dengue virus NS4B indicated
that amino acids 1 to 77 failed to mediate the IFN signaling
block (37). Our results demonstrated that amino acids in the
N-terminal domain are required for this inhibition in WNV. It
is important that both regions are predicted to reside in the
lumen of the ER (35). The WNV NS4B-E22K24mutation may
prevent important charge-charge interactions necessary for
protein binding. Further, the mutation might alter the confor-
mation of a critical domain for function. This possibility is
intriguing due to the finding that dengue NS4B can have mul-
tiple conformations, which are dictated by the N terminus (35).
Our results demonstrate that IFN inhibition by WNV is
more complex than previously envisioned. Although NS4B is
capable of blocking IFN signaling in transient expression and
replicon systems, the exact mechanism is unclear. There are a
FIG. 9. Mutant viruses exhibit growth kinetics comparable to wild type. Wild-type (A and C) or IFN-?/? receptor knockout (B and D) B6 MEFs
were infected at an MOI of 0.2 (A and B) or 0.002 (C and D) with WNII, WNII-ER, WNII-KUN4B, or WNII-KUN4B-EK for 12, 24, 48, and 72 h.
Virus titers in medium from infected cells were determined by plaque assay on BHK-21 cells. Means are shown with standard error bars.
11814EVANS AND SEEGER J. VIROL.
number of possibilities, both direct and indirect, through which
NS4B can block IFN signaling. First, reducing the amount of
IFN receptor would cause an inhibition. However, we have
found that expression of IFNAR is unaffected by the presence
of the replicon (15). Second, NS4B could bind to the receptor
or the JAK proteins to physically block activation. Third, NS4B
could activate a negative regulatory protein such as a phos-
phatase or the suppressor of cytokine signaling proteins to act
at the level of the receptor. Finally, NS4B could cause a gen-
eralized cellular stress that leads to down-regulation of a num-
ber of cell signaling pathways. The mechanism of NS4B-in-
duced inhibition may be determined by dissecting the
biochemical composition of the IFN receptor in the presence
Although mutations in NS4B in subgenomic replicons al-
lowed for IFN signaling, infections with full-length viruses con-
taining corresponding mutations abrogated IFN pathway acti-
vation. As mentioned above, the replicon cells were pools, and
the effects seen were global; thus, it is unlikely that other WNV
proteins were mutated in the replicon. It is more plausible that
a structural protein not present in the replicon cells is involved
in blocking STAT phosphorylation. However, apart from their
role in virus entry and egress, not much has been discovered to
date about the structural proteins during virus replication.
Even though they are not necessary to maintain functionally
replicating replicons, they may play an as yet unknown role in
virus pathogenesis (25). For example, it has been reported that
capsid proteins could cause apoptosis and inflammation under
certain experimental conditions (60). Also, Munoz-Jordan and
colleagues demonstrated that the inhibition of IFN signaling
by NS4B could be augmented by the presence of NS2A and
NS4A (38). In the case of infectious virus, it is possible that
NS2A or NS4A can compensate for NS4B during the viral life
cycle. In other words, protein interactions may dictate the
degree of IFN response inhibition. Further, the level of repli-
cation may affect the IFN signaling pathway. However, we
think that this possibility is unlikely, since Northern blot anal-
ysis showed that levels of WNV genomic RNA were similar in
infected and replicon cells (data not shown).
It is important to note that the replicon is maintained in cells
without the CPE that is a hallmark of WNV infection (40, 43,
46, 49). Interestingly, the mutant WNII viruses were less cyto-
pathic than the wild type, yet virus titers were not reduced.
These data would directly link NS4B as a mediator of cell
death separate from other functions. NS4B is known to expand
and modify the ER, which facilitates virus replication (56). It
may be that NS4B alters the ER such that cells incur death
from an unfolded protein response or ER stress (50, 61).
Whatever the mechanism might be, our data suggest that in-
hibition of IFN innate immunity during infection is more com-
plex than previously suggested. Future studies will elucidate
the mechanism(s) through which WNV can evade and influ-
ence the immune system.
