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Dengue virus NS4B interacts with NS3 and
dissociates it from single-stranded RNA
Indira Umareddy, Alex Chao, Aruna Sampath, Feng Gu
and Subhash G. Vasudevan
Correspondence
Subhash G. Vasudevan
subhash.vasudevan@novartis.
com
Novartis Institute for Tropical Diseases, 10 Biopolis Road, #05-01 Chromos Building,
Singapore 138670
Received 20 January 2006
Accepted 24 April 2006
Dengue virus, a member of the family Flaviviridae of positive-strand RNA viruses, has seven
non-structural proteins: NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5. Except for enzymic
activities contained within NS3 and NS5, the roles of the other proteins in virus replication
and pathogenesis are not well defined. In this study, a physical interaction between NS4B and the
helicase domain of NS3 was identified by using a yeast two-hybrid assay. This interaction was
further confirmed by biochemical pull-down and immunoprecipitation assays, both with purified
proteins and with dengue virus-infected cell lysates. NS4B co-localized with NS3 in the perinuclear
region of infected human cells. Furthermore, NS4B dissociated NS3 from single-stranded RNA and
consequently enhanced the helicase activity of NS3 in an in vitro unwinding assay. These results
suggest that NS4B modulates dengue virus replication via its interaction with NS3.
INTRODUCTION
Dengue fever and its more severe form, dengue haemorrha-
gic fever, are mosquito-borne viral diseases that are caused
by one of the four antigenically distinct serotypes of Dengue
virus, DENV-1–DENV-4. Dengue fever affects 50–100
million people in the tropical and subtropical regions
annually (Gubler, 1998, 2002). Contemporary demogra-
phical and lifestyle trends, such as population explosion and
urbanization, have led to the spread of this disease to non-
endemic regions. The pathogenesis of dengue fever remains
poorly characterized and there are no antivirals or vaccines
available to counter this emerging disease.
Dengue virus belongs to the family Flaviviridae that consists
of enveloped, positive-sense, single-stranded RNA (ssRNA)
viruses, such as those that cause yellow fever, Japanese
encephalitis, West Nile fever and hepatitis C. Its RNA
genome is encapsulated in an icosahedral nucleocapsid
(30 nm) that is enveloped in a lipid bilayer (10 nm) (Kuhn
et al., 2002) consisting of the membrane and envelope pro-
teins. The 11 kb, capped RNA genome encodes a single
polyprotein that is processed co- and post-translationally
by host signalases, as well as the virus-encoded serine pro-
tease, into the three structural and seven non-structural
proteins (NS) in the order C (Core)–prM (pre-Membrane)–
E (Envelope)–NS1–NS2A–NS2B–NS3–NS4A–NS4B–NS5
(Chambers & Rice, 1987; Lindenbach & Rice, 2003).
The polymerase, helicase and protease enzymic acti-
vities encoded by the dengue virus genome ensure virus
replication and polyprotein processing. NS3 (618 aa) is a
multifunctional protein with protease, helicase, NTPase
and 59-terminal RNA triphosphatase activities (Arias et al.,
1993; Benarroch et al., 2004; Falgout et al., 1991; Li et al.,
1999; Zhang et al., 1992), whilst NS5 (900 aa) has RNA-
dependent RNA polymerase and methyltransferase activities
(Ackermann & Padmanabhan, 2001; Chu & Westaway,
1987; Egloff et al., 2002; Kapoor et al., 1995; Tan et al., 1996).
These two proteins form a functional complex that is vital
for flavivirus replication (Brooks et al., 2002; Johansson
et al., 2001; Yon et al., 2005). The role of other non-
structural proteins is not clear, except for NS2B, which is
a cofactor for the protease activity of NS3 (Clum et al.,
1997; Falgout et al., 1993). Interestingly, dengue virus NS4B
has been reported to interfere with the interferon res-
ponse in host cells by blocking the activation and nuclear
translocation of Stat-1 (Mun
˜oz-Jorda
´net al., 2003, 2005).
NS4B of members of the Flaviviridae is a small (248 aa),
hydrophobic protein. NS4B proteins of dengue virus sero-
types share 78–85 % amino acid sequence identity, whereas
those of Yellow fever virus,West Nile virus and Dengue
virus share 35 % identity. Hepatitis C virus (HCV) NS4B
bears a negligible resemblance. Despite this divergence, the
topology of NS4B, containing several endoplasmic reti-
cular (ER) and cytoplasmic domains separated by trans-
membrane regions (Miller et al., 2006), is strikingly similar
among members of the Flaviviridae, suggesting a conserved
function of NS4B in the viral life cycle (Lundin et al., 2003).
Deletion of NS4B, as well as insertions in its sequence, inhi-
bit replication of both Bovine viral diarrhea virus (BVDV)
and Kunjin viruses (Balint et al., 2005; Grassmann et al.,
Supplementary material is available in JGV Online.
