JVI Accepted Manuscript Posted Online 7 January 2015 J. Virol. doi:10.1128/JVI.03453-14 Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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Characterization of dengue virus NS4A and NS4B protein interaction

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Jing Zou1,2,3, Xuping Xie2, Qing-Yin Wang2, Hongping Dong2, Michelle Yueqi Lee4, Congbao

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Kang4,†, Zhiming Yuan1,*,†, Pei-Yong Shi2,*,†

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State Key Laboratory of Virology, Wuhan Institute of Virology, University of Chinese Academy

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of Sciences, Wuhan, China1; Novartis Institute for Tropical Diseases, Singapore2; University of

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Chinese Academy of Sciences, Beijing, China3; Experimental Therapeutics Centre, Agency for

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Science, Technology and Research (A*STAR), Singapore4

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*Address correspondence to Pei-Yong Shi: [email protected]; or Zhiming Yuan:

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[email protected]

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†

C.K., Z.Y., and P.Y.S. are co-senior authors of the study

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Running title: Dengue virus NS4A/NS4B interaction

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Keywords: flavivirus replication, membrane protein, dengue virus

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ABSTRACT Flavivirus replication is mediated by a membrane-associated replication complex where

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viral membrane proteins NS2A, NS2B, NS4A, and NS4B serve as the scaffold for the replication

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complex formation. Here we used dengue virus serotype-2 (DENV-2) as a model to characterize

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viral NS4A/NS4B interaction. NS4A interacts with NS4B in virus-infected cells and in cells

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transiently expressing NS4A and NS4B in the absence of other viral proteins. Recombinant

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NS4A and NS4B proteins directly bind to each other with an estimated Kd of 50 nM. Amino

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acids 40-76 (spanning the first transmembrane domain [amino acids 50-73]) of NS4A and amino

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acids 84-146 (also spanning the first transmembrane domain [amino acids 101-129]) of NS4B

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are the determinants for NS4A/NS4B interaction. NMR analysis suggests that NS4A residues17-

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80 form two amphipathic helices (helix α1 [residues 17-32] and helix α2 [residues 40-47]) that

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associate with the cytosolic side of endoplasmic reticulum (ER) membrane and helix α3

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(residues 52-75) that transverses ER membrane. In addition, NMR analysis identified NS4A

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residues that may participate in NS4A/NS4B interaction. Amino acid-substitution of these NS4A

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residues exhibited distinct effects on viral replication. Three of the four NS4A mutations (L48A,

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T54A, and L60A) that affected NS4A/NS4B interaction abolished or severely reduced viral

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replication; in contrast, two NS4A mutations (F71A and G75A) that did not affect NS4A/NS4B

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interaction had marginal effects on viral replication, demonstrating the biological relevance of

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NS4A/NS4B interaction to DENV-2 replication. Taken together, the study has provided

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experimental evidence to argue that blocking the NS4A/NS4B interaction could be a potential

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antiviral approach.

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SIGNIFICANCE

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Flavivirus NS4A and NS4B proteins are essential components of the ER membrane-

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associated replication complex. The current study systematically characterizes the interaction

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between flavivirus NS4A and NS4B. Using DENV-2 as a model, we show that NS4A interacts

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with NS4B in virus-infected cell, in cells that transiently expressing NS4A and NS4B proteins,

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or in vitro with recombinant NS4A and NS4B proteins. We mapped the minimal regions

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required for NS4A/NS4B interaction to be amino acids 40-76 of NS4A and amino acids 84-146

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of NS4B. NMR analysis revealed the secondary structure of amino acids 17-80 of NS4A and the

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NS4A amino acids that may participate in the NS4A/NS4B interaction. Functional analysis

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showed a correlation between viral replication and NS4A/NS4B interaction, demonstrating the

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biological importance of NS4A/NS4B interaction. The study has advanced our knowledge of the

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molecular function of flavivirus NS4A and NS4B proteins. The results also suggest that

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inhibitors of NS4A/NS4B interaction could be pursued for flavivirus antiviral development.

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INTRODUCTION

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The four serotypes of dengue virus (DENV 1-4) are the causative pathogens of dengue

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disease, which has become a major public health threat. DENV infection causes flu-like illness

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known as dengue fever (DF). Some DENV-infected patients could develop life-threatening

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diseases known as dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS) (1).

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DENV causes about 390 million human infections annually, leading to 96 million cases with

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manifest symptoms (2). Neither approved vaccine nor antiviral is clinically available for

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prevention and treatment of DENV infection. Better understanding of the molecular mechanisms

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of DENV replication will benefit vaccine and antiviral development.

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DENV is a member of genus Flavivirus within family Flaviviradae. Besides DENV,

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many flaviviruses are important human pathogens, including West Nile virus (WNV), Japanese 3

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encephalitis virus (JEV), yellow fever virus (YFV), and tick-borne encephalitis virus (TBEV).

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The plus-sense, single-strand genomic RNA of flavivirus contains a 5’ untranslated region (UTR)

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with a type-I cap structure, a single open-reading frame (ORF), and a non-polyadenylated 3’UTR.

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The genome encodes a polyprotein that is processed co- and post-translationally by cellular and

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viral proteases into three structural proteins (capsid [C], pre-membrane [prM], and envelope [E])

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and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). DENV

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infection induces ER-derived vesicles to accommodate viral replication complexes (3).

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Nonstructural proteins are mainly involved in viral replication. NS3 serves as a protease (using

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NS2B as a cofactor) (4), RNA helicase, nucleotide triphosphatase and RNA triphosphatase (5-7).

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NS5 functions as a methyltransferase (8-10), weak guanylyltransferase (11) and RNA-dependent

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RNA polymerase (12). The glycosylated NS1 protein is essential for viral replication (13, 14).

