The C-terminal 50 Amino Acid Residues of Dengue NS3 Protein Are Important for NS3-NS5 Interaction and Viral Replication^*

Cell Lines and Virus

Baby hamster kidney cells (BHK-21) were cultured in RPMI 1640 medium (Invitrogen) supplemented with 10% fetal bovine serum (FBS), 100 units/ml penicillin, and 100 μg/ml streptomycin (1% P/S) in 5% CO₂ at 37 °C.

Human hepatoma (Huh-7) cells were cultured in DMEM (Invitrogen) supplemented with 10% FBS and 1% P/S at 37 °C, with 5% CO₂. Aedes albopictus mosquito (C3/36) cells were cultured in RPMI 1640 medium supplemented with 25 mm HEPES, 10% FBS, and 1% P/S at 28 °C, in the absence of CO₂.

DENV2 of cosmopolitan genotype (GenBank^TM number EU081177.1) that was used in this study was grown in C3/36 cells and titered in BHK-21 cells before storage at −80 °C. This virus was isolated during a local dengue outbreak that occurred in 2005 as part of Early Dengue Infection and Outcome (EDEN) Study in Singapore (38).

Plasmid Construction

Plasmids for the expression of DENV3 NS5 RdRP (residues 273–900), NS3 full-length protein (NS2B₁₈NS3; residues 49–66 of NS2B and 1–618 of NS3 connected by a flexible linker G₄SG₄), and DENV4 NS3 helicase domain (NS3(172–618)) have been described previously (Fig. 1A and Table 1) (11, 25, 39).

DENV3 NS3(172–618), NS3(482–618), and NS3(566–618) fragments were amplified from DENV3 NS2B₁₈NS3 construct (as mentioned above). NS3(172–618) fragment was amplified with forward primer 5′-CAAACAAATGCACATATGGATGGACCGACACC-3′ and the reverse primer 5′-GTGCTCGAGTGCGGATCCAAGCTTTTACTTTC-3. The underlined sequence corresponds to NdeI and BamHI sites, respectively. The PCR fragment was digested with NdeI and BamHI and cloned into pET14b (Novagen, Germany). The NS3(482–618) fragment was amplified with the forward primer 5′-CCAGCCTCTCGGATCCGATGAAGACCATG-3′ and the reverse primer 5′-CACAAGATCAAGCTCGAGTCACTTTCTGCC-3′. The NS3(566–618) fragment was amplified with the forward primer 5′-GAAAATGGTGCGGATCCGGGCAACGCAATAATC-3′ and the same reverse primer as above. The underlined sequence corresponds to BamHI and XhoI sites, respectively. Both PCR products were digested with BamHI and XhoI and cloned into pGEX-4T-1 (GE Healthcare) (Fig. 1A and Table 1).

DENV4 NS3(172–618)-NS5(320–341) fragment (connected by a flexible linker G₄SG₄, Fig. 3A and Table 1) was amplified from DENV4 NS2B₁₈NS3 construct (as mentioned above). The full-length PCR product was generated by first amplifying the helicase region with the forward primer 5′-GAAAACCTGTATTTTCAGGGCGCCGGCGGTGAGCCAGATTATGAAGTGG-3′ and the reverse primer 5′-CTCCATTCACCATTCCTCCTCCTCCTGATCCTCCTCCTCCCTTTCTTCCACTGGC-3′, followed by two separate PCRs that added the flexible linker sequence and NS5(320–341) sequence to the helicase via two reverse primers, 5′-GGAATCACATCCCAGGGTTTTGTCAGCAGTTTCACCACTCCATTCACCATTCCTC-3′ and 5′-GCGGCCGCGACTAGTGAGCTCGTCGACTTACTGGGTCACCATTGGAATCACATCCCAGGG-3′. The underlined sequence corresponds to NaeI and SalI sites, respectively. The PCR product was digested with NaeI and SalI and cloned into pProEx HTb vector.

Mutation of NS3 Asn-570 to alanine in DENV3 NS2B₁₈NS3 was done using QuikChange II XL site-directed mutagenesis kit (Stratagene), according to the manufacturer's protocol. The following primers were used: NS3 N570A forward (5′-GATGGGCAACGCAATGCTCAAATTTTAGAGGAG-3′) and reverse (5′-CTCCTCTAAAATTTGAGCATTGCGTTGCCCATC-3′). The underlined nucleotide corresponds to the mutation that was being made. Mutations were confirmed by automated DNA sequencing.

Protein Expression and Purification

For ELISA and ATPase activity assay, NS3 and NS5 constructs (Table 1) were transformed into Escherichia coli BL21 CodonPlus (DE3)-RIL cells (Stratagene) for protein expression and purified as previously published (11, 25, 39).