0008-1844 G2006 SGM Printed in Great Britain 2605
Journal of General Virology (2006), 87, 2605–2614 DOI 10.1099/vir.0.81844-0
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2001; Khromykh et al., 2000; Li & McNally, 2001). BVDV
NS4B interacts with NS3 and NS5A (Qu et al., 2001) and
HCV NS4B plays a role in viral RNA replication, possibly
by inducing morphological changes in the ER membrane
(Egger et al., 2002; Gretton et al., 2005; Piccininni et al.,
2002). Whilst these studies indicate that NS4B is a com-
ponent of the replication complex of members of the
Flaviviridae, co-immunoprecipitations of cell lysates using
antibodies to double-stranded RNA (dsRNA) failed to
reveal the presence of NS4B in the Kunjin virus replica-
tion complex (Chu & Westaway, 1992; Westaway et al.,
2003).
In this study, an interaction between the dengue virus non-
structural proteins NS4B and NS3 was identified by using
a yeast two-hybrid assay and validated in pull-down and
immunoprecipitation studies. Furthermore, recombinant
NS4B dissociated ssRNA from NS3 and consequently
enhanced the overall helicase activity of NS3 in in vitro
assays. Our results suggest a novel role for NS4B in dengue
virus replication.
METHODS
Cloning. Non-structural protein sequences were amplified from
cDNA of the dengue virus strain TSV01 and cloned into respec-
tive plasmids. The P104L mutation in NS4B was generated by
site-directed mutagenesis of C to T at nucleotide position 7136 of
TSV01 (GenBank accession no. AY037116), which corresponds to
nucleotide position 7129 (P101L mutation) reported in rDEN4-2A-5
(Hanley et al., 2003). Yeast expression constructs for NS3 and NS5,
full-length NS3 (NS3-FL) and NS3 pro constructs were described
previously (Brooks et al., 2002; Johansson et al., 2001; Li et al., 2005;
Xu et al., 2005). For other constructs and primers used in this study,
see Supplementary Table S1 (available in JGV Online).
Yeast two-hybrid screening. The two-hybrid screens were per-
formed as described in the Matchmaker GAL4 Two-hybrid System 3
User Manual (Clontech). Briefly, an interaction between two pro-
teins is indicated by the activation of the reporter genes HIS3 and
ADE2, which allow growth on media lacking histidine (His) and
adenine (Ade), respectively, or MEL1, which secretes a-glucosidase
that can be assayed on X-a-gal indicator plates. The pGBKT7- and
pGADT7-derived constructs encoding dengue virus NS proteins
were co-transformed into AH109 cells and plated onto culture
plates lacking tryptophan (Trp) and leucine (Leu) to select for co-
transformants. After 72–96 h, the co-transformants were streaked
onto plates lacking Trp, Leu, His and Ade and containing X-a-gal to
allow selection of interacting partners.
In vitro translation and immunoprecipitation. Radiolabelled
NS4B was generated from the pGBK4B plasmid by using the
TNT T7-coupled reticulocyte lysate system (Promega) and [
35
S]Met
(Amersham Biosciences). In vitro-translated NS4B (10 ml) was incu-
bated at 4 uC for 1 h with or without 5 mg His-tagged NS3
303–618
protein. Ni–NTA agarose beads were added to capture the NS4B–NS3
complex and incubated again for 1 h. The complex was resolved by
SDS-PAGE (12 % gel) and visualized by autoradiography.
Pull-downs. Bacterial cell lysates expressing glutathione S-transferase
(GST), GST–NS4B and GST–NS4BM were incubated with glutathione–
Sepharose beads (Amersham Biosciences) for 2 h at 4 uC, washed
with PBS and these beads were used for pull-down experiments. Ten
microlitres of these beads was incubated with or without 7 mg NS3
with cofactor (CF NS3) for 3 h at room temperature, washed three
times with PBS and the proteins were eluted by boiling the beads in
40 ml SDS loading buffer. Ten microlitres of these reactions was
resolved by SDS-PAGE (12 % gel) and stained with Coomassie blue.
