Mapping the Interactions of Dengue Virus NS1 Protein
with Human Liver Proteins Using a Yeast Two-Hybrid
System: Identification of C1q as an Interacting Partner
Emiliana M. Silva1., Jonas N. Conde1., Diego Allonso1, Mauricio L. Nogueira2, Ronaldo Mohana-Borges1*
1Laborato ´rio de Geno ˆmica Estrutural, Instituto de Biofı ´sica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Rio de Janeiro, Brazil,
2Departamento de Doenc ¸as Dermatolo ´gicas, Infecciosas e Parasita ´rias, Faculdade de Medicina de Sa ˜o Jose ´ do Rio Preto, Sa ˜o Jose ´ do Rio Preto, Sa ˜o Paulo, Brazil
Dengue constitutes a global health concern. The clinical manifestation of this disease varies from mild febrile illness to
severe hemorrhage and/or fatal hypovolemic shock. Flavivirus nonstructural protein 1 (NS1) is a secreted glycoprotein that
is displayed on the surface of infected cells but is absent in viral particles. NS1 accumulates at high levels in the plasma of
dengue virus (DENV)-infected patients, and previous reports highlight its involvement in immune evasion, dengue severity,
liver dysfunction and pathogenesis. In the present study, we performed a yeast two-hybrid screen to search for DENV2 NS1-
interacting partners using a human liver cDNA library. We identified fifty genes, including human complement component 1
(C1q), which was confirmed by coimmunoprecipitation, ELISA and immunofluorescence assays, revealing for the first time
the direct binding of this protein to NS1. Furthermore, the majority of the identified genes encode proteins that are
secreted into the plasma of patients, and most of these proteins are classified as acute-phase proteins (APPs), such as
plasminogen, haptoglobin, hemopexin, a-2-HS-glycoprotein, retinol binding protein 4, transferrin, and C4. The results
presented here confirm the direct interaction of DENV NS1 with a key protein of the complement system and suggest a role
for this complement protein in the pathogenesis of DENV infection.
Citation: Silva EM, Conde JN, Allonso D, Nogueira ML, Mohana-Borges R (2013) Mapping the Interactions of Dengue Virus NS1 Protein with Human Liver Proteins
Using a Yeast Two-Hybrid System: Identification of C1q as an Interacting Partner. PLoS ONE 8(3): e57514. doi:10.1371/journal.pone.0057514
Editor: Luciano A. Moreira, Centro de Pesquisas Rene ´ Rachou, Brazil
Received November 22, 2012; Accepted January 22, 2013; Published March 14, 2013
Copyright: ? 2013 Silva et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the following funding agencies: the Brazilian Health Ministry, CNPq, FAPERJ, IMBEBB, FINEP (GENOPROT Dengue), and the
National Institutes of Science and Technology in Structural Biology and Bioimaging (INCT-INBEB). The funders had no role in the study design, data collection and
analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
. These authors contributed equally to this work.
Dengue constitutes a major global health concern. It is
estimated that nearly half of the worldwide population lives in
risk areas and that fifty to one hundred million infections occur
each year, including 500,000 hospitalizations of patients with
severe dengue illness [1,2]. Dengue virus (DENV) is a member of
the Flaviviridae family, and it cocirculates as four distinct antigenic
serotypes (DENV1–4). Infection with DENV may induce a
spectrum of symptoms varying from none to severe plasma
leakage, hemorrhage and organ impairment . The mechanism
underlying endothelial cell dysfunction and vascular leakage is of
primary importance; however, it is far from being understood.
Several studies have been published attempting to elucidate the
principal phenomenon that leads to severe disease. Indeed, it has
been established that the risk of developing severe dengue may be
associated with secondary heterologous infection, leading to the
phenomenon of antibody-dependent enhancement (ADE) , in
addition to high viral loads [5–7] and multiple host factors
including age, gender, genotype and prior immunity, among
others [8,9]. Disease severity can also be correlated to circulating
levels of certain cytokines and chemokines such as tumor necrosis
factor-alpha (TNF-a), interleukin 1b (IL-1b), interleukin 6 (IL-6),
interleukin 10 (IL-10), interferon-gamma (IFN-c), interleukin 8
(IL-8), macrophage inflammatory protein 1 (MIP-1) [10–18], and
complement components (C3a, C5a, factor D and factor H) [19–
22]. Despite the fact that several cell types and tissues have been
described as potential sites for DENV replication and release of
plasma immune mediators, the liver is one of the most important
infection target organs [23,24].
