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Quantification of the interaction forces between dengue virus and dopamine type-2 receptor using optical tweezers

Virology Journal volume 21, Article number: 215 (2024) Cite this article

Abstract

Background

Dengue virus (DENV) causes the most significant mosquito-borne viral disease with a wide spectrum of clinical manifestation, including neurological symptoms associated with lethal dengue diseases. Dopamine receptors are expressed in central nervous system, and dopamine antagonists have been reported to exhibit antiviral activity against DENV infection in vivo and in vitro. Although identification of host-cell receptor is critical to understand dengue neuropathogenesis and neurotropism, the involvement of dopamine receptors in DENV infection remains unclear.

Results

We exploited the sensitivity and precision of force spectroscopy to address whether dopamine type-2 receptors (D2R) directly interact with DENV particles at the first step of infection. Using optical tweezers, we quantified and characterized DENV binding to D2R expressed on Chinese hamster ovary (CHO) cells. Our finding suggested that the binding was D2R- and DENV-dependent, and that the binding force was in the range of 50–60 pN. We showed that dopamine antagonists prochlorperazine (PCZ) and trifluoperazine (TFP), previously reported to inhibit dengue infection, interrupt the DENV-D2R specific binding.

Conclusions

This study demonstrates that D2R could specifically recognize DENV particles and function as an attachment factor on cell surfaces for DENV. We propose D2R as a host receptor for DENV and as a potential therapeutic target for anti-DENV drugs.

Graphical abstract

Introduction

Dengue infection is the most critical mosquito-borne viral disease worldwide [1]. It is caused by one of four serotypes of dengue virus (DENV), which belongs to the genus of Flavivirus [2]. The disease covers a broad spectrum of clinical manifestation, from asymptomatic infection or self-limited febrile illness named dengue fever (DF) to life-threatening diseases including dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) [3, 4]. Although DENV primarily targets immune cells, liver, and endothelial cells [5]; neurological manifestation associated with dengue cases have increased [6]. Brain autopsy of fatal dengue victims showed traces of dengue replication in neurons, astrocytes, and microglial cells, indicating a direct infection of neurons by DENV [7]. Since 2009, the World Health Organization (WHO) has included neurological symptoms as a warning sign of severe dengue, prompting further research into dengue infection in central neuro system (CNS) [1].

Cellular receptors are the key to virus infection. They may serve as an attachment factor that anchors virus particles and prolongs their stay to activate entry, as a signaling receptor that triggers the required biochemical cascade in the viral life cycle, or as an entry receptor that induces conformational change followed by viral endocytosis [8, 9]. Hence, identifying the viral receptors and elucidating the interaction between virus and its receptor are crucial for understanding the virus tropism and pathogenesis. Several putative DENV receptors have been proposed, but only TIM-1 has been demonstrated to be a DENV entry receptor and a signaling receptor for activating DENV-induced autophagy [10,11,12]. However, TIM-1 has not been detected on neuron cell membranes [13]. Only a few studies have investigated DENV cellular receptors in CNS [14]. Recently, type II dopamine receptor (D2R) has been proposed as a cellular receptor required for DENV infection [15].

Dopamine receptors are the most abundant receptor in CNS. The receptors are expressed on the primary targets of DENV, such as immune cells (macrophages, monocytes and dendritic cells) and the liver cells (HepG2) [16, 17]. In macrophage, dopamine receptors increase the susceptibility for HIV infection [18, 19]. In neuroblastoma cells, dopamine receptors improve the susceptibility against Japanese encephalitis virus (JEV) infection [20]. Previous studies have reported that several dopamine-receptor-targeting molecules and anti-psychotic drugs could inhibit dengue infection in vivo and in vitro, and D2R was the most prevalent target in these studies [15, 21,22,23,24,25]. However, most of the antagonists were phenothiazine-derived drugs inhibiting clathrin-mediated endocytosis (CME), the most common pathway for DENV entry [15, 26,27,28,29]. To date, the direct involvement of D2R in DENV infection remains unclear.

