2.1.

Cell Cultures

Microtubule-associated protein 1A/1B-light chain 3 (LC3) is the reputable marker of autophagosomes in mammalian cells.³⁶ Stably expressed green fluorescence protein-fused microtubule-associated protein 1A/1B-light chain 3 (GFP-LC3) Huh7.5 cell line produced by transduction of cells with lentiviruses expressing GFP-LC3 was generously provided by Dr. Michael M.C. Lai (Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan).³⁷ GFP-LC3 Huh7.5 cells were maintained in high-glucose Dulbecco’s modified Eagle’s medium (DMEM, GIBCO, USA) supplemented with 10% fetal bovine serum (FBS) (HyClone, Thermo, USA), 1% nonessential amino acids (NEAA, GIBCO, USA), $4\mathrm{\mu g}/\mathrm{ml}$ blasticidin (Invitrogen, USA). The cells were cultivated at 37°C in a humidified atmosphere with 5% ${\mathrm{CO}}_2$. The Aedes albopictus C6/36 cell line, useful for propagation of flaviviruses, was cultured in RPMI 1640 medium (GIBCO, USA) supplemented with 10% FBS, 1% NEAA, 2% penicillin/streptomycin (GIBCO, USA), and 1 mM sodium pyruvate (Biological Industries, Kibbutz Beit-Haemek, Israel) and cultivated at 28°C in a humidified atmosphere with 5% ${\mathrm{CO}}_2$. The baby hamster kidney cell line BHK-21 was maintained in high-glucose DMEM supplemented with 10% FBS and 2% penicillin/streptomycin at 37°C and 5% ${\mathrm{CO}}_2$.

2.2.

DENV Propagation and Infection

DENV serotype 2 strain 16681 was propagated in confluent monolayers of C6/36 cells. Cells were inoculated with virus supernatant and incubated for 90 min at 28°C with gentle rocking at 15-min intervals for virus adsorption. Afterward, maintenance medium (RPMI 1640 with 5% FBS) was added and incubated at 28°C for 5 to 7 days. Infected cell culture medium was collected; cell debris was removed by centrifugation, and virus supernatant was aliquoted and stored. For virus infection, Huh7.5 cells were adsorbed with DENV at a multiplicity of infection (MOI) of 10 at 4°C for 30 min to synchronize viral entrance. The cells were then washed once with PBS to remove unbound viral particles and incubation temperature was switched to 37°C. To repress or stimulate cellular autophagic response, GFP-LC3 Huh7.5 cells were either pretreated with 10 mM of 3-methyladenine (3-MA; Sigma–Aldrich, USA) or with 100 nM of rapamycin (Sigma–Aldrich, USA) in serum free DMEM at 4°C for 30 min prior to DENV infection.

2.3.

Plaque Quantitation Assay

A sample vial of virus suspension to be quantified was diluted in 10-fold step with serum-free DMEM. BHK-21 was seeded in 6-well plates ($4\times {10}^5\text{cells}/\text{well}$) and incubated overnight at 37°C. The medium was then aspirated and 100 μl of serially diluted viral suspension were added. After 2-h incubation at 37°C with gentle rocking at 15-min intervals, the cells were overlaid DMEM containing 1% low-melting agarose (Lonza, Switzerland) and 2% heat-inactivated FBS. Six-days postinfection, the overlaid medium was then removed, and cells were fixed with 10% formaldehyde (Sigma–Aldrich, USA) and stained with the 1% crystal violet (Sigma–Aldrich, USA). The end-point titer was determined by macroscopic counting of plaques.

2.4.