Preparation of dsRNA/ssRNA substrate. Plasmid pGEM4Z was
linearized by digestion with XbaI and was in vitro transcribed in the
presence of [a-
32
P]GTP by using a Riboprobe kit (Promega). After
incubation for 1 h at 37 uC, the reaction mixtures were treated with
DNase I and extracted with phenol/CHCl
3
. Unincorporated NTPs
were separated by a Chromaspin-10 spin column (BD Clontech)
and RNA was precipitated with ethanol. Radiolabelled in vitro tran-
scription product of pGEM4Z was used as ssRNA substrate for
electrophoretic mobility-shift assays (EMSAs). dsRNA substrate pre-
paration has been described elsewhere (Xu et al., 2005).
dsRNA-unwinding assay. The dsRNA-unwinding assay was per-
formed as described previously (Xu et al., 2005). Briefly, the reaction
mixture for this assay contained 25 mM HEPES (pH 7?5), 1 mM
ATP, 3 mM MnCl
2
, 2 mM dithiothreitol (DTT), 100 mg BSA, 5 U
RNasin, 0?25 pmol RNA substrate and 3 mM NS3 in a final volume
of 20 ml. The mixture was incubated for 30 min at 37 uC and the
reaction was terminated by adding 2?5ml termination mix [100 mM
Tris/HCl (pH 7?5), 50 mM EDTA, 0?1 % Triton X-100, 0?5 % SDS,
50 % glycerol, 0?1 % bromophenol blue]. The helicase assay mixtures
were resolved on a 10 % native polyacrylamide gel and analysed
with a Typhoon phosphorimager (Amersham Biosciences). For each
value, the background from the negative control was subtracted and
the fold variation of ssRNA release from each lane was calculated
against ssRNA release by NS3-FL and plotted on a graph (Fig. 6b).
Pvalues were calculated by performing a two-tailed ttest on raw
data.
EMSA. The reaction mixture for this assay (20 ml) contained
20 mM HEPES (pH 7?5), 50 mM KCl, 1 mM EDTA, 5 % glycerol,
1 mM DTT and 200 mg BSA ml
21
, along with
32
P-labelled ssRNA
substrate and 1–3 mM NS3-FL or CF NS3 and 1–6 mM GST or
GST–NS4B proteins. The mixtures were incubated for 5 min at
37 uC, 5 ml loading buffer (20 % glycerol) was added and the mix-
tures were resolved on an 8 % native polyacrylamide gel at 4 uC.
Bands were identified by a Typhoon phosphorimager (Amersham
Biosciences).
Cell culture, transfection and immunofluorescence. BHK-21,
C6/36 [maintained in RPMI medium containing 10 % fetal bovine
serum (FBS)]and A549 (maintained in Dulbecco’s modified Eagle’s
medium containing 10 % FBS) cell lines were purchased from the
ATCC. Medium components were purchased from Gibco/Invitrogen
Corporation. Monolayers of A549 cells were cultured on coverslips
in 24-well plates and co-transfected with 1 mg each of pXJ-NS4B and
pXJ-NS3-FL plasmids by using Lipofectamine 2000 (Invitrogen).
The cells were fixed in cold methanol 24 h post-transfection. For
virus infections, A549 cells were seeded 24 h before infection with
5 m.o.i. TSV01 and fixed in cold methanol 3 days post-infection.
Anti-NS3 and anti-NS4B antisera, generated in house, were used as
primary antibodies. Texas red-conjugated anti-rabbit and fluorescein
isothiocyanate (FITC)-conjugated anti-mouse secondary antibodies
were used (Jackson Laboratories). Co-transfection images were cap-
tured by using a Leica fluorescent microscope, whereas the images
of infected cells that were morphologically slightly different from
uninfected cells were captured by using a confocal microscope
(Olympus).
Antibody production. Anti-NS4B antiserum was prepared in mice
against NS4B expressed as a GST fusion protein and eluted from
polyacrylamide gels. This was purified by using an Escherichia coli
lysate column to remove any non-specific antibodies and was tested
on virus-infected C6/36, BHK-21 and A549 cell lines, as well
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as transient transfections of NS4B in 293T, HeLa and A549 cells by
Western blotting, immunoprecipitation and immunofluorescence.
Polyclonal rabbit anti-DENV-2 NS3 was generated by injecting
purified NS3 into rabbits. The serum was collected after 4 weeks and
tested as described above.
Virus infection and immunoprecipitation assays. C6/36 and
A549 cell lines were seeded in T75 cell-culture flasks 24 h prior to
infection and, when about 80 % confluent, infected with 10 m.o.i.
TSV01. Infected and mock-infected cells were lysed in 2 ml cold m-
RIPA buffer [50 mM Tris/HCl (pH 7?4), 1 % NP-40, 0?25 % sodium
deoxycholate, 150 mM NaCl, 1 mM EDTA, 16protease inhibitor
cocktail (Sigma)]72 h post-infection. Lysates were pre-cleared with
Protein A–agarose beads and normalized for protein concentration.
About 10 ml NS4B antibody (roughly 1 mg) was added to 500 mg
extract and incubated overnight at 4 uC with gentle agitation. The
complexes were captured by incubation for 1 h with 50 ml Protein
A–agarose beads. Beads were washed three times each with m-RIPA
buffer and PBS, boiled in 20 ml loading buffer and Western blotting
was performed with anti-NS3 antibody.