Optical tweezers, a non-invasive optical method, use an intensely focused laser beam to trap and manipulate particles ranging from nanometer to micrometer [30, 31]. With proper calibration, optical tweezers function as a sensitive force transducer capable of measuring biological forces and molecular bonds at pico-Newton scale [32,33,34]. In this study, we used optical-tweezers-based force spectroscopy complemented with fluorescence imaging and traditional biochemical techniques to explore the interactions between D2R and DENV particles. Through precise measurement of the unbinding force between DENV particles and D2R, we showed that D2R directly interacted with DENV-2 particles and established D2R as a direct cellular receptor for DENV. Additionally, we investigated the inhibitory effects of phenothiazine-derived drugs on the DENV-D2R specific binding [15, 21]. The use of optical tweezers allowed us to elucidate the physical interaction of DENV particles with D2R on living cells at single-virion level.

Materials and Methods

DENV-coated bead preparation and verification

Virus strains and staining methods are described in the Supplementary Data. DENV particles were labeled with DiI (1,1′-Dioctadecyl-3,3,3′,3′-Tetramethylindocarbocyanine Perchlorate) (Invitrogen, USA) followed by an incubation with carboxyl-modified polystyrene beads (Invitrogen, USA) in 50 mM 2-(N-morpholino)ethanesulfonic acid (MES) buffer (Sigma Aldrich, Germany) in a rotator at 4 °C overnight. The beads were further incubated in 1% bovine serum albumin (BSA) (Merck KGaA, Germany) dissolved in MES buffer to cover surface gaps on the bead and minimize non-specific binding. Unbound DENV particles and BSA molecules were removed with centrifugation, and the beads were resuspension in MES buffer. Adsorption of DENV particles on the bead surface was verified through ground state depletion (GSD) super-resolution imaging (Leica SR GSD, Germany) equipped with an oil immersion objective (100 × , NA = 1.47, HCX APO, Leica, Germany) and a high sensitive electron multiplying charge-couple device (EMCCD, Andor iXon DU-897, Oxford Instruments, United Kingdom). The GSD imaging used a high-power laser to temporarily inactivate fluorophores in a region except for one and captured an image of the single active fluorophore. The process was repeated until every fluorophore was imaged one-by-one, and a super-resolution image was constructed from the localized single molecule images, achieving a spatial resolution of 20 nm.

Optical tweezers setup

A customized optical tweezers setup was coupled to a commercial inverted microscope (TiE Eclipse, Nikon, Japan) equipped with an EMCCD (Andor iXon DU-888E, Oxford Instruments, United Kingdom). To trap a bead, a 1064 nm laser (Cobolt Rumba, HÜBNER Photonics GmbH, Germany) beam was collimated via a pair of concave-convex lenses and focused via an oil-immersion objective (100 × , NA = 1.45, CFI Plan Apo Lambda, Nikon, Japan) to the sample mounted on a piezo stage (P-730, Physik Instrumente, Germany). The trapping beam intensity was adjusted through a rotatable half-wave plate followed by a polarizing beam splitter, and the axial position of the focal spot was adjusted via a pair of convex lenses. A HeNe laser beam (LGR 7628, LASOS Lasertechnik GmbH, Germany) was used to track the trajectory of the trapped bead. The tracking beam was aligned to project interferences images of the trapped bead on a quadrant photodiode (QPD) sensor (OT-301, On-Trak, USA). The position of a trapped bead was determined by back-focal plane interferometry [35].

Binding force measurement

Cell lines and culture conditions are described in the Supplementary Data. One day prior to the optical trapping experiment, cells were seeded on 30-mm glass coverslips in 6-well plates (2 to 3 × 105 cells/well). The coverslip was mounted on a custom sample holder filled with bead solution in serum-free F-12 (Ham’s Nutrient Mixture, GIBCO, USA) medium and covered with a 22-mm glass coverslip, creating a sealed environment. A custom LabVIEW (National Instrument) program was used to control a two-axis piezo-electric translation stage to move at a constant velocity of 2 µm/s and to record the trajectory of the bead. Optical trapping force can be modeled as a Hookean spring, which obeys the Hooke’s Law:

$$F = - k \,x$$

where F, k, and x denote force, spring constant, and the displacement of the trapped bead (relative to the trapping center), respectively. The spring constant (or the trapping force stiffness) k was calibrated via the power spectrum analysis with a MATLAB (MathWorks) program according to previous studies [36, 37].

