Dengue Virus Subverts Host Innate Immunity by Targeting Adaptor Protein MAVS

Dengue Virus Subverts Host Innate Immunity by Targeting Adaptor

Protein MAVS

Zhenjian He,a,b,cXun Zhu,b,c,dWeitao Wen,b,c,dJie Yuan,b,c,eYiwen Hu,b,c,dJiahui Chen,b,c,dShu An,b,c,dXinhuai Dong,b,c,d Cuiji Lin,b,c,dJianchen Yu,b,c,dJueheng Wu,b,c,dYi Yang,b,fJunchao Cai,b,c,dJun Li,b,c,eMengfeng Lib,c,d

School of Public Health, Sun Yat-sen University, Guangzhou, Chinaa

; Key Laboratory of Tropical Disease Control (Sun Yat-sen University), Ministry of Education, Guangzhou, Chinab

; Guangdong Province Key Laboratory of Functional Molecules in Oceanic Microorganism (Sun Yat-sen University), Bureau of Education, Guangzhou, Chinac

; Department of Microbiology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Chinad

; Department of Biochemistry, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Chinae

; Department of Pharmacology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Chinaf

ABSTRACT

Dengue virus (DENV) is the most common mosquito-borne virus infecting humans and is currently a serious global health

chal-lenge. To establish infection in its host cells, DENV must subvert the production and/or antiviral effects of interferon (IFN). The

aim of this study was to understand the mechanisms by which DENV suppresses IFN production. We determined that DENV

NS4A interacts with mitochondrial antiviral signaling protein (MAVS), which was previously found to activate NF-

␬

B and IFN

regulatory factor 3 (IRF3), thus inducing type I IFN in the mitochondrion-associated endoplasmic reticulum membranes

(MAMs). We further demonstrated that NS4A is associated with the N-terminal CARD-like (CL) domain and the C-terminal

transmembrane (TM) domain of MAVS. This association prevented the binding of MAVS to RIG-I, resulting in the repression of

RIG-I-induced IRF3 activation and, consequently, the abrogation of IFN production. Collectively, our findings illustrate a new

molecular mechanism by which DENV evades the host immune system and suggest new targets for anti-DENV strategies.

IMPORTANCE

Type I interferon (IFN) constitutes the first line of host defense against invading viruses. To successfully establish infection,

den-gue virus (DENV) must counteract either the production or the function of IFN. The mechanism by which DENV suppresses

IFN production is poorly understood and characterized. In this study, we demonstrate that the DENV NS4A protein plays an

important role in suppressing interferon production through binding MAVS and disrupting the RIG-I–MAVS interaction in

mitochondrion-associated endoplasmic reticulum membranes (MAMs). Our study reveals that MAVS is a novel host target of

NS4A and provides a molecular mechanism for DENV evasion of the host innate immune response. These findings have

impor-tant implications for understanding the pathogenesis of DENV and may provide new insights into using NS4A as a therapeutic

and/or prevention target.

D

engue virus (DENV) (family

Flaviviridae

, genus

Flavivirus

) is

an enveloped virus with a positive-sense, single-stranded

RNA genome that is responsible for dengue fever (DF) and the

more severe, life-threatening dengue hemorrhagic fever (DHF)

and dengue shock syndrome (DSS) (

1

). It has been estimated that

50 million to 100 million people are infected by DENV worldwide

each year, and 500,000 patients progress to severe DHF (

2

). DENV

is categorized into four antigenically related but distinct serotypes,

designated DENV1, -2, -3, and -4. The DENV genome is

⬃

11 kb

in length and encodes a single polyprotein that can be processed

co- and posttranslationally by cellular and viral proteases into

three structural (C, prM, and E) and seven nonstructural (NS1,

NS2A, NS2B, NS3, NS4A, NS4B, and NS5) proteins in the

endo-plasmic reticulum (ER) (

3

).

As an early response to viral infection, mammalian cells

pro-duce type I interferons (IFNs), mainly IFN-

␣

and IFN-

␤

, which

repress virus replication (

4

,

5

). This early innate antiviral response

is initiated by viral recognition through pathogen recognition

re-ceptors (PRRs), including Toll-like rere-ceptors (TLRs) and retinoic

acid-inducible gene I (RIG-I)-like receptors (RLRs). RIG-I, a

member of the RLRs, binds viral RNA, which triggers

conforma-tional changes that expose CARD domains for subsequent

signal-ing (

6

). Signaling by RIG-I requires the adaptor protein

desig-nated mitochondrial antiviral signaling adaptor (MAVS) (also

known as IPS-1/VISA/Cardif) (

7–10

). MAVS then transduces

sig-nals from RIG-I through CARD-CARD domain interactions,

which activate IFN regulatory factor 3 (IRF3) and NF-

␬

B through

a signaling cascade involving the cytosolic protein kinases

TANK-binding kinase 1 (TBK1) and I

␬

B kinase

ε

(IKK

ε

) (

11

),

conse-quently leading to type I IFN production.

Previous studies have shown that DENV infection usually leads

to low levels of IFN-

␣

and -

␤

, thus suggesting that DENV may

inhibit IFN production after DENV infection (

12–16

). Several

re-search groups have reported that prevention of STAT1

phosphor-ylation or induction of STAT2 degradation is a mechanism

possi-bly underlying the antagonistic effects of DENV on IFN signaling

Received5 February 2016Accepted23 May 2016

Accepted manuscript posted online1 June 2016

CitationHe Z, Zhu X, Wen W, Yuan J, Hu Y, Chen J, An S, Dong X, Lin C, Yu J, Wu J, Yang Y, Cai J, Li J, Li M. 2016. Dengue virus subverts host innate immunity by targeting adaptor protein MAVS. J Virol 90:7219 –7230.doi:10.1128/JVI.00221-16.

Editor:J. U. Jung, University of Southern California

Address correspondence to Mengfeng Li, limf@mail.sysu.edu.cn. Z.H., X.Z., and W.W. contributed equally to this work.

Supplemental material for this article may be found athttp://dx.doi.org/10.1128 /JVI.00221-16.

Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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(

17–22

). However, previous studies focused mainly on the effects

of DENV on antiviral signaling downstream of IFN, leaving the

molecular mechanism underlying the suppression of IFN

produc-tion by DENV largely unknown. Recently, two groups reported

similar results showing that human mediator of IRF3 activation

(MITA) or stimulator of interferon genes (STING) is cleaved by

the DENV protease NS2B/3 (

23

,

24

).

In the present study, we report that DENV NS4A potently

dis-rupts the induction of IFN by targeting MAVS. We found that

NS4A binds MAVS and prevents the binding of MAVS with

RIG-I, hence abrogating IFN induction. Furthermore, we

demon-strate that NS4A is associated with the N-terminal CARD-like

(CL) domain and the C-terminal transmembrane (TM) domain

of MAVS, and the third transmembrane (TM3) domain of NS4A

is essential for binding to MAVS. Thus, our findings collectively

suggest that NS4A contributes to DENV immune evasion through

the inhibition of MAVS-mediated cellular responses.

MATERIALS AND METHODS

Plasmids.A plasmid encoding human MAVS with an N-terminal Flag epitope tag was amplified by reverse transcription-PCR (RT-PCR) and cloned into the KpnI and XbaI sites of the pcDNA3.1 vector. The plasmid expressing Flag-tagged RIG-I was obtained from Addgene (Addgene plas-mid 27236). For the green fluorescent protein (GFP)-IRF3 construct, human IRF3 was amplified from plasmid pcDNA3-V5-IRF3 (Addgene plasmid 32713) and subcloned into the XhoI and EcoRI sites of the pEGFP-C3 vector. To generate plasmids pFN10A(ACT)-RIG-I, MAVS, TBK1, and pFN10A(ACT)-IKKε, each of the RIG-I, MAVS, TBK1, and IKKεfragments was amplified by PCR and subcloned into the pFN10A(ACT) vector (Promega, San Luis Obispo, CA), respectively. cDNA encoding each DENV protein, including C, prM, E, NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5, was amplified by RT-PCR using DENV2 strain New Guinea C (NGC) cDNA as a tem-plate and cloned into the pFN11A(BIND) vector (Promega, San Luis Obispo, CA). Truncated forms of MAVS (amino acids [aa] 1 to 77, 74 to 173, and 174 to 540) with N-terminal Flag epitope tags were amplified from the full-length template and cloned into the pcDNA3.1 vector. Plas-mids expressing Myc-tagged full-length NS4A and deletion mutants (aa 1 to 49, 1 to 74, 1 to 100, and 1 to 127) were also amplified by PCR and cloned into the pcDNA3.1 vector. Plasmids encoding DENV2 prM and influenza virus PB1-F2 (A/Puerto Rico/8/1934 [PR8] strain) with N-ter-minal Myc epitope tags were amplified by RT-PCR and cloned into the KpnI and BamHI sites of the pcDNA3.1 vector. Plasmid pEF-BOS-NS4A with a C-terminal 6⫻His tag was constructed by cloning the NS4A frag-ment into the pEF-BOS vector using the XhoI and BamHI sites. The pIFN-␤-luc reporter plasmid was constructed by cloning a 125-bp frag-ment of the IFN-␤promoter into the pGL3-Basic vector using the NheI and HindIII sites, as previously described (25). All constructs were veri-fied by DNA sequencing. The PCR primers used in this study are summa-rized in Table S1 in the supplemental material.

Cell culture and virus.HeLa (purchased from ATCC) and 293T (ob-tained from the Cell Bank of the Chinese Academy of Sciences, Shanghai, China) cells were cultured at 37°C with 5% CO₂in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS) (Gibco, Carlsbad, CA), 2 mML-glutamine, 100␮g/ml streptomycin, and 100 U/ml penicillin (Invitrogen, Carlsbad, CA).Aedes albopictusC6/36 cells (ATCC CRL-1660) (26) were main-tained at 28°C with 5% CO2in DMEM supplemented with 10% FBS.

DENV2 strain NGC (GenBank accession numberM29095) was kindly provided by the Guangzhou Center for Disease Control and Prevention (CDC) (27) and propagated in the mosquito cell line C6/36. Virus stocks were titrated by fluorescence-activated cell sorter (FACS) assays with C6/36 cells according to a previously described method (28). Sendai virus

(SeV) was grown in 10-day-old embryonated chicken eggs and titrated by a hemagglutination assay as previously described (29,30).

Luciferase reporter assays.293T cells seeded into 24-well plates were transiently transfected with plasmids encoding IFN-␤and the internal control pRL-TK together with NS4A (250 and 500 ng), prM (500 ng), or PB1-F2 (500 ng). Cells were then infected with SeV at 100 hemagglutinat-ing units (HAU)/ml for 16 h, followed by analysis of cell lysates for lucif-erase activity with a Dual-Luciflucif-erase Reporter Assay System kit (Promega, San Luis Obispo, CA) according to the manufacturer’s protocol.

Mammalian two-hybrid assay.293T cells were seeded into a 24-well plate 24 h prior to transfection. Next, 100 ng (each) of the pFN11A(BIND) vector expressing an individual DENV protein with a GAL4 DNA binding domain (GAL4-BD) fusion protein and 100 ng (each) of the pFN10A(ACT) vector expressing the RIG-I, MAVS, TBK1, or IKKε pro-tein was cotransfected with 250 ng of reporter plasmid pGL4.31 into 293T cells by using Lipofectamine 2000 (Invitrogen, Carlsbad, CA). The pFN11A(BIND) vector contained aRenillaluciferase gene, which was used as an internal control to normalize the DNA transfection efficiency. The pBIND and pACT vectors were used as the negative controls, and the pBIND-Id and pACT-MyoD vectors were used as the positive controls, according to the manufacturer’s instructions (Promega, San Luis Obispo, CA). After 48 h, firefly andRenillaluciferase activities were determined by using a Dual-Luciferase Reporter Assay System kit (Promega, San Luis Obispo, CA).