Preparation of Fluorescence-Labeled DENV Particles

DENV particles were labeled with the lipophilic fluorescent dye DiD (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate salt) (Invitrogen, USA), with the maximum absorption at 630 nm and the maximum emission at 670 nm, as described previously with the minor modification.^8,28,38 Briefly, $1\times {10}^7\mathrm{pfu}/\mathrm{ml}$ of DENV was mixed with 2 nmol of DiD dissolved in dimethyl sulfoxide in HNE buffer [5 mM 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES), 150 mM NaCl, 0.1 mM ethylenediamine tetra-acetic acid, pH 7.4] for 10 min. The unincorporated dye was separated from DiD-labeled DENV particles by gel filtration on a Sephadex G-50 column in HNE buffer (GE Heathcare, Sweden). Labeled-DENV particles were store at 4°C.

2.5.

Fluorescence Confocal Microscopy Imaging

Images of fluorescent samples were acquired with an Olympus FluoView 1000 confocal microscope equipped with a $60\times$ oil immersion objective with a numerical aperture (N.A.) of 1.4. Huh7.5 cells were inoculated on a 3.5 cm glass-bottom plate (Mettek, USA). DENV-infected cells were fixed with 3.7% formaldehyde for 5 min at room temperature. After PBS washed, samples were kept in imaging buffer containing phenol red free DMEM, 75 mM HEPES, and 2% glucose and added DAPI for nucleus staining. Lysosomes were stained with 1 μM of Lysotracker (Invitrogen, USA) for 30 min. Images were captured in the green (GFP), red (Lysotracker), and far-red (DiD) channels, respectively. All images were analyzed by Olympus software FV10-ASW Viewer version 3.1. The selected confocal images were further deconvoluted by the Huygens Essential software (Scientific Volume Imaging B.V., Hilversum, The Netherlands).

2.6.

Real-Time Epi-Fluorescence and Differential Interference Contrast (DIC) Microscopy System

The tracking experiments were conducted as described previously.^8,26,39 To capture a simultaneous image of DiD-labeled DENV particles and GFP-tagged LC3 puncta, DiD was excited with a 633 nm helium–neon laser (Melles-Griot, USA), whereas GFP was excited with a 488-nm laser (Coherent, USA). Live-cell imaging was recorded by using an inverted microscope (IX 71, Olympus, Japan) equipped with an imaging split system (U-SIP, Olympus, Japan), a high sensitivity monochrome charge-coupled devide (CCD camera (CoolSNAP HQ2, Photometrics, USA). A triple-band dichromatic mirror (Semrock, USA) was used to reflect the laser lines onto the samples. The fluorescent emission was collected by a $100\times$ oil immersion objective with a N.A. of 1.40 (Olympus, Japan). The emission signals of the samples were first spectrally separated by a 660 nm long-pass dichromatic mirror (Chroma Technology, USA). A 512/630-25 nm band-pass filter (Semrock, USA) was used for GFP fluorescent emission and a 665 nm long-pass filter (Chroma Technology, USA) was used for DiD emission. The separated emission signals were imaged onto the two half areas of the CCD camera. To make sure that the shapes of the cells were not significantly altered during the recording period, the nucleus and plasma membrane of the cell were identified using the differential interference contrast (DIC) optics (Olympus, Japan) before and after the fluorescence imaging. Live-cell recordings were conducted in the live cell instrument (Leica, Germany) at 37°C with 5% ${\mathrm{CO}}_2$ supplement. Time-lapse image sequences were recorded by RS Image version 1.9.2 (Roper Scientific, Inc., USA).

2.7.

Particle Tracking

The positioning processing of DENV particles was described previously,⁴⁰ as implemented in a MATLAB-based Polyparticletracker program. Briefly, it is comprised of five steps: First, image noise is reduced and smoothed by convolving the image with a Gaussian function: $I(x,y)=\sum _{i=-\omega }^{\omega }\sum _{j=-\omega }^{\omega }{I}_{\mathrm{raw}}(x+i,y+j)\exp \{-[(i^2+j^2)/4{\lambda }^2]\}$. Second, single particles are tracked and particle center coordinates are estimated. Third, subpixel refinement of the particle coordinates by polynomial fitting with Gaussian weight (PFGW). Next, particle discrimination and parameter are calculated. Finally, the particle positions between individual frames are connected by straight lines to form the trajectories of particles by the Polyparticletracker program.⁴⁰ Following these procedures, over 50 DENV particles in 20 cells were tracked in the present work.