Immunofluorescence imaging

Cells were cultured on a 3.5 cm glass bottom dish (Mettek, USA) for 30 to 36 h. The cells were then fixed with 4% paraformaldehyde for 15 min in room temperature and were washed with phosphate-buffered saline (PBS, 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4). Subsequently, the cells were incubated in antibody solution consisting primary antibody and blocking buffer (3% BSA in PBS) for overnight at the 4 °C with gentle shaking. Anti-hDC-SIGN (CD209) clone # 120507 (R&D System, USA) and anti D2DR (sc-5303, Santa Cruz Biotechnology, USA) were used as the primary antibody to detect DC-SIGN and D2R receptors, respectively. After PBS wash, the cells were labeled with the secondary antibody Alexa Fluor 532 (Thermo Fisher Scientific, USA) for 4 h in room temperature, followed by PBS wash and WGA-488 staining (W11261, Invitrogen) for 15 min. Finally, the WGA-488 solution was replaced with PBS for confocal imaging (FluoView 1000, Olympus, Japan).

Virus binding assay

Cells were inoculated on a 3.5 cm glass bottom dish for 30 to 36 h. The adhered cells were pre-treated in the presence or absence of drugs for 1 h at 37 °C, followed by adsorption of DiI-labeled DENV at 4 °C on a shaker for 1 h. The cells were washed with PBS and were stained with WGA-488 (W11261, Invitrogen) for 15 min at room temperature. After PBS wash, the cells were fixed with 4% paraformaldehyde and were kept in PBS buffer for confocal imaging.

Data and statistical analysis

FV31S-SW Viewer version 2.5 and ImageJ were used to analyze images. Binding force was calculated with a customized MATLAB program. Plots and statistical analysis were carried out via OriginPro 2016 Software (Origin Lab). Unless stated otherwise, data are presented as mean ± SD.

Results

Quantification of DENV-cell receptor binding force from the displacement–time curves using a custom-built optical tweezers system

As illustrated in Fig. 1A, we applied optical tweezers to quantify the binding force between a single virus particle and cellular receptors by trapping a polystyrene bead bound with virus particles and using it as a probe. A single cell expressing a particular receptor was moved towards the trapped bead (Fig. 1A, Step I), and after the cell established a contact with the bead (Step II), the cell along with the attached bead was retracted without delay to reduce the probability of multiple bindings (Step III). The trapping force exerted on the bead increased as the bead was pulled away from its equilibrium position and eventually detached the bead from the cell at a certain distance x (Step IV), prompting the bead to return to its initial position (Step V). The maximum displacement reached by the bead x determined the binding forces between DENV particle and the cell. A typical bead trajectory corresponding to one approach-and-retraction cycle is shown in Fig. 1B. The experiment was conducted using a custom optical tweezers platform merged with a fluorescence imaging system as described in detail in the method section (Fig. 1C).

Fig. 1
figure 1

Force measurement using optical tweezers. A A schematic drawing illustrates the five major steps of force measurement to quantify the virus-cell interaction using optical tweezers (I-V). The images show a cell (green) moving towards and away from a laser-trapped polystyrene bead (blue) coated with DiI-labeled DENV-2 (yellow). Arrows indicate the direction of the movements of and the trapped bead attached on the cell, and x denotes the maximum displacement with respect to the trapping center of the bead before it detaches from the cell. B A displacement-vs-time graph shows a typical bead trajectory, corresponding to the procedure depicted in (A). C A schematic diagram represents an optical tweezers system coupled to an inverted fluorescence microscope. Insets show bright field (left) and epifluorescence images (right) of a trapped bead coated with fluorescence-labeled-DENV near a cell

