Dengue reporter viruses reveal viral dynamics in interferon receptor-deficient mice and sensitivity to interferon effectors in vitro

Abstract

Dengue virus (DENV) is a global disease threat for which there are no approved antivirals or vaccines. Establishing state-of-the-art screening systems that rely on fluorescent or luminescent reporters may accelerate the development of anti-DENV therapeutics. However, relatively few reporter DENV platforms exist. Here, we show that DENV can be genetically engineered to express a green fluorescent protein or firefly luciferase. Reporter viruses are infectious in vitro and in vivo and are sensitive to antiviral compounds, neutralizing antibodies, and interferons. Bioluminescence imaging was used to follow the dynamics of DENV infection in mice and revealed that the virus localized predominantly to lymphoid and gut-associated tissues. The high-throughput potential of reporter DENV was demonstrated by screening a library of more than 350 IFN-stimulated genes for antiviral activity. Several antiviral effectors were identified, and they targeted DENV at two distinct life cycle steps. These viruses provide a powerful platform for applications ranging from validation of vaccine candidates to antiviral discovery.

Dengue virus (DENV) is an arthropod-borne pathogen of the Flaviviridae family that causes significant morbidity and mortality worldwide. Each year, over 50 million people are affected by dengue fever, and ∼500,000 are hospitalized with the more severe dengue hemorrhagic fever (1). The virus is endemic to tropical environments in Southeast Asia, the Pacific, and the Americas, and has recently reemerged as far north as southern Florida (2). Currently, no vaccines or antivirals have been approved for prevention or treatment of DENV infection.

Four genetically and antigenically distinct DENV serotypes circulate, and each can be isolated from infected human sera and propagated in cell culture. The pathogenesis and immune response to patient-derived virus can be studied in vitro and in vivo by quantifying viral genomes or antigens. These detection methods are also used to study the molecular virology of DENV with reagents such as virus-like particles (VLPs) and subgenomic replicons. In some cases, VLPs and subgenomic replicons have been engineered to take advantage of reporter proteins, such as luciferase, which can be used in high-throughput screening platforms for discovery of inhibitors of viral entry or replication (3). A caveat to these tools is the inability to fully recapitulate the entire viral life cycle. This can be overcome by launching virus production from full-length infectious molecular clones, which have been generated for DENV serotypes 1, 2, and 4 (4–6).

Full-length flavivirus infectious clones are often difficult to work with, largely due to instability in various bacterial cell lines and the inability to rescue sufficient quantities of DNA for downstream applications (7). Additionally, mutagenesis of viral sequences may result in disruption of the various long-range interactions required for establishment of a productive replication complex (8–10). Thus, strategies to generate infectious viruses expressing heterologous sequences have to contend with both suboptimal growth conditions in Escherichia coli and the complex secondary RNA structural requirements of the viral genome.

Recently, several groups reported the development of infectious serotype 2 DENV (strain TSV01) expressing Renilla luciferase (11–13). Though this luciferase variant has utility in a number of applications, such as drug discovery, it is considered inferior to firefly luciferase with respect to bioluminescence imaging (14). We sought to generate infectious DENV based on serotype 2 strain 16681 that express either GFP or firefly luciferase for cell-based screening assays and bioluminescence imaging of DENV infection in mice. Fully infectious viruses expressing these reporter proteins could be rescued and were infectious in vitro and in vivo. A luciferase-expressing virus was used to characterize the dynamics of DENV infection in living mice, revealing infection primarily in immune organs and gut-associated tissues. The GFP-expressing recombinant virus was used as a tool in a cell-based screen to probe a collection of 350+ type I IFN-induced genes for inhibitors of viral replication. Several anti-DENV effectors were identified, and they selectively targeted two distinct life cycle stages. These viruses provide a platform for future screens to identify antiviral molecules and probe the mechanisms of action of individual IFN effectors against DENV.

Results

Construction and Characterization of Reporter DENV.