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Four nonstructural proteins (NS2A, NS2B, NS4A, and NS4B) are integral membrane

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proteins. NS2A contains five transmembrane domains (TMD) and is important for viral

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replication and assembly (15, 16). NS2B is a cofactor for NS3 protease (17). Membrane proteins

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NS4A and NS4B are linked by a conserved 2K signal peptide. The cleavage between NS4A and

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2K by NS2B/NS3 proteinase is a prerequisite for the cleavage between 2K and NS4B by a host

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signalase (18). DENV-2 NS4A consists of 127 amino acids and contains two TMDs (19); the

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first 48 amino acids of DENV-2 NS4A were reported to form an amphipathic helix that mediates

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oligomerization (20). DENV-2 NS4B consists of 248 amino acids and contains three TMDs (21).

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NS4A and NS4B play multiple roles in viral replication and virus-host interactions: (i) WNV

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NS4A and its C-terminal 2K peptide induce ER membranes rearrangement. Removal of 2K

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results in a redistribution of NS4A to the Golgi apparatus (22); however, expression of DENV-2

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NS4A without 2K causes the ER membrane alterations resembling virus-induced structures, 4

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whereas expression of NS4A in tandem with 2K fails to induce membrane rearrangement (19).

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This evidence suggests 2K regulates NS4A’s function in modulating ER membrane through

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distinct mechanisms in different flaviviruses. (ii) Interaction between DENV NS4A and cellular

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vimentin regulates the formation of virus replication complexes (23). (iii) Flavivirus NS4A

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protein induces autophagy to prevent cell death and facilitate viral replication (24). (iv) WNV

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NS4A regulates the ATPase activity of NS3 helicase (25), while DENV NS4B interacts with the

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helicase domain of NS3 and dissociates it from single-strand RNA (26). (v) NS4A and NS4B

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genetically interact with NS1 to modulate viral replication. The replication defect of YFV

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containing NS1 mutations could be restored by an adaptive mutation in viral NS4A (27). The

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replication defect of WNV containing NS1 mutations could be compensated by a mutation in

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viral NS4B (28). (vi) An N-terminal cytoplasmic mutant of DENV-1 NS4A is defective in viral

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replication. The replication defect can be rescued by mutations in the TMD3 of NS4B (29). (vii)

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Both NS4A and NS4B can induce host unfolded protein response (16) and inhibit IFN signaling

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(30-32). These results suggest that NS4A and NS4B may function cooperatively in viral

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replication and host response. However, the direct interaction between NS4A and NS4B remains

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to be established and characterized.

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In this study, we demonstrated a direct interaction between DENV-2 NS4A and NS4B.

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We mapped amino acids 40-76 of NS4A and amino acids 84-146 of NS4B to be the determinants

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for the NS4A/NS4B interaction. NMR studies revealed the secondary structure of NS4A residues

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17-80, and identified the residues within this region that may participate in the NS4A/NS4B

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interaction. Functional analysis using a DENV-2 genome-length RNA showed that the identified

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NS4A residues were important for viral replication. Furthermore, the replication defects of

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NS4A mutant viruses correlated with the mutational effect on NS4A/NS4B interaction,

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demonstrating the biological relevance of NS4A/NS4B interaction.

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MATERIALS AND METHODS

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Cells, viruses, and antibodies. Baby hamster kidney (BHK-21) cells were purchased

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from the American Type Culture Collection (ATCC) and maintained in high glucose Dulbecco

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modified Eagle medium (DMEM) (Life technologies) supplemented with 10% fetal bovine

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serum (FBS) (HyClone Laboratories, Logan, UT) and 1% penicillin/streptomycin (Life

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technologies). HEK 293T cells were grown in low-glucose DMEM (Life technologies)

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containing 10% FBS and 1% penicillin/streptomycin. DENV-2 (strain NGC; GenBank number

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AF038403) was generated from its corresponding infectious cDNA clone (pACYC-NGC FL)

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(15). The following antibodies were used in this study: negative control mouse IgG2b (Millipore),

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mouse monoclonal antibody (mAb) against DENV-2 NS4B protein (clone 44-4-7) (33), mouse

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mAb against DENV-2 NS4A protein (generated in house, unpublished), mouse mAb D2C1

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against DENV-2 capsid (34), mouse mAb 4G2 (ATCC) against DENV E protein, mouse mAb

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anti-EGFP (Roche), rabbit polyclonal antibody (pAb) anti-EGFP (Abcam), rabbit pAbs anti-HA

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and anti-Flag (Sigma), mouse mAb anti-Flag (Sigma), mouse mAb anti-DENV-2 NS3, rabbit

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pAbs anti-DENV 2 NS3 and NS4A (GeneTex), Alexa Fluor® 488 goat anti-mouse IgG and

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Alexa Fluor® 594 goat anti-rabbit IgG (Life technologies), goat anti-mouse or goat anti-rabbit

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antibody conjugated to horseradish peroxidase (HRP) (Sigma), and protein A conjugated to HRP

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(Sigma).

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Plasmid construction. Standard molecular biology procedures were performed for all plasmid constructions. The fragments encoding full length (FL) or peptides of NS4A and NS4B

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were amplified from plasmid pACYC-NGC FL (15) and fused in-tandem with an HA or Flag tag

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by PCR using corresponding primer pairs. The tag position of individual construct is indicated in

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each experiment (see below). The PCR products were digested with EcoRI and XhoI restriction

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enzymes, and cloned into a mammalian expression vector pXJ (15) to generate constructs pXJ-

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NS4A-Flag (encoding full-length NS4A protein with a C-terminal Flag tag), pXJ-Flag-NS4A

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(50-76) (encoding NS4A peptide 50-76 with an N-terminal Flag tag), pXJ-Flag-NS4A (40-76)

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(encoding NS4A peptide 40-76 with an N-terminal Flag tag), pXJ-2K-NS4B-HA (encoding full-

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length NS4B protein with an N-terminal 2K signal peptide and a C-terminal HA tag) and pXJ-

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NS4B (84-146)-HA (encoding NS4B peptide 84-146 with a C-terminal HA tag). For C-

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terminally EGFP-tagged NS4A or NS4B constructs, the NS4A or NS4B fragments were

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amplified by PCR, digested with EcoRI and BamHI restriction enzymes and inserted into the

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plasmid pXJ-EGFP (15) to generate constructs pXJ-4A-peptides-EGFP and pXJ-NS4B-peptides-

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EGFP.