3.1.

DENV Induces Host Cellular Autophagic Responses

In order to examine whether DENV infection triggers autophagy responses of host cells at the early steps of infection, DENV was infected into the Huh7.5 cells that stably express GFP-fused LC3 and autophagy was detected by the formation of LC3 puncta, an indicator of autophagosome. Compared to the control group [Fig. 1(a), top panel], confocal images clearly showed that DENV induced the formation of GFP-LC3 puncta at 30 and 60-min postinfection [Fig. 1(a), green dots in the bottom panel], suggesting that the formation of autophagosome could be induced by DENV during the early stage of infection. In addition, Lysotracker staining revealed that lysosomes were engulfed into LC3 puncta dramatically at 30 and 60-min postinfection, indicating the formation of autophagolysosomes, the ultimate vesicle of autophagy progression [Fig. 1(a), red dots in the bottom panel). The numbers of the autophagolysosomes were elevated by DENV infection in a time-dependent manner, compared to the DENV-free groups [Fig. 1(b)]. Taken together, these results implied that DENV infection could induce autophagy responses and form autophagolysosomes at the early step of infection.

Fig. 1

Induction of autophagy by Dengue virus (DENV) in Huh7.5 cells. (a) Stably expressed GFP-LC3 Huh7.5 cells were grown on a 3.5 cm glass-bottom plate. Cells were mock-infected (top panel) or DENV-infected (bottom panel) at a multiplicity of infection (MOI) of 10 for the indicated time. Fluorescence signals were observed using an Olympus FluoView 1000 confocal microscopy. GPF-LC3 puncta was shown in green. Lysosomes were visualized by lysotracker and shown in red. White arrows indicate the location of autolysosomes. The scale bar represents 20 μm. (B) The formation of autophagolysomes was quantified in the absence (white bar) and presence (black bar) of DENV at 0, 30, and 60-min postinfection, respectively. The percentage of the autolysome was calculated by the number of lysosomes overlapped with autophagosomes divided by the total number of autophagosomes. At least 10 cells were examined in each group. The error bars represent standard deviation.

3.2.

Visualization of DENV Particles by a Lipophilic Fluorescent Dye

To investigate whether DENV was transported into autophagosomes during infection, a single-virus tracking experiment was conducted. Fluorescent DENV particles were first prepared by labeling with a lipohilic fluorescence dye, DiD.^28,29,39 Fluorescence-labeled viral particles were separated from excess free DiD dye by a Sephadex G-50 column. Compared to the fraction of the 40 nm fluosphere signals, the fluorescent signal of DiD appeared from fraction 5 to fraction 7 [Fig. 2(a), red triangles and blue squares]. No fluorescence signal was detected in the same range of fractions from neither the DiD-free DENV group nor the DiD only group [Fig. 2(a), green circles and gray diamonds]. These results indicated that DENV particles were successfully labeled with DiD. The infectivity of DiD-labeled DENV was then determined by the plaque assay. Results showed that the DiD-positive fraction indeed contained infectious DENV particles (Table 1). Furthermore, compared to the label-free viruses, DiD-labeled DENV particles possessed similar infectious activities, suggesting that the DiD-labeling procedure has no significant influence on the infectivity of DENV (Table 1). To further characterize the properties of DiD-labeled DENV, DiD-labeled viral particles were added to a glass coverslip, and the size and the fluorescent intensity of DiD-labeled virus were measured. Since clusters of DENV particles formed in the preparation or the counting procedure may be considered as a single particle, the correlation between fluorescent intensity and pixel size of DiD-labeled DENV particles was evaluated. The majority of the fluorescent bright dots had relatively smaller sizes (10 to 20 pixels) and lower fluorescence intensity [250 to 750 arbitrary units (AU)], presumably indicating these DiD-labeled viral particles possess a similar brightness, and most probably represent a single DENV particle [Fig. 2(b)]. However, there were approximately one-fourth of fluorescent particles that exhibited stronger fluorescence and larger pixel sizes, indicating that they might be aggregated viral particles. The combination of the fluorescent image of single DENV particles with the DIC image of Huh7 cells revealed that DiD-labeled DENV particles possess a good signal-to-noise (S/N) ratio allowing single viral particles to be clearly detected [Fig. 2(c)].