The flavivirus nonstructural protein 1 (NS1) is a 50 kDa
intracellular homodimeric glycoprotein that plays a pivotal role in
DENV replication , and there is evidence that it also plays an
important role in dengue severity and pathogenesis [6,26].
Although lacking a membrane-anchoring domain, the NS1
protein associates with organelle membranes and in particular
with lipid-rafts, suggesting that it is involved in signal transduction
pathways . This association likely occurs via a GPI anchor
. The DENV NS1 protein is also secreted into the plasma as a
lipid-associated barrel-shaped hexamer that is detectable in patient
serum in the first few days after the onset of clinical symptoms in
both primary and secondary infections [29,30]. Recent reports
highlight the involvement of the NS1 protein in the modulation of
the complement system and the vascular leakage process, which
facilitate immune complex formation . Moreover, the NS1
protein elicits autoantibodies that react with platelets and
extracellular matrix proteins  or that interfere with endothelial
PLOS ONE | www.plosone.org1 March 2013 | Volume 8 | Issue 3 | e57514
antibody-dependent, complement-mediated cytolysis . DENV
NS1 also exhibits complement antagonism by binding directly to
complement proteins, including C4 and C1s, which leads to the
degradation of C4 in solution and, consequently, to the inhibition
of complement activation . Alcon-LePoder and coworkers
(2005) demonstrated that the liver is the major site for NS1 protein
accumulation and preincubation of hepatocytes with soluble NS1
enhances subsequent infection by a homologous strain of DENV
. However, the mechanism by which NS1 is involved in
dengue pathogenesis remains unclear.
To understand the role of the NS1 protein in DENV infection
and pathogenesis, a yeast two-hybrid system was used to screen for
the interacting partners of the DENV NS1 protein using a human
liver cDNA library. We identified 50 different NS1-interacting
partners, including the C1q protein. Coimmunoprecipitation,
ligand binding ELISA, and immunofluorescence assays were also
performed to confirm the direct binding of NS1 to human C1q.
These results indicate the association of the DENV NS1 protein
with another complement component, which suggests a role for
this complement protein in DENV pathogenesis.
Identification of DENV NS1-interacting partners using the
yeast two-hybrid system
Considering the importance of the NS1 protein during DENV
infection and its possible involvement with liver dysfunction ,
we aimed to understand its role in DENV infection and
pathogenesis. Therefore, we performed a yeast two-hybrid
screening to detect novel putative NS1 interacting-partners using
a liver cDNA library. First, to determine whether the recombinant
yeast AH109 cells expressed the NS1 protein, we performed a
Western blot assay using the anti-NS1 polyclonal antibody (as
described elsewhere ). We observed that the NS1 protein was
properly expressed in these cells and did not interfere with cell
growth (data not shown). Previous studies from our laboratory
revealed that the NS1 protein expressed in yeast cells preserved its
structural properties and was found as a glycosylated dimer
Next, to select putative positive clones, we first grew the
recombinant cells containing the bait and prey plasmids in triple
drop-out media. This selection yielded 2,080 colonies. We
identified the recombinant colonies containing a positive NS1
protein-interacting partner by HIS3, ADE2 and lacZ reporter gene
activation, as visualized by cell growth on triple and quadruple
drop-out media plates and by blue color staining following the
colony-lift assays (Fig. 1). This analysis led to the identification of
50 different genes, including genes that encode the complement
proteins C1q and C4 in addition to genes that encode several
acute-phase proteins (APPs), such as plasminogen, haptoglobin,
and hemopexin, among others (Table 1). Interestingly, the
interaction of NS1 with the complement C4 protein has been
described previously .
Thus, to identify the corresponding proteins of these identified
genes, we subjected their sequences to BLASTX analysis (available
at the NCBI website). Next, we arranged them according to their
primary cellular localization, as shown in Figure 2. We determined
that NS1-interacting partners belonged to a wide range of protein
classes including extracellular milieu-released proteins (35% of all
proteins screened by the yeast-two hybrid system) that includes, for
example, the apolipoprotein A2 and H, C1q, C4, haptoglobin,
hemopexin, plasminogen, and transferrin; cytoplasmic-resident
proteins (23% of all proteins) such as alcohol dehydrogenase 1B,
aldehyde dehydrogenase 1 and 7, and eukaryotic translation
elongation factor 1; mitochondrial-resident proteins (11% of all
proteins), such as, for example, 3-hydroxybutyrate dehydrogenase,
monoamine oxidase B and phosphoenolpyruvate carboxykinase 2;
plasma membrane proteins (11% of all proteins) such as CD14;
endoplasmic reticulum-resident proteins (4% of all proteins) such
as carboxylesterase 1; and lysosomal proteins (4% of all proteins)
such as prosaposin. These results indicate the ability of the NS1
protein to interact with different classes of host proteins, primarily
those localized in the extracellular region.