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Verification of DENV-coated polystyrene beads as a probe to investigate the binding interaction at the single-virus resolution

To confirm DENV particles adsorbed on the surface of the polystyrene beads, we labeled the DENV particles with lipophilic fluorescence membrane dye, DiI, prior to immobilizing them on the polystyrene beads surface (Supplemental Fig. 1) [38]. Super-resolution images showed successful adsorption of the DENV particles on each polystyrene bead in a dose-dependent manner. The size of individual fluorescent puncta on the bead surface (Fig. 2A) was around 60 nm (Fig. 2B). Most fluorescent puncta have a full-width-at-half-maximum (FWHM) of 50–60 nm (Fig. 2C). Analysis of 19 DiI puncta revealed a punctum size of 56.9 ± 7.8 nm (mean ± SEM), consistent with the known DENV particle size measured by electron microscopy [39]. Collectively, these data showed the adsorption of DENV on polystyrene bead surface could reach a single-virion resolution.

Fig. 2
figure 2

The size determination of DiI-DENV particles adsorbed on a polystyrene bead. A Super-resolution (GSD imaging) images of a single DiI-labeled-DENV-coated bead show a dose-dependent adsorption of DiI-labeled DENV (red) generated by the ratio of polystyrene beads to DENV as indicated. Scale bars: 1 µm. B Analysis of individual puncta resulted in a FWHM ~ 50 to 60 nm. c Particle size analysis of 19 DiI-fluorescence puncta on the beads indicates the size distribution of DENV particles, indicating that the DENV particle size was approximately 56.9 ± 7.8 nm (mean ± SD)

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Evaluation of the optical tweezers approach to quantify DENV-DC-SIGN interaction

We then assessed optical tweezers suitability to detect the interaction between DENV and cellular receptors on a living cell using dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) as a model. DC-SIGN is a well-established putative DENV receptor and its binding interaction with DENV has been resolved at the molecular level with cryo-EM images [40]. CHO-A745 cell-line (CHO cell) was chosen because it has a high transfection efficiency rate and a low susceptibility against DENV infection [41]. Moreover, it is deficient of glycosaminoglycans (GAGs) and thus, it lacks of heparan sulfate (HS) that can bind with DENV [42,43,44]. Since CHO-A745 cells lack of natural expression of DC-SIGN, the cells were over-expressed with DC-SIGN (DC-SIGN-CHO cells). We confirmed the surface expression of DC-SIGN on DC-SIGN-CHO cells with immunofluorescence (Fig. 3A) and quantified its binding force with DENV by optical tweezers. The interaction was probed using three kinds of beads having different DENV quantities on their surface: BSA beads without DENV served as a control, low-DENV beads optimized for single-virion investigation, and high-DENV beads served to increase the probability of DENV-specific interaction. Force quantification revealed that, in the absence of DENV particles, most binding interactions exhibited forces weaker than 30 pN, ranging from 5 to 30 pN (Fig. 3B-I, gray bars). The force distribution in the histogram that was fit with a Gaussian model yielded a force of 14 ± 9 pN (Fig. 3B-I, in red). When low-DENV beads were used, there were stronger forces in the range of 30–50 pN detected (Fig. 3B-II, gray bars). Two Gaussian peaks were yielded, 8 ± 3 pN (in red) and 31 ± 13 pN (in blue), in the force distribution (Fig. 3B-II). In addition, when high-DENV beads were used, compared with Fig. 3B-II, two force distributions became more apparent (Fig. 3B-III, gray bars). Gaussian fitting gave similar two peaks, 15 ± 6 pN (in red) and 37 ± 7 pN (in blue), in the force distribution (Fig. 3B-III). Accordingly, the blue peak recognized as a stronger force only appeared in the presence of DENV, suggesting a specific interaction force induced by DENV (Fig. 3B-II and B-III). In contrast, compared to Fig. 3B-I, the red peak recognized as a weaker force might represent non-specific bindings (Fig. 3B-II and 3B-III). We then counted occurrence of the non-specific binding forces and DENV-specific binding forces and compared it to total number of approach-retraction cycle. Notably, the bindings occurred more frequently as the DENV particles on the bead surface increased. The frequency of the weak-binding force increased from 5.5% (14/254) to 25.9% (57/220) of the cycles, while the frequency of the strong binding-force increased slightly from 11.8% (30/254) and 14.6% (32/220) of the total cycles. The binding frequency increment indicated that both weak and strong forces were DENV-dependent. To further verify whether DENV interact with CHO cells, we quantified the interaction forces between high-DENV beads with parental CHO cells that did not express DC-SIGN. The major interaction forces were distributed from 10 to 34 pN, and Gaussian fitting analysis only yielded a single force peak with 22 ± 12 pN accounting for 11.2% (34/302) (Supplemental Figure S2), suggesting that DENV could interact with CHO cells without DC-SIGN expression. These findings strongly indicated that the stronger binding force of 37 ± 7 pN corresponded to the interaction between a DENV and DC-SIGN, and the weaker forces corresponded to the non-specific interaction, such as interaction of DENV particles with cell surface or BSA with the cell surface regardless of the presence of DC-SIGN. Therefore, we confirmed that our optical tweezers setup could characterize DENV-cellular receptor interaction on living cells and distinguished it from non-specific interactions.