Protein purification. NS3-FL protein purification has been des-
cribed elsewhere (Xu et al., 2005). Briefly, BL21-RIL E. coli cells
expressing NS3-FL and CF NS3 were induced for 16 h at 16 uC with
10 mM IPTG and lysed in 50 mM HEPES (pH 7?5), 300 mM NaCl,
5 % glycerol in a cell disrupter. The supernatant was purified by
using a HiTrap Ni
2+
–NTA affinity column (Amersham Biosciences)
and proteins were eluted from the column in the same buffer con-
taining 500 mM imidazole, then desalted with PD-10 columns
[buffer exchanged with 10 mM Tris/HCl (pH 7?5)]. Desalted frac-
tions were then pooled and concentrated in an Amicon filter
(Millipore). NS3-FL protein was cleaved from thioredoxin with
enterokinase, purified by using Talon spin columns and concen-
trated. Note that NS3-FL, described in Fig. 5(c), was not cleaved.
NS4B is considered a membrane protein. We expressed NS4B and
NS4BM as N-terminal fusions of GST in the BL21 strain of E. coli
cells. Induction with 20 mM IPTG for 20 h at 16 uC greatly enhanced
their solubility. Cells were lysed in 20 mM Tris/HCl (pH 7?5),
0?3 M NaCl, 0?25 % NP40, 5 % glycerol by sonication for 20 min.
Clarified supernatant was loaded onto a GST column (5 ml;
Amersham Biosciences) pre-equilibrated with 50 mM Tris (pH 8?0)
and eluted with 10 mM reduced glutathione. Peak fractions were
pooled and concentrated by ultrafiltration at 3000 g(Centricon-
30; cut-off, 30 kDa) and passed through a gel-filtration column
(Sephadex-75; Amersham Biosciences) using Tris buffer to obtain
pure GST–NS4B and GST–NS4BM.
RESULTS
Identification of a specific interaction between
NS4B and NS3 using the yeast two-hybrid
system
The yeast two-hybrid interaction method is a powerful tool
to detect molecular interactions (Fields & Song, 1989). To
elucidate the role of NS4B in virus replication, we carried
out yeast two-hybrid screening to find interacting part-
ners of NS4B among the other non-structural proteins of
Dengue virus. Full-length NS4B (NS4B), N-terminal aa 1–
135 (NS4B N) and C-terminal aa 136–248 of NS4B (NS4B
C) were engineered into the yeast bait vector (Table 1).
A single point mutation of a conserved residue in NS4B
(P104L) of DENV-4 has been reported to enhance virus
replication in mosquito cells while decreasing its replica-
tion in mammalian cells (Hanley et al., 2003). We there-
fore also included this NS4B mutant (NS4BM) in the
screen. Full-length NS1, NS2A, NS2B, NS4A and NS4B
Table 1. Yeast two-hybrid results
Screening was done with NS4B, NS4BM, NS4B N and NS4B C as bait (represented in rows) and non-structural proteins as prey (repre-
sented in columns). The grid shows strong (+++), weak (+)orno(2) interaction in the yeast two-hybrid screen. NS4B shows a strong
homo-association and the region of this interaction was narrowed down to aa 91–136 of its N-terminal domain (data not shown). NS4B
interacts strongly with NS3
303–618
and very weakly with NS3
1–303
. NS4B N, NS4B C and NS4BM do not interact with NS3
303–618
, suggesting
that this interaction is conformation-dependent. ND, Not determined.
Bait NS1 NS2A NS2B NS3
1–303
NS3
303–618
NS4A NS4B NS5
1–405
NS5
405–900
NS4B 2+2+ +++ 2+++ 22
NS4B N ++ + 222+++ 2+
NS4B C +22 +2222 +
NS4BM ND ND ND 22ND ND ND ND
Fig. 1. Identification of an interaction between NS4B and NS3
by yeast two-hybrid screening. NS4B, but not NS4BM, inter-
acts with the C terminus of NS3. Dual transformants of NS4B
and NS3
303–618
grow on X-a-gal plates lacking leucine, trypto-
phan, adenine and histidine. NS4BM and NS3
303–618
dual
transformants do not grow on these plates.
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and different domains pertaining to NS3
1–303
, NS3
303–618
and NS5
1–405
, NS5
405–900
were cloned into the prey vector
and tested for expression. Co-transformants were then
grown either on low-stringency plates (lacking leucine,
tryptophan and adenine) to identify weak interactions or
on high-stringency plates (X-a-gal plates lacking leucine,
tryptophan, adenine and histidine) to allow identification of
strong interactions.