DENV NS1 directly interacts with C1q
Because the interaction of flavivirus NS1 with complement
system proteins and its regulators appears to play an important
role in the immune evasion process [32,35,36], we focused our
studies on the confirmation of the DENV NS1 and C1q
interaction. First, we performed a coimmunoprecipitation assay
in which the anti-NS1 polyclonal antibody was immobilized to the
resin and subsequently incubated with NS1, which was purified in
its hexameric form from the supernatants of DENV-infected BHK
cells, and purified human C1q protein. Because C1q is able to
bind the Fc region of antibodies , we also assessed the
interaction of purified C1q with the anti-NS1-coated resin
(control). The elution fractions were then analyzed by Western
blot using another specific anti-NS1 polyclonal antibody  and
an anti-C1q monoclonal antibody. Two bands of approximately
30 and 50 kDa corresponding to C1q and NS1, respectively, were
obtained, indicating that C1q coimmunoprecipitated with the NS1
protein (Fig. 3A). As expected, a band of approximately 30 kDa
was also observed in the control experiment (Fig. 3A). However,
the band intensity for C1q in the presence of NS1 was more than
four-fold higher than that in the absence of NS1 (control), and this
difference is statistically significant (p=0.0177, Fig. 3B). These
findings confirm the interaction between the NS1 and C1q
proteins as determined by the yeast two-hybrid screening.
Previous studies have shown that anti-NS1 antibodies are able
to cross-react with some human proteins, such as blood clotting
factors and adhesion molecules [31,38]. Therefore, to verify
whether the C1q precipitation in the control experiment was
caused by its binding to the Fcregion of the anti-NS1 antibody or
its cross interaction, we performed an identical experiment as
described above using a nonspecific IgG1-coated resin. We
observed a sharp band of approximately 30 kDa in the elution
fraction, indicating that the C1q protein was also immunoprecip-
itated by IgG1at low levels (Fig. 3A). Analysis of the band intensity
revealed that there is no significant difference between coimmu-
noprecipitation using IgG1and the control experiments, but there
is a significant difference between coimmunoprecipitation using
IgG1 and coimmunoprecipitation in the presence of NS1
(p=0.0111, Fig. 3B). These results indicate that the anti-NS1
antibody does not cross-react with C1q.
To assess whether NS1 directly binds C1q, an ELISA assay was
designed in which the purified C1q was immobilized onto the
plate and incubated with increasing concentrations of NS1
purified from the supernatant of DENV-infected BHK cells. As
a control, we used the supernatant of mock-infected BHK cells
subjected to an identical protocol used in the NS1 purification. To
detect the interaction, we used a specific conformational anti-NS1
monoclonal antibody (DN1). We observed a significant increase in
the optical density (OD) values when both proteins were incubated
together, whereas no difference in the OD value was observed in
the control experiment (Fig. 4A). Statistical analysis using two-way
ANOVA revealed a significant difference between the control and
binding curves. This result clearly indicates that NS1 binds C1q
directly in a dose-dependent manner.
Dengue Virus NS1 Protein Interacts with C1q
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The NS1 protein is glycosylated in at least two sites  and
coexists as different oligomeric states (dimers and/or hexamers).
However, there is considerable discussion concerning which
oligomeric state of NS1 interacts with other proteins and whether
these interactions occur through its carbohydrate moiety. Previous
studies from our group demonstrated that bacterial recombinant
NS1 protein is able to form dimers in a conformation similar to
those produced in insect cells depending on the refolding protocol
used . To evaluate whether recombinant NS1 purified from
bacteria was able to bind C1q, we performed an identical
experiment as described above for NS1 purified from BHK cells.
As a control, we used purified bovine serum albumin (BSA). We
observed that recombinant NS1 was also able to bind C1q directly
in a dose-dependent manner similar to what was observed for NS1
purified from BHK cells. Note that the peak of the OD value
measured for each NS1 protein was similar. We also performed an
identical experiment but in the opposite order, i.e., immobilizing
the NS1 protein (either from DENV-infected BHK or from
recombinant bacterial cells) onto the plate followed by incubation
with increasing concentrations of purified human C1q. These data
were similar to those shown in Figure 4 (data not shown).