To generate serotype 2-based DENV expressing reporter proteins, we used the IC30P-A full-length infectious clone of strain 16681 as a template (5). A series of genetic modifications were made to enable insertion of sequences for enhanced GFP and firefly luciferase (Fluc) (Fig. 1A and SI Materials and Methods). To overcome commonly encountered challenges when modifying DENV infectious clones, we used MDS42 reduced-genome E. coli to propagate full-length clones under optimized growth conditions (Materials and Methods) (15). Rescued plasmid DNA was used as a template for T7-driven transcription of viral RNAs, which were electroporated into World Health Organization (WHO) Vero cells to generate viral stocks. DENV-GFP stocks were used to infect Vero cells, which were stained by indirect immunofluorescence for expression of the viral envelope E protein using the 4G2 monoclonal antibody. Analysis of infected cells by fluorescence microscopy and flow cytometry revealed that more than 90% of cells expressing E protein were also positive for GFP (Fig. 1 B and C). We determined that maximum yields of DENV-GFP were obtained 8 d postelectroporation using a flow cytometry-based titering assay (Fig. S1 A and B). Compared with the parental, non-GFP virus, yields of DENV-GFP were ∼10× lower, as reflected by both infectivity and titering assays (Fig. 1D). Serially passaging viral supernatants resulted in diminished GFP fluorescence, demonstrating the GFP reporter-containing genome is unstable beyond several life cycles (Fig. S1C), which is consistent with previous reports on heterologous gene insertion into DENV (11), Sindbis virus (16), West Nile virus (17), chikungunya virus (18), and influenza virus (19). Thus, DENV-GFP will be most useful for applications that monitor single rounds of infection.

Fig. 1.

Construction and characterization of reporter DENV. (A) Schematic of the DENV genome expressing heterologous reporter proteins GFP and Fluc. The first 25 amino acids of DENV capsid were duplicated and placed upstream of the reporter gene fused to sequences encoding a 2A cleavage site and ubiquitin. The native cyclization sequence (CS) in full-length capsid was conservatively mutated to preserved amino acid identity but limit long-range interactions with the 3′ CS. Asterisk (*) denotes site of L52F mutation in NS4B. (B) Immunofluorescence microscopy images of WHO Vero cells infected with DENV-GFP and stained with 4G2 anti-E antibody. (Left) GFP channel and (Center) immunostaining for E-protein; both panels are grayscale and merged in color (Right), with E protein shown as magenta. (C) FACS plots showing the coexpression of GFP and E protein in cells infected with DENV-GFP. (D) Comparison of infectivity (left y axis) and titers (right y axis) of parental DENV or DENV-GFP (left y axis) in Huh7 cells. Data are presented as mean ± SD, n = 3. (E) Sensitivity of DENV-Fluc to chemical inhibition by MPA and NITD008 in Huh7 cells. (F) Sensitivity of DENV-Fluc to inhibition by type I and III IFNs in Huh7 cells. For E and F, data are presented as mean ± SD of one of two independent experiments, n = 6.

A reporter virus expressing firefly luciferase, DENV-Fluc, was used to demonstrate viral sensitivity to well-characterized antiviral compounds mycophenolic acid (MPA), NITD008, and type I and type III interferons. MPA and NITD008 inhibited DENV-Fluc with an IC₅₀ of 0.09 μg/mL and 1.5 μM, respectively (Fig. 1E), consistent with previously published studies on nonreporter DENV (20, 21). DENV-Fluc replication was potently inhibited by human type I IFN (IFN-α2), and modestly suppressed by human type III IFNs (IL28B and IL29; Fig. 1F). These data demonstrate that serotype 2 DENV is amenable to insertion of a variety of reporter genes, and that heterologous reporters express at dynamic ranges suitable for testing both chemical and biological antiviral molecules.

In Vivo Dynamics and Tissue Localization of DENV-Fluc.

We next evaluated the in vivo utility of DENV-Fluc for monitoring viral dynamics and testing the efficacy of neutralizing antibodies and antiviral compounds. Using the previously characterized AG129 mouse model of DENV infection (22, 23), which lacks receptors for IFN-α/IFN-β and IFN-γ, we infected adult mice i.v. with DENV-Fluc and measured luciferase activity 3 d postinfection using bioluminescence imaging. Consistent with previous reports demonstrating that adaptive mutations are needed for DENV to establish infection even in highly immunocompromised AG129 mice (24), luminescence signal in DENV-Fluc mice did not rise over the background of an uninfected AG129 mouse (Fig. S2A). To generate a DENV-Fluc virus suitable for mouse infection, we took advantage of the recent observation that a single point mutation (L52F) in the viral nonstrucutural 4B protein (NS4B) was sufficient to confer virulence to the nonvirulent TSV01 strain in mice (25). The NS4B L52F mutation was engineered into DENV-Fluc to generate DENV-Fluc(NS4B:L52F), and both viruses were characterized for growth and infectivity properties (Materials and Methods). Maximum titers of both viruses were obtained 10–11 d after electroporation of viral RNA into Vero cells (Fig. S3 A and B). When comparing DENV-Fluc(NS4B:L52F) to DENV-Fluc, we consistently observed a 10- to 100-fold increase in titers, which correlated to similar increases in luciferase activity. These data indicate that, in cell culture lines, the NS4B L52F mutation confers a dramatic enhancement in virus infectivity and production of strain 16681-based DENV-Fluc, consistent with previous studies on strain TSV01 (25).