Fig. 2

Purification and characterization of DiD-labeled DENV particles. (a) DiD-labeled DENV particle was purified within the fraction layer 5 to 7 from gel filtration (blue squares). No fluorescence signal was detected neither in DiD only (gray diamonds) nor DiD-free DENV (dark red circles) within the same fraction layers. The 40 nm fluorescence microspheres were used as a size marker (red triangles). The error bars represent standard deviation. (b) The intensity and size of DiD-labeled DENV were determined by fluorescence microscopy and plotted in a graph. Particles with low fluorescence intensity (0 to 750 AU) were selected for image analysis. (c) Huh7.5 cells were infected with DiD-labeled DENV virus at a MOI of 10. The morphology of the cell was detected using differential interference contrast (DIC) optics (Olympus, Japan) after the fluorescence imaging. Fluorescence images of single DENV particles acquired by fluorescence microscopy were indicated in red (white arrow). Merge image from both DIC image and fluorescent image clearly demonstrated the DENV particle spots. The scale bar represents 10 μm.

Table 1

Effect of DiD-labeling on the infectivity of DENV. Plaque assays were performed to determine the infectivity of DENV particles incubated with DiD for indicated time periods. DiD-free DENV was used as a control. All virus particles were purified from the same fractions of the Sephadex G-50 column. The results were determined by at least three independent experiments.

3.3.

DENV Particles Co-Localized with LC3 Puncta During Virus Infection

To confirm the localization of DENV particles during the early stage of infection, GFP-LC3 stably expressed Huh-7.5 cells were infected with DiD-labeled DENV particles at MOI of 10. At the initial time point, confocal microscopy images showed that DiD-labeled DENV particles surrounded Huh7.5 cells, indicating the attachment of DENV particles on the cellular surface of Huh7.5 cells [Fig. 3(a)]. In addition, only a few GFP-LC3 puncta were observed at this time point. Consistent with the previous results in Fig. 1, the number of GFP-LC puncta was dramatically increased by DENV infection from 15 to 60 min [Fig. 3(a)]. DiD-labeled DENC particles were found to co-localize with GFP-LC3 puncta at 15 min after internalization. Quantitative analyses further showed that the number of DENV particles co-localized with autophagosome significantly increased with the infectious time ($p<0.0001$) [Fig. 3(b)], suggesting that DENV particles entered into autophagosome during the early stage of infection. Furthermore, three-dimensional (3-D) images from confocal Z-stack images revealed that DENV particles co-localized with LC3 puncta and were surrounded by GFP-LC3 [Fig. 3(c)]. Cross sectional analyses using fluorescence intensity profiling displayed the signal distribution of GFP-LC3 and DiD-labeled virus particles, suggesting that DENVI-induced autophagosomes indeed engulfed DENV particles [Fig. 3(c)]. These results demonstrated that DENV particles were translocated into autophagosomes within 15-min postinfection.