Western blotting.Cells were lysed with sampling buffer (50 mM Tris-HCl [pH 7.4], 1 mM phenylmethylsulfonyl fluoride [PMSF], 10% glyc-erol, 6% SDS, 5% beta-mercaptoethanol, and 0.1% bromophenol blue), and protein concentrations were measured with a bicinchoninic acid (BCA) protein assay (Thermo Fisher Scientific, Rockford, IL). Protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene difluoride (PVDF) membrane. Nonspecific antibody binding sites were blocked with 5% nonfat milk in Tris-buffered saline (TBS) (20 mM Tris-HCl [pH 7.6], 135 mM NaCl, and 0.1% Tween 20) for 1 h at room temperature and then reacted with the following primary antibodies: anti-MAVS (Bethyl Laboratories, Montgomery, TX), anti-anti-MAVS (T-20) (Santa Cruz Biotechnology, Santa Cruz, CA), anti-phospho-IRF3 (S396) (Cell Signaling, Danvers, MA), anti-IRF3 (Cell Signaling, Danvers, MA), anti-NS4A (GeneTex Inc., Irvine, CA), anti-Flag M2, anti-c-Myc, and anti-␤-actin (Sigma-Aldrich, St. Louis, MO). Membranes were incubated with horseradish peroxidase-conjugated secondary antibody, and signals were detected by enhanced chemiluminescence using a commercial kit (Thermo Fisher Scientific, Rockford, IL) according to the manufacturer’s suggested protocols.

Immunofluorescence assay.Cells were plated onto coverslips in a 24-well plate and transfected with the indicated plasmids (500 ng). At 24 h posttransfection, cells were washed once with phosphate-buffered saline (PBS) and fixed in 4% paraformaldehyde in PBS. Cells were permeabil-ized with 0.2% Triton X-100 and blocked for 30 min at room temperature with 10% bovine serum albumin (BSA) in PBS, followed by incubation with the primary antibody for 1 h. After three washes with PBS containing 0.1% Tween 20 (PBST), cells were incubated with fluorescein isothiocya-nate (FITC)- or rhodamine-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA) or with Alexa Flour 488 dye- and Alexa Flour 647 dye-conjugated secondary antibodies (Life Technologies, Grand Island, NY) for 30 min and then incubated with 4=,6-diamidino-2-phenylindole (DAPI) for 10 min. Finally, coverslips were washed extensively and fixed onto slides. Images were taken under an LSM780 confocal microscope (Carl Zeiss MicroImaging GmbH, Jena, Germany).

Coimmunoprecipitation assay.293T cells were transfected with the indicated plasmids (10␮g/ml) by using a standard calcium phosphate coprecipitation method. Twenty-four hours later, cells were lysed with protein lysis buffer containing 25 mM HEPES, 150 mM NaCl, 1 mM EDTA, 2% glycerol, 1% NP-40, and a cocktail of protease and

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tase inhibitors (Roche, Basel, Switzerland). Lysates were incubated with the indicated antibodies or IgG antibody overnight at 4°C. Subsequently, precipitates were washed five times with wash buffer containing 20 mM HEPES, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 2% glycerol, and 0.1% NP-40; resuspended in sampling buffer; and examined by Western blot-ting. For the endogenous immunoprecipitation (IP) experiment, lysates were incubated with the indicated antibodies, and protein G Dynabeads (Life Technologies, Grand Island, NY) were subsequently added to cap-ture the protein complexes according to the manufaccap-turer’s suggested protocol.

Biacore analysis.Surface plasmon resonance (SPR) data were ob-tained with a Biacore T100 instrument and research-grade CM5 sensor chips (General Electric Company [GE]) according to the manufacturer’s protocol. Briefly, MAVS was immobilized on a CM5 sensor chip. Differ-ent concDiffer-entrations (0, 62.5, 125, 250, 500, 1,000, and 2,000 nM) of NS4A were injected at a flow rate of 30␮l/min for 3 min. Association data were collected for 3 min, followed by a 20-min dissociation period. The binding surfaces were regenerated after each injection by injection of 10␮l of 10 mM NaOH for 20 s. All procedures were performed at 25°C by using standard HBS (HEPES-buffered saline; GE) as a running buffer. A buffer injection control was included for each experiment and was subtracted from the data before analysis. The equilibrium dissociation constant (KD)

was calculated as previously described (31). Data analysis to obtain bind-ing curves was performed by usbind-ing BIAevaluation 3.1 (Biacore), by glob-ally fitting the data to a simple 1:1 Langmuir ligand binding model defined by the formula Bound⫽ (Bmax⫻ [NS4A])/(KD ⫻[NS4A]), where

“Bound” is measured in response units (RU),Bmaxis the maximum

re-sponse (in rere-sponse units), and [NS4A] is the concentration of the NS4A protein. Recombinant human MAVS protein was purchased from OriGene (catalog no. TP308175). Recombinant NS4A was produced from 293T cells and purified by using Ni-nitrilotriacetic acid (NTA) affinity chromatography (QIAexpressionist; Qiagen, Chatsworth, CA), followed by Superdex 200 (GE) gel filtration chromatography, as previously de-scribed (32,33).

Real-time PCR.Total cellular RNA was prepared with TRIzol (Invit-rogen, Carlsbad, CA) according to the manufacturer’s instructions. First-strand cDNA synthesis was performed by using random hexamer primers, and real-time PCR was carried out by using FastStart Universal SYBR green master mix (Roche, Basel, Switzerland). All readings were normal-ized to the level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. The primer sets used for real-time PCR are shown in Table S1 in the supplemental material.

Isolation of monocytes.Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood by Ficoll-Hypaque density gradient cen-trifugation according to the manufacturer’s instructions (Lymphoprep; Nycomed, Oslo, Norway). Primary monocytes were purified from PBMCs by using Monocyte Isolation kit II (Miltenyi Biotec GmbH, Ber-gisch Gladbach, Germany) according to the protocol provided by the manufacturer. Purified monocytes were resuspended and cultured in RPMI 1640 medium (Invitrogen, Carlsbad, CA) supplemented with 10% FBS (HyClone, Logan, UT), 15 mM HEPES, 2 mML-glutamine, 100

␮g/ml streptomycin, and 100 U/ml penicillin (Invitrogen, Carlsbad, CA).