For SAXS, NS3(172–618) and NS3(172–618)-NS5(320–341) constructs were transformed into E. coli BL21 CodonPlus (DE3)-RIL cells (Stratagene) for protein expression and purified as described below. The cells were disrupted on ice by sonication three times for 1 min in buffer A (20 mm sodium phosphate, pH 7.4, 500 mm NaCl, 0.8 mm DTT, and 2 mm Pefabloc^SC (Biomol)). Precipitated material was separated by centrifugation at 12,500 × g for 25 min, and the supernatant was filtered (0.45 μm; Millipore). The filtered supernatant was incubated with 2 ml of nickel-nitrilotriacetic acid-agarose (Qiagen) for 1.5 h at 4 °C, and the His₆-tagged protein was eluted with an imidazole gradient from 20 to 200 mm in buffer A. Fractions containing the protein of interest were pooled and subjected to overnight cleavage with thrombin for NS3(172–618) or tobacco etch virus protease for NS3(172–618)-NS5(320–341) and dialyzed overnight in buffer B (20 mm sodium phosphate, pH 7.4, 200 mm NaCl) at 4 °C. Following incubation, the dialyzed sample was incubated with 1 ml of nickel-nitrilotriacetic acid-agarose for 1 h at 4 °C, and the flow-through containing cleaved NS3(172–618) or NS3(172–618)-NS5(320–341) was collected and applied to a gel filtration column (Superdex^TM 200 HR 10/300 column, GE Healthcare) in buffer C (20 mm Tris/HCl, pH 7.4, 200 mm NaCl, 5% glycerol, 1 mm DTT). Fractions containing NS3(172–618) or NS3(172–618)-NS5(320–341) were pooled and concentrated using Amicon Ultra-4 centrifugal unit (10-kDa molecular mass cutoff; Millipore).

Peptide Synthesis

Peptides were synthesized at the Nanyang Technological University peptide synthesis core facility (Singapore).

Competitive NS3-NS5 Interaction ELISA

The ELISA (Fig. 1B) was performed as published previously (33). The concentration of NS2B₁₈NS3 was fixed at 60 or 80 nm and mixed with increasing concentrations of truncated NS3 proteins (DENV3 NS3(172–618), GST-NS3(482–618), GST-NS3(566–618), or GST (negative control)) or NS3 peptides (NS3(566–585) and NS(566–585) (N570A), NS(571–585), or NS(86–100) (negative control); listed in Table 1). The concentration of NS3 protein at which 50% inhibition of NS2B₁₈NS3 protein binding occurs represents the apparent K_D value for the DENV3 NS5 RdRP-NS3 protein interaction. Data were fitted to the sigmoidal dose-response equation (variable slope) by nonlinear regression using GraphPad Prism 5 from triplicate measurements.

Viral Inhibition Assay

2 × 10⁵ Huh-7 cells were seeded into a 12-well plate and incubated overnight at 37 °C with 5% CO₂. Cells were infected with DENV2 at a multiplicity of infection of 1 for 1 h, after which the virus inocula were then removed and replaced with 5% FBS/DMEM maintenance media. At 6 h post-infection, the infected cells were treated with 7.5 μm NS3 peptides complexed with 22.5 μm penetratin in a molar ratio of 1:3 or 7.5 μm NS5-penetratin fusion peptides (Table 1). At 24 h post-infection, cells were washed once with PBS prior to lysis by TRIzol for cellular viral RNA quantification by real time RT-PCR analysis with primers that binds to the NS1 gene (forward 5′-CCGCTGACATGAGTTTTGAGTC-3′ and reverse 5′-CATGACAGGAGACATCAAAGGA-3′) (40).

ATPase Activity Assay

The assay was carried out as described (13), with slight modifications. Purified NS2B(320–368)NS3 WT or N570A protein of 2.5 nm was preincubated at 37 °C with poly(U) (10 μg/ml) in 40 μl of reaction buffer (50 mm Tris/HCl, pH 7.5, 2 mm MgCl₂, 1.5 mm dithiothreitol, 0.05% Tween 20, 0.25 μg/ml bovine serum albumin (Sigma)) for 5 min. The reaction was initiated by the addition of 10 μl of varying ATP concentrations (2-fold serial dilution, starting from 2000 μm) and carried out for 10 min at 37 °C. 10 μl of malachite green reagent (BioAssay Systems) was added to stop the reaction. Absorbance was read at 635 nm after 30 min at room temperature. The K_m of the protein was determined with GraphPad Prism 5, with Michaelis-Menten Equation 1,