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For site-directed mutagenesis, all NS4A mutants were first introduced into a subclone

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TA-E (containing cDNA from nucleotide 5,426 to the 3’ end of the genome of DENV-2, strain

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NGC) individually by using a QuickChange II XL site-directed mutagenesis kit (Agilent

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Technlologies, Inc.). The DNA fragment containing each NS4A mutation was cloned into the

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plasmid pACYC-NGC FL(15). To construct pXJ-NS4A-EGFP encoding mutant NS4A-EGFP

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proteins, the corresponding NS4A fragments were amplified from the plasmids pACYC-NGC

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FL containing designed NS4A mutations, digested with EcoRI and BamHI restriction enzymes

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and inserted into the plasmid pXJ-EGFP, resulting in constructs pXJ-NS4A-mutants-EGFP. All

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constructs were validated by restriction enzyme digestion and cDNA sequencing. Primers

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sequences are available upon request. 7

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Immunofluorescence assay (IFA). Cells grown on an 8-well Lab-Tek chamber slide

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(Thermo Fisher Scientific) were washed once by phosphate-buffered saline (PBS), fixed with 4%

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paraformaldehyde in PBS at room temperature for 30 min, and then permeabilized with 0.1%

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(v/v) Triton X-100 in PBS for 10 min. After blocking with PBS containing 1% bovine serum

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albumin (BSA) for 1 h, the cells were incubated with primary antibody for 1 h and followed by 3

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washes with PBST buffer (PBS containing 0.05% Tween 20). Afterwards, the cells were

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incubated with secondary antibodies Alexa Fluor® 488 goat anti-mouse IgG or Alexa Fluor® 594

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goat anti-rabbit IgG (1:1000) for 1 h in PBST buffer containing 1% BSA. After 3 washes by

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PBST, the cells were mounted in a mounting medium with DAPI (4', 6-diamidino-2-

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phenylindole; Vector Laboratories, Inc.). Fluorescence images were acquired with a Leica

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DM4000 B system.

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For confocal study, BHK-21 cells were infected with DENV-2 NGC (MOI 10) in wells

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of an 8-well chamber slide at 37°C for 24 hours. After that, the cells were fixed, permeabilized

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and incubated with antibodies described above. Confocal images were acquired by using a Zeiss

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LSM 510 META laser scanning confocal microscope armed with oil immersion 100× objective

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lens (Biopolis Shared Facilities, Singapore). Images were merged using Fiji image software.

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Co-immunoprecipitation (Co-IP). HEK 293T cells in a 10-cm dish were transfected

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with indicated plasmid DNA using an X-tremeGENE 9 DNA transfection reagent (Roche). At 48

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h post-transfection (p.t.), the cells were lysed in 1 ml IP buffer (20 mM Tris, pH 7.5, 100 mM

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NaCl, 0.5% DDM (n-Dodecyl β-D-Maltopyranoside), and 1× EDTA-free protease inhibitor

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cocktail [Roche]) by rotation at 4°C for 1 h. Cell lysates were clarified by centrifugation at

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20,000 × g 4°C for 30 min and the supernatants were subjected to co-IP using protein G

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conjugated magnetic beads according to the manufacturer’s instructions (Millipore). Briefly, 200

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µl~400 µl of the cell lysates were mixed with 2 µg antibodies in a 500-µl volume containing

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250~400 mM sodium chloride to form immune-complexes at 4°C overnight. Subsequently, the

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immune-complexes were precipitated by protein G conjugated magnetic beads at 4°C for 1 h

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with rotation. After five washes with PBS containing 0.1% Tween 20, the bound proteins were

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eluted in 4 × lithium dodecyl sulfate (LDS) sample buffer (Life technologies) containing 100

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mM dithiothreitol (DTT) by heating at 70°C for 15 min on an ThermoMixer (Eppendorf) with a

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shaking speed at 1200 × rpm. Eluates were analyzed by SDS-PAGE & Western blotting.

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Expression and purification of recombinant NS4A and NS4B proteins. DENV-2

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NGC NS4B protein was expressed and purified by following a protocol as described previously

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(35). Similar protocol was used to express and purify DENV-2 NS4A protein with some

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modifications. Briefly, the cDNA encoding the full-length NS4A was amplified from pACYC-

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NGC FL, fused N-terminally with a hexahistidine (His)6, a TEV cleavage site and a thrombin

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cleavage site, and cloned into a vector pNIC28-Bsa4 (GenBank accession EF198106) by NdeI

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and HindIII restriction enzymes, resulting in the construct pNIC28-NS4A. The plasmids were

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transformed into E. coli Rossetta (Stratagene) cells. The expression of NS4A protein was

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induced by adding isopropyl β-D-1-thiogalactopyranoside (IPTG) to a final concentration of 0.3

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mM when the cell density reached OD600 0.6~0.8. After growing for 16 h at 18°C, the cells are

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harvested, resuspended in a lysis buffer (20 mM Tris-HCl, 300 mM NaCl, 2 mM Tris-[2-

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carboxyethyl] phosphine [TCEP], 0.5 × EDTA-free protease inhibitor cocktail, pH 8.5) and

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disrupted by sonication using a Digital Sonifier 450 (Branson). The cell debris was removed by

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centrifugation at 10,000 × rpm for 10 min at 4°C. The supernatants were collected and applied to

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ultra-centrifugation at 45,000 × rpm for 1 h at 4°C using a Beckman Type 70 Ti rotor. The

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pellets containing the membrane fractions were collected and solubilized with 1% (w/v) 69

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Cyclohexyl-1-hexyl-β-D-maltoside (Cymal-6) (Anatrace) in lysis buffer overnight at 4°C.

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Following another ultra-centrifugation step at 45,000 × rpm for 1 h, the supernatants were

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collected and loaded to a HisTrap Fast Flow column (GE Healthcare) which was pre-equilibrated

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with buffer A (20 mM Tris, 300 mM NaCl, 2 mM TCEP, and 0.1 % Cymal-6, pH 8.5). The

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protein was eluted using a linear gradient of imidazole concentration from 40 to 300 mM. The

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fractions containing (His)6-NS4A proteins were pooled, concentrated and subjected to gel

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filtration using a HiLoad Superdex 200 16/60 column (GE Healthcare) in buffer A. The peak

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fractions containing (His)6-NS4A proteins were pooled and concentrated to approximately 5~10

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mg/ml before storage at -80°C.