Fig. 3
figure 3

Quantification of forces required to break the binding interaction between DENV and DC-SIGN. A Parental (left) and DC-SIGN-CHO cells (right) were immunostained with WGA-488 for cell membrane (green) and Alexa 532 (red) for DC-SIGN. Confocal microscopy images show colocalization of WGA-488 with Alexa 532 on DC-SIGN-CHO, confirming the presence of DC-SIGN on the cell surface. Pearson’s co-localization coefficient of the images is 0.031 and 0.816 for parental and DC-SIGN-CHO, respectively. Scale bars indicate a length of 50 µm. B Force histograms of the interaction between DENV and DC-SIGN-CHO cells show that optical tweezers were able to distinguish DENV-DC-SIGN specific binding (blue) from non-specific binding (red). Gaussian fitting yields the DENV-DC-SIGN specific binding force of 37 ± 7 pN and non-specific binding forces of 15 ± 6 pN (mean ± SD). The “n” in the histograms refers to the number of the approach-retraction cycles performed in the experiment

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Characterization of DENV-D2R specific binding interactions

Next, we investigated the DENV binding on D2R using D2R-overexpressed CHO (D2R-CHO) cells. The surface expression of D2R on the cells was confirmed via immunofluorescence (Fig. 4A). Similar to the previous experiment, we used three types of beads to probe the DENV-D2R interaction. Force quantification revealed that the histogram of high-DENV beads displays two peaks (Fig. 4B-III, gray bars). Gaussian fitting yielded a weak force of 20 ± 8 pN (Fig. 4B-III, in red) and a strong force of 55 ± 11 pN (Fig. 4B-III, in blue). Replacing the beads to low-DENV beads did not significantly change the magnitude of the forces (Fig. 4B-II, gray bars). Similar two Gaussian peaks were obtained, a weak force 26 ± 13 pN (in red) and a strong force of 60 ± 9 pN (in blue). However, only the strong-binding-force frequency decreased significantly, from 11.6% (42/363) to 2.5%; (12/484). The weaker binding-force frequency changed slightly, from 10.4% (38/363) to 10.9% (53/484). Depleting the beads of DENV eliminated forces stronger than 50 pN and reduced its binding frequency to 4.9% (9/182) (Fig. 4B-I). The control experiment carried on parental CHO cells (Supplemental Figure S2) did not differ from the no-DENV-beads experiment (Fig. 4B-1). Taken together, the stronger forces (blue color, Fig. 4B-II and B-III) correspond to the specific DENV-D2R binding, and the weak forces (red color, Fig. 4B-I to B-III) in these experiments were attributed to non-specific interactions. All these outcomes strongly suggested that DENV interact with D2R specifically.