We thank Kerry Campbell and William Mason for their helpful
comments and critical reading of the manuscript. We acknowledge the
services provided by the Fox Chase Cancer Center tissue culture and
DNA sequencing facilities. We thank Alexander Khromykh (Brisbane,
Australia) for the C20DXrep/neo plasmid, Vladimir Yamshchikov
(University of Kansas) for the pSP6WN/Xba plasmid, Curt Horvath
(Northwestern University) for the pEF-HA-HPIV2 plasmid, and Mi-
chael Diamond (Washington University, St. Louis, MO) for the wild-
type and IFN-?/? receptor knockout MEFs essential to conduct this
study. We acknowledge J. David Stiffler for producing replicon mu-
tants and Paul Gramlich for screening NS antibodies.
This work was supported by grants from the NIH and an appropri-
ation from the Commonwealth of Pennsylvania. J.D.E. was supported
by grant T32 CA-09035-30.
1. Babb, R., C. C. Huang, D. J. Aufiero, and W. Herr. 2001. DNA recognition
by the herpes simplex virus transactivator VP16: a novel DNA-binding struc-
ture. Mol. Cell. Biol. 21:4700–4712.
2. Berthet, F. X., H. G. Zeller, M. T. Drouet, J. Rauzier, J. P. Digoutte, and V.
Deubel. 1997. Extensive nucleotide changes and deletions within the enve-
lope glycoprotein gene of Euro-African West Nile viruses. J. Gen. Virol.
3. Best, S. M., K. L. Morris, J. G. Shannon, S. J. Robertson, D. N. Mitzel, G. S.
Park, E. Boer, J. B. Wolfinbarger, and M. E. Bloom. 2005. Inhibition of
interferon-stimulated JAK-STAT signaling by a tick-borne flavivirus and
identification of NS5 as an interferon antagonist. J. Virol. 79:12828–12839.
4. Brinton, M. A. 2002. The molecular biology of West Nile Virus: a new
invader of the western hemisphere. Annu. Rev. Microbiol. 56:371–402.
5. Burke, D. S., and T. P. Monath. 2001. Flaviviruses, p. 1043–1125. In D. M.
Knipe, P. M. Howley, D. E. Griffin, R. A. Lamb, M. A. Martin, B. Roizman,
and S. E. Straus (ed.), Fields virology, 4th ed. Lippincott Williams and
Wilkins, Philadelphia, PA.
6. Chan-Tack, K. M., and G. Forrest. 2005. Failure of interferon alpha-2b in a
patient with West Nile virus meningoencephalitis and acute flaccid paralysis.
Scand. J. Infect. Dis. 37:944–946.
7. Colamonici, O., H. Yan, P. Domanski, R. Handa, D. Smalley, J. Mullersman,
M. Witte, K. Krishnan, and J. Krolewski. 1994. Direct binding to and
tyrosine phosphorylation of the alpha subunit of the type I interferon recep-
tor by p135tyk2 tyrosine kinase. Mol. Cell. Biol. 14:8133–8142.
8. Colamonici, O. R., H. Uyttendaele, P. Domanski, H. Yan, and J. J.
Krolewski. 1994. p135tyk2, an interferon-alpha-activated tyrosine kinase, is
physically associated with an interferon-alpha receptor. J. Biol. Chem. 269:
9. Der, S. D., A. Zhou, B. R. Williams, and R. H. Silverman. 1998. Identification
of genes differentially regulated by interferon alpha, beta, or gamma using
oligonucleotide arrays. Proc. Natl. Acad. Sci. USA 95:15623–15628.
10. Egloff, M. P., D. Benarroch, B. Selisko, J. L. Romette, and B. Canard. 2002.
An RNA cap (nucleoside-2?-O-)-methyltransferase in the flavivirus RNA
polymerase NS5: crystal structure and functional characterization. EMBO J.
11. Fredericksen, B. L., M. Smith, M. G. Katze, P. Y. Shi, and M. Gale, Jr. 2004.
The host response to West Nile virus infection limits viral spread through the
activation of the interferon regulatory factor 3 pathway. J. Virol. 78:7737–
12. Gauzzi, M. C., L. Velazquez, R. McKendry, K. E. Mogensen, M. Fellous, and
S. Pellegrini. 1996. Interferon-alpha-dependent activation of Tyk2 requires
phosphorylation of positive regulatory tyrosines by another kinase. J. Biol.
13. Grun, J. B., and M. A. Brinton. 1986. Characterization of West Nile virus
RNA-dependent RNA polymerase and cellular terminal adenylyl and
uridylyl transferases in cell-free extracts. J. Virol. 60:1113–1124.
14. Grun, J. B., and M. A. Brinton. 1988. Separation of functional West Nile
virus replication complexes from intracellular membrane fragments. J. Gen.