SAXS

SAXS data of the NS3(172–618) and NS3(172–618)-NS5(320–341) were measured by the NanoStar^TM instrument (Bruker), equipped with a METALJET^TM x-ray source and Vantec 2000-detector system. The METALJET^TM source uses the liquid gallium source to deliver a high intensity x-ray beam at the wavelength of λ = 1.34 Å. The SAXS measurements were carried out with the source to sample distance of 145 cm, a two-pinhole collimation system, and the sample to detector distance of 67 cm (41). SAXS experiments of both proteins were carried out at 1.2, 2.2, and 4 mg/ml in a sample volume of 40 μl at 15 °C. For each sample, a total of nine measurements at 5-min intervals were recorded. The data were flood-field and spatially corrected, and processed using the built-in SAXS software. We tested the possible radiation damage by comparing all data sets, and no changes were detected. The scattering intensity of the buffer was subtracted, and the difference curves were scaled for the concentration. All the data processing steps were performed automatically using the program package PRIMUS (42). The forward scattering I(0) and the radius of gyration R_g were evaluated using the Guinier approximation (43). These parameters were also computed from the entire scattering patterns using the indirect transform package GNOM (44), which also provides the distance distribution function p(r). Ten low resolution models of NS3(172–618) or NS3(172–618)-NS5(320–341) were independently built by the program GASBOR (45). The spatial discrepancy (NSD), which is a measure of similarity between sets of three-dimensional points, was computed between all 10 reconstructions using the DAMAVER program (46). The reconstruction with the least NSD was selected for NS3(172–618) or the fusion protein NS3(172–618)-NS5(320–341). The ensemble optimization method (EOM) suite was used to select an ensemble of conformations that best fit the experimental data, and the dimensions of selected conformations were compared with the random pool to evaluate the flexibility and compactness of NS3(172–618)-NS5(320–341) (47, 48).

DENV2 Full-length cDNA Clone Construction and Site-directed Mutagenesis

To construct a full-length DENV2 cDNA clone (GenBank^TM accession EU081177.1, cosmopolitan genotype; Fig. 4A), low passage virus stock was subjected to viral RNA extraction by RNeasy kit (Qiagen), and three cDNA fragments (fragment boundaries indicated by nucleotide numbers; Fig. 4A) covering the complete genome were amplified from viral RNA by RT-PCR using SuperScript III one-step RT-PCR kits (Invitrogen). Fragment 1 contained the SphI restriction site, a T7 promoter sequence, and DENV2 cDNA nucleotides 1–4498, which also contained the KpnI restriction site. Fragment 2 spanned from the KpnI site (nucleotide position 4493) to the XbaI site (nucleotide position 6008). Fragment 3 spanned from XbaI site (nucleotide position 6003) to the 3′ end of the genome (nucleotide position 10,723), containing the SacI site at the end of the genome. The PCR product of each cDNA fragment was digested and cloned into a pre-digested and modified low copy number plasmid pWSK29 (49). The plasmid was modified by the replacement of the BssHII site that was located before the T7 promoter with the SphI site by site-directed mutagenesis of the plasmid with the following primer: forward 5′-GGCCAGTGAGCATGCGTAATACGAC-3′ and reverse 5′-GTCGTATTACGCATGCTCACTGGCC-3′. The underlined nucleotide corresponds to the mutation that was being made. The subclone that maintained each fragment was named accordingly as follows: pWSK29 D2 fragment 1, fragment 2, and fragment 3, respectively, and each subclone was validated by DNA sequencing by 1st BASE DNA Sequencing Services (Singapore) before proceeding for assembly. Subsequently, fragment 2 was inserted into the subclone pWSK29 D2 fragment 1 at the KpnI and XbaI site to generate subclone pWSK29 D2 fragment 1 + 2. Finally, fragment 3 was inserted to generate subclone pWSK29 D2 fragment 1 + 2 at XbaI and SacI sites to generate the full-length cDNA clone, pWSK29 D2 full length. The E. coli XL-1 Blue chemically competent cell (Stratagene) was used for construction and propagation of the cDNA clones. Standard cloning procedures were performed with the exception that the cDNA clones were propagated at 30 °C for at least 20 h. All restriction enzymes were purchased from New England Biolabs.

The genome-length cDNA clones with NS3:N570A and NS5:K330A mutations were constructed using the subclone pWSK29 D2 fragment 3. The mutations were generated using QuikChange II XL site-directed mutagenesis kit (Stratagene) and performed according to the manufacturer's protocol. The following primers were used for the generation of both mutants: NS3:N570A forward (5′-CTTTGATGGAGTCAAGAACGCCCAAATCTTGGAAGAAAATG-3′) and reverse (5′-CATTTTCTTCCAAGATTTGGGCGTTCTTGACTCCATCAAAG-3′); NS5:K330A forward (5′-GTGGTTAGGCTGCTAACAGCACCTTGGGATGTCATCCCC-3′) and reverse (5′-GGGGATGACATCCCAAGGTGCTGTTAGCAGCCTAACCAC-3′). The underlined nucleotides correspond to the mutation that was being made. Mutations were confirmed by automated DNA sequencing, and fragment 3 bearing the mutation was excised from the plasmid by XbaI and SacI and inserted into subclone pWSK29 D2 fragments 1 + 2 that were similarly cut with XbaI and SacI.