Fig. 4
figure 4

Characterization of DENV and D2R specific unbinding force via optical-tweezers-based force spectroscopy. A CHO cells were over-expressed with D2R (D2R-CHO), and the expression was verified via immunostaining with Alexa 532 (red) for D2R and with WGA-488 (green) for cell membrane. Confocal microscopy images of parental (left) and D2R-CHO (right) are shown. Pearson’s co-localization coefficient of the images is 0.007 and 0.861 for parental and D2R-CHO, respectively. Scale bars represent a length of 50 µm. B Force histograms of the interaction between DENV and D2R-CHO cells indicate a specific DENV-D2R binding force of 55 ± 11 pN (blue, Gaussian fitting, mean ± SD) and a non-specific binding force of 20 ± 8 pN (red, Gausian fitting, mean ± SD). The “n” in the histograms refers to the number of the approach-retraction cycles performed in the experiment

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Examination of dopamine antagonists effects on DENV interaction with D2R

Prochlorperazine (PCZ) and trifluoperazine (TFP) are D2R antagonists that effectively inhibit dengue infection and the main entry pathway of DENV [15, 21]. To investigate this effect, we further quantified D2R-DENV binding force using high-DENV beads on D2R-CHO cells treated with various concentration of PCZ or TFP. Both PCZ and TFP treatment specifically reduced the DENV-D2R interaction (Fig. 5A), whereas the non-specific binding forces were not affected (Supplemental Figure S3); their magnitude remained at 20–30 pN (Supplemental Figure S3A) and accounted for 7–13% in the entire experiments (Supplemental Figure S3B). Interestingly, the drugs did not weaken the DENV-D2R binding strength, and the DENV-D2R binding forces were consistently in the range of 50 to 60 pN throughout the experiments (Fig. 5B); only its frequencies were reduced in a dose-dependent manner (Fig. 5C). The reduction was also evident in fluorescence images (Fig. 6). The confocal microscopy images clearly show that PCZ and TFP treatment reduced the number of DENV particles bound to the D2R-CHO cells (Fig. 6A). Likewise, the number of bound virus-particles also decreased (Fig. 6B), consistent with the findings from optical tweezers. All these results suggest that the drugs worked on the specific DENV-D2R binding and affirmed the hypothesis that D2R facilitates DENV infection.

Fig. 5
figure 5

Inhibition of DENV-D2R binding on cells treated with prochlorperazine (PCZ, top) and trifluoperazine (TFP, bottom). A The force histograms of DENV-D2R interaction pre-treated with the drugs (gray) show a particular depletion of DENV-D2R binding forces in the untreated experiments (white), suggesting that the drugs prevented specific DENV-D2R binding interaction. B The box chart (25th and 75th percentile, mean, and standard deviation) shows that the DENV-D2R binding force did not depend on the drug concentration. The binding forces remained at 50–60 pN (mean) without significant differences; “NS” indicates not significant (p > 0.05 by the ANOVA test). C DENV-D2R binding frequency was negatively correlated with the PCZ and TFP concentrations; the Pearson correlation coefficient (r = 0.90) is provided in the upper panel of (B)

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Fig. 6
figure 6

Reduction of DENV particles bound on PCZ- and TFP-pre-treated D2R-CHO cell surfaces. A Confocal images of binding assays on D2R-CHO cells showed that PCZ and TFP treatments reduced the number of DENV bound on the cell surfaces. The cells were pre-treated with indicated drugs for 1 h followed by adsorption of DiI-labeled DENV (red; multiplicity of infection (MOI, 10). Cells were then stained with WGA-488 conjugated with cell membrane (green). B The bar chart shows the reduction in the bound-DENV to the cells after D2R antagonist treatment. The chart represents data from one independent experiment (5 fields of view) with a total cells ranging from 84 to 124. *p < 0.05, **p < 0.01 by the Mann–Whitney U test