Subcellular fractionation. Mitochondrion-associated endoplasmic reticulum membranes (MAMs), mitochondria, and cytosolic fractions containing lysosomes and microsomes were isolated from primary mono-cytes or 293T cells by using Percoll gradient fractionation as described previously (34). Equivalent amounts of protein from each fraction were determined by Western blotting.

Statistical analysis.The results are expressed as means⫾standard deviations (SD). Statistical analyses were performed on data from tripli-cate experiments by using two-tailed Student’sttest.

RESULTS

DENV NS4A interacts with MAVS, which mediates the type I

IFN induction pathway.

To investigate whether DENV proteins

interfere with the IFN induction pathway by interacting with the

pathway components, we employed the mammalian two-hybrid

system to identify molecular targets of DENV proteins in PRR

signaling. Each of the 10 DENV proteins was expressed as a fusion

protein with the yeast GAL4-BD, and the 4 key PRR signaling

molecules, i.e., RIG-I, MAVS, TBK1, and IKK

ε

, were fused to the

transcriptional activation domain of herpes simplex virus VP16

(VP16-AD). 293T cells were then cotransfected with 10 DENV

proteins and the 4 key PRR signaling proteins, and the expression

of each transfected gene was verified by Western blotting using

available commercial antibodies or antibodies against the Flag,

Myc, or hemagglutinin (HA) tag (see Fig. S1 in the supplemental

material). As shown in

Fig. 1

, there was no interaction between

DENV proteins and RIG-I, TBK1, or IKK

ε

(

Fig. 1A

,

C

, and

D

),

whereas MAVS and NS4A interacted with each other (

Fig. 1B

),

suggesting that MAVS may be an interactive partner of DENV

NS4A. Furthermore, we employed an IFN-

␤

luciferase reporter

assay to determine whether NS4A could reverse the activation of

the IFN-

␤

promoter driven by MAVS. Overexpression of the

NS4A and MAVS proteins was confirmed by Western blotting

(

Fig. 1E

). Our results showed that DENV2 NS4A markedly

de-creased the reporter luciferase activity induced by MAVS (

Fig.

1E

). To further test whether the NS4A-MAVS interaction might

also function to inhibit type I IFN production, we examined the

effects of the DENV3 and DENV4 NS4A proteins on IFN-

␤

re-porter activity, and our results showed that the NS4A proteins of

both DENV3 and DENV4 had inhibitory effects on IFN-

␤

re-porter activities driven by MAVS (

Fig. 1F

), indicating that the

identified NS4A-MAVS interaction represents a common

anti-IFN mechanism across different serotypes of dengue virus.

DENV NS4A physically colocalizes with MAVS during

DENV infection.

Based on the finding that NS4A interacts with

MAVS, we next asked whether the two molecules colocalize. For this

purpose, HeLa cells were infected with DENV2 and then stained for

NS4A and MAVS. As shown in

Fig. 2A

, confocal microscopy revealed

costaining of NS4A and MAVS in cells infected by DENV2,

suggest-ing colocalization of the two proteins (

Fig. 2A

). We next used the

DENV protein prM, which did not interact with MAVS in the

mam-malian two-hybrid assay (

Fig. 1B

), as a negative control and the

in-fluenza virus PB1-F2 protein, which has been reported to antagonize

type I IFN induction by targeting MAVS, as a positive control (

35

,

36

). Our results showed that PB1-F2, but not DENV prM, also

colo-calized with MAVS (

Fig. 2A

).

To address whether the NS4A protein physically interacts with

MAVS, we performed endogenous IP assays on DENV2-infected

cell lysates. Our results showed that NS4A, but not prM, could be

precipitated by the MAVS antibody (

Fig. 2B

), suggesting that

NS4A and MAVS form a complex in DENV-infected cells. To

better elucidate the interaction between NS4A and MAVS, we

per-formed

in vitro

co-IP assays based on MAVS and NS4A

overex-pression, and the results showed that MAVS interacted with NS4A

(

Fig. 2C

). Additionally, we performed IP to precipitate

endoge-nous MAVS using Myc-tagged NS4A and cells transfected with

prM or PB1-F2 as controls. We found that endogenous MAVS was

pulled down together with Myc-tagged NS4A or PB1-F2 but not

prM (

Fig. 2D

and

E

), further confirming the ability of DENV

NS4A to form a complex with MAVS. The binding affinities

be-tween MAVS and NS4A were also analyzed by Biacore surface

plasmon resonance assays with MAVS immobilized on the chip

surface and injections containing analytes consisting of serial

di-Targeting MAVS by Dengue Virus NS4A

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lutions of NS4A proteins. The data showed a significant increase

in NS4A binding to MAVS in solution, with an apparent

KD

of

1.12

⫻

10

M (

Fig. 2F

). These data indicate that NS4A directly

interacts with MAVS.

DENV NS4A translocates to the MAM and interacts with

MAVS.

The ER contains specialized intracellular components,

known as MAMs, that physically connect the ER to mitochondria

(

37

,

38

). Because DENV NS4A is an ER membrane-bound protein

FIG 1Mammalian two-hybrid analyses of interactions between DENV proteins and IFN signaling proteins. Protein-protein interactions were determined by the mammalian two-hybrid system. (A to D) 293T cells in 24-well plates were cotransfected with a pGL4.31 vector, a pFN11A(BIND) vector expressing a fusion protein of the GAL4-BD and individual DENV proteins, and a pFN10A(ACT) vector expressing the RIG-I (A), MAVS (B), TBK1 (C), or IKK_ε(D) protein. The pFN11A(BIND) vector contained aRenillaluciferase gene that was used as an internal control to normalize DNA transfection efficiency. At 48 h posttransfection, the levels of luciferase activity were examined. The results are shown as relative luciferase activity after normalization toRenillaluciferase activity. The pBIND and pACT vectors were used as negative controls, and the pBIND-Id and pACT-MyoD vectors were used as positive controls (PC), according to the manufacturer’s instructions. (E and F) Luciferase activities of 293T cells transfected with an IFN-_␤luciferase reporter, the pRL-TK (internal control) reporter, and the plasmid expressing MAVS. An empty vector or a vector expressing DENV2 NS4A (500 ng) (E), DENV3 NS4A (500 ng) (F), or DENV4 NS4A (500 ng) (F) was also cotransfected. At 36 h posttransfection, cell lysates were harvested for luciferase activity assays. Overexpression of NS4A and MAVS proteins was confirmed by Western blotting. The data are shown as the means⫾SD derived from three repeat experiments. ** indicates aPvalue of⬍0.01 (Student’sttest). RLU, relative luciferase units.