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Expression and purification of NS4A (17-80). The cDNA encoding residues M17 to

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K80 of NS4A of DENV-2 (NGC strain) was synthesized and cloned into the NdeI and XhoI sites

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of pET29b. The resulting plasmid pET29-NS4A encodes NS4A (17-80) with a C-terminal

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(His)6-tag to facilitate protein purification. Plasmid was transformed in E. coli competent cells

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and cells were plated onto a LB plate containing kanamycin. The NS4A (17-80) was induced and

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purified using the similar protocol to that used for other membrane proteins (36). Briefly, two to

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three colonies were inoculated in 20 ml of M9 medium and incubated at 37°C overnight with

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shaking. The culture was then transferred into M9 medium supplied with kanamycin. When

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OD600 reached 0.6-0.8, protein was induced overnight at 37°C by adding IPTG to a final

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concentration of 1 mM. The E. coli cells were collected and re-suspended into a lysis buffer that

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contained 20 mM Tris-HCl, pH 7.8, 300 mM NaCl, and 2 mM β-mercaptoethanol. Cells were

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then broken by sonication in an ice bath and cell lysate was cleared by centrifugation at 40,000 ×

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g for 20 min. The pellet was solubilized in a urea buffer containing 8 M urea, 300 mM NaCl, 10

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mM SDS and 20 mM Tris-HCl, pH 7.8. The solution was cleared by centrifugation at 40,000 × g 10

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for 20 min at room temperature. The resulting supernatant was then mixed with nitrilotriacetic

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acid saturated with nickel (Ni2+-NTA) resin and the resin was then loaded on a gravity column.

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The resin was washed with 10 column volumes of urea buffer containing 20 mM imidazole.

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Resin was further washed with 10 column volumes of lysis buffer with 10 mM SDS and another

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10 column volumes of lysis buffer with 15 mM dodecylphosphocholine (DPC). Protein was

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eluted in an elution buffer that contained 20 mM sodium phosphate, pH 6.5, 300 mM imidazole,

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and 15 mM DPC. Purified protein from Ni2+-NTA resin was concentrated from 8 ml to 1 ml

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using a 3-kDa molecular weight cutoff concentrator and loaded on a SuperdexTM 200 10/300 GL

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column that was pre-equilibrated with a gel filtration buffer containing 20 mM sodium phosphate,

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pH 6.5, 15 mM DPC, and 1 mM DTT. Sample was concentrated to 200-400 µl before it was

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transferred into an NMR tube for data acquisition.

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Enzyme-linked immunosorbent assay (ELISA). ELISA was performed according to a

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previous report (37) with minor modifications. In brief, purified DENV-2 NS4A protein was

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diluted in coating buffer (50 mM carbonate/bicarbonate buffer, pH 9.6) and incubate in 96-well

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ELISA plates (Nunc) (250 ng/well) at 4°C overnight. Next day, the plates were incubated at

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room temperature for 2 hours in a blocking buffer (TBS buffer [10 mM Tris-HCl, pH 8.0, 150

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mM NaCl] supplemented with 2% FBS). After three washes with TBS buffer plus 0.05% Triton

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X-100, blocked wells were incubated with serial dilutions of DENV-2 NS4B protein in blocking

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buffer at room temperature for 2 hours. After another three washes with TBS buffer plus 0.05%

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Triton X-100, the plates was incubated with a mouse mAb anti-NS4B (clone 44-4-7), followed

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by incubating with a secondary antibody (goat anti-mouse antibody conjugated to HRP). After

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extensive washing, the plates were incubated with the substrate 3,3’,5,5’-tetramethylbenzidine

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(TMB) (Thermo scientific) at room temperature for less than 3 minutes. The reactions were 11

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stopped by adding equal volumes of 1 M sulfuric acid. The absorbance at 450 nm was measured

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using a Safire II plate reader (Tecan). Two sets of controls were used for background subtraction:

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i) wells coated with NS4A were not sequentially incubated with NS4B; ii) and NS4A-free wells

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were sequentially incubated with NS4B. The concentration of NS4B at which half of NS4A

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proteins were bound by NS4B was defined as Kd, which was calculated using a “one-site

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specificity” model in Prism 5 software.

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In situ proximity of ligation assay (PLA). PLA was performed using a Duolink in situ

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kit (Olink Bioscience) according to the manufacturer’s instruction. Briefly, 2.5×104 BHK-21

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cells were seeded into each well of an 8-well chamber. Next day, cells were co-transfected with

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pXJ-Flag-NS4A (50-76) and pXJ-NS4B (84-146)-HA, or pXJ-Flag-NS4A (40-76) and pXJ-

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NS4B (84-146)-HA. Cells transfected with pXJ-NS4B (84-146)-HA alone were set as a negative

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control. At 48 h p.t., the cells were fixed by 4% paraformaldehyde in PBS for 30 min at room

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temperature, and then permeabilized by 0.1% Triton X-100 in PBS for 10 min. After blocking in

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PBS containing 1% FBS and 0.05% Tween 20 for 1 h, the cell were simultaneously incubated

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with two different primary antibodies (mouse mAb anti-Flag and rabbit pAb anti-HA) for 1 h.

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The following steps including incubation with PLA probes, ligation, amplification and

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preparation for imaging were performed according to the manufacturer’s instructions. Images

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were acquired using a Leica DM4000 B microscope system armed with 20 × and 63 × objectives.

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For each experiment, fifteen images per well were acquired under the identical parameters of

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camera settings. About thirty PLA-positive cells were randomly chosen and the means of

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fluorescence intensity in each cell was quantified and calculated using Fiji image software. The

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unpaired two-tailed Student’s t-test was performed in Prism 5 software.