To determine if the NS4B point mutation conferred infectivity in vivo, we injected AG129 mice i.p. with 6 × 10⁵ TCID50 DENV-Fluc(NS4B:L52F) and measured bioluminescence over 6 d. At 24 h, striking Fluc signals were observed in the forelimb region and on the left side of the main body cavity, consistent with limb regional lymph nodes and spleen, respectively (Fig. 2 A and D). At 48 h and 72 h, Fluc signals were variable among individual mice and typically seen near splenic and gut regions. When we spatially separated and quantitated Fluc dynamics across three distinct body regions, we consistently observed initial infection in the upper body, followed by the appearance of infection in middle and lower body regions at later time points (Fig. S2 B–E). Beyond 72 h, luminescence in other anatomical sites was not observed, and signals eventually reached background levels across the whole body at day 6 (Fig. 2A). These kinetic data support previous observations that immune compartments are primary sites of DENV infection (26–28), and highlight the dynamic nature of DENV infection in living mice. Our data suggest a model in which virus administered i.p. initially infects lymph nodes near the forelimbs and then disseminates to the spleen and lower lymph nodes. However, we cannot rule out the possibility that infection in visceral tissues at later time points results from a kinetic delay in the onset of replication or is due to migration of infected cells from upper body lymph nodes.

Fig. 2.

Bioluminescent imaging of DENV dynamics, localization, and inhibition in vivo. (A and B) Dynamics and localization of AG129 mice injected with 6 × 10⁵ TCID₅₀ DENV-Fluc(NS4B:L52F). Infected animals were imaged at indicated time points by injecting 1.5 mg luciferin substrate and monitoring bioluminescence in the whole animal over a 6-d time course (A) or in isolated organs at 48 h postinfection (B). (C) Neutralization of DENV infection by preincubation of 6 × 10⁵ TCID₅₀ DENV-Fluc(NS4B:L52F) with 4G2 anti-DENV antibody. Signals were quantified with IVIS software. Results are presented as mean ± SD (n = 10 for mock, n = 8 for DENV, n = 3 for DENV + NAb). (D) Chemical inhibition of DENV infection with 25 mg/kg body weight NITD008. Drug was administered upon initial infection with 3 × 10⁶ TCID₅₀ DENV-Fluc(NS4B:L52F) and every 12 h throughout the course of the experiment. (E) Quantitation of whole-animal Fluc signals from NITD008 inhibition. Results are presented as mean ± SD (n = 3 for DENV, n = 4 for DENV + NITD008).

Given the sharply delineated signals emanating from both lateral and central lower gut regions, we speculated that in addition to mesenteric or other lymph nodes, a luciferase signal might also be emanating from gut associated tissues. A cohort of six mice was injected with DENV-Fluc(NS4B:L52F), and lymphoid and nonlymphoid tissues were harvested for ex vivo luciferase imaging at 48 h and 72 h postinfection (Fig. 2B). Strong luciferase activity was reproducibly detected in spleen, whereas signals emanating from heart, brain, lung, and thymus were similar to those of uninfected animals. In some mice, low luciferase signals were observed in liver and kidney, but this result was not reproducible across the cohort (Fig. 2B). Surprisingly, punctae of luminescence were observed in isolated intestines from all mice at both time points, and signals were more robust at 72 h (Fig. 2B). These data suggest that gut-associated lymphoid tissue may be a target of DENV infection or a destination for infected migrating cells in this mouse model.