In Vitro Transcription, RNA Electroporation, Plaque Assay, Real Time RT-PCR (Reverse Transcription-PCR), Immunofluorescence Assay (IFA), and Western Blot

BHK-21 cells were trypsinized, washed twice with cold PBS, and resuspended in Opti-MEM (Invitrogen) at a cell density of 1 × 10⁷ cells/ml. 10 μg of in vitro transcribed RNA with T7 mMESSAGE mMACHINE kit (Ambion) of DENV2 WT and mutants were mixed with 800 μl of cell suspension in a pre-chilled 0.4-cm cuvette and electroporated at settings of 850 V and 25 microfarads, 2 pulses with an interval of 3 s. Electroporated cells were allowed to recover at room temperature for 10 min before resuspending in complete RPMI 1640 medium for cell recovery. Cells (3 × 10⁵) were then seeded into a 12-well plate and incubated at 37 °C in the presence of 5% CO₂. Media were changed to 2% FBS maintenance media after 6 h post-transfection. Samples were harvested every 24 h post-transfection until 120 h. Supernatants were collected and clarified for titering of the infectious virus particle by standard plaque assay and extracellular viral RNA quantification by real time RT-PCR analysis (40). Cells were then washed once with PBS prior to lysis by TRIzol reagent (Invitrogen) or 1× SDS-PAGE reducing loading dye for cellular viral RNA quantification and Western blot, respectively.

For cellular viral RNA quantification, total RNA was isolated using the TRIzol extraction method from the cell lysate. 500 ng of total RNA was used for cDNA synthesis using the Improm II reverse transcription system (Promega) with random primer in accordance to manufacturer's instructions. 40 ng of cDNA was used for real time RT-PCR analysis of viral RNA in Bio-Rad iQ-5 real time thermal cycler with the use of SYBR Green supermix (Bio-Rad) as described previously with primers that bind to the NS1 gene (forward 5′-CCGCTGACATGAGTTTTGAGTC-3′ and reverse 5′-CATGACAGGAGACATCAAAGGA-3′) (40). Absolute numbers of intracellular viral RNA genome copy were quantitated using the DENV standard curve, normalized to actin levels, and reported as absolute number of viral RNA genome copy per μg of RNA used for real time RT-PCR.

For intracellular viral RNA genome copy of both positive and negative strands, 5′-tagged primers that bind to the E gene (Eden2_forward_RT, 5′-GGCCGTCATGGTGGCGAATAACAGGCTATGGCACTGTCACGAT-3′, and Eden2_reverse_RT,5′-GGCCGTCATGGTGGCGAATAACCATTTGCAGCAACACCATCTC-3′) were used for transcribing cDNAs of both polarities (50, 51). The forward primer was used to transcribe cDNA from the negative-strand RNA, whereas the reverse primer was used to transcribe cDNA from positive-strand RNA by using Improm II reverse transcription system (Promega). 40 ng of cDNA was used for real time RT-PCR analysis of viral RNA in Bio-Rad iQ-5 real time thermal cycler with the use of SYBR Green supermix (Bio-Rad) with the appropriate primer pair for either negative-strand (forward 5′-GGCCGTCATGGTGGCGAATAA-3′ and reverse 5′-CCATTTGCAGCAACACCATCTC-3′) or positive-strand (forward 5′-GGCCGTCATGGTGGCGAATAA-3′ and reverse 5′-CAGGCTATGGCACTGTCACGAT-3′) detection. The underlined sequence corresponds to the 5′-tagged sequence (51). Absolute positive- and negative-strand copy numbers were quantitated and reported as described above.

For extracellular viral RNA quantification, viral RNA from the supernatant was extracted by Qiagen viral RNA extraction kit according to the manufacturer's instructions, and for its quantification, a SYBR Green one-step real time RT-PCR (Bio-Rad) was conducted using the same PCR conditions as cellular RNA quantification, with primers that bind to the NS1 gene (as mentioned above). Absolute viral RNA genome copy was calculated based on the DENV standard curve generated and reported as absolute number of viral RNA genome copy per ml of supernatant. The detection limit of each real time RT-PCR assay is indicated as a gray line in the graph.