As indicated in Table 1 and Fig. 1, full-length NS4B
interacted strongly with the C-terminal region of NS3
(NS3
303–618
), which encompasses its helicase motif. The
interaction is specific, because NS4B interacted very weakly
with N-terminal NS3 (NS3
1–303
), which contains the pro-
tease and NTPase motifs, and with NS2A, whilst no inter-
action was detected with NS1, NS2B, NS4A or NS5. Neither
the NS4B N nor NS4B C termini interacted with NS3
303–618
,
suggesting that protein conformation is important for
this interaction, whilst NS4BM did not interact with NS3
(Fig. 1). We identified a strong, homotypic interaction of
NS4B through its N-terminal domain, indicating that NS4B
may exist as an oligomer. The significance of this interaction
was not investigated in this study. Also, there was no strong
interaction of the small non-structural proteins NS2A,
NS2B and NS4A with NS4B in our yeast two-hybrid assay.
However, we cannot exclude the possibility that, being
highly hydrophobic, these proteins could not be transported
into the yeast nucleus.
Verification of interaction by pull-down and
immunoprecipitation assays
In order to corroborate our yeast two-hybrid results, pull-
down experiments were performed. Equal amounts of
35
S-
labelled, in vitro-translated, myc-tagged, full-length NS4B
were incubated with or without His-tagged NS3. Proteins
pulled down by Ni–NTA beads were resolved by SDS-PAGE
and detected by autoradiography. As shown in Fig. 2(a),
NS4B could be pulled down specifically by NS3
303–618
.In
a reversal of the pull-down, GST–NS4B, but not GST–
NS4BM, could pull down bacterially expressed full-length
NS3 with 40 aa of NS2B cofactor CF NS3 (Fig. 2b) and
in vitro-translated NS3
303–618
(see Supplementary Fig. S1,
available in JGV Online), supporting the yeast two-hybrid
interaction data.
To confirm the interaction between endogenously expressed
NS4B and NS3, a co-immunoprecipitation assay was carried
out using dengue virus-infected mosquito and mammalian
cell lysates. C6/36 and A549 cells were infected with DENV-
2 (TSV01 strain) at an m.o.i. of 10 and harvested 48 and
60 h post-infection, respectively. The presence of both NS3
and NS4B was detected in the infected cells by immuno-
blotting with the respective antibodies, which did not
cross-react (see Supplementary Fig. S2, available in JGV
Online). NS4B was immunoprecipitated with a mouse
polyclonal anti-NS4B antibody raised in house. The co-
immunoprecipitated material was separated by SDS-PAGE
and immunoblotted with anti-NS3 antibody. NS3 could
be co-immunoprecipitated by anti-NS4B antibody from
infected C6/36 (Fig. 3b) and A549 (Fig. 3c) cell lysates, but
not from uninfected lysates. Together, the in vivo co-
immunoprecipitation and the in vitro pull-down results
provide evidence that a specific interaction exists between
NS4B and NS3.
Fig. 2. Pull-down analyses of the NS4B and NS3 interaction.
(a) Pull-down of NS4B by NS3. Ni–NTA agarose beads were
incubated with
35
S-labelled, in vitro-translated NS4B with or
without recombinant His-tagged NS3
303–618
protein. Lane 1
depicts in vitro-translated NS4B protein as a positive control.
Ni–NTA beads do not pull down NS4B in the absence of
NS3
303–618
protein (lane 2) and NS3
303–618
pulls down NS4B
specifically (lane 3). One microlitre of in vitro-translated NS4B
(from a total reaction volume of 50 ml) was loaded in lane 1
and one-third of the material from the pull-downs was loaded in
lanes 2 and 3. (b) Pull-down of NS3 by NS4B. Ten microlitres
each of glutathione–Sepharose beads that were pre-incubated
with crude cell lysates of bacteria expressing GST, GST–NS4B
or GST–NS4BM was incubated with or without 7 mg recombi-
nant CF NS3 protein, run on an SDS-PAGE gel and
Coomassie-stained for detection of proteins. Lane 1, 7 mlCF
NS3; lanes 2 and 3, 10 ml GST beads without and with CF
NS3, respectively; lanes 4 and 5, 10 ml GST–NS4B beads
without and with CF NS3, respectively; lanes 6 and 7, 10 ml
GST–NS4BM beads without and with CF NS3 respectively.
Note that GST–NS4B, but not GST–NS4BM, pulls down CF
NS3.
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NS3 and NS4B share similar
subcellular-localization patterns
Kunjin virus NS4B localizes to the nucleus in the early
stages of infection (Westaway et al., 1997), whilst HCV
NS4B is an integral ER membrane protein (Hu
¨gle et al.,
2001). Interestingly, dengue virus NS4B does not translocate
into the nucleus even in later stages of infection, but resides
primarily in cytoplasmic foci of ER origin (Miller et al.,
2006). NS4B expressed along with an N-terminal 2K
fragment shows a reticular staining and co-localizes with
ER markers (Mun
˜oz-Jorda
´net al., 2005).