To test whether bioluminescent imaging of DENV-infected mice could serve as a readout to rapidly identify neutralizing antibodies or antiviral drugs, we established proof-of-concept studies with the broadly neutralizing 4G2 monoclonal antibody and the antiviral compounds MPA and NITD008. Preincubation of DENV-Fluc(NS4B:L52F) with a neutralizing antibody resulted in a dose-dependent block to infection in cell culture (Fig. S4A). Similarly, when the virus was incubated with a 1:10 dilution of 4G2 antibody before infection in mice, we observed Fluc signals that were similar to uninfected animals at 24 h (Fig. 2C). The adenosine nucleoside inhibitor NITD008 administered at 25 mg/kg body weight twice daily resulted in a complete block to infection, with luciferase signals similar to background over a 3-d time course (Fig. 2D). In this experiment, luciferase signals were markedly higher than the first time course because we used ∼5× more virus to demonstrate drug efficacy (compare Fig. 2A with Fig. 2D). These results complement in vitro data showing potent inhibition of DENV by NITD008 in cell culture (Fig. 1E). Identical dosing of MPA did not significantly inhibit infection in mice at early time points, but provided modest protection at 72 h (Fig. S4C). These data contrast the strong suppression of DENV replication by MPA in cell culture (Fig. 1E), suggesting that compounds having anti-DENV activity in cell-based assays may not necessarily be effective in mice.

Identification and Characterization of IFN Effectors Targeting DENV-GFP.

In cell culture, DENV is sensitive to type I IFN (29) (Fig. 1F), and type I and II IFN receptor-deficient mice are highly susceptible to DENV infection (22) (Fig. 2). These observations suggest that the IFN system contributes significantly to controlling DENV replication in vivo. Antiviral IFNs are induced upon viral infection, signal through the Janus activated kinase/signal transducer and activator of transcription (JAK-STAT) pathway, and induce de novo transcription of hundreds of IFN-stimulated genes (ISGs). The products of these genes facilitate viral clearance and protect uninfected cells from incoming virus. However, few ISGs with anti-DENV activity have been reported. We recently developed a cell-based expression screen to identify human antiviral effectors induced by type I IFN (30). The screen relies on lentiviral delivery of a bicistronic mRNA consisting of an ISG and TagRFP. ISG-expressing cells are challenged with a GFP-expressing virus, and replication is quantified by FACS (Materials and Methods and Fig. S5).

We first determined whether DENV-GFP was suitable for this screen by testing the IFN effector IFITM3, which was previously shown to inhibit DENV infection (31). Huh7 cells were transduced with lentiviruses expressing IFITM3 or Fluc as a negative control. ISG-expressing cells were challenged with DENV-GFP, and replication was monitored 48 h postinfection by FACS. IFITM3 robustly inhibited DENV-GFP replication by at least 50% compared with the control Fluc (Fig. 3A). These data confirm previous findings and validate DENV-GFP as a screening tool for IFN effector activity.

Fig. 3.

Screening reporter DENV against a library of 350+ ISGs. (A) Inhibition of DENV-GFP by Huh7 cells transduced with lentivirus expressing IFITM3. Data are represented by FACS plots showing uninfected (gray line) and infected (black line) cells. (B) Screening 350+ ISGs for antiviral activity against DENV-GFP. Huh7 cells were transduced with lentiviral stocks for 3 d and then infected with DENV-GFP. Cells were analyzed for ISG-mediated inhibition of DENV-GFP replication by high-throughput FACS. Data are represented as a dot plot, with replication levels normalized to a Fluc control. Selected ISGs are indicated in blue. The red line indicates the population mean. (C) Confirmation assays of select antiviral ISGs in Huh7 (Left) cells or STAT1^−/− fibroblasts (Right). Replication levels were normalized to the Fluc control. Data are presented as box and whisker plots: gray boxes extend from the 25th to the 75th percentile, with a black line at the population median; whiskers extend to show the highest and lowest values, n = 6. (D) Time course of ISG-mediated inhibition of DENV-Fluc(NS4B:L52F). ISG-expressing cells were infected with virus and Fluc levels were monitored over time. Results are presented as mean ± SD of two independent experiments, each performed in quadruplicate, n = 8 at each data point. (E–H) Individual time points of data shown in D. Statistical significance was determined by one-way ANOVA (***P < 0.001; ns, not significant).