15. Guo, J. T., J. Hayashi, and C. Seeger. 2005. West Nile virus inhibits the signal
transduction pathway of alpha interferon. J. Virol. 79:1343–1350.
16. Guo, J. T., Q. Zhu, and C. Seeger. 2003. Cytopathic and noncytopathic
interferon responses in cells expressing hepatitis C virus subgenomic repli-
cons. J. Virol. 77:10769–10779.
17. Guyatt, K. J., E. G. Westaway, and A. A. Khromykh. 2001. Expression and
purification of enzymatically active recombinant RNA-dependent RNA
polymerase (NS5) of the flavivirus Kunjin. J. Virol. Methods 92:37–44.
18. Hanna, S. L., T. C. Pierson, M. D. Sanchez, A. A. Ahmed, M. M. Murtadha,
and R. W. Doms. 2005. N-linked glycosylation of West Nile virus envelope
proteins influences particle assembly and infectivity. J. Virol. 79:13262–
19. Horvath, C. M., G. R. Stark, I. M. Kerr, and J. E. Darnell, Jr. 1996.
Interactions between STAT and non-STAT proteins in the interferon-stim-
ulated gene factor 3 transcription complex. Mol. Cell. Biol. 16:6957–6964.
20. Kalil, A. C., M. P. Devetten, S. Singh, B. Lesiak, D. P. Poage, K. Bargen-
quast, P. Fayad, and A. G. Freifeld. 2005. Use of interferon-alpha in patients
with West Nile encephalitis: report of 2 cases. Clin. Infect. Dis. 40:764–766.
21. Khromykh, A. A., M. T. Kenney, and E. G. Westaway. 1998. trans-comple-
mentation of flavivirus RNA polymerase gene NS5 by using Kunjin virus
replicon-expressing BHK cells. J. Virol. 72:7270–7279.
VOL. 81, 2007INHIBITION OF IFN RESPONSE BY WNV11815
22. Khromykh, A. A., P. L. Sedlak, and E. G. Westaway. 2000. cis- and trans-
acting elements in flavivirus RNA replication. J. Virol. 74:3253–3263.
23. Khromykh, A. A., P. L. Sedlak, and E. G. Westaway. 1999. trans-comple-
mentation analysis of the flavivirus Kunjin ns5 gene reveals an essential role
for translation of its N-terminal half in RNA replication. J. Virol. 73:9247–
24. Khromykh, A. A., A. N. Varnavski, and E. G. Westaway. 1998. Encapsidation
of the flavivirus Kunjin replicon RNA by using a complementation system
providing Kunjin virus structural proteins in trans. J. Virol. 72:5967–5977.
25. Khromykh, A. A., and E. G. Westaway. 1997. Subgenomic replicons of the
flavivirus Kunjin: construction and applications. J. Virol. 71:1497–1505.
26. Lanciotti, R. S., J. T. Roehrig, V. Deubel, J. Smith, M. Parker, K. Steele, B.
Crise, K. E. Volpe, M. B. Crabtree, J. H. Scherret, R. A. Hall, J. S. Mac-
Kenzie, C. B. Cropp, B. Panigrahy, E. Ostlund, B. Schmitt, M. Malkinson,
C. Banet, J. Weissman, N. Komar, H. M. Savage, W. Stone, T. McNamara,
and D. J. Gubler. 1999. Origin of the West Nile virus responsible for an
outbreak of encephalitis in the northeastern United States. Science 286:
27. Li, X., S. Leung, I. M. Kerr, and G. R. Stark. 1997. Functional subdomains
of STAT2 required for preassociation with the alpha interferon receptor and
for signaling. Mol. Cell. Biol. 17:2048–2056.
28. Lin, R. J., B. L. Chang, H. P. Yu, C. L. Liao, and Y. L. Lin. 2006. Blocking
of interferon-induced Jak-Stat signaling by Japanese encephalitis virus NS5
through a protein tyrosine phosphatase-mediated mechanism. J. Virol. 80:
29. Lin, R. J., C. L. Liao, E. Lin, and Y. L. Lin. 2004. Blocking of the alpha
interferon-induced Jak-Stat signaling pathway by Japanese encephalitis virus
infection. J. Virol. 78:9285–9294.
30. Lindenbach, B. D., and C. M Rice. 2001. Flaviviridae: the viruses and their
replication, p. 991–1041. In D. M. Knipe, P. M. Howley, D. E. Griffin, R. A.