Fig. 3

Co-localization of DENV particles and autophagosomes. (a) Huh7.5 cells were infected with DiD-labeled DENV at an MOI of 10 for the indicated time. Fluorescence images were acquired using an Olympus FluoView 1000 confocal microscopy. GPF-LC3 puncta was shown in green. Nuclei were visualized by DAPI staining. The scale bar represents 10 μm. (b) Quantification of co-localized DiD-labeled DENV particles with GFP-LC3 puncta. Percentage of co-localization was determined by the number of viral particles with GFP-LC3 puncta divided by total DENV particles. A statistical analysis was undertaken by one-way analysis of variance using SPSS with $p<0.0001$ for significance. The numbers of the examined cells at each time point were 28, 26, and 22. (c) The three-dimensional reconstruction of the Z-stacked deconvolved image showed the engulfed DENV particles by autophagosomes. In fluorescence intensity profiling, the signal distribution of GFP-LC3 and DiD-labeled virus particles was indicated in green and red, respectively.

3.4.

Trajectory Analysis of Single DENV Particle and GFP-LC3 Puncta Interaction

Time-lapse dual-channel fluorescence microscopy images allowed us to perform single-virus tracking in living cells.²⁶ The setup of a home-built real-time dual-fluorescence microscopy was shown in Fig. 4(a) and described in detail in Sec. 2. To examine the mechanical stability and to exclude surrounding vibration, we calculated the standard deviations of positions by the time-lapsed imaging of an immobile specimen. Fluorescent microspheres with a 200-nm diameter were spread onto glass coverslips and fixed by a mounting medium, and the fluorescence images were captured for 5 min at a frame rate of $2\text{frames}/\min$. The central fluorescence intensity distribution [Fig. 4(b), gray line] of the fluorescent microsphere spots [Fig. 4(b), upper right] were examined by ImageJ software and fitted by Gaussian function [Fig. 4(b), red line]. The standard deviation of the diameter of an identified spot was 0.21 pixels or 13.95 nm in real scale. The relative error was smaller than 2.8%, suggesting that the microscope system would be stable for single-virus tracking. Prior to tracking single viral particles in live cells, we also determined the tracking accuracy of DiD-labeled DENV particles by examining the central fluorescence intensity distribution [Fig. 4(c), gray line] of viral spots [Fig. 2(d), upper right] fitted by Gaussian function [Fig. 2(d), green line]. The tracking accuracy ($\sigma$) was calculated as follows:, where $s$ is the standard deviation of the point spread function in pixels, and S/b is the S/N ratio. According to the measurement of 30 individual viral particles, the tracking accuracy was determined at 0.078 pixels, or 4.98 nm in real scale. The relative error was smaller than 0.4%, indicating that DiD-labeling provided an SN ratio high enough for estimating particle positions. In order to visualize the movement of a single DENV particle in a live cell, cells were infected with DiD-labeled DENV in the presence or absence of chlorpromazine (CPZ), a cationic amphiphilic drug that inhibited the formation of clathrin-coated pits to block clathrin-mediated cytocytosis. Compared to the CPZ-free group, the treatment of CPZ displayed significant effects to restrict trajectories of DENV particles [Fig. 4(d), left panel]. Furthermore, we plotted the mean-square displacement (MSD) of each viral particle of CPZ treatment [Fig. 4(d), right panel]. Simple diffusion should display a linear relationship between MSD and time, and an upward curvature would indicate a directed motion.⁸ In the absence of CPZ, an upward curvature suggested that DENV particles were transported within the cells [Fig. 4(d), top right panel]. In contrast, a linear mode of MSD in the presence of CPZ indicated that DENV moved on the cell surface in a simple diffusion manner [Fig. 4(d), right bottom panel], suggesting that DENV particles were unable to enter cells but were attached on the cell surface.

Fig. 4

Single-virus particle tracking by real-time epi-fluorescence microscopy. (a) Schematic diagram of the setup of the microscopy system. (b) Represented the central one-dimension (1-D) fluorescence intensity distribution of 200 nm fluorescence microspheres spot (gray line) and Gaussian fitting (red line). The upper right image was shown a single fluorescence microsphere spot. The scale bar represents 1 μm. (c) Represented the central 1-D fluorescence intensity distribution of DiD-labeled DENV-2 particle spot (gray line) and