Together, CoIP and ELISA results not only confirm the previous
Figure 1. Plasmid linkage assays for transformants identified using DENV2 NS1 as bait in the yeast two-hybrid screening.
Transformants containing the bait and prey plasmids were visualized by their growth on double drop-out media (SD–Leu–Trp; column A). Putative
interacting partners were visualized by their growth on triple (SD–His–Leu–Trp; column B) and quadruple drop-out media (SD–Ade–His–Leu–Trp;
column C) and by the blue color staining of the colony-lift filter assay (column D) indicating HIS3, ADE2 and lacZ reporter gene activation,
respectively. AH109 yeast cells cotransformed with the plasmids pGBKT7-53 (murine p53 fused to the GAL4 DNA-binding domain) and pGADT7-T
(SV40 large T-antigen fused to the GAL4 activation domain) served as positive controls (C+). AH109 cotransformed with the plasmids pGBKT7-NS1
and pGADT7-AD (C1), pGBKT7-NS1 and pGADT7-T (C2), pGBKT7 and pGADT7 (C3), pGBKT7 and pGADT7-T (C4), pGBKT7-Lam (laminin C) and pGADT7
(C5), and pGBKT7-Lam and pGADT7-T (C6) served as negative controls. The gene name for each acronym is detailed in Table 1.
Dengue Virus NS1 Protein Interacts with C1q
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Table 1. Human liver proteins that interact with DENV2 NS1 protein identified by yeast two-hybrid screening.
Abbreviation Gene nameNCBI ID No. of clones detected
ACTG1actin, gamma 1NG_011433.12
ADH1B alcohol dehydrogenase 1B (class I), beta polypeptideNG_011435.14
ALDH1L1 aldehyde dehydrogenase 1 family, member L1NG_012260.12
ALDH7A1 aldehyde dehydrogenase 7 family, member A1 NG_008600.25
ALDOB aldolase B, fructose-bisphosphateNG_012387.113
AMBP alpha-1-microglobulin/bikunin precursorNM_001633.35
APEH N-acylaminoacyl-peptide hydrolaseNM_001640.32
APOA2 apolipoprotein A-IINM_001643.11
APOH apolipoprotein H (beta-2-glycoprotein I)NG_012045.15
AZGP1 alpha-2-glycoprotein 1, zinc-binding NM_001185.31
BCKDHA branched chain keto acid dehydrogenase E1, alpha polypeptideNM_000709.31
BDH13-hydroxybutyrate dehydrogenase, type 1 NM_004051.41
C1QA complement component 1, q subcomponent, A chainNG_007282.12
C4A complement component 4ANM_007293.22
C7orf10 C7orf10 chromosome 7 open reading frame 10NM_001193311.11
CCS copper chaperone for superoxide dismutaseNM_005125.11
CD14 CD14 molecule NG_023178.12
CES1 carboxylesterase 1NG_012057.11
COL18A1 collagen, type XVIII, alpha 1NM_030582.31
CTSB cathepsin BNG_009217.11
CUTA cutA divalent cation tolerance homolog (E. coli)NM_001014840.11
ECHDC1 enoyl CoA hydratase domain containing 1NM_001002030.12
EEF1A1 eukaryotic translation elongation factor 1 alpha 1NM_001402.53
EMR2 egf-like module containing, mucin-like, hormone receptor-like 2NM_152916.11
ENTPD5 ectonucleoside triphosphate diphosphohydrolase 5NM_001249.21
FAH fumarylacetoacetate hydrolase (fumarylacetoacetase)NG_012833.11
FHfumarate hydratase NG_012338.11
GPX2glutathione peroxidase 2 NM_002083.21
HP haptoglobin NG_012651.11
HPX hemopexin NM_000613.21
LSR lipolysis stimulated lipoprotein receptorNM_205834.21
MAOB monoamine oxidase BNG_008723.11
MPNDMPN domain containing NM_001159846.11
MT2A metallothionein 2ANM_005953.3 13
MTERFD2MTERF domain containing 2 NM_182501.31
MTHFD1 methylenetetrahydrofolate dehydrogenase (NADP+ dependent) 1,
methenyltetrahydrofolate cyclohydrolase, formyltetrahydrofolate synthetase
PARP12poly (ADP-ribose) polymerase family, member 12 NM_022750.22
PCK2phosphoenolpyruvate carboxykinase 2 (mitochondrial) NG_008162.12
PKD1 polycystic kidney disease 1NG_008617.11
RBP4 retinol binding protein 4, plasmaNG_009104.12
RELN reelin NG_011877.14
SAT1spermidine/spermine N1-acetyltransferase 1 NG_012929.11
SEPP1selenoprotein P, plasma, 1NM_001085486.11
SIVA1 SIVA1, apoptosis-inducing factorNM_006427.37
Dengue Virus NS1 Protein Interacts with C1q
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identification of C1q as an NS1-interacting partner but also reveal
that this interaction occurs directly. Moreover, the finding that
bacterial recombinant NS1 protein is also able to bind C1q
indicates that this interaction is independent of posttranslational
Colocalization of the NS1 protein with endogenous C1q
To evaluate whether these proteins colocalize during DENV
infection in vivo, we performed an immunofluorescence assay of
mock- and DENV-infected human acute monocytic leukemia
(THP-1) cells using both anti-NS1 and anti-C1q antibodies.