IFA against E protein by anti-E mouse antibody 4G2, NS3 protein by anti-NS3 human antibody 3F8, and dsRNA by anti-dsRNA mouse antibody J2 (Scicons), and Western blot against NS3 protein by anti-NS3 3F8 were performed as described previously (33). IFA images were captured on an inverted fluorescence microscope (Olympus IX71, Center Valley) at ×20 magnification, and image analysis was performed with ImageJ software (52).

Statistical Analysis

Student's t test was used to determine statistical significance. p values of ≤0.05 were considered as significant.

NS3(566–618) Interacts with NS5 RdRP

Even though NS3(303–618) of the helicase domain (residues 172–618, see Fig. 1A) was previously shown by Y2H study to interact with NS5(320–368) (bNLS) (36) of the RdRP domain (residues 273–900) (35, 36), the amino acid residues of NS3 helicase that are responsible for binding to NS5 RdRP have not been pinpointed. However, it was hypothesized that helicase subdomain III, NS3(482–618), contains the interaction site (13, 22). To test this, we first established that the binding of NS2B₁₈NS3 to coated NS5 RdRP increased in a dose-dependent manner as described previously (33) and carried out the NS3-NS5 interaction assay in a competitive ELISA format, with an increasing amount of the following competing proteins, namely NS3(172–618)_, GST-NS3(482–618) (subdomain III, or GST-NS3(566–618) (Fig. 1, A and B). From these data, all three NS3 truncated proteins were found to be able to compete with NS2B₁₈NS3 protein binding to coated NS5 RdRP (Fig. 1C). The apparent K_d values of the NS3-NS5 interaction for NS3(172–618), GST-NS3(482–618), and -(566–618) were found to be comparable (5.33 ± 0.35, 10.18 ± 0.91, and 10.25 ± 1.14 μm (mean ± S.D., n = 3) respectively) and also similar to the K_d values for NS3(172–618) as in previous reports (33, 37). Next, we also determined the IC₅₀ value for NS3(172–618), which was 6.07 ± 2.60 μm. The IC₅₀ values for GST-NS3(482–618) and GST-NS3(566–618) were not determined as we were unable to reach 100% inhibition due to limited availability of truncated protein. Protein expression constructs spanning other NS3 subdomain regions (NS3(172–482), -(307–619), and (172–566)) were also generated in this study, but due to protein stickiness, protein instability, or low protein yield, these proteins could not be purified to sufficient quantity for the competition assay.

NS3 Asn-570 Is Critical for the Interaction

To map the NS3 sequence that interacts with NS5 more precisely, we proceeded to screen an array of overlapping 15-mer peptides (Mimotopes) (53, 54) that spanned subdomain III in the same competitive ELISA format (Fig. 2A), and we identified two peptides (NS3(566–580) and -(571–585)) that moderately (p value = 0.06 and 0.009, respectively) blocked NS3-NS5 interaction, thus narrowing down the interaction region to residues 566–585 of NS3. We next tested the synthetic peptide NS3(566–585) in ELISA and showed that it could also disrupt NS3-NS5 interaction in a dose-dependent manner with an IC₅₀ value of 128.8 ± 2.57 μm (Fig. 2B). At the same time, another peptide, NS3(86–100) with a similar charge as NS3(566–585), did not compete. This indicates NS3-NS5 interaction involves sequence-specific residues on the NS3 protein. Additionally, the same peptide also inhibited NS3-NS5 interaction in the AlphaScreen assay format where the interacting partners were synthesized in vitro using the wheat germ expression system (data not shown) (55).

Next, we also used the peptide in the viral inhibition assay to determine whether blocking the interaction site could reduce viral replication (Fig. 2C). Uptake of NS3(566–585) peptide into infected cells was facilitated by a well characterized cell-penetrating peptide, penetratin (56), which forms a nonconvalent complex with the peptide (57). As shown in Fig. 2C, NS3(566–585) peptide could reduce viral replication by ∼33% (p value = 0.0195, Fig. 2C, panel i). As a positive control, we also tested the NS5(320–341) peptide that was covalently linked to penetratin, which can also facilitate the uptake of the peptide into infected cells (58). NS5(320–341) peptide could also reduce viral replication by ∼33% (p value = 0.0043, Fig. 2C, panel ii). This suggests that blocking interaction between NS3 and NS5 could be a potential therapeutic target.

Sequence alignment of NS3(566–585) of DENV1–4 and other flaviviruses (Fig. 2D) suggested that Asn-570 (highlighted in gray) is highly conserved within this region and may be critical for the NS3-NS5 interaction. To test this, we synthesized NS3(566–585)(N570A) peptide and carried out the same competitive ELISA. We found that the replacement of asparagine by alanine at position 570 of NS3 resulted in a null-peptide with respect to its ability to block the NS3-NS5 interaction (Fig. 2B). Next, we expressed and purified the NS3:N570A full-length protein to measure its ATPase activity (59), and we found that it was comparable with the WT NS3 protein (Fig. 2E). The affinity of ATP for NS3 WT and NS3 N570A was similar (K_m = 185.8 ± 10.86 and 176.1 ± 8.73 μm, respectively). Both proteins also had similar turnover numbers (k_cat = 3.15 and 3.11 s⁻¹, respectively). From these results, we surmised that Asn-570 of NS3 helicase subdomain III appears to be critical for the NS3-NS5 interaction without affecting the in vitro ATPase activity.