We next used the DENV-GFP virus to screen a library of more than 350 ISGs for their ability to inhibit virus replication in human hepatoma (Huh7) cells. The data are represented by the level of replication in ISG-expressing cells normalized to Fluc-expressing control cells (Fig. 3B) and by z score (Fig. S5C). We found 12 genes that were able to inhibit DENV replication with a z score of less than −2.0 (Table S1). The hit list includes genes previously shown to inhibit dengue virus (IFITM2 and IFITM3) (31), genes that participate in antiviral signaling (RIG-I, IRF1, IRF7, STAT2, and IL28RA), and genes whose antiviral functions are either previously unidentified or identified but uncharacterized (IFI6, HPSE, NAPA, ADM, and CD9).

To confirm the primary screening hits, we generated independent lentiviral stocks for nine effectors and tested their ability to inhibit DENV-GFP in Huh7 cells and immortalized human STAT1^−/− fibroblasts (32). We also included two genes (LY6E and MCOLN2) that were previously shown to enhance the replication of yellow fever virus, a related member of the Flaviviridae family (30). Most genes were confirmed to inhibit dengue virus replication to similar levels as the primary screen in Huh7 (Fig. 3C). In STAT1^−/− fibroblasts, we observed stronger inhibitory or enhancing effects on a per gene basis (Fig. 3C), consistent with our previous observation that these cells are more sensitive than Huh7 cells for detecting ISG-mediated effects (30). Only one gene, NAPA, did not inhibit DENV-GFP in follow-up assays in Huh7 cells, suggesting a false positive. However, NAPA expression in STAT1^−/− fibroblasts inhibited viral replication by ∼30%. Thus, the antiviral properties of this effector are modest and may depend on the cellular background.

To gain insight into ISG-mediated mechanisms of action with respect to the virus life cycle, we assessed the kinetics of DENV-Fluc(NS4B:L52F) replication in STAT1^−/− fibroblasts stably expressing IFI6, IFITM3, IRF1, STAT2, or an empty cassette. Stable cell lines were generated by lentiviral transduction followed by drug selection (SI Materials and Methods). Gene expression of each ISG was verified by RT–quantitative PCR (Fig. S6A). Using MPA, which inhibits virus replication but not incoming viral genome translation (20), we first determined viral kinetics that would distinguish these two life cycle steps. STAT1^−/− fibroblasts transduced with an empty lentiviral cassette were infected with DENV-Fluc(NS4B:L52F) and simultaneously treated with MPA or DMSO control. For the first 24 h of infection, cells treated with MPA had similar Fluc signals compared with DMSO-treated control cells (Fig. S4B). After 24 h, MPA robustly inhibited virus replication, as shown by complete suppression of Fluc signal. Thus, we chose 12 h as the time point to uncouple the initial round of translation from later stages of viral replication.

Cell lines stably expressing ISGs were infected with DENV-Fluc(NS4B:L52F), and Fluc levels were monitored from 12 to 72 h (Fig. 3D). At 12 h, IFITM3 and IRF1 reduced Fluc levels by more than 50% compared with infected control cells (Fig. 3E). This level of inhibition indicated a block at or before primary translation, which is consistent with previous mechanistic studies on these two genes (30, 31, 33). IFI6- and STAT2-expressing cells were indistinguishable from control cells at the 12-h time point. However, these effectors conferred inhibition at 24 h and at later time points, suggesting a block at the level of genome amplification (Fig. 3 F–H). Because IRF1 and STAT2 are both involved in antiviral signaling, we wanted to determine their contribution to an antiviral state. We previously showed that IRF1 confers antiviral activity in a STAT1^−/− background by transcriptionally inducing a subset of 100–150 ISGs independently of IFN induction (30). To determine if STAT2 confers a similar phenotype, we harvested total RNA from cells stably expressing IFI6, IFITM3, IRF1, and STAT2, and quantified mRNA levels for ISGs MX1, OAS1, and IFIT1. Only IRF1-expressing cells induced significant levels of these ISGs over control cells (Fig. S6B). STAT2 did not confer ISG expression, regardless of whether the cells were infected with DENV. STAT2 is, therefore, not driving an antiviral program like IRF1.

Discussion

DENV is a significant global disease threat with few therapeutic interventions. The successful development of therapeutics for DENV would likely benefit from viral tools that have robust readouts for in vitro and in vivo screening. A major hurdle in dissecting the molecular virology of DENV in the context of a complete viral life cycle is the relative intractability of full-length infectious clones. By manipulating the DENV genome and taking advantage of mouse-adaptive mutations in NS4B, we have generated unique viruses for monitoring DENV infectivity in cell culture and in mice. These recombinant viruses have yielded insight with respect to DENV dynamics in vivo and susceptibility to IFN effectors.