Although the liver is the principal production site of complement
components, hepatocytes do not express the C1q protein . In
fact, it is the liver-resident macrophages (Kupffer cells) that are
responsible for the production of C1q . Alternatively, we used
a mononuclear phagocytic cell line (THP-1) that has been shown
to express C1q . We observed that infected cells were labeled
for both C1q (green) and NS1 (red) proteins (Fig. 5). When the
images were merged, distinct yellow regions were observed,
indicating the colocalization of NS1 with C1q in these areas (Fig. 5,
detail). The C1q labeling was also observed in mock-infected cells,
and it appeared at an identical position as that observed in DENV-
infected cells in which no NS1 was detected. These results clearly
indicate the colocalization of DENV NS1 with C1q in cell culture.
In the present study, we identified 50 putative interacting
partners of DENV2 NS1 by screening a human liver cDNA
library using a yeast two-hybrid system. In this screening, we
detected the NS1 interaction with a key protein of the complement
system, C1q. According to recent reports, the flavivirus NS1 seems
to interact with complement system proteins and its regulators
exhibiting immune-evasion functions, which contributes somehow
to disease pathogenesis [32,35,36]. Based on this information, we
focused our efforts on the confirmation of the DENV NS1 and
C1q interaction by coimmunoprecipitation, ELISA and immuno-
fluorescence assays. The complement system consists of more than
30 proteins that are present as either soluble blood-borne or
membrane-associated proteins. This collection of proteins orga-
nizes into a hierarchy of proteolytic cascades beginning with the
identification of pathogenic surfaces and leading to i) the
generation of potent proinflammatory mediators (anaphylatoxins);
ii) the opsonization (coating) of the pathogenic surface using
various complement opsonins (e.g., C3b); and iii) targeted lysis of
the pathogenic surface by assembling membrane-penetrating
pores known as the membrane attack complex (MAC) [43,44].
Viruses have developed strategies to limit or retard their
recognition by the complement cascade and inhibit the immune
response. These strategies include i) secretion of molecules that
Table 1. Cont.
Abbreviation Gene nameNCBI ID No. of clones detected
THNSL1threonine synthase-like 1 NM_024838.41
Figure 2. Cellular localization of DENV2 NS1 interacting-
partners identified by yeast two-hybrid screening.
Figure 3. Coimmunoprecipitation of human C1q and NS1 proteins. (A) Purified NS1 from the supernatants of DENV-infected BHK cells and
purified human C1q protein were immunoprecipitated with anti-NS1 polyclonal antibody, and the eluted fractions 1 and 2 (E1 and E2) were
subjected to Western blot analysis. Bands of approximately 30 and 50 kDa corresponding to C1q and NS1, respectively, were observed in the elution
fraction of the coimmunoprecipitation. (B) C1q was capable of binding anti-NS1 antibody, although the band intensity in the DENV lane E1 was more
than four-fold intense than that in the control lane E1. The IgG1-coated resin eluted a similar amount of C1q as the control. Error bars indicate the
standard deviation from three independent experiments, and the p value denotes significant differences from the control.
Dengue Virus NS1 Protein Interacts with C1q
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mimic or recruit host complement regulators and expression of
surface proteins that interact with the antibody Fcregion, thereby
preventing C1q-dependent action; ii) incorporation of comple-
ment components on the virion; and iii) upregulation of
complement regulatory proteins on the surface of infected cells
. It is known that C1q binds to the Fcregion of the antibody
on antigen-antibody complexes and activates the classical pathway
in which C3 convertase promotes C3b-mediated opsonization and
assembly of C5b-C9 MAC [37,45,46]. This pathway is also
activated in the absence of antibodies due to the direct interaction
of C1q with viral surface proteins, as observed for the p15E
protein of oncornavirus  and gp41 and gp120 of the human
immunodeficiency virus (HIV-1) [48,49].