We examined the localization of transiently expressed
NS4B and NS3 in A549 cells by immunofluorescence. Co-
transfected cells were double-labelled with rabbit anti-NS3
and mouse anti-NS4B antibodies and observed under a
fluorescent microscope. Forty-eight hours after transfection,
both NS3 and NS4B showed a reticular staining pattern
that surrounded the nucleus and extended through the
cytoplasm, typical of the ER localization, and they co-
localized with each other when the two labellings were
merged (Fig. 4). Further confirming the co-localization of
the two molecules in vivo, A549 cells infected with DENV-2
showed a similar co-localization pattern 48 h post-infection
(Fig. 4). Cells infected with Dengue virus showed marked
morphological changes in the ER compartment, similar to
those seen in HCV infection (Egger et al., 2002; Gretton
et al., 2005). Our results are in agreement with another
report that NS4B co-localizes with NS3 and dsRNA, arguing
that NS4B is part of the membrane-bound virus replication
complex (Miller et al., 2006).
NS4B dissociates NS3 from ssRNA
The C-terminal domain of flaviviral NS3 has been proposed
to function in RNA and protein recognition (Xu et al.,
2005; Yon et al., 2005). In order to test whether its inter-
action with NS4B would affect the RNA-binding pro-
perty of NS3, we carried out EMSA. A radiolabelled ssRNA
probe was generated by incorporation of
32
P during in
vitro transcription and EMSA was performed as described
in Methods. Briefly, equal amounts of the ssRNA were
incubated with proteins at 37 uC for 5 min to allow bind-
ing and the protein–RNA complexes were resolved on
an 8 % native polyacrylamide gel under non-denaturing
conditions.
Increasing amounts of GST–NS4B decreased the binding
of NS3-FL (bacterially expressed full-length NS3) to ssRNA,
whilst GST and GST–NS4B did not bind to ssRNA. Inter-
estingly, this dissociation of NS3 from ssRNA occurred
only when the stoichiometric ratio of NS4B to NS3 was at
least 2 : 1 (Fig. 5a). Also, CF NS3, but not NS3-FL, formed
higher-order complexes, possibly protein–RNA concata-
mers [denoted by * in Figs 5(a, b)], that disappeared when
the concentration of CF NS3 was reduced to accommodate
the stoichiometry of GST/GST–NS4B versus CF NS3 (lanes
12 and 13). This suggests that the formation of these con-
catamers is dependent on concentration of the protein, as
well as the presence of its cofactor. However, dissociation of
NS3 from ssRNA in the presence of NS4B was independent
of the NS2B cofactor, as shown in Fig. 5(b), where NS4B
abolished ssRNA binding of both NS3 (NS3-FL) and NS3
with cofactor (CF NS3). In a control experiment, GST–
NS4BM did not show any effect on ssRNA binding of NS3
(Fig. 5c).
Fig. 3. Co-immunoprecipitation studies of the NS4B and NS3
interaction. (a) Pull-down with infected C6/36 cell lysates.
Uninfected and infected C6/36 cell lysates were incubated with
or without NS4B antibody and Protein A beads were added to
capture the complex. Western blotting with NS3 antibody
shows an approximately 68 kDa band that corresponds to the
full-length NS3 being captured specifically by the anti-NS4B
antibody in infected cell lysates (lane 4). Lanes 1 and 2 corre-
spond to uninfected cell lysates (negative control) without and
with anti-NS4B antibody, respectively, whilst lanes 3 and 4 cor-
respond to infected cell lysates without or with anti-NS4B anti-
body. (b) Pull-down with infected A549 cell lysates. Uninfected
and infected A549 cell lysates were incubated with or without
NS4B antibody and Protein A beads were added to capture
the complex. Western blotting with NS3 antibody shows full-
length NS3 (68 kDa, see arrow) being immunoprecipitated by
the NS4B antibody in infected cell lysates (lane 2). Lanes 1
and 3, uninfected cell lysates with and without anti-NS4B anti-
body, respectively; lanes 2 and 4, infected cell lysates with and
without anti-NS4B antibody, respectively.
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NS4B modulates the dsRNA-unwinding activity
of NS3
As NS4B interfered with the RNA binding of NS3, we asked
whether this affects the helicase activity of NS3 in a dsRNA-
unwinding/helicase assay (Xu et al., 2005). Briefly, a radio-
labelled dsRNA substrate was incubated with NS3-FL alone
or with GST, GST–NS4B or GST–NS4BM at 37 uC for
30 min; the mixture was then run on a gel to separate the
ssRNA from the dsRNA. A 1 : 1 or 1 : 2 molar ratio of GST
or GST–NS4B to NS3 was employed in the assay and
ssRNA release was measured by autoradiography. As seen
in Fig. 6(a), there was an approximately twofold increase
in the helicase activity of NS3 upon addition of GST–
NS4B, as opposed to the addition of GST. These experi-
ments were repeated three times, the autoradiography
signals were quantified and the helicase activity is repre-
sented in Fig. 6(b). Statistical relevance of these results was
calculated by using a two-tailed ttest (Fig. 6b). GST, GST–
NS4B and GST–NS4BM did not exhibit any unwinding
activity on their own. GST–NS4BM did not enhance the
helicase activity of NS3 (data not shown). Taken together,
these results suggest that NS4B enhanced the overall dsRNA-
unwinding activity of NS3 by dissociating it from ssRNA
and thereby enabling it to bind to a new duplex.
