The neurovirulent RP-9 strain of JEV was used for both in vitro and in vivo studies and was propagated in mosquito C6/36 cells as described (Chen et al., 1996). Viruses were titrated by plaque-forming assay in baby hamster kidney BHK-21 cells (ATCC: CCL-10). Dopaminergic human neuroblastoma BE(2)C cells (ATCC CRL-2268) were cultured in RPMI 1640 medium (Gibco) supplemented with 10% fetal bovine serum. Primary antibodies included anti-JEV-NS3, anti-phospho-tyrosine hydroxylase (Cell Signaling, #2791), anti-tyrosine hydroxylase (Cell Signaling, #2792), anti-D2R (Santa Cruz Biotechnology, sc-9113), anti-D1R (Santa Cruz Biotechnology, sc-1434), anti-phospho-CaMKII (Thermo Scientific, 22B1), anti-integrin β3 (BD Biosciences, 611140), anti-vimentin (Sigma, V6389), anti-β-actin (Chemicon), and anti-α-tubulin (Sigma–Aldrich).

Mouse experiments were approved and performed in accordance with the guidelines of the Academia Sinica Institutional Animal Care and Use Committee. Groups of 4-week-old AG129 mice were used to evaluate the effect of D2R agonist and antagonists on animal survival. Mice were challenged intraperitoneally with 5 × 10⁴ PFU/mouse of JEV and simultaneously injected with 30 μl PBS intracranially into the right hemisphere of the brain. Immediately, mice received PBS (vehicle control, n = 5) or 8 mg QH (D2R agonist)/kg body weight intravenously in the absence (QH, n = 5) or presence of either 8 mg D1R antagonist/kg body weight (QH+SCH23390, n = 5) or 8 mg D2R antagonist/kg body weight (QH+Haloperidol, n = 5). The animal survival was monitored daily.

Cells were adsorbed with JEV [multiplicity of infection (MOI), 1 or 5] for 2 h. Cells were washed to remove unbound virus and incubated for the indicated times. For antiviral assays, cells were adsorbed with JEV (MOI, 0.1) with the indicated doses of chemical compounds for 2 h, washed thoroughly, then incubated for 24 h with and without chemical compounds.

The lentivirus vector pLKO.1, which carries a short hairpin RNA (shRNA) targeting the human D2R (5′-GTCCTGGGAGACCCATGTAAA-3′, TRCN0000315421) or LacZ (5′-TGTTCGCATTATCCGAACCAT-3′, TRCN0000072223), obtained from the Taiwan National RNAi Core Facility, was cotransfected with pMD.G and pCMVΔR8.91 into HEK293T cells by use of Lipofectamine 2000 (Invitrogen). The lentiviruses were harvested from culture supernatants and concentrated by ultracentrifugation at 35,000 rpm in a Beckman SW41 rotor for 3.5 h at 4°C. The viral pellets were re-suspended and used to transduce BE(2)C cells. The D2R-deficient BE(2)C cells (shD2R-BE(2)C) and LacZ-control cells (shLacZ-BE(2)C) were selected with puromycin (10 μg/ml).

Quantitative assay of dopamine was performed according to the manufacturer’s protocol (Dopamine ELISA kit, KA3838-Abnova). Briefly, 250 μl of cell culture medium was used for enzymatic conversion. Sample absorbance was determined by use of an ELISA reader (Molecular Devices) at 450 nm.

cAMP level in cell lysate (10 μg) was measured by chemiluminescent enzyme-linked immunosorbent assay kit (Cell Biolabs). Quantitative assay of IP₁ was performed according to the manufacturer’s protocol (IP-One Tb Cisbio Bioassays). Briefly, cells were plated in a 96-well black clear-bottom plate (50,000 cells/well) overnight. The level of IP₁ in cell lysate was measured by use of SpectraMax M5 (Molecular Devices; Fluorescence ratio: 668/620 nm). Changes in intracellular cytosolic calcium level were evaluated by using calcium-sensitive dye Fluo-4AM (Calcium assay kit, Molecular Probes) with a fluorescence microplate reader (Spectramax, Molecular Devices). Cells were loaded with Fluo-4AM dye in HEPES-buffered Modified Hanks’ Balanced Salt Solution without phenol red and calcium. Cells were incubated for 10 min at 37°C. Quinpirole hydrochloride (QH) at the indicated doses was added to cells and fluorescence intensity was recorded at 5 min intervals over 30 min at room temperature.

Japanese encephalitis virus was purified and labeled with Lightning-Link Atto-488 (Innova Biosciences) as described (Liang et al., 2011). Briefly, virus was ultracentrifuged through a 35% sucrose cushion at 35,000 rpm in a Beckman SW41 rotor for 3.5 h at 4°C. The viral pellets were re-suspended in PBS and labeled with Lightning-Link Atto-488 (Innova Biosciences).