The flavivirus NS1 protein has been described as an immune
evading protein  that binds complement regulators such as
human clusterin, which inhibits MAC formation , and C4b-
binding protein (C4BP), which regulates complement activation by
interacting with C4b . In addition, the NS1 protein binds
complement proteins such as C4, also identified in this work,
forming a complex with proC1s/C1s and leading to C4
degradation . These interactions may help the virus to reduce
complement activation via the classical and lectin pathways
[32,36]. Moreover, several studies have indicated that the
complement system can restrict ADE. ADE is a phenomenon
associated with dengue severity that occurs when non-neutralizing
antiviral antibodies enhance viral entry into host cells, leading to
increased efficiency of infection . Experiments with mouse sera
lacking individual complement components have indicated that
C1q is sufficient to restrict ADE in West Nile virus (WNV)
infection , and this has also been demonstrated using human
sera in DENV infection . C1q can also increase the potency of
antibodies targeted against WNV by modulating the stoichiomet-
ric requirements for virus neutralization . Our results
indicated that DENV2 NS1 can directly bind the complement
protein C1q, and this interaction is not dependent on the
glycosylated form of DENV NS1. Nevertheless, further experi-
ments are required to ascertain whether this interaction will
modulate complement activation.
Interestingly, most of the identified NS1-interacting partners are
proteins that are secreted to the extracellular milieu, such as
plasminogen, haptoglobin, hemopexin, a-2-HS-glycoprotein, ret-
inol binding protein 4, transferrin, and C4 and are classified as
APPs. They actively participate in the acute-phase response (APR),
and their plasma concentration varies during the inflammatory
process. The APR is an important pathophysiological phenome-
non that supersedes the normal homeostatic mechanisms during
infection. The APR is an essential component of the innate
immune system and plays an important role in limiting hepatic
tissue injury. Several APPs can initiate, amplify or sustain
inflammation, whereas others can attenuate it [54,55]. Increasing
the circulating levels of proinflammatory cytokines, chemokines,
and immune effectors, for example, can lead to initiation of the
inflammatory process. APR proteins crosstalk with proteins
involved in the coagulation process, as observed during septic
shock. This crosstalk is of great importance in the induction of
episodes of DHF/DSS [56,57]. Therefore, the NS1-plasminogen
interaction would be a promising start point to study the abnormal
activation of the fibrinolytic system associated with DHF/DSS
events. There has been considerable discussion on the origin and
maintenance of signals/effectors that trigger the vascular leakage
phenomenon in DENV infection. However, the hypothesis that
several factors, such as host genetic background, virus character-
istics and immune response, contribute to dengue severity is
commonly accepted . During the course of DENV infection,
increased levels of proinflammatory cytokines and chemokines are
observed, including TNF-a, IL-1b, IL-6, IL-8, IL-10, IFN-c, and
more recently, the HMGB-1  [10–17]. High levels of TNF-a
and IL-6 influence disease severity and are directly correlated to
the increase in vascular permeability . It is known that the
NS1 protein interacts with STAT-3b  and also activates the
transcriptional regulator NF-kB . These two proteins (NF-kB
and STAT3) are likely to play important roles in the liver
inflammatory response and in the maintenance of homeostasis,
induction of APP synthesis by stimulating cytokines such as IL-6
and TNF-a, and in inducing liver dysfunction [54,61]. However,
further studies are required to elucidate the mechanism by which
NS1 modulates APP and APR phenomena.
In summary, our results demonstrate that DENV NS1 directly
interacts with C1q, a key protein of the complement system. The
Figure 4. DENV NS1 directly binds human C1q in an ELISA
assay. (A) Microtiter plates were coated with purified human C1q
(10 mg/mL). After incubation with increasing concentrations of purified
DENV NS1 that was purified from the supernatant of BHK cells, bound
NS1 was detected using a specific conformational monoclonal anti-NS1
antibody. (B) Microtiter plates were coated with purified human C1q
(10 mg/mL). After incubation with increasing concentrations of purified
DENV NS1 that was purified from E. coli cells, bound NS1 was detected
with a specific polyclonal anti-NS1 antibody. Error bars indicate
standard deviation from three independent experiments, and asterisks
indicate significant difference from the control mock or BSA. *p,0.05,
Dengue Virus NS1 Protein Interacts with C1q
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specific C1q-NS1 interaction may enable the virus to avoid
complement activation through the classical pathway by possibly
preventing antibody interaction and/or enabling C1-complex
assembly. This hypothesis could be confirmed with in vivo studies
by assessing complement activation in C1q, C1s or C1r-deficient
mice challenged with DENV and/or inoculated with NS1 protein.