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SDS-PAGE and Western blot. Proteins were separated on 12% Mini-PROTEAN®

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precast gels (Biorad) or Bolt™ 4–12% Bis-Tris Gels (Life technologies), and transferred onto a

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PVDF membrane using the Trans-Blot® Turbo™ Transfer System (Bio-Rad). The blots were

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blocked in TBST buffer (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 0.1% Tween 20)

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supplemented with 5% skim milk for 1 h at room temperature, and followed by 1 h incubation

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with primary antibodies. After three washes with TBST buffer, the blots were incubated with

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secondary antibodies (goat-anti-mouse or goat-anti-rabbit antibodies conjugated to HRP) or

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protein A conjugated to HRP in TBST buffer plus 5% milk for 1 h. After another three washes

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with TBST buffer, the blots were incubated with the Amersham ECL Prime Western Blotting

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detection reagent (GE Healthcare). Chemiluminescence signals were detected by exposing the

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blots to Kodak X-ray films or a C-DiGit Blot Scanner (LI-COR).

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Resonance assignment. Uniformly 13C/15N-labeled NS4A (17-80) was prepared using

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aforementioned method and concentrated to 0.5 mM in a buffer containing 20 mM sodium

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phosphate, pH 6.5, 1 mM DTT, 200 mM DPC and 10% D2O. All the NMR spectra were

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recorded at 313 K on a Bruker Avance 600 MHz spectrometer with a cryogenic triple resonance

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probe. The pulse programs were from Topspin 2.1 program library. Data acquired were

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processed with NMRPipe (38) and analyzed using NMRView (39). Backbone resonances

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assignment was obtained based on the experiments including 2D-1H-15N-HSQC, 3D HNCACB,

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HNCA, HN(CO)CA, HN(CO)CACB, HNCO and HBHACONH experiments. Secondary

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structure was analyzed by analysis of 13C chemical shift (40) and TALOS+ (41).

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Structural model of NS4A (17-80). The structural model of NS4A (17-80) was

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generated by XPLORE-NIH (42) using mainly backbone dihedral angles restraints derived from

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TALOS+ (41). Structure determination was carried out using a randomized template. Simulated 13

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annealing was performed and energy minimization were carried out as previously described (43).

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Simulated annealing was carried out with starting temperature of 3, 500 K and 15, 000 K cooling

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steps. The structure was energy-minimized with Powell energy minimization. One model out of

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50 calculated ones was selected for presentation.

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RNA transcription, electroporation and virus production. Wild type (WT) and mutant

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DENV-2 genome-length RNAs were transcribed in vitro using a T7 mMessage mMachine kit

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(Ambion) from cDNA plasmids pre-linearized by XbaI. The RNA transcripts (10 µg) were

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electroporated into BHK-21 cells following a protocol described previously (44). After

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electroporation, cells were immediately seeded on an 8-well chamber slide. At indicated time

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points, the cells were subjected to immunofluorescence assay as described above. For virus

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production, the transfected cells were seeded in one T-75 flask. After incubation at 37°C for 24 h,

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the medium was replaced with fresh DMEM medium plus 2% FBS to remove extracellular input

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RNAs. Afterwards, the cells were maintained at 30°C for another four days. On every day from

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day 1 to 5 p.t., 500 µl culture fluids were collected, clarified by centrifugation at 415 × g for 5

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min, and stored at -80°C. Virus titers were quantified by a standard CPE-based plaque assay (45).

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Surrogate plaque assay by immunostaining. This surrogate assay is used to quantify

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those viruses did not generate obvious plaque by above standard plaque assay (45). In brief, 1.5

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× 105 BHK-21 cells per well were seeded into a 24-well plate. Next day, one hundred microliters

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of undiluted or series of ten-fold diluted virus samples were added to individual wells. After 1h

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incubation at 30°C with 5% CO2, the inoculums in each well were replaced with about 0.6 ml

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“overlay” medium (RPMI 1640 medium plus 0.8% methylcellulose[Aquacide II; Merck, CA]

306

and 2% FBS). After incubation at 37°C with 5% CO2 for 5.5 days, the cells were washed twice

307

with PBS to completely remove the “overlay”, followed by fixation with 3.7% formaldehyde in 14

308

PBS for 30 min. After that, cells were washed once with PBS and permeabilized with 0.1% (v/v)

309

Triton X-100 in PBS for 10 min. After blocking with PBS containing 1% FBS for 1 h at room

310

temperature, cells were incubated with a mouse mAb against E protein (4G2) for 1 h and

311

followed by 3 washes with PBST buffer. Afterwards, cells were incubated with a goat-anti-

312

mouse IgG conjugated to HRP (1:1000 dilution) for 1 h. After 3 washes with PBST, cells were

313

stained by DAB-Plus Substrate Kits (Life technologies) according to the manufacturer’s

314

instructions.

315

RESULTS

316

Intracellular NS4A/NS4B interaction. Three experiments were performed to

317

demonstrate the NS4A/NS4B interaction in cells. Firstly, we examined co-localization of NS4A

318

and NS4B in DENV-2 infected BHK-21 cells using a confocal microscopy. Both NS4A and

319

NS4B showed similar ER staining pattern that extended throughout the cytoplasm, which

320

resembled the distribution of NS3 in infected cells (Fig. 1A). Statistics analysis showed that the

321

IFA signals of NS4A and NS4B significantly overlapped with each other (Pearson coefficient >

322

0.5). This result confirms the previous reports that NS4A and NS4B co-localize with other viral

323

proteins (e.g. NS3) and dsRNA at viral replication site (19, 21).

324

Secondly, we performed a co-IP experiment to examine the NS4A/NS4B interaction in

325

the context of viral infection. BHK-21 cells infected with DENV-2 were subjected to co-IP

326

analysis, where mouse mAb anti-NS4A, mouse mAb anti-NS4B, or mouse IgG2b (negative

327

control) was used for pull-down. As shown in Fig. 1B, anti-NS4A mAb pulled down NS4A

328

protein together with NS4B and NS3 proteins, but not capsid protein; in reciprocal, anti-NS4B

329

mAb pulled down NS4B protein together with NS4A and NS3 proteins, but not capsid protein.