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Discussion

D2R antagonists have been reported to inhibit dengue infection in cell models and mouse models [15, 21, 23, 24]. Several conventional cell biology techniques, including immunofluorescence staining, plaque assay, confocal microscopy, and viral-entry assay have indicated that the inhibition occur in a D2R-dependent manner [15, 23, 24]. However, these approaches did not directly resolve either the DENV binding on D2R or the interference of D2R antagonist with DENV-D2R binding, leaving DENV-D2R interaction unclear. In the present study, we dissected the infection steps further and focused on quantifying the binding force between DENV with D2R using optical tweezers. The quantification allowed us to characterize and to distinguish DENV-receptor specific bindings from non-specific bindings and provided direct evidences of DENV interaction with D2R (Fig. 4), consistent with previous study that showed D2R knockdown reduced DENV attachment in N18 cells [15]. Furthermore, optical tweezers revealed that PCZ directly inhibit the binding of DENV to D2R (Fig. 5), aligning well with the results from confocal imaging (Fig. 6) as well as the previous study [15]. Similarly, both optical tweezers and confocal imaging data elucidated that TFP also directly inhibits DENV-D2R binding. This finding contrasts with the time-of-addition assay, which suggested that TFP inhibits viral entry rather than viral-receptor binding in Vero cells [21]. The difference might be due to the cell line used in the study. Given that D2R was not detected among the membrane proteins in Vero cells from proteomic analysis using shotgun liquid chromatography-tandem mass spectrometry (LC–MS/MS), the effects observed by the time-of-addition assay might be independent from D2R [45]. Therefore, the ability of D2R antagonists, PCZ and TFP, to directly inhibit the binding of DENV-D2R affirmed the D2R services as an attachment factor for DENV.

More importantly, previous study showed that optical tweezers could investigate virus-receptor binding at brief contact time and at single-virion level, closer to what happens when the virus encounters a cell [46]. The transient contact time reduced the probability of multiple DENV-D2R interactions. At the single-virion level, stochastic behaviors of the detachment hidden in ensemble analysis from bulk conventional methods could be detected. This is evident in the differences in the characterization of DENV-DC-SIGN binding (Fig. 3) and DENV-D2R binding (Fig. 4). The binding frequency of DENV to DC-SIGN saturated at lower DENV quantity than the DENV-D2R binding, suggesting that DENV particles bound to D2R at lower efficiency than DC-SIGN. The lower efficiency of D2R could be attributed to structural difference between D2R and DC-SIGN. D2R is a G-protein-coupled receptor (GPCR) that is mainly composed of transmembrane and intracellular domains; its longest extracellular part is the N-terminus comprised of 37 amino acids [47]. In comparison, DC-SIGN mainly consists of an extracellular domain with an extended neck featuring at least 161 amino acids, followed by carbohydrate recognition domain (CRD) responsible for DENV binding; its length is estimated to be 20–30 nm [48]. Such sticking-out structural arrangement might offer more accessible contact points for the virions. Our study also suggested that DENV particles bound to D2R more strongly than to DC-SIGN. However, further studies, especially structural imaging, are required to draw further conclusion. Cryo-EM and in-silico investigation have provided insight on DC-SIGN-DENV binding site [40, 49,50,51]. Unfortunately, no such study on D2R-DENV has been reported. It would be worth investigating the D2R-DENV binding further because D2R is expressed in the primary target cells of DENV, suggesting D2R might facilitate DENV infection in those cells.

Conclusions

Our research has successfully demonstrated the role of D2R in facilitating DENV infection, functioning as an attachment factor on cell surfaces as determined by optical-tweezers-based force spectroscopy. The interaction forces between DENV and D2R-expressing cells were quantified to be ~ 50 to 60 pN. Furthermore, treating the cells with D2R antagonists, PCZ and TFP, can effectively inhibit DENV binding into cells by interrupting the DENV-D2R specific binding interactions. These findings establish D2R as a potential DENV receptor and highlight its importance as a target for developing anti-DENV therapeutics. Taken together, this study presents new insight into D2R involvement in DENV infection and offers a photonics tool that provides direct and straightforward evidence regarding virus-cellular receptor interaction. Force characterization at single-virion level provided by optical tweezers allowed us to focus on the first step of infection and pinpoint the origin of binding interaction. This approach benefits virus-host cell investigation in living cells while preserving the natural structure of the receptors. We believe that optical tweezers could serve as a promising complement to conventional biochemical assay, imaging, and structural analysis techniques to study virus-host cell interaction in general.