Lamb, M. A. Martin, B. Roizman, and S. E. Straus (ed.), Fields virology, 4th
ed. Lippincott Williams and Wilkins, Philadelphia, PA.
31. Liu, W. J., H. B. Chen, and A. A. Khromykh. 2003. Molecular and functional
analyses of Kunjin virus infectious cDNA clones demonstrate the essential
roles for NS2A in virus assembly and for a nonconservative residue in NS3
in RNA replication. J. Virol. 77:7804–7813.
32. Liu, W. J., P. L. Sedlak, N. Kondratieva, and A. A. Khromykh. 2002.
Complementation analysis of the flavivirus Kunjin NS3 and NS5 proteins
defines the minimal regions essential for formation of a replication complex
and shows a requirement of NS3 in cis for virus assembly. J. Virol. 76:10766–
33. Liu, W. J., X. J. Wang, V. V. Mokhonov, P. Y. Shi, R. Randall, and A. A.
Khromykh. 2005. Inhibition of interferon signaling by the New York 99
strain and Kunjin subtype of West Nile virus involves blockage of STAT1 and
STAT2 activation by nonstructural proteins. J. Virol. 79:1934–1942.
34. Mackenzie, J. M., and E. G. Westaway. 2001. Assembly and maturation of
the flavivirus Kunjin virus appear to occur in the rough endoplasmic retic-
ulum and along the secretory pathway, respectively. J. Virol. 75:10787–
35. Miller, S., S. Sparacio, and R. Bartenschlager. 2006. Subcellular localization
and membrane topology of the dengue virus type 2 non-structural protein
4B. J. Biol. Chem. 281:8854–8863.
36. Morrey, J. D., C. W. Day, J. G. Julander, L. M. Blatt, D. F. Smee, and R. W.
Sidwell. 2004. Effect of interferon-alpha and interferon-inducers on West
Nile virus in mouse and hamster animal models. Antivir. Chem. Chemother.
37. Munoz-Jordan, J. L., M. Laurent-Rolle, J. Ashour, L. Martinez-Sobrido, M.
Ashok, W. I. Lipkin, and A. Garcia-Sastre. 2005. Inhibition of alpha/beta
interferon signaling by the NS4B protein of flaviviruses. J. Virol. 79:8004–
38. Munoz-Jordan, J. L., G. G. Sanchez-Burgos, M. Laurent-Rolle, and A. Garcia-
Sastre. 2003. Inhibition of interferon signaling by dengue virus. Proc. Natl.
Acad. Sci. USA 100:14333–14338.
39. Parisien, J. P., J. F. Lau, J. J. Rodriguez, B. M. Sullivan, A. Moscona, G. D.
Parks, R. A. Lamb, and C. M. Horvath. 2001. The V protein of human
parainfluenza virus 2 antagonizes type I interferon responses by destabilizing
signal transducer and activator of transcription 2. Virology 283:230–239.
40. Parquet, M. C., A. Kumatori, F. Hasebe, K. Morita, and A. Igarashi. 2001.
West Nile virus-induced Bax-dependent apoptosis. FEBS Lett. 500:17–24.
41. Pont-Kingdon, G. 1994. Construction of chimeric molecules by a two-step
recombinant PCR method. BioTechniques 16:1010–1011.
42. Puig-Basagoiti, F., M. Tilgner, C. J. Bennett, Y. Zhou, J. L. Munoz-Jordan,
A. Garcia-Sastre, K. A. Bernard, and P. Y. Shi. 2007. A mouse cell-adapted
NS4B mutation attenuates West Nile virus RNA synthesis. Virology 361:
43. Ramanathan, M. P., J. A. Chambers, P. Pankhong, M. Chattergoon, W.
Attatippaholkun, K. Dang, N. Shah, and D. B. Weiner. 2006. Host cell killing
by the West Nile virus NS2B-NS3 proteolytic complex: NS3 alone is sufficient
to recruit caspase-8-based apoptotic pathway. Virology 345:56–72.
44. Rossi, S. L., Q. Zhao, V. K. O’Donnell, and P. W. Mason. 2005. Adaptation
of West Nile virus replicons to cells in culture and use of replicon-bearing
cells to probe antiviral action. Virology 331:457–470.
45. Samuel, M. A., and M. S. Diamond. 2005. Alpha/beta interferon protects
against lethal West Nile virus infection by restricting cellular tropism and
enhancing neuronal survival. J. Virol. 79:13350–13361.