15

330

On the contrary, negative control mouse IgG2b could not pull down any viral proteins (capsid,

331

NS3, NS4A, or NS4B). The results suggest that NS4A interacts with NS4B in virus infected cells;

332

such interaction could be mediated through a direct NS4A/NS4B binding or mediated through an

333

intermediate viral/host protein.

334

Thirdly, we used co-IP experiment to demonstrate NS4A/NS4B interaction in the absence

335

of other viral proteins. HEK 293T cells were co-transfected with two plasmids: one plasmid

336

expressing full-length NS4B with an N-terminal 2K signal peptide and a C-terminal EGFP tag

337

(construct 2K-NS4B-EGFP); another plasmid expressing full-length NS4A with a C-terminal

338

Flag tag (construct NS4A-Flag). The 2K peptide was included in construct 2K-NS4B-EGFP to

339

ensure the correct topology of NS4B; when expressed inside cell, a host protease cleaves 2K-

340

NS4B-EGFP to generate 2K and NS4B-EGFP. As shown in Fig. 1C, the EGFP antibody was

341

able to pull down NS4B-EGFP together with NS4A-Flag (lane 3). Besides NS4B-EGFP, a band

342

corresponding to the molecular mass of EGFP was also detected; this band varied from

343

experiments to experiments (see below), possibly representing a degradation product of NS4B-

344

EGFP. On the contrary, when EGFP was co-expressed with NS4A-Flag, the EGFP antibody

345

pulled down EGFP, but not together with NS4A-Flag (lane 2). As a control, NS4A-Flag alone

346

did not bind to beads or EGFP antibody under the same co-IP conditions (lane 1). Next, we

347

performed a reciprocal co-IP experiment using full-length NS4A with a C-terminal EGFP tag

348

(construct NS4A-EGFP) and full-length NS4B with an N-terminal 2K signal peptide and a C-

349

terminal HA tag (construct 2K-NS4B-HA). The EGFP antibody was able to pull down NS4A-

350

EGFP together with NS4B-HA (Fig. 1C, lane 5). In contrast, the EGFP antibody could pull down

351

EGFP, but not NS4B-HA (lane 4). Taken together, the results suggest that NS4A interacts with

352

NS4B in the context of viral replication or when expressed in the absence of other viral proteins. 16

353

Interaction of recombinant NS4A and NS4B proteins. ELISA was used to examine the

354

in vitro NS4A/NS4B interaction. Recombinant DENV-2 NS4A and NS4B proteins were

355

expressed and purified from E.coli. SDS-PAGE analysis showed that both proteins reached

356

purities of > 90% (Fig 2A). For NS4A proteins, besides monomer (17.2 kDa), several SDS-

357

resistant oligomer species (dimer or higher order oligomers) were detected, suggesting that

358

recombinant NS4A protein can oligomerize in vitro. This result is in agreement with a recent

359

report that DENV NS4A protein could oligomerize (20). For NS4B protein, two bands,

360

representing monomer (28 kDa) and dimer (56 kDa), were observed. The result is consistent with

361

our previous result that NS4B could dimerize (35). Western blot showed that antibodies against

362

DENV-2 NS4A or NS4B could specifically detect various oligomeric forms of recombinant

363

proteins (Fig. 2B). Next, the recombinant NS4A and NS4B proteins were used for an ELISA

364

analysis. The ELISA plate was coated with NS4A proteins, blocked with 2% FBS, and incubated

365

with a serial of dilutions of NS4B proteins. The captured NS4B was detected using an NS4B

366

mouse mAb and a goat-anti-mouse IgG conjugated to HRP as the primary and secondary

367

antibody, respectively. As shown in Fig. 2C, NS4B bound to NS4A in a dose-dependent manner,

368

with an estimated Kd of 50 nM. The ELISA result demonstrates that recombinant NS4A and

369

NS4B proteins could interact with each other in vitro.

370

Amino acids 40-76 of NS4A are responsible for interaction with NS4B. To define the

371

regions that are responsible for the NS4A/NS4B interaction, we performed a co-IP assay using a

372

series of truncated peptides of NS4A and full-length NS4B. Based on the topology of NS4A (Fig.

373

3A), we prepared a panel of C-terminally truncated NS4A peptides and tested their interaction

374

with full-length NS4B. HEK 293T cells were co-transfected with two plasmids: one plasmid

375

expressing a full-length 2K-NS4B protein with a C-terminal HA tag (construct 2K-NS4B-HA); 17

376

another plasmid expressing NS4A peptide with a C-terminal EGFP tag (construct NS4A-peptide-

377

EGFP). As shown in Fig. 3B, an EGFP antibody pulled down various NS4A-peptides-EGFP

378

together with different amounts of 2K/NS4B-HA (see definition below). Similar levels of

379

2K/NS4B-HA were pulled-down by NS4A-EGFP peptides 1-76, 1-101, 1-122, and 1-127 (full-

380

length NS4A). In contrast, only trace amounts of 2K/NS4B-HA were pulled-down by NS4A-

381

EGFP peptides 1-50 and 1-60. Notably, when 2K-NS4B-HA was expressed in HEK 293T cells,

382

two bands can be detected in the cell lysate and IP eluates (Figs. 3B-D and Fig 9A). This may be

383

caused by an inefficient cleavage between 2K and NS4B that is mediated by a host signalase. For

384

simplicity, these two bands were collectively labeled as “2K/NS4B-HA.” The cleavage

385

efficiency at the 2K and NS4B junction varied between experiments (Compare Fig. 1C with Figs.

386

3A-D), possibly due to the HEK 293T cells with different passage numbers which could affect

387

the amount/efficiency of the host signalase. In addition, the variation in EGFP cleavage was

388

observed for different NS4A peptides through an unknown mechanism. Nevertheless, the results

389

demonstrate that deletion of the C-terminal 77-127 residues of NS4A does not significantly

390

affect NS4A/NS4B interaction.