Moreover, yeast two-hybrid screening revealed that DENV NS1
interacts with a wide range of proteins including some APPs that
are important effectors of APR, which is an orchestrated
mechanism focused on the reestablishment of normal liver and
plasma homeostasis. Binding to and altering the properties of
APPs may provide the molecular basis to link dengue infection and
hemostatic abnormalities such as vascular leakage, thrombocyto-
penia and hemorrhage. However, further studies are necessary to
confirm the role of NS1 interaction with C1q and APPs in the
pathogenesis of DENV infection.
Materials and Methods
Cloning the ns1 gene into the pGBKT7 plasmid
The ns1 gene, from the cDNA of the DENV2 New Guinea C
(NGC) strain, was amplified by PCR using the forward primer 59-
containing the BamHI restriction site (underlined bases) and the
CAAGGAGTTGAC-39 containing the NotI restriction site
(underlined bases) and the TAA stop codon. The PCR conditions
were as follows: 94uC for 2 min, 35 cycles of 94uC for 30 s, 58uC
for 1 min and 68uC for 2 min followed by a final extension at
68uC for 7 min. The amplified gene was cloned in frame with the
GAL4 DNA-binding domain (BD) of the yeast expression vector
pGBKT7 (Clontech, CA, USA) to construct the bait plasmid
Yeast two-hybrid screening
Using the full-length NS1 as bait, a yeast two-hybrid screening
was performed against a human liver cDNA library fused to the
GAL4 activation domain (AD) using the pACT2 vector and the
Matchmaker GAL4 Two-Hybrid System 3 (Clontech, CA, USA).
The yeast strain AH109 was transformed with the pGBKT7-NS1
plasmid using the lithium acetate method and then grown in SD
(synthetic defined) medium lacking tryptophan (SD–Trp). Auto-
activation of the HIS3 reporter was confirmed by the growth of
clones in SD medium lacking histidine, leucine and tryptophan
(SD–His–Leu–Trp). The transformed cultures were then plated
onto SD–His–Leu–Trp and SD–Ade–His–Leu–Trp media to
select putative positives clones. The activity of the lacZ reporter
gene was evaluated by the b-galactosidase assay (colony-lift filter)
using the substrate X-gal on nitrocellulose membranes. To
eliminate false positives, a plasmid linkage assay was also
performed. The positive plasmids were sequenced, and their gene
sequences were analyzed using the BLASTN and BLASTX
software available at NCBI (www.ncbi.nlm.nih.gov).
Cell culture and infection
Baby hamster kidney fibroblast (BHK; ATCC, USA) and
human acute monocytic leukemia (THP-1; ATCC, USA) cell lines
were cultured in a-MEM Medium (Gibco) and RPMI Medium
1640 (Gibco, NY, USA), respectively, supplemented with 10%
fetal bovine serum (Invitrogen, NY, USA), 0.22% sodium
bicarbonate and 0.2% HEPES, pH 7.4, in a humid CO2
incubation chamber at 37uC. After 2 days, BHK and THP-1
cells were mock-infected or infected with DENV2 strain 16681 at
a multiplicity of infection of 2. All experiments were performed at
48 h post infection.
DENV2 NS1 was produced and isolated from the supernatant
of BHK cells that were infected with the DENV2 strain 16881.
The supernatant was harvested, and NS1 was purified as described
previously . DENV NS1 from E. coli was expressed and
purified as described previously .
Coimmunoprecipitation and Western blot
The coimmunoprecipitation was performed using a kit (Pierce,
IL, USA). Purified anti-NS1 polyclonal antibody or IgG1control
antibody was attached to an N-terminal-binding resin. Purified
Figure 5. Colocalization of NS1 and C1q proteins by confocal microscopy. DENV2-infected THP-1 cells were labeled by incubation with
polyclonal anti-NS1 (red stained) or monoclonal anti-C1q (green stained) antibodies. NS1 and C1q proteins were localized in vesicle-like structures in
the cytoplasm, which is characteristic of secretory proteins. When the images were merged, distinct yellow regions were revealed, indicating
colocalization of NS1 with C1q in these areas (detail). The subcellular localization of C1q was also analyzed in mock-infected cells, and it appeared at
an identical position as that observed in DENV-infected cells, whereas no NS1 protein was detected in these cells. The cells were also incubated with
DAPI for nuclear staining.