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FIG 2DENV NS4A colocalizes and interacts with MAVS during DENV infection. (A) HeLa cells infected with DENV2 at an MOI of 10 or mock-infected HeLa cells were fixed with 4% paraformaldehyde and costained with anti-MAVS and anti-NS4A or anti-prM antibodies as well as DAPI. HeLa cells transfected with a plasmid expressing Myc-tagged influenza virus PB1-F2 (500 ng) or an empty vector were stained with anti-Myc and anti-MAVS antibodies as well as DAPI. HeLa cells expressing Myc-tagged NS4A (500 ng) were stained with anti-Myc antibody and an antibody against the ER marker calnexin. Secondary antibodies conjugated to rhodamine and Alexa Fluor 488 dye were used to visualize the indicated proteins. Images are representative of results from three independent experiments, and a semiquantitative colocalization analysis of the indicated proteins was performed by using the line profile of ZEN imaging software. Fluorescence intensities along the red lines sectioning the cells are plotted for the indicated proteins. (B) 293T cells were infected with DENV2 at an MOI of 10, followed by immunoprecipitation using anti-MAVS antibody or control mouse IgG, and protein G Dynabeads were added to capture the immunocomplexes. The immunocomplexes were analyzed by Western blotting using anti-NS4A, anti-prM, or anti-MAVS antibodies (Abs). The arrow indicates the position of NS4A, and IgG-L indicates the IgG light chain. IB, immunoblotting. (C) 293T cells cotransfected with plasmids encoding Flag-tagged MAVS (10␮g/ml) and Myc-tagged NS4A (10_␮g/ml) were used in a co-IP assay. Cell lysates were precipitated with anti-Flag antibody or control mouse IgG, and immunocomplexes were analyzed with the indicated antibodies by Western blotting. (D and E) 293T cells transfected with plasmids encoding Myc-tagged NS4A (D) or prM or PB1-F2 (E) (10_␮g/ml) were used for precipitation experiments with anti-Myc antibody or control mouse IgG, and immunocomplexes were determined with anti-MAVS antibody by Western blotting. A small aliquot of the cell lysate was also prepared before IP for equal input of Flag-MAVS, Myc-NS4A, Myc-prM, or Myc–PB1-F2. (F) SPR analysis of interactions between MAVS and NS4A. Direct binding was measured by Biacore assays. MAVS was immobilized on a CM5 chip. The analytes consisted of serial dilutions of NS4A proteins at concentrations of between 0 nM and 2,000 nM. The data shown are representative of data from three independent experiments with similar results.

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(

39

), we assessed whether MAMs were able to position DENV

NS4A in proximity to MAVS and thus facilitate their interaction.

To address this issue, HeLa cells overexpressing Myc-tagged NS4A

were examined for the localization pattern of MAVS and NS4A.

Calnexin and MitoTracker Red were used as ER and

mitochon-drion markers, respectively, and MAMs and the peroxisome were

labeled by antibodies against fatty acid-coenzyme A ligase 4

(FACL4) and the 70-kDa peroxisomal membrane protein

(PMP70), respectively. We found that a significant fraction of

NS4A colocalized with MAVS in the MAM, and a small fraction of

NS4A colocalized with MAVS in the mitochondria but not in the

peroxisome (

Fig. 3A

to

C

). Moreover, when we isolated MAMs

from primary monocytes infected with DENV2 and performed

Western blotting to analyze NS4A, we detected both NS4A and the

MAM marker FACL4 in the MAM fraction (

Fig. 3D

). We also

isolated MAMs from 293T cells transfected with Myc-tagged

NS4A and detected Myc-tagged NS4A in the MAM fractions (

Fig.

3E

). Together, these data suggested that NS4A may translocate to

the MAM and interact with MAVS via this platform.

DENV infection and NS4A prevent RIG-I from forming

complexes with MAVS.

MAVS transduces signals from RIG-I

through CARD-CARD domain interactions, leading to the

ac-FIG 3DENV NS4A translocates to the MAM and interacts with MAVS. (A) Following transfection with Myc-tagged NS4A (500 ng) or an empty vector, HeLa cells were fixed with 4% paraformaldehyde and stained with anti-Myc antibody for NS4A, anti-FACL4 antibody for MAMs, and an anti-MAVS antibody. Secondary antibodies conjugated to Alexa Fluor 488, rhodamine, and Alexa Fluor 647 were used to visualize the stained NS4A, FACL4, and MAVS proteins, respectively. All images for all panels are representative of results from three independent experiments, and a semiquantitative colocal-ization analysis of the indicated proteins was performed by using the line profile of ZEN imaging software. Fluorescence intensities along the red lines sectioning the cells are plotted for the indicated proteins. (B) HeLa cells were transfected with a Myc-tagged NS4A expression plasmid (500 ng) or an empty vector. At 24 h posttransfection, cells were incubated with MitoTracker Red dye, fixed with 4% paraformaldehyde, and then stained with anti-Myc and anti-MAVS antibodies as well as DAPI, followed by secondary antibodies conjugated to Alexa Fluor 488 and Alexa Fluor 647 to visualize the NS4A and MAVS proteins, respectively. (C) HeLa cells expressing Myc-tagged NS4A (500 ng) or an empty vector were stained with anti-Myc for NS4A, anti-PMP70 antibody for the peroxisome, and anti-MAVS antibody. Secondary antibodies conjugated to rhodamine, Alexa Fluor 488, and Alexa Fluor 647 were used to visualize stained NS4A, PMP70, and MAVS proteins, respectively. (D) Primary monocytes that were infected with DENV2 at an MOI of 10 or mock infected were used to isolate a MAM, and the isolated MAM was analyzed by Western blotting. (E) 293T cells transfected with Myc-tagged NS4A (10_␮g/ml) were used to isolate MAMs and were analyzed by Western blotting. WCL, whole-cell lysates; Cyto, cytosol. The Western blot shown is representative of data from three independent experiments with similar results.