NS5(320–341) of RdRP bNLS Binds to NS3 Helicase in Solution

To gain some insights on the NS3-NS5 interaction at the structural level, we constructed a synthetic fusion of NS3(172–618) with NS5(320–341) (within bNLS) connected by a flexible linker (G₄SG₄, NS3(172–618)-NS5(320–341); Fig. 3A) for SAXS study. NS5(320–341) was selected based on surface plasma resonance data for peptide binding and NS5 peptide-phage ELISA that supported the interaction of NS5(320–341) to NS3(172–618) (data not shown). Through modifications of the purification strategy, NS3(172–618) and NS3(172–618)-NS5(320–341) were purified to high purity and used in the SAXS experiments, which provide three-dimensional low resolution structures in solution (60) as described for single proteins (41, 61, 62), as well as multidomain complexes like ATPase (63, 64) and the NS3 protease-helicase complex of DENV (11). SAXS data of both proteins at three different concentrations were collected to yield the final composite scattering curves shown in Fig. 3, B and C, which indicate that both proteins are monodispersed in solution. Inspection of the Guinier plots at low angles revealed good data quality and no protein aggregation (Fig. 3, B and C, insets). NS3(172–618) has a radius of gyration (R_g) of 25.16 ± 0.6 Å and a maximum dimension (D_max) of 72.95 Å (Fig. 3D), whereas the NS3(172–618)-NS5(320–341) has an R_g of 25.42 ± 0.6 Å and a D_max of 75.64 Å (Fig. 3D). Comparison of the forward scattering of both proteins with the values obtained from a reference solution of lysozyme (14.4 kDa) yielded a molecular mass of 44.4 kDa for NS3(172–618) and 47.7 kDa for NS3(172–618)-NS5(320–341), indicating that both proteins are monomeric at the concentrations used.

The low resolution solution structures of NS3(172–618) and NS3(172–618)-NS5(320–341) were restored ab initio using the program GASBOR (45). The normalized spatial discrepancy (NSD) of 10 independent reconstructions of NS3(172–618) is 0.95 ± 0.02 Å, and the NSD of 10 reconstructions of NS3(172–618)-NS5(320–341) is 0.99 ± 0.03 Å. The reconstructions with the least NSD from both proteins were selected, and the obtained solution shapes for both proteins yielded a good fit to the experimental data in the entire scattering range. The corresponding fits for NS3(172–618) and NS3(172–618)-NS5(320–341) have discrepancies of χ = 1.15 and χ = 1.04, respectively. NS3(172–618) and NS3(172–618)-NS5(320–341) revealed globular shapes, with the dimensions of about 59.9 × 54.4 × 34 and 65.6 × 57.7 × 36.8 Å, respectively. When superimposed, the crystallographic structure of NS3(176–618) (Protein Data Bank code 2JLS) accommodated very well in the solution form of the protein (Fig. 3E), indicating the high quality of the solution data. When the solution form of NS3(172–618) (green) and NS3(172–618)-NS5(320–341) (cyan) were superimposed (Fig. 3F), two protrusions were observed and are denoted by arrows in Fig. 3F. Protrusion 1 that is at the bottom of the NS3(172–618)-NS5(320–341) solution shape leads to a more elongated conformation in NS3(172–618)-NS5(320–341) than NS3(172–618). This is also reflected by the increased D_max in NS3(172–618)-NS5(320–341) (75.64 Å) when compared with the D_max of NS3(172–618) (72.95 Å) (Fig. 3D). The protrusion is in close proximity to residue Asn-570 (shown as blue sticks in the inset of Fig. 3F) and to the NS3 peptide region, NS3(566–585) that used in ELISA (shown as black schematic in the inset of Fig. 3F). The arrangement of NS3(566–585) enables an interaction with NS5(320–341) to occur.

Comparison of the NS3(172–618) and NS3(172–618)-NS5(320–341) low resolution structures also revealed a second protrusion of the NS3(172–618)-NS5(320–341) that may reflect a conformational alteration due to the NS3(172–618) and NS5(320–341) interaction. Taken together, these results appear to be consistent with the ELISA data and suggest a physical interaction between NS3(172–618) and NS5(320–341) in solution.