46. Samuel, M. A., J. D. Morrey, and M. S. Diamond. 2007. Caspase 3-depen-
dent cell death of neurons contributes to the pathogenesis of West Nile virus
encephalitis. J. Virol. 81:2614–2623.
47. Samuel, M. A., K. Whitby, B. C. Keller, A. Marri, W. Barchet, B. R. Wil-
liams, R. H. Silverman, M. Gale, Jr., and M. S. Diamond. 2006. PKR and
RNase L contribute to protection against lethal West Nile Virus infection by
controlling early viral spread in the periphery and replication in neurons.
J. Virol. 80:7009–7019.
48. Shi, P. Y., M. Tilgner, M. K. Lo, K. A. Kent, and K. A. Bernard. 2002.
Infectious cDNA clone of the epidemic West Nile virus from New York City.
J. Virol. 76:5847–5856.
49. Shrestha, B., D. Gottlieb, and M. S. Diamond. 2003. Infection and injury of
neurons by West Nile encephalitis virus. J. Virol. 77:13203–13213.
50. Su, H. L., C. L. Liao, and Y. L. Lin. 2002. Japanese encephalitis virus
infection initiates endoplasmic reticulum stress and an unfolded protein
response. J. Virol. 76:4162–4171.
51. Takaoka, A., and H. Yanai. 2006. Interferon signalling network in innate
defence. Cell Microbiol. 8:907–922.
52. Varnavski, A. N., and A. A. Khromykh. 1999. Noncytopathic flavivirus rep-
licon RNA-based system for expression and delivery of heterologous genes.
53. Varnavski, A. N., P. R. Young, and A. A. Khromykh. 2000. Stable high-level
expression of heterologous genes in vitro and in vivo by noncytopathic
DNA-based Kunjin virus replicon vectors. J. Virol. 74:4394–4403.
54. Wengler, G. 1991. The carboxy-terminal part of the NS 3 protein of the West
Nile flavivirus can be isolated as a soluble protein after proteolytic cleavage
and represents an RNA-stimulated NTPase. Virology 184:707–715.
55. Wengler, G., G. Czaya, P. M. Farber, and J. H. Hegemann. 1991. In vitro
synthesis of West Nile virus proteins indicates that the amino-terminal seg-
ment of the NS3 protein contains the active centre of the protease which
cleaves the viral polyprotein after multiple basic amino acids. J. Gen. Virol.
56. Westaway, E. G., A. A. Khromykh, M. T. Kenney, J. M. Mackenzie, and
M. K. Jones. 1997. Proteins C and NS4B of the flavivirus Kunjin translocate
independently into the nucleus. Virology 234:31–41.
57. Westaway, E. G., J. M. Mackenzie, M. T. Kenney, M. K. Jones, and A. A.
Khromykh. 1997. Ultrastructure of Kunjin virus-infected cells: colocalization
of NS1 and NS3 with double-stranded RNA, and of NS2B with NS3, in
virus-induced membrane structures. J. Virol. 71:6650–6661.
58. Wicker, J. A., M. C. Whiteman, D. W. Beasley, C. T. Davis, S. Zhang, B. S.
Schneider, S. Higgs, R. M. Kinney, and A. D. Barrett. 2006. A single amino
acid substitution in the central portion of the West Nile virus NS4B protein
confers a highly attenuated phenotype in mice. Virology 349:245–253.
59. Yamshchikov, V. F., G. Wengler, A. A. Perelygin, M. A. Brinton, and R. W.
Compans. 2001. An infectious clone of the West Nile flavivirus. Virology
60. Yang, J. S., M. P. Ramanathan, K. Muthumani, A. Y. Choo, S. H. Jin, Q. C.
Yu, D. S. Hwang, D. K. Choo, M. D. Lee, K. Dang, W. Tang, J. J. Kim, and
D. B. Weiner. 2002. Induction of inflammation by West Nile virus capsid
through the caspase-9 apoptotic pathway. Emerg. Infect. Dis. 8:1379–1384.
61. Yu, C. Y., Y. W. Hsu, C. L. Liao, and Y. L. Lin. 2006. Flavivirus infection
activates the XBP1 pathway of the unfolded protein response to cope with
endoplasmic reticulum stress. J. Virol. 80:11868–11880.
11816EVANS AND SEEGER J. VIROL.