Cells were adsorbed with JEV for 2 h with rocking on a linear shaker at room temperature for virus binding/entry assay. The cells were washed three times with cold PBS. Virus binding/entry was evaluated by confocal microscopy with fluorescently labeled (MOI, 20∼30) and plaque-forming assay with unlabeled JEV (MOI, 5). For virus binding assay, cells were adsorbed with JEV for 1 h at 4°C with rocking on linear shaker. For the virus entry assay, cells were adsorbed with JEV for 1 h at 4°C. After a gentle wash with cold medium, cells were shifted to 37°C incubator for 1 h to allow virus entry. Cells were then treated with acid glycine (0.1 M, pH = 3) to inactivate non-internalized virus for 5 min and washed with cold HBSS. Cells were harvested with use of cell scrapers, and the adsorbed viruses were released by passing the cells through a 27-G needle 3 times. Cell lysates were centrifuged at 12,000 rpm for 1 min, and the supernatant was used for plaque-forming assay.

Cells were plated in a 6-well plate for overnight. After treatments, cells were immunostained with anti-integrin β3 (BD Biosciences, 611140) or anti-vimentin (Sigma, V6389) without cell permeabilization for 1 h, then washed thoroughly and incubated with Alexa Fluor-488-conjugated secondary antibody (Molecular Probes) for 1 h at room temperature. After washing, cells were harvested with use of cell scrapers and passed through cell strainer. FACSCanto flow cytometry was used to measure signal intensity of respective proteins.

Data are presented as mean ± standard deviation (SD). For comparison of three or more categorical data, ANOVA-Bonferroni post hoc test was used. Two categorical data were compared by independent Student t-test. The statistical tests were two-tailed and significance was set at P < 0.05 and < 0.01. For immunoblotting, the band density was quantified by use of ImageJ (US National Institutes of Health). A survival curve was descriptively analyzed by use of SigmaPlot v10.0 (Systat Software). Determination of the median survival time (T₅₀) and P-values, by the log-rank test, involved used of Prism v5.0 (GraphPad Software).

To investigate whether JEV affects dopamine production, we used dopaminergic human neuroblastoma BE(2)C cells, which show a high level of TH activity for dopamine synthesis (Kim et al., 1996). JEV-infected BE(2)C cells released a significantly higher level of dopamine at 3 and 6 h post-infection (hpi) as compared with mock-infected cells; however, at later times of 24 and 36 hpi dopamine release was reduced by JEV infection (Figure 1A), probably due to cytopathic effect caused by JEV. Consistently, JEV also modulated the rate-limiting enzyme of dopamine biosynthesis, with an increase of phospho-TH levels at 3 and 6 hpi and decrease at later times, when JEV-NS3 was abundant (Figure 1B). Notably, UV-inactivated JEV (UV-JEV) also significantly increased dopamine and phospho-TH levels at 3 and 6 hpi but different from that of JEV, the levels were not decreased by UV-JEV at 24 and 36 hpi (Figures 1A,B). These data suggest that JEV increases dopamine levels at the early stage of infection probably through viral binding/entry.

The biological effect of dopamine is mediated by DRs and to elucidate their role in JEV infection, we used selective antagonists targeting dopamine D1- or D2-like receptors (Missale et al., 1998). Similar to our previous study with D2R antagonist prochlorperazine (Simanjuntak et al., 2015), the D2R antagonists haloperidol and risperidone dose-dependently reduced JEV-NS3 protein expression and viral progeny production as compared with solvent-treated cells (Figures 1C,D). SCH23390, a D1R antagonist, also inhibited JEV infection, although it required a relatively high dose (Figures 1C,D). Thus, our data indicate the major role of D2R in JEV infection as compared with D1R.

To address the role of D2R activation in JEV infection, we used a D2R-specific agonist with putative dopaminergic properties, QH (Koller et al., 1987). Treatment of QH for 2 h before JEV infection dose-dependently enhanced JEV-NS3 expression and viral progeny production (Figures 2A–C). To verify the requirement of D2R in QH-enhanced JEV infection, we knocked down D2R expression in BE(2)C cells by transduction with a lentivirus expressing shRNA targeting human D2R (shD2R). The knockdown of D2R did not affect D1R expression and cellular localization (Figure 2D). The enhancement effect of QH on JEV-NS3 expression and viral progeny production was significantly lower in shD2R-BE(2)C cells than parental BE(2)C cells and knockdown control shLacZ-BE(2)C cells (Figures 2E–G). To further confirm the biological significance of D2R activation on JEV infection, we performed animal challenge experiment. QH treatment significantly accelerated animal mortality as compared with PBS treatment in JEV-challenged AG129 mice (T₅₀: 4 days vs. 8 days) (Figure 2H). Notably, this acceleration of animal mortality was abolished in the presence of D2R antagonist (haloperidol; T₅₀ = 9 days) but not D1R antagonist (SCH23390; T₅₀ = 4 days) (Figure 2H). These data demonstrate that stimulation of D2R positively regulates JEV infection.