To eliminate the possibility that the peptide may be flexible in solution, the x-ray scattering data set of NS3(172–618)-NS5(320–341) had been further analyzed using the Ensemble Optimization Method (EOM) (47, 48) to assess the compactness and flexibility of NS3(172–618)-NS5(320–341) in solution. Based on the width and position of the selected ensemble peak relative to random pool in R_g distribution, the flexibility and compactness of the protein in solution can be determined. In the case of NS3(172–618)-NS5(320–341), the R_g distribution of the ensemble contains a narrow peak, indicating that NS3(172–618)-NS5(320–341) is rigid in solution. This peak is centered at 24.6 Å, which is slightly smaller than the center R_g of a random pool, 24.8 Å, suggesting that NS3(172–618)-NS5(320–341) is compact in solution. The EOM data demonstrate that peptide NS5(320–341) is bound to NS3(172–618).

NS3:N570A Mutant Has Reduced Infectious Virus Production and Viral Protein Synthesis

To study the impact of NS3:N570A mutation on NS3-NS5 interaction during the virus life cycle, we first generated a DENV2 WT cDNA clone by standard molecular cloning techniques. DENV2 of strain D2/SG/05K3295DK1/2005 of the cosmopolitan genotype (GenBank^TM accession number EU081177.1) was isolated during a local dengue outbreak that occurred in 2005 as part of Early Dengue Infection and Outcome (EDEN) Study in Singapore, and it was picked as the template for construction of the DENV2 full-length cDNA clone because of the well documented history of the patients (38). The overall schematic representation of cloning strategy is shown in Fig. 4A. The cDNA clone was subdivided into three fragments based on unique restrictions that are present within the genome to facilitate the assembly of the full-length clone. To prevent unwanted rearrangement of the clone, both full-length cDNA and cDNA fragments were maintained in low copy plasmid pWSK29 and E. coli XL-1 Blue that were transformed with these plasmids grown at 30 °C (49). The assembled full-length cDNA clone contained a T7 promoter at the 5′ end for in vitro transcription and a SacI site at the 3′ end for linearization of cDNA. The use of SacI site generated the in vitro RNA transcript, which contains an additional nonviral nucleotide at the 3′ end, instead of two additional nonviral nucleotides for DENV cDNA that used the XbaI site (65). Prior to linearization for in vitro transcription, the full-length cDNA was checked for possible rearrangement by EcoRI digestion, and the predicted fragments of 9609, 4015, 1284, and 1122 bp were observed on 0.6% agarose gel (data not shown), indicating no sign of recombination (65).

IFA of BHK-21 cells that were transfected with in vitro transcribed genome-length RNA showed increasing numbers of cells expressing E protein from days 1 to 3 post-transfection, with ∼50–60% of cells being E-positive on day 3 (Fig. 4B). Plaque assay for supernatant showed an increase in infectious viral particles from days 1 to 3, peaking at 1 × 10⁵ pfu/ml on day 3 (Fig. 4C). The size of the plaque on BHK-21 cells, which was produced by the supernatant of BHK-21-transfected cells, was comparable with those produced by the supernatant of C6/36-transfected cells (Fig. 4C, left and right insets, respectively). These results show that the RNA transcribed from the cDNA clone is highly infectious and that infectious virus can be produced in both BHK-21 mammalian cells and C6/36 mosquito cells.

After determining the growth kinetics of cDNA clone-derived DENV2 WT virus, the DENV2 NS3:N570A cDNA mutant clone was generated by site-directed mutagenesis, and its phenotype in infectious virus production and viral protein synthesis was examined over the course of 5 days' post-transfection. For comparison, we also generated the NS5:K330A mutant (37), a known NS3-NS5 interaction-defective mutant that failed to replicate by reverse genetics studies, as a control. Similar to the NS5:K330A mutant, no infectious virus was recovered from the NS3:N570A mutant even when neat supernatant was used for titering (Fig. 5B). In agreement with the lack of infectious virus, extracellular viral RNA levels for both mutants were at the limit of detection of DENV RNA by RT-PCR and did not change over 5 days, suggesting that the mutants are inviable (Fig. 5A). The RNA detection for WT increased over 72 h when it reached maximal virus RNA detection that stabilized until 120 h. Next, to determine whether polyprotein synthesis from the transfected RNA is occurring and that the absence of infectious virus particles may be due to a lack of packaging and/or release of infectious virion, we checked the level of NS3 protein by both Western blot and IFA. Surprisingly, for the cells transfected with NS3:N570A mutant transcript, we could detect NS3 protein using humanized monoclonal antibody 3F8 (33) in the 24- and 48-h samples (Fig. 5C, red arrows) but not in the 72-h sample. The highest percentage of NS3-positive cells (5–10%) was observed on day 2 post-transfection, which declined to ≤5% of NS3-positive cells on day 3 post-transfection (Fig. 5D), and finally to an undetectable level at later time points on days 4 and 5 (data not shown). On the other hand, for the NS5:K330A mutant, the NS3 protein was almost undetectable by Western blot and IFA (≤1% of NS3-positive cells) at all time points post-transfection (Fig. 5, C and D). Taken together, the results indicate that reduced NS3-NS5 interaction in NS3:N570A mutant impairs infectious virus production and viral protein synthesis.