In this study, we optimized conditions for infectious clone propagation and engineered fully infectious serotype 2 (strain 16681) DENV expressing heterologous GFP and firefly luciferase reporters. A key experimental strategy in manipulating DENV infectious clones was the use of a recently developed E. coli strain, MDS42. This bacterial line is based on the standard K-12 E. coli but has 15% of the genome removed, including nonessential genes and recombinogenic or mobile DNA elements, which allows propagation of plasmids that may be otherwise unstable (15). We found that MDS42 and the RecA-deficient variant, MDS42Rec, were superior to standard laboratory strains such as DH5α with respect to faithfully propagating full-length dengue virus clones. Stbl2 cells could also be used, but total plasmid yields were lower, suggesting that MDS42-based lines may be superior for both clone stability and DNA yield.

DENV reporter viruses were infectious in cell culture and sensitive to neutralizing antibodies, antiviral compounds, and antiviral interferons. To study DENV-Fluc infection in living mice, we incorporated a single amino acid substitution (L52F) in NS4B. This virus could be grown to high titer and exhibited robust infectivity in vitro and in vivo. Bioluminescence imaging was used to probe DENV dynamics in the AG129 mouse model. Our data suggest that DENV localizes predominantly to lymph nodes and spleen, but we also observed infection of gut-associated tissue in all infected animals. Interestingly, the kinetics of luciferase expression indicate that infection may initiate in lymph nodes and spread to spleen and gut tissues at later time points. The presence of luciferase punctae in the intestines of animals infected with DENV-Fluc suggests that DENV can localize to Peyer’s patches or other gut-associated lymphoid tissues. This observation is consistent with a recent study on DENV serotype 2 strain S221 infection in AG129 mice (34). In that study, viral RNA could be detected in the small intestines of infected animals, and a fraction of isolated laminar propria macrophages stained positive for DENV prM protein. Further characterization of the cell types harboring DENV within these punctae will be critical for determining whether gut lymphoid tissues play a role in DENV pathogenesis.

The Fluc-expressing virus was also used to demonstrate sensitivity to a neutralizing antibody and known anti-dengue compounds. As expected, the adenosine nucleoside inhibitor NITD008 potently suppressed DENV-Fluc replication in cell culture and in mice. In contrast, mycophenolic acid, a known inhibitor of DENV replication in cell culture, had little suppressive effect on the virus in vivo. These data suggest that bioluminescence imaging may be useful as an orthogonal screen in drug discovery pipelines to rapidly identify bioavailable compounds that target DENV in living animals.

The innate immune system is strongly implicated in the control of DENV in vivo. To characterize further the nature of innate immune control on DENV replication, we first confirmed that our reporter viruses were sensitive to type I and III IFN. To identify which antiviral effectors may have activity against DENV, we screened DENV-GFP against a library of more than 350+ ISGs. This screen yielded at least 10 genes that significantly inhibited viral replication, the majority of which were confirmed in two distinct cell types. Interestingly, several antiviral effectors were genes that are known to be involved in antiviral responses (e.g., IRF1 and IRF7). We previously uncovered each of these genes while screening other viruses (30), and showed that their antiviral activity is retained even in a STAT1^−/− cell line. Similar results were found with DENV, further strengthening the observation that antiviral effector functions are able to operate independently of a fully functioning IFN system.

Mechanistically, our studies indicate that IRF1 and IFITM3 target DENV at an early life cycle stage, whereas STAT2 and IFI6 impact later stages of virus replication. Gene expression studies indicated that STAT2 overexpression is not driving antiviral signaling like IRF1. Previous studies have shown that the DENV NS5 polymerase potently antagonizes IFN signaling by mediating STAT2 degradation (35, 36). Our data fit this mechanism and suggest that STAT2 overexpression may be sufficient to squelch DENV replication by stoichiometrically overcoming the viral NS5 polymerase. Thus, the STAT2:NS5 interaction may be a unique target for therapeutic intervention. Moreover, using reporter DENV to further probe the cellular mechanisms of action of other antiviral effectors may reveal pathways that can be targeted for anti-DENV drug discovery.