To determine the D2R signaling event involved in JEV life cycle, we evaluated the major pathways of AC and PLC downstream of D2R stimulation (Figure 3A). Through Gαi/αo, D2R may inhibit AC to negatively regulate cAMP level (Beaulieu and Gainetdinov, 2011), while via Gαq/βγ D2R may activate PLC to hydrolyzes phosphatidylinositol 4,5-biphosphate (PIP₂) to inositol 1,4,5-triphosphate (IP₃). IP₃ then stimulates the release of calcium from intracellular stores before it is rapidly degraded to IP₂ and then IP₁ (Trinquet et al., 2006). At the early time of JEV infection when dopamine level was elevated (3∼6 h), cAMP levels were similar between mock and JEV-infected BE(2)C cells (Figure 3B). However, the levels of IP₁ were significantly increased by JEV infection (Figure 3C). Furthermore, treatments with PLC signaling inhibitors at non-cytotoxic doses and in the range of their IC₅₀ (Supplementary Figure S1) dose-dependently reduced JEV-NS3 expression and viral progeny production (Figure 3D). Thus, PLC signaling pathway is involved in JEV infection in dopaminergic BE(2)C cells.

Next, we verified the activation of PLC pathway by the D2R agonist QH. Treatment with QH dose-dependently increased IP₁ production as compared with solvent treatment (Figure 3E). This QH-induced IP₁ production was associated with D2R because IP₁ level was lower in shD2R-BE(2)C than BE(2)C cells and knockdown control shLacZ-BE(2)C cells. Similarly, JEV infection induced IP₁ production in a D2R-associated mechanism, since lower IP₁ level was noted in cells with D2R knockdown (Figure 3E). In addition, QH dose-dependently increased cytosolic calcium level as measured by Fluo-4 AM calcium indicator assay (Figure 3F). Notably, cytosolic calcium level was lower in shD2R-BE(2)C than control cells with QH treatment, so QH-mediated calcium flux was associated with D2R. This D2R-dependent effect on cytosolic calcium level was also observed in JEV-infected cells (Figure 3F).

Calcium flux may activate calcium-dependent enzymes such as CaMKII, the most abundant neuronal protein kinase in the brain (Berridge et al., 2003; Okamoto et al., 2009). Calcium flux and CaMKII activation triggered by DR-mediated signaling can affect cell morphology by regulating cytoskeletal dynamics (Rosenberg and Spitzer, 2011). Treatment with QH and JEV infection for 5 h greatly increased phosphorylated CaMKII T286 (pCaMKII) level, a marker of CaMKII activation (Coultrap and Bayer, 2012), and also altered the cell shape to form neurite-like structures with high actin composition (Figure 3G). Together, these data demonstrate that D2R stimulation and JEV infection induces a PLC signaling cascade in BE(2)C cells.

To dissect the viral life cycle affected by D2R-PLC activation, we performed viral binding/entry study in BE(2)C cells with or without QH pretreatment for 5 h. The amount of JEV binding/entry was significantly increased with QH treatment (Figures 4A,B). Furthermore, this enhancement in QH-treated cells was concomitantly reduced with PLC signaling cascade inhibitors KN93, 2APB, and U73122 (Figures 4A,B). To further address the effect on viral binding and/or viral entry, we performed virus binding and virus entry assays as described in the section “Materials and Methods.” Both JEV binding and entry were significantly increased by QH treatment and this enhancement could be repressed by PLC signaling inhibitors and with D2R knockdown in shD2R-BE(2)C cells (Figures 4C,D). However, D2R stimulation had no effect on post-entry step of JEV infection because treatments with QH after virus adsorption did not significantly change JEV-NS3 expression, virus progeny production, and intracellular JEV RNA level (Figures 4E–G). Therefore, D2R-mediated PLC signaling plays a role in enhancing JEV binding/entry.

Phospholipase C activation increases cytosolic calcium level, which in turn activates calcium-dependent kinases and/or directly enhances actin assembly to facilitate the formation of focal adhesion on the cell surface (Prevarskaya et al., 2011). One of the main components of focal adhesion, αvβ3 integrin adhesion receptor, has been reported to mediate entry of flavivirus such as West Nile virus and JEV (Chu and Ng, 2004). Furthermore, integrin β3 is involved in recruiting vimentin to the cell surface (Bhattacharya et al., 2008) and our previous work indicated the role of vimentin in JEV binding and virulence (Liang et al., 2011). Thus, we tested whether the surface expression of integrin β3 and vimentin was increased with treatment of the D2R agonist, QH. Treatment with QH for 5 h greatly increased the surface expression of integrin β3 and vimentin as compared to solvent-treatment in BE(2)C cells (Figures 5A,B) dependent on PLC because the PLC inhibitor U73122 reversed this effect (Figure 5B). The increased surface expression of integrin β3 and vimentin was not likely due to the effect on protein expression because their total protein levels were not changed by QH (Figure 5C).

To support the role of integrin β3 and vimentin in D2R-PLC-enhanced JEV binding/entry, we performed antibody-blockage experiments. BE(2)C cells-treated with QH for 5 h underwent viral binding/entry assay with and without antibodies against integrin β3 and vimentin. The enhanced JEV binding/entry with QH was significantly repressed by anti-integrin β3 and anti-vimentin antibodies as compared with mouse IgG control (Figures 5D,E). These data suggest that D2R-PLC-enhanced JEV binding/entry is associated with the increased surface expression of JEV binding/entry molecules including integrin β3 and vimentin.