DISCUSSION
The roles of dengue virus NS3, NS5 and NS2B in replication
have been fairly well characterized, but those of the other
non-structural proteins, NS1, NS2A, NS4A and NS4B, have
remained somewhat elusive. We sought to ascertain the
role of NS4B in replication by searching for its interacting
partner among the other non-structural proteins. The yeast
two-hybrid interaction-trap method has been used success-
fully to characterize the interactions of HCV NS4B, which
(similar to dengue NS4B) is a hydrophobic protein with
several transmembrane domains (Dimitrova et al., 2003).
Hence, we used this assay to search for interacting partners
of dengue virus NS4B.
In this study, we identified an interaction between NS4B
and the C-terminal part of NS3 (aa 303–618) that contains
a helicase motif. Full-length NS4B, but neither the N- nor
C-terminal truncations of NS4B, interacted with NS3 (aa
303–618) in our yeast two-hybrid assay, suggesting that
this interaction is dependent on NS4B conformation. We
validated this interaction by using biochemical pull-downs
with recombinant proteins and co-immunoprecipitations
of endogenously expressed proteins in infected cell lysates
and have shown that they co-localize to similar subcellular
compartments.
Structural analysis of NS3 helicase suggests that it binds to
RNA as well as proteins through its C-terminal region (Wu
et al., 2005; Xu et al., 2005). As NS4B interacted with the
C-terminal region of NS3, it seemed likely that RNA bind-
ing of NS3 might be affected by this interaction. Our RNA-
binding experiments have shown that wild-type NS4B, but
not the mutant (NS4BM), dissociates NS3 from ssRNA.
Interestingly, the dissociation is dependent on stoichiome-
try of the molecules. At least two molecules of NS4B per
molecule of NS3 are needed to have a pronounced effect on
ssRNA binding of NS3. These data are supported by our
yeast two-hybrid results, wherein NS4B interacted with
itself, suggesting that a functional NS4B molecule may
be an oligomer. Dengue virus proteins are translated as
a polyprotein, wherein a 1 : 1 stoichiometry of molecules
seems logical. However, there are examples of other dengue
virus proteins, such as NS1, which acts as a hexamer
(Flamand et al., 1999; Winkler et al., 1988), and E protein,
which forms a heterodimer with the prM protein (Zhang
et al., 2003), that are known to form functional oligomers,
suggesting the existence of such stoichiometry of molecules
in vivo.
In the case of HCV, NS4A increases the ability of NS3
to bind to RNA and thereby enhances its helicase activity
(Gallinari et al., 1999; Howe et al., 1999; Morgenstern et al.,
1997; Pang et al., 2002). As dengue virus NS4B dissociates
NS3 from RNA, we hypothesized that NS4B might act
as a negative modulator of NS3. Surprisingly, NS4B did
(a) (b) (c)
(d) (e) (f)
Fig. 4. Subcellular co-localization of NS3
and NS4B proteins. NS3 and NS4B exhibit
similar subcellular-distribution patterns in
A549 cells co-transfected with NS4B and
NS3 plasmids (a–c) and also in TSV01
virus-infected A549 cells (d–f). Mouse anti-
NS4B antibody was detected with FITC-
labelled anti-mouse secondary antibody
(green) and rabbit anti-NS3 antibody was
detected with Texas red-labelled anti-rabbit
secondary antibody (red). The right-hand
images show a merge of the other two
panels, demonstrating co-localization of NS3
and NS4B.
2610 Journal of General Virology 87
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not decrease the dsRNA-unwinding activity of NS3, but
enhanced it in an in vitro helicase assay. The helicase acti-
vity of dengue virus NS3 protein is coupled functionally
to its NTPase activity (reviewed by Rocak & Linder,
2004). However, NS4B had no effect on the ATP hydrolysis
activity of NS3-FL in a colorimetric assay described pre-
viously (Lanzetta et al., 1979; Silverman et al., 2003; Xu et al.,
2005) (data not shown). This suggests that NS4B does not
regulate the helicase activity of NS3 via its NTPase func-
tion. We hypothesize that NS3 is displaced from ssRNA in
Fig. 5. NS4B abolishes ssRNA binding of NS3. (a) NS4B dissociates NS3 from ssRNA in a dose-dependent manner. Equal
amounts of the
32
P-labelled ssRNA substrate were incubated with proteins at 37 6C for 5 min to allow binding and the
complexes were resolved on an 8 % native polyacrylamide gel under non-denaturing conditions. Gels were scanned by using
a Typhoon phosphorimager (Amersham Biosciences). Arrows represent free probe and shifted bands. Lanes 1 and 2,
negative control and NS3 pro; lane 3, CF NS3; lanes 4 and 5, GST and GST–NS4B; lanes 6–13, increasing molar ratios of
GST and GST–NS4B with CF NS3. (b) Dissociation of NS3 from ssRNA by NS4B is independent of the NS2B cofactor.