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tivation of IRF3 and IFN induction (

11

). To investigate

whether the suppressed binding of endogenous RIG-I to MAVS

contributes to the inhibition of IFN by DENV infection, we

examined the formation of RIG-I–MAVS complexes after

DENV infection. Primary monocytes were infected with

DENV2 at different multiplicities of infection (MOIs) or mock

infected for 24 h and then stimulated with SeV for 16 h. Cell

lysates were harvested, immunoprecipitated with an

anti-MAVS antibody, and analyzed by Western blotting using an

anti-RIG-I antibody. As shown in

Fig. 4A

, RIG-I interacted

with MAVS following SeV stimulation, but cells preinfected

with DENV2 exhibited a significant decrease in the interaction

between RIG-I and MAVS, and such an inhibition correlated

with the MOI of DENV2 used for infection. To address whether

NS4A affects the interaction between RIG-I and MAVS, we

examined the formation of RIG-I–MAVS complexes in the

presence or absence of NS4A. 293T cells were transfected with

different amounts of Myc-tagged NS4A or the control vector

and then stimulated with SeV prior to IP assays. Our results

showed that cells expressing NS4A exhibited a significantly

re-pressed interaction between RIG-I and MAVS in a

dose-depen-dent manner (

Fig. 4B

). Together, these data indicate that NS4A

binds MAVS, which may in turn prevent its interaction with

RIG-I, modulate downstream signaling, and suppress IFN

pro-duction.

NS4A interacts with both the CL and TM domains of MAVS.

MAVS is a 540-aa protein consisting of three structural and

functional domains, namely, the N-terminal CARD-like (CL)

domain, the middle proline-rich (PR) domain, and the

C-ter-minal transmembrane (TM) domain (

Fig. 5A

). To map the

MAVS domains involved in NS4A binding, we generated the

following serial MAVS deletion mutants: Flag-MAVS(CL),

ex-pressing aa 1 to 77 and containing the CARD-like domain;

Flag-MAVS(PR), expressing aa 78 to 173 and containing the

proline-rich domain; Flag-MAVS(TM), expressing aa 174 to

540 and containing the transmembrane domain; and

Flag-MA-VS(FL), expressing full-length MAVS (

Fig. 5A

). Co-IP assays

showed that either the CL or TM domain of MAVS could pull down

NS4A (

Fig. 5B

). Furthermore, the results of mammalian two-hybrid

experiments also confirmed that the CL or TM domain of MAVS

could interact with NS4A (

Fig. 5C

). Taken together, these results

sug-gest that both domains contribute to the formation of the

MAVS-NS4A complex.

The TM3 domain of NS4A is required for interaction with

MAVS.

To identify the NS4A region(s) required for binding with

MAVS, we performed serial deletion mutagenesis analysis of

NS4A (

Fig. 5D

). Myc-tagged full-length NS4A and four deletion

constructs of NS4A were cotransfected with Flag-tagged MAVS.

The subsequent co-IP assays showed that NS4A lacking the

C-ter-minal TM4 domain was still able to interact with MAVS, similarly

to full-length NS4A (

Fig. 5E

). However, further deletion of the

TM3 domain completely abrogated the interaction between NS4A

and MAVS (

Fig. 5E

), suggesting that the TM3 domain is critical

for NS4A to bind MAVS. Next, we asked whether the TM3

do-main has an inhibitory effect on MAVS, and an IFN-

␤

luciferase

reporter assay was performed. Our results showed that the TM3

domain alone markedly decreased reporter luciferase activity

driven by MAVS (

Fig. 5F

), further demonstrating that the

C-ter-minal TM3 domain of NS4A is required for a specific

NS4A-MAVS interaction.

DENV NS4A downregulates type I IFN production by

inhib-iting IRF3 activation.

To further investigate how NS4A inhibits

the signaling pathway mediating type I IFN production, we used

SeV as a stimulus and determined the effect of NS4A on IFN-

␤

luciferase reporter activity. Our results showed that both NS4A

and PB1-F2, but not prM, inhibited SeV-induced activation of the

reporter. Overexpression of the NS4A, prM, or PB1-F2 protein

was confirmed by Western blotting (

Fig. 6A

).

The transcription factor IRF3 is a key innate immune system

component that mediates IFN-

␤

induction. Once IRF3 is

phos-phorylated, it forms a dimer, translocates into the nucleus from

the cytoplasm, and induces the expression of IFN-

␤

and

IFN-stimulated genes (ISGs) through specifically binding to their

pro-moter regions (

40

). To further investigate how NS4A inhibits the

signaling that mediates type I IFN production, we assessed the

translocation of IRF3 in 293T cells that expressed GFP-tagged

IRF3 or NS4A following infection with SeV. In cells transfected

with a blank control vector, 32.46% (

⫾

3.85%) of IRF3 rapidly

FIG 4DENV2 infection or ectopic NS4A prevents RIG-I from binding MAVS. (A) Primary monocytes were infected with DENV2 at the indicated MOIs (0, 5, and 10) or mock infected for 24 h and then infected with SeV for 16 h. Cell lysates were harvested, subjected to immunoprecipitation using an anti-MAVS antibody, and analyzed by Western blotting. (B) 293T cells were transfected with different amounts of Myc-tagged NS4A (0, 10, and 20␮g/ml) or a blank vector control for 24 h and then infected with SeV for 16 h. Whole-cell lysates were subjected to immunoprecipitation using an anti-MAVS antibody and analyzed by Western blotting. Expression of precipitated proteins was determined by Western blotting using the indicated antibodies (bottom). The data shown are representative of data from three independent experiments with similar results.