Dengue Virus NS1 Protein Interacts with C1q
PLOS ONE | www.plosone.org7 March 2013 | Volume 8 | Issue 3 | e57514
NS1 from BHK supernatants and purified human C1q (Sigma-
Aldrich, USA) were mixed and added to the anti-NS1-coated
resin. C1q protein was also added to the anti-NS1- and IgG1-
coated resin to serve as controls and to assess nonspecific binding.
The samples obtained from the CoIP assay were separated by 12%
SDS-PAGE and transferred onto a Hybond ECL nitrocellulose
membrane (GE Healthcare, Sweden). The membrane was then
blocked with 5% BSA in TBST (0.1% Tween 20 in TBS [25 mM
Tris-HCl, pH 7.6, 3 mM KCl, and 140 mM NaCl]) for 2 h
followed by overnight incubation with a mouse polyclonal anti-
NS1 or mouse monoclonal anti-C1q antibody (Abcam, USA) in
blocking solution. The membrane was then washed three times
with TBST and incubated with anti-mouse IgG conjugated to
horseradish peroxidase (Promega, USA) in blocking solution for
2 h. The membrane was washed again, developed with a
Supersignal West Pico kit (Pierce, IL, USA) and exposed to
Kodak MXG/PLUS film. The band intensity was measured by
Scion Image software.
DENV2 NS1 and C1q ligand binding ELISA
Purified human C1q (Sigma-Aldrich, USA) at a concentration
of 10 mg/mL was adsorbed to wells in MaxiSorp microtiter plates
(Nunc) at 4uC overnight. After five washes with phosphate-
buffered saline (PBS), nonspecific binding sites were blocked with
200 mL of 1% BSA in PBS containing 0.05% Tween 20 (PBST)
for 1.5 h at 37uC followed by five washes with PBS. Purified NS1
from BHK or E. coli at specific concentrations were added to each
well and incubated for 2 h at 37uC. Plates were then washed five
times with PBST followed by a 1-h incubation with a DENV2
NS1-specific monoclonal (Abcam) or polyclonal antibody (1:1000
dilution). After five washes with PBST, anti-mouse IgG conjugated
to horseradish peroxidase (Promega, USA) was added sequentially
for 1 h at 37uC. After five final washes with PBST, the signal was
developed by adding 100 mL of 0.4 mg/mL o-phenylenediamine
(OPD) and 50 mL of 9 N H2SO4stop solution to each well. The
OD at 490 nm was determined by a 96-well plate reader.
Datasets were compared by a two-tailed, unpaired Student t test
and statistical significance was achieved when p values were
,0.05. Multiple comparisons were performed using two-way
ANOVA (Bonferroni post-test), and asterisks indicate significant
difference from the control (*p,0.05, **p,0.01, ***p,0.001).
Immunofluorescence confocal microscopy
THP-1 cells were washed in PBS, pH 7.2, and fixed for 30 min
with 4% freshly prepared formaldehyde diluted in PBS. Cells were
deposited on poly-L-lysine-treated microscope slides and permea-
bilized with 1.5% Triton X-100 in PBS, pH 7.2, for 25 min. The
slides were incubated in blocking solution containing 1.5% BSA in
PBS, pH 7.2, and then with purified anti-NS1 polyclonal and anti-
C1q monoclonal antibodies diluted 1:100 in blocking solution for
1 h. The cells were washed and incubated for 45 min with Alexa
488-conjugated goat anti-mouse IgG antibody (Invitrogen, USA)
or with Alexa 546-conjugated goat anti-rabbit IgG antibody
(Invitrogen, USA) diluted 1:400 in blocking solution. The cells
were subsequently incubated with 5 mM 49,6-diamidino-2-pheny-
lindole (DAPI, Sigma, USA) for 20 min at room temperature. The
slides were mounted in N-propyl gallate and observed using a
Leica TCS SP5 confocal microscope. All images were collected
with LAS AF Lite 2.6 software (Leica Microsystems).
We thank Iamara Andrade for her assistance in cell culture. We would also
like to thank the Genomic Platform for DNA sequencing of PDTIS/
FIOCRUZ and the Confocal Microscopy Unit of the Biomedical Science
Institute (ICB, UFRJ).
Conceived and designed the experiments: EMS JNC RMB. Performed the
experiments: EMS JNC DA. Analyzed the data: EMS JNC DA RMB.
Contributed reagents/materials/analysis tools: MLN RMB. Wrote the
paper: EMS JNC DA RMB.
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