Lane 1, negative control; lanes 2 and 3, 3 mM each of CF NS3 and NS3-FL, respectively; lanes 4 and 6, 6 mM GST with
NS3-FL and CF NS3, respectively; lanes 5 and 7, 6 mM GST–NS4B with NS3-FL and CF NS3, respectively. (c) Mutant
NS4B does not alter RNA binding of NS3. Lane 1, negative control; lanes 2 and 3, GST and GST–NS4BM, respectively; lane
4, NS3-FL; lane 5, GST–NS4BM with NS3-FL.
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the presence of NS4B, enabling it to interact with the next
duplex and thereby increasing the overall processivity of
the enzyme in vitro. In this light, it will be interesting to
see whether ssRNA and NS4B compete to bind to the same
region of NS3.
A single amino acid mutation in NS4B (P104L), which has
been reported previously to have pleiotropic effects on
dengue virus replication in mosquito versus human cells
(Hanley et al., 2003), disrupted the interaction between
NS3 and NS4B in both yeast two-hybrid and pull-down
assays. This NS4B mutant had no effect on the RNA-
binding or helicase activities of NS3. Proline at amino
acid position 104 of NS4B is conserved in DENV-1–DENV-
4, but, interestingly, Japanese encephalitis virus, Kunjin
virus and West Nile virus each possess a leucine at that
position. Therefore, Hanley et al. (2003) proposed that
the substitution of proline by leucine in position 104 of
NS4B causes a change in its structure or conformation that
results in altered replication in vivo. This hypothesis might
explain why, in our yeast two-hybrid and in vitro studies,
neither truncated NS4B nor the P104L mutant interacted
with NS3.
An in vivo implication of the NS4B–NS3 interaction is in
the formation of a functional complex that holds the two
strands of the RNA apart. It has been proposed that flaviviral
NS3 and NS5 act as a functional complex (Brooks et al.,
2002; Yon et al., 2005). The physical interaction of NS3 and
NS4B demonstrated in this study might imply that all three
molecules (NS3, NS4B and NS5) form a complex that holds
the separated strands apart as the helicase moves along the
duplex. Further in vivo studies will be needed to verify this
hypothesis and to determine the role of the non-structural
protein complex in flavivirus replication.
Finally, it is evident that an understanding of the flavivirus
replication cycle will require characterization of the physical
and functional interactions of the proteins that form the
replication complex, including unidentified host proteins.
Whereas many studies have indicated how NS3 and NS5
might participate in the replication process, this is the first
report of the role of flaviviral NS4B in virus replication.
Further work on the finely balanced interactions between
all of these components should ultimately provide a working
model for the control of flavivirus replication.
ACKNOWLEDGEMENTS
We would like to thank Dr Siew Pheng Lim for the kind gift of plasmid
pXJ-NS3-FL, Wei Liu for help with virus and antibody production, Drs
Yen Liang Chen and Mark Schreiber for useful discussions and Wai Yee
Phong and Daying Wen for NS3 protein.
Fig. 6. Modulation of RNA-unwinding activity of NS3 by NS4B. (a) NS4B enhances unwinding activity of NS3. Lane 1,
negative control; lane 2, heat-denatured duplex; lane 3, 3 mM NS3-FL; lanes 4 and 5, 6 mM each of GST and GST–NS4B;
lanes 6 and 7, 3 mM GST and GST–NS4B, respectively, with 3 mM NS3-FL; lanes 8 and 9, 6 mM GST and GST–NS4B,
respectively, with 3 mM NS3-FL. In all reactions, 0?25 pmol dsRNA substrate and 1 mM ATP were used, incubated at 30 6C
for 30 min, terminated and resolved on a 10 % native gel. (b) Quantification of autoradiography signals. Amount of ssRNA in
each lane was quantified by using ImageQuant software and represented as fold variation with respect to the ssRNA released
by NS3-FL. The variation in the unwinding activity of NS3 in the presence of GST–NS4B is significant (P=0?0025 and
0?0029) as opposed to that seen in the presence of GST (P=0?33058 and 0?0637). The values in the graph represent
means of three staggered experiments.
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