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translocated into the nucleus after SeV infection. In contrast, IRF3

was retained in the cytoplasm of cells expressing ectopic NS4A

after SeV stimulation, and only 4.91% (

⫾

0.66%) of IRF3 was

translocated into the nucleus (

Fig. 6B

), indicating a suppressive

function of NS4A in IRF3 translocation. We next determined the

phosphorylation of endogenous IRF3 in cells with or without the

expression of NS4A. Our data showed that SeV-induced

phos-phorylation of IRF3 was repressed by NS4A or PB1-F2 but not

prM (

Fig. 6C

). Additionally, NS4A or PB1-F2, but not prM,

mark-edly diminished mRNA production of

IFNB

,

OAS1

, and

IFITM1

following SeV infection (

Fig. 6D

). These data demonstrate that

NS4A inhibits the expression of type I IFN induced by viral

infec-tion or MAVS through blocking the phosphorylainfec-tion and

trans-location of IRF3.

Previous studies have shown that a recombinant Newcastle

disease virus (NDV)-GFP or vesicular stomatitis virus (VSV)-GFP

FIG 5Both the CL and TM domains contribute to MAVS complex formation with NS4A, and the TM3 domain of NS4A is required for binding to MAVS. (A) Schematic diagram of the MAVS protein and the following functional domains: the CARD-like domain (aa 10 to 77), the proline-rich domain (aa 103 to 173), and the transmembrane domain (aa 514 to 535). (B) Co-IP and Western blotting of 293T cells transfected with Myc-tagged NS4A (10␮g/ml) along with vectors expressing the indicated Flag-tagged MAVS truncation forms or full-length MAVS (10␮g/ml). An empty vector was used as a negative control. (C) 293T cells in 24-well plates were cotransfected with the pGL4.31 vector; a pFN11A(BIND) vector expressing NS4A; and a pFN10A(ACT) vector expressing MAVS(FL), MAVS(CL), MAVS(PR), or MAVS(TM). At 48 h posttransfection, levels of luciferase activity were examined. pBIND and pACT vectors were used as negative controls. (D) Schematic illustration of the DENV NS4A protein and its serial deletion mutants. (E) 293T cells were cotransfected with a Flag-MAVS plasmid and individual Myc-tagged NS4A truncated plasmids or full-length NS4A (10␮g/ml). A blank vector served as a negative control. At 24 h posttransfection, cells were lysed, co-IP was performed by using the anti-Flag antibody, and immunocomplexes were analyzed by Western blotting. The presence of transfected proteins in cell lysates (input) was verified by detection with the indicated antibodies (bottom). The numbers at the top indicate amino acid positions. The data shown are representative of results from three independent experiments. (F) Luciferase activities of 293T cells were measured after transfection with an IFN-␤luciferase reporter and the pRL-TK reporter together with a plasmid expressing MAVS. A blank vector control or increasing amounts of a vector expressing the TM3 domain of NS4A (NS4A-TM3) (250 and 500 ng) were also cotransfected. At 36 h posttransfection, cell lysates were harvested for luciferase activity assays and Western blotting. Data are presented as the means⫾ SD derived from three repeat experiments. **,P⬍0.01 (Student’sttest).

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system can be used as a strategy to screen proteins possessing

IFN-antagonizing activity (

41

,

42

). In the present study, we

em-ployed recombinant VSV-GFP to investigate whether DENV

NS4A is an antagonist of IFN production. When 293T cells

ex-pressed NS4A, a high level of VSV-GFP replication was present,

which was consistent with the notion that NS4A inhibits IFN

pro-duction in host cells (

Fig. 7A

to

C

). Upon VSV-GFP stimulation,

NS4A reduced IFN-

␤

reporter activity, suggesting that the

inhib-itory effect of DENV NS4A on IFN production is also present

during actual viral infection.

DISCUSSION

The innate immune system is an evolutionarily conserved system

functioning as the first line of defense against invading pathogens

and is essential for the subsequent activation of adaptive

immu-nity. Central to early antiviral defense is the induction of IFN,

FIG 6NS4A negatively regulates the IFN signaling pathway by inhibiting IRF3 activation. (A) 293T cells transfected with the IFN-␤reporter together with NS4A (250 and 500 ng), prM (500 ng), or PB1-F2 (500 ng) were infected with SeV at 100 HAU/ml for 16 h, followed by analysis of cell lysates for luciferase activity. The protein expression levels of NS4A, prM, and PB1-F2 used in this reporter assay were determined by Western blotting. (B) Representative fluorescence micrographs of IRF3 in 293T cells transfected with GFP-IRF3 together with an empty vector or Myc-tagged NS4A (500 ng) for 24 h and then infected with SeV for 16 h. DAPI is a DNA-intercalating dye. (C) Western blotting of phosphorylated and total IRF3 expression in 293T cells transfected with a blank control vector or a vector expressing Myc-tagged NS4A, prM, or PB1-F2 and infected with SeV. (D)IFNB,OAS1, andIFITM1mRNA levels in 293T cells transfected with a blank control vector or a vector expressing Myc-tagged NS4A, prM, or PB1-F2 (1␮g/well) and then infected with SeV were determined by real-time PCR. IVA, influenza virus A. The data are presented as the means⫾SD derived from three repeat experiments. **,P⬍0.01 (Student’sttest).

Targeting MAVS by Dengue Virus NS4A

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especially type I IFN, which further leads to the expression of a set

of antiviral ISGs (

43

). DENV is one of the most important

mos-quito-borne viruses affecting humans, causing dengue fever or

more severe dengue symptoms such as DHF and DSS. Like other

viruses, DENV evades host immune defenses. In the present study,

a novel function of NS4A—suppression of IFN-

␤

by interacting

with MAVS and subsequently blocking IRF3 phosphorylation and

nuclear translocation— has been identified, highlighting an

im-portant role for NS4A in the establishment of DENV infection in

humans.