NS3:N570A Mutant Showed Accumulation of Negative-strand RNA

The reduction in viral protein synthesis for the NS3:N570A mutant and the lack of detectable infectious plaques suggest that attenuated RNA replication could occur at an early time point for this mutant. Therefore, we examined the RNA replication kinetics by real time RT-PCR and IFA for detection of dsRNA. The results showed that intracellular RNA can be detected for NS3:N570A and WT but, intriguingly, not for NS5:K330A-transfected cells (Fig. 6A and B). The RNA copy for WT increased from around 10⁶ copies/μg at 6 h post-transfection to 10⁹ copies/μg of RNA at 120 h, as detected by real-time RT-PCR. Although less than WT, a 10-fold increase in intracellular viral RNA copy was observed from day 1 to 2 (p value = 0.008) for NS3:N570A mutant and thereafter, the level declined till day 5. The RNA copy for the NS5:K330A mutant declined from 6 to 48 h post-transfection and remained relatively stable from 48 to 120 h, suggesting that there was no viral RNA replication, and only the input RNA that was transfected by electroporation was being detected. Interestingly, the detection of the peak intracellular RNA level for NS3:N570A mutant on day 2 correlated with the highest level of NS3 protein detected by Western blotting (Fig. 5C).

To explore this further, we quantified the level of intracellular positive- and negative-strand viral RNA with strand-specific tagged primers in the real time RT-PCR experiment (Fig. 6C). Essentially, the tag sequence that was described by Plaskon et al. (51) was fused to the sequence that binds to E gene (50) to create a primer that can bind to either the positive or negative strand during the cDNA synthesis step. During real time RT- PCR, the tag and E-specific primers were used to distinguish cDNAs that were transcribed from either the positive- or negative-strand template for accurate quantification of both strands. As expected, for WT viral RNA-transfected cells, the level of both positive- and negative-strand RNAs increased over time until day 3 and declined at slightly different rates after that until day 5. The quantification was consistent with previous data that an excess of positive- over negative-strand RNA can be detected in WT virus-infected cells (66). The RNA quantification for the NS3:N570A mutant revealed a different trend to WT and NS5:K330A mutants. Overall, the level of positive- and negative-strand RNA synthesis for NS3:N570A was lower than WT. However, the level of negative-strand RNA synthesis (rate = 2.06 × 10⁴ ± 9.77 × 10² negative-strand/h) for NS3:N570A appeared to increase faster than positive-strand RNA (rate = −2.17 × 10⁵ ± 5.82 × 10⁴ positive-strand/h) during a 6–24-h period, and at a somewhat similar rate for both strands (rate = 3.55 × 10⁵ ± 3.74 × 10³ negative-strand/h and 7.48 × 10⁵ ± 1.28 × 10⁵ negative-strand/h) during the 24–48-h period, suggesting that the negative-strand RNA may be accumulating as a double-stranded replicative form by base-pairing with the transfected positive-sense RNA. Interestingly, beyond 48 h, the rates at which both positive- and negative-strand RNA accumulated for NS3:N570A mutant over time were almost identical. For NS5:K330A mutant, the level of detected positive- and negative-strand RNA corresponds to the amount of transfected positive- and negative-strand RNA that degraded over time. (Note: T7 transcribed positive-sense RNA used for transfection contained ∼0.1–0.3% negative-sense RNA that could be detected by the primers that bind to E gene and is the basis for the gray cutoff line for the residual negative-strand from transfected RNA in Fig. 6C.) The NS3:N570A mutant probably results in either reduced or completely abolished NS3-NS5 interaction and does not support further RNA synthesis from the dsRNA template that is formed (67). This is consistent with the detection of highest percentage of dsRNA-positive cells on day 2 (5–10%) by intracellular RNA staining that does not accumulate as seen in WT at the later time points (Fig. 6B). Interestingly, our detailed analysis suggests that NS5:K330A mutant cannot synthesize negative-strand RNA, although the mutation has been shown to have no effect on the in vitro enzymatic activities (37). These results suggest that the impairment of infectious virus production and viral protein synthesis in the NS3:N570A mutant may be due to the inability of the mutant NS3 to engage with NS5 and use the dsRNA template to produce more positive-strand genomic RNA that can be translated and processed.