391

Next, we performed N-terminal deletion analysis of NS4A (Fig. 3C). NS4A-EGFP

392

peptide 40-127 and full-length NS4A-EGFP pulled down similar amounts of 2K/NS4B-HA,

393

demonstrating that the N-terminal 39 residues of NS4A are not required for NS4A/NS4B

394

interaction. In contrast, NS4A-EGFP peptide 50-127 almost lost its ability to pull down NS4B

395

protein, suggesting that residues 40-50 of NS4A contribute to NS4A/NS4B interaction. The

396

results presented in Figs. 3B and C suggest that amino acids 40-76 of NS4A represent the

397

minimal region required for NS4B interaction. To confirm this conclusion, we prepared two

398

more NS4A-EGFP peptides (40-76 and 50-76) and compared their abilities to bind NS4B (Fig. 18

399

3D). As expected, NS4A-EGFP peptide 40-76 bound NS4B as efficiently as the full-length

400

NS4A-EGFP 1-127. In contrast, NS4A-EGFP peptide 50-76 almost lost its binding to NS4B.

401

The results clearly indicate that amino acids 40-76 of NS4A are responsible for the NS4A/NS4B

402

interaction.

403

Amino acids 84-146 of NS4B are required for interaction with NS4A. We used a

404

similar approach as described above to define the region of NS4B required for NS4A interaction.

405

In this case, HEK 293T cells were co-transfected with two plasmids: one plasmid expressing a

406

full-length NS4A with a C-terminal Flag tag (construct NS4A-Flag); another plasmid expressing

407

truncated NS4B with a C-terminal EGFP tag (construct NS4B-peptide-EGFP). Based on the

408

topology of NS4B (Fig. 4A), we first tested the effect of C-terminal truncation of NS4B on

409

NS4A/NS4B interaction (Fig. 4B). For the C-terminally truncated NS4B-peptide-EGFP, the 2K

410

sequence was retained at the N-terminus of each construct to ensure a correct topology on ER

411

membrane. As shown in Fig. 4B, NS4B-EGFP full-length 1-248 and peptide 1-146 exhibited

412

similar binding activities to NS4A-Flag; in contrast, NS4B-EGFP peptide 1-130 almost lost the

413

NS4B-binding activity. These results suggest that the C-terminal 147-248 of NS4B is not

414

required for the NS4A/NS4B interaction.

415

Next, we prepared two NS4B-EGFP peptides (84-146 and 130-165) to further map the

416

region of NS4B required for the NS4A/NS4B interaction. We did not engineer the 2K sequence

417

to the N-termini of NS4B (84-146)-EGFP and NS4B (130-165)-EGFP. Based on the topology

418

model of NS4B, NS4B (84-146)-EGFP should form the right topology on the ER membrane; and

419

NS4B (130-165)-EGFP should locate in cytosol. As shown in Fig. 4C, NS4B-EGFP full-length

420

1-248, peptide 1-146, and peptide 84-146 yielded equivalent levels of NS4A-binding activity;

19

421

however, NS4B-EGFP peptide 130-165 lost the NS4A-binding (Fig. 4C). Collectively, the

422

results indicate that residues 84-146 of NS4B are required for the NS4A/NS4B interaction.

423

NS4A 40-76 and NS4B 84-146 peptides are in close proximity when co-expressed

424

inside cells. The above results prompted us to directly examine the interaction between NS4A

425

40-76 and NS4B 84-146 peptides. Due to the small size of these peptides (< 7 kDa), it is

426

technically difficult to detect their interaction through fusion with small tags (such as HA and

427

Flag) for co-IP and Western blot analysis. Initially, we attempted to overcome this difficulty by

428

fusing the NS4A and NS4B peptides with bigger tags (EGFP and glutatione S-transferase [GST]).

429

Unfortunately, we found that GST showed a strong nonspecific binding to EGFP in the absence

430

of any NS4A and NS4B peptides when co-expressed in cells (data not shown), thus excluding

431

the feasibility of this approach. As an alternative method, we used in situ proximity of ligation

432

assay (PLA) to examine the spatial proximity of NS4A/NS4B peptides intracellularly. PLA

433

allows for detection of two molecules in proximity of < 40 nm in cells (46, 47). The PLA was

434

performed by transfecting BHK-21 cells with two plasmids: One plasmid expressing Flag-NS4A

435

(40-76) peptide or Flag-NS4A (50-76) peptide (Fag tag was at the N-terminus of NS4A peptides);

436

another plasmid expressing NS4B (84-146)-HA peptide (HA tag was at the C-terminus of NS4B

437

peptide). Fig. 5A shows equivalent number of transfected cells expressing Flag-tagged NS4A

438

and/or HA-tagged NS4B peptides, indicating that similar transfection efficiencies were achieved

439

for different plasmids. The PLA showed very weak fluorescence signals (red) in cells transfected

440

with Flag-NS4A (50-76) and NS4B (84-146)-HA (Fig. 5B, left panel). In contrast, cells

441

transfected with Flag-NS4A (40-76) and NS4B (84-146)-HA produced about 4-fold stronger

442

fluorescence signals (Figs. 5B and C). No fluorescence signal was detected in cells without

443

plasmid transfection or in cells transfected with NS4B (84-146)-HA plasmid alone (data not 20

444

shown). Overall, the PLA results suggest that NS4A 40-76 and NS4B 84-146 peptides are in

445

close proximity when co-expressed intracellularly.

446

Secondary structure of NS4A 17-80 amino acids. We used NMR to analyze the

447

structure of an NS4A fragment representing amino acids 17-80 that included the mapped NS4B

448

binding site (40-76), referred as NS4A (17-80). NS4A (17-80) was selected from a series of N-

449

terminal deletion peptides of NS4A (including peptide [1-80]); only peptide (17-80) could be

450

successfully expressed and purified, and gave good NMR spectra for further study. Recombinant

451

NS4A (17-80) was expressed in E. coli and purified in DPC micelles. The C-terminally (His)6-

452

tagged peptide was purified through a Ni2+ column followed by a gel filtration. SDS-PAGE

453

analysis of the purified NS4A (17-80) showed a single band of >98% purity (Fig 6A). 1H-15N-

454

HSQC spectrum of NS4A (17-80) was obtained. The dispersion of cross-peaks in the spectrum

455

suggested the feasibility to analyze the structure of NS4A (17-80) using NMR spectroscopy. The

456

backbone resonances of NS4A (17-80) were assigned using 3D triple-resonance and NOESY

457

experiments. Almost complete backbone assignments for NS4A (17-80) in DPC micelles were

458

obtained (Fig. 6B).