Materials and Methods

In Vitro Transcription, Virus Production, and Titering.

Plasmids bearing full-length DENV genomes were constructed as described in SI Materials and Methods. Plasmids were digested with XbaI and the linearized DNA was used as a template for T7-driven RNA transcription using T7 mMESSAGE mMACHINE (Ambion) according to the manufacturer's recommendations with addition of cap analog. Five micrograms of viral RNAs were electorporated into WHO Vero cells using a BTX 830 Electroporator (860V, 99-μs pulse length, five pulses), and virus-containing supernatants were collected 6–13 d postelectroporation. Virus titration was performed by seeding WHO Vero cells in poly-l-lysine–coated 96-well plates. Samples were serially diluted 10-fold in complete growth medium and used to infect the seeded cells (typically 6–8 wells per dilution). Following 2 or 3 d of incubation, the cells were immunostained for E protein. Wells that expressed at least one E-expressing cell were counted as positive, and the median tissue culture infective dose (TCID₅₀) was calculated according to the method of Reed and Muench (37). For DENV-GFP, infectious unit titers were also determined by limiting dilution and quantitation of GFP positivity by FACS, as described previously (38).

DENV in Vitro Assays.

Typical infection assays were carried out in 24-well plates. Cell lines were infected in a total volume of 200 μL virus for 2 h at 37 C. After 2 h, 800 μL complete media was added to the cells, and infections proceeded for a total of 48 h. For indirect immunofluorescence assays, DENV-GFP–infected cells were stained for E protein with the 4G2 monoclonal antibody using the Whole Cell Stain kit (Cellomics) according to manufacturer’s instructions. For DENV-GFP FACS-based assays, media was removed and cells were detached using 200 μL Accumax Cell Aggregate Dissociation Medium (eBiosciences). Cells were pelleted by centrifugation, fixed in 1% paraformaldehyde for at least 20 min, and stored in 1× PBS containing 3% FBS. GFP fluorescence was monitored by FACS using an LSRII flow cytometer (BD Biosciences). For DENV-Fluc luciferase-based assays, media was removed and cells were washed once with 1× PBS before lysis in 1× cell culture lysis buffer (Promega). Cell lysates were assayed for luciferase activity using the Luciferase Assay System (Promega). For inhibitor experiments, interferons, drugs, or DMSO vehicle were added to the virus inoculum and maintained in the media until the cells were harvested. For antibody inhibition assays, virus inoculum was incubated on ice with antibody dilutions for 1 h before infection. After a 2-h infection, the antibody-containing inoculum was removed, cells were washed 1× with PBS, and fresh media was added to the cells.

For ISG inhibition assays, construction and characterization of lentiviral plasmids and pseudoparticles has been described previously (30). ISG-expressing lentiviruses were used to transduce Huh7 or STAT1^−/− fibroblasts by spinoculation. Briefly, 7 × 10⁴ cells were infected with lentiviral pseudoparticles and transduced by spinning plates at 1,000 × g for 45 min at 37 °C in media containing 3% FBS, 20 mM Hepes, and 4 μg/mL polybrene. At 48 h posttransduction, cells were split 1:2 or 1:3. The next day, ISG-expressing cells were infected with DENV and assayed 48 h later as described above.

DENV in Vivo Assays.

Age-matched 129J or AG129 mice were challenged with DENV-Fluc or DENV-Fluc(NS4B:L52F) at the indicated doses. At regular time intervals postinfection, mice were anesthetized and injected i.p. with 1.5 mg luciferin (Caliper Life Sciences). Bioluminescence was measured using an IVIS Lumina II platform, which was equipped with a supercooled CCD camera (Caliper Life Sciences; 5-min acquisition time, f/stop 1, binning 4). For antibody inhibition experiments, virus stocks were preincubated with a 1:10 dilution of 4G2 antibody on ice for 1 h before virus injection. For chemical inhibitor experiments, mice were injected i.p. with virus inoculum containing DMSO vehicle or 25 mg/kg body weight NITD008 solubilized in DMSO. Mice were dosed with 200 μL 1× PBS containing DMSO vehicle or 25 mg/kg body weight NITD008 every 12 h until the experiment was terminated at 72 h postinfection.

Statistical Analysis.

All statistical analyses were performed using the Student t test, with the exception of ISG confirmation assays, which were analyzed by one-way ANOVA using Dunnett’s correction.