Availability of data and materials

The datasets used and/or analyzed are available from the corresponding author on reasonable request.

Abbreviations

BSA:

Bovine serum albumin

CHO:

Chinese hamster ovary

CME:

Clathrin-mediated endocytosis

CNS:

Central nervous system

Cryo-EM:

Cryogenic electron microscopy

CRD:

Carbohydrate recognition domain

D2R:

Dopamine type-2 receptor

DC-SIGN:

Dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin

DENV:

Dengue virus

DF:

Dengue fever

DHF:

Dengue hemorrhagic fever

DiI:

1,1′-Dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate

DSS:

Dengue shock syndrome

dSTORM:

Direct stochastic optical reconstruction microscopy

EMCCD:

Electron-multiplying charged-coupled device

FWHM:

Full width at half maximum

GAGs:

Glycosaminoglyclans

GPCR:

G-protein-coupled receptor

GSD:

Ground state depletion

HS:

Heparan sulfate

HIV:

Human immunodeficiency virus

JEV:

Japanese encephalitis virus

LC–MS/MS:

Liquid chromatography coupled with tandem mass spectrometry

MES:

2-(N-Morpholino)ethanesulfonic acid

NA:

Numerical aperture

PBS:

Phosphate-buffered saline

PCZ:

Prochlorperazine

QPD:

Quadrant photodiode

TFP:

Trifluoperazine

TIM-1:

T cell immunoglobulin mucin domain-1

WGA:

Wheat germ agglutinin

WHO:

World Health Organization

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Acknowledgements

The authors thank Dr. Yogy Simanjuntak (Inserm, French Institute of Health and Medical Research, Strasbourg, France) for the fruitful discussions. We also thank the Imaging Core Facility at the Institute of Cellular and Organismic Biology, Academia Sinica, Taipei, Taiwan, for the technical support in the super-resolution image acquisition. Graphical abstract was created with BioRender.com.

Funding

This work was supported by the grant from the Ministry of Science and Technology, Taiwan (MOST 109-2320-B-010-034-MY3, NSTC 112-2320-B-A49-044, NSTC 113-2327-B-002-005, NSTC 113-2320-B-A49-024). L.W.C. was supported by the Ministry of Science and Technology, Taiwan (MOST 110-2811-B-A49A-037, NSTC 111-2811-B-A49A-047).

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Authors and Affiliations

  1. Institute of Biophotonics, National Yang Ming Chiao Tung University, Taipei, 11221, Taiwan

    Jane C. Arifin, Bo-Ying Tsai, Chun-Yu Chen, Chau-Hwang Lee, Arthur Chiou & Yueh-Hsin Ping

  2. Department and Institute of Pharmacology, National Yang Ming Chiao Tung University, Taipei, 11221, Taiwan

    Li-Wei Chu & Yueh-Hsin Ping

  3. Institute of Biomedical Sciences, Academia Sinica, Taipei, 11529, Taiwan

    Yi-Ling Lin

  4. Research Center for Applied Sciences, Academia Sinica, Taipei, 11529, Taiwan

    Chau-Hwang Lee

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Contributions

JCA: conceptualization, methodology, software, formal analysis, acquisition, writing—original draft, and visualization. BYT: methodology, formal analysis, and acquisition. CYT: conceptualization and acquisition. LWC: methodology, writing—editing. YLL: conceptualization and resource. CHL: resource and writing—editing. AC—conceptualization, methodology, resource, and writing—editing. YHP: conceptualization, methodology, supervision, resource, funding acquisition, writing—original draft, writing—editing. All authors reviewed the manuscript.

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Arifin, J.C., Tsai, BY., Chen, CY. et al. Quantification of the interaction forces between dengue virus and dopamine type-2 receptor using optical tweezers. Virol J 21, 215 (2024). https://doi.org/10.1186/s12985-024-02487-8

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