DENV is known to be a weak inducer of type I IFN, and

DENV-infected human dendritic cells exhibit an impaired type I IFN

response to infection with several viruses and to stimulation with

poly(I·C) (

14

,

44

,

45

). Thus far, the functional significance of

NS4A in viral replication remains poorly characterized. NS4A was

previously suggested to harbor viral replication complexes by

in-ducing curvature of the ER membrane and promoting membrane

rearrangements (

39

). Our findings reveal that NS4A may have

evolved to be a multifunctional protein that is directly involved in

the DENV replication machinery and also contributes to the

eva-sion of host immunity. It was demonstrated previously that

DENV NS2B/3 blocks the type I IFN response by targeting MITA/

STING (

23

,

24

). Our work extends these findings by showing that

NS4A proteins derived from various serotypes of DENV also

block type I IFN production through targeting MAVS-mediated

signaling pathways, implying that the inhibition of

MAVS-depen-dent IFN activation by DENV NS4A is not limited to only one

DENV serotype. Further investigation of how this effect of NS4A

cooperates with other counter-IFN effects of DENV, such as IFN

inhibition by NS2B/3 via proteolytic cleavage of MITA (

23

), is

warranted. The knowledge gained by investigating the

mecha-nisms of DENV immune evasion may facilitate the future use of

attenuated viruses as potential live vaccines. Furthermore, small

molecules directed against signaling antagonists, such as

inhibi-tors of the NS4A-MAVS interaction, may be developed as

anti-DENV therapeutics.

We report that DENV NS4A, an ER membrane-bound

pro-tein, interacts with the mitochondrial protein MAVS. This raises

the question of whether NS4A is a strict membrane-bound protein

or whether it can localize to other intracellular compartments

such as MAMs. A recent study showed that MAM localization of

HCV NS3/4A governs its dual distribution in the ER and

mito-chondria (

46

); therefore, it would be of interest to study whether

MAM could also position DENV NS4A in proximity to the

adap-FIG 7NS4A inhibits VSV-induced IFN. (A) 293T cells were transfected with an empty vector or NS4A-, prM-, or PB1-F2-expressing vectors. Twenty-four hours later, 293T cells were infected with VSV-GFP for 24 h, and the number of GFP-positive cells was scored by flow cytometry. (B) 293T cells transfected with the IFN-␤reporter together with NS4A, prM, and PB1-F2 (1␮g/well) were infected with VSV-GFP for 24 h, and cell lysates were then analyzed by a luciferase activity assay. The data are shown as the means⫾SD derived from three repeat experiments. **,P⬍0.01 (Student’sttest). (C) Representative fluorescence micrographs of VSV-GFP in 293T cells transfected with a blank control vector or an NS4A-, prM-, or PB1-F2-expressing vector for 24 h followed by infection with VSV-GFP for 24 h. Mock-infected cells were used as a control. IVA, influenza virus A; DIC, differential interference contrast.

He et al.

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tor protein MAVS. Indeed, in our study, DENV NS4A was found

to colocalize and interact with MAVS in the MAM, which may

represent a new cellular localization and function for this

impor-tant DENV nonstructural protein. In addition, in our study, the

transmembrane domain of NS4A could bind the CARD domain

of MAVS. It is possible that the MAM is able to position DENV

NS4A in proximity to MAVS and thus facilitate their interaction

with each other.

Our findings reveal that NS4A binds MAVS through

interact-ing with both the N-terminal and C-terminal domains of MAVS

and subsequently suppresses the binding of endogenous RIG-I.

Notably, the N-terminal CARD-like domain of MAVS was

previ-ously shown to interact with RIG-I and to mediate IFN signaling

(

11

). Additionally, as the C-terminal transmembrane

domain-containing region of MAVS has been found to mediate

oligomer-ization (

47

) and interactions with other adaptor proteins, such as

tumor necrosis factor receptor-associated factor 3 (TRAF3) (

48

)

and TRAF6 (

49

), it is possible that NS4A also disrupts MAVS

oligomerization or the formation of complexes with other adaptor

proteins. Furthermore, it should be noted that another member of

the RLR family, melanoma differentiation-associated gene 5

(MDA5), was previously reported to be a sensor of DENV and to

synergistically mediate type I IFN responses with RIG-I (

50

).

No-tably, MDA5 also has two CARD domains at its N terminus and

transduces its signal through MAVS (

8

), and thus, it may be of

interest to further investigate whether NS4A can disrupt the

inter-action between MAVS and MDA5.

In summary, the present study demonstrates that the adaptor

protein MAVS is a target of DENV NS4A, revealing a novel

func-tion of the DENV NS4A protein in modulating host innate

immu-nity and possibly facilitating DENV infection. The physiological

significance of NS4A in DENV replication and its pathological

role in dengue diseases warrant further investigation into the

po-tential value of this molecule as a therapeutic and/or prevention

target.

ACKNOWLEDGMENTS

This work was supported by the Natural Science Foundation of China (grants 81330058, 81272417, 81501744, and 81571992), the National Mega Project on Major Infectious Disease Prevention (grant 2012ZX10004-213), the Key (Key grant) Project of the Chinese Ministry of Education (grant 311030), the National Science and Technique Major Project (grant 201305017), Guangdong Natural Science Funds for Distin-guished Young Scholar (grant 2014A030306023), and the Guangdong Recruitment Program of Creative Research Groups (grant 2009010058).

FUNDING INFORMATION

This work was funded by National Mega Project on Major Infectious Disease Prevention (2012ZX10004-213). This work was funded by Key Project of Chinese Ministry of Education (311030). This work was funded by National Science and Technique Major Project (201305017). This work was funded by Guangdong Natural Science Funds for Distinguished Young Scholars (2014A030306023). This work was funded by Guangdong Recruitment Program of Creative Research Groups (2009010058). This work was funded by National Natural Science Foundation of China (NSFC) (81330058, 81272417, 81501744, 81571992).

The funders had no role in study design, data collection and interpreta-tion, or the decision to submit the work for publication.

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