459

Secondary structure analysis was conducted using both Cα chemical shift difference from

460

random coil values and TALOS+ (Figs. 6C and D). The NS4A (17-80) contains three helical

461

fragments: Helix α1 consists of residues 17-32; helix α2 contains residues 40-47; helix α3

462

includes residues 52-75 and transverses membrane (Figs. 6C and D). Inter-proton NOE

463

connectivity was plotted against residue number based on a 15N-HSQC-NOESY experiment (Fig.

464

6E). Although we did not observe a large number of NOE restrains, which might be due to the

465

dynamic nature or the size of the protein/micelle complex. The observation of NOEs between the

466

Hα of residue i and HN of residue i+3/4 suggests the presence of helical structures under the 21

467

current experimental conditions. One structural model of NS4A (17-80) was generated using

468

XPLOR-NIH package with the dihedral angles restraints derived from TALOS+ (Fig. 6F). Helix

469

α3 should be buried in the membrane, while helices α1 and α2 may interact with ER membrane.

470

Although the tertiary structure of NS4A (17-80) could not be determined due to the lack of long-

471

range distance restrains, the secondary structural information is still useful to understand the

472

function of NS4A. Charge surface representation of the model of NS4A (17-80) was obtained

473

and shown in Fig. 6G. As expected, the transmembrane domain helix α3 is hydrophobic. The

474

surface charges of helices α1 and α2 are very similar because both helices are amphipathic. Helix

475

wheel representations of the three helices were generated to further understand their structures

476

(Fig. 6H): Helices α1 and α2 exhibit an amphipathic feature with one surface formed by

477

hydrophobic residues and another surface formed by hydrophilic residues. The above results are

478

in general agreement with a previous report that the first 48 amino acids of DENV-2 NS4A form

479

amphipathic helix structure (20).

480

NMR analysis of the interaction between NS4A (17-80) and NS4B (84-147). With the

481

resonance assignment of NS4A (17-80), we tested its interaction with a peptide corresponding to

482

residues 84-147 of DENV-2 NS4B (strain NGC; synthesized by AAT Bioquest, Inc; purity of

483

90%). Fig. 7A shows the 1H-15N-HSQC spectra of NS4A (17-80) in the absence and presence of

484

NS4B (84-147). In the presence NS4B (84-147), line broadening of cross-peaks in the 1H-15N-

485

HSQC of NS4A (17-80) was observed, suggesting that the two peptides interact with each other

486

in DPC micelles. Affected residues of NS4A (17-80) in the presence of NS4B peptide were

487

mapped onto the structural model (Fig. 7B). In the HSQC spectrum, cross peaks of residues

488

(purple sphere), including Q19, A21, V30, A34, L48, L52, E53, T54, L56, L57, L61, A62, T63,

489

T65, G66, G67, F69, F71, L72, and G75, exhibited line-broadening when NS4B peptide was 22

490

added, suggesting that the interacting NS4A and NS4B peptides were undergoing intermediate

491

exchange. Such intermediate exchange suggests that the binding affinity between NS4A and

492

NS4B peptides might be in the range of micro-molar to nano-molar, which is consistent with the

493

ELISA binding Kd (Fig. 2). Cross peaks of NS4A residues (cyan sphere), including K20, R22,

494

A40, A44, and L60, exhibited chemical shift perturbation when NS4B peptide was added,

495

suggesting that these residues may participate in the NS4A/NS4B interaction or that they are

496

affected by the NS4A/NS4B interaction. Interestingly, most affected residues were located in the

497

transmembrane domain, suggesting that the transmembrane helix α3 of NS4A is critical for

498

interaction with NS4B. Only few residues from the two amphipathic helices α1 and α2 were

499

affected by the addition of NS4B peptide, again suggesting that the transmembrane helix α3 of

500

NS4A is the main binding site with NS4B (84-147) peptide.

501

Functional analysis of NS4A mutations. To characterize the biological relevance of the

502

NMR-identified NS4A residues that may participate in the NS4A/NS4B interaction, we analyzed

503

the conservation of these residues within NS4A (40-76) among the four serotypes of DENV. The

504

NS4A (40-76) region was selected for such analysis because this region was defined to be

505

essential for NS4B interaction (Fig. 3). As shown in Fig. 7C, eleven residues within NS4A (40-

506

76) are identical among DENV-1 to -4, including two alanine residues (A40 and A44) and nine

507

non-alanine residues (L48, E53, T54, L57, L60, T65, G67, F71, and G75).

508

To analyze the function of these amino acids, we mutated each of the eleven residues

509

(Ala to Gly; non-Ala residue to Ala) in a DENV-2 genome-length RNA. The mutational effect

510

on viral replication was examined. Equal amounts of mutant and WT RNAs were electroporated

511

into BHK-21 cells. The transfected cells were examined for viral E protein expression by IFA

512

(Fig. 8A), the plaque morphology of resulting virus (Fig. 8B), and virus production by plaque 23

513

assay (Fig. 8C). The eleven mutants showed distinct levels of viral replication and could be

514

divided into four groups. (i) Compared with the WT, mutants F71A and G75A exhibited similar

515

IFA-positive cells, plaque morphology, and viral yields. Sequencing the genomic RNAs of

516

resulting viruses confirmed that the engineered mutations were retained without any extra

517

mutations throughout their genomes. (ii) Mutants E53A, L57A, and G67A reduced IFA-positive

518

cells and decreased viral yields (especially on day 1 p.t.). The plaques formed by mutants L57A

519

and G67A were smaller than the WT virus by a standard plaque assay. Interestingly, mutant

520

E53A did not form plaques using the standard cytopathic effect (CPE)-based plaque assay, but

521

could be detected using an immune-staining assay (Fig. 8B, lower panel). Sequencing analysis

522

showed that mutant E53A, L57A, and G67A viruses kept the engineered mutations without extra

523

mutations throughout the viral genomes. (iii) Mutants A40G and A44G generated few IFA-

524

positive cells on day 3-4 p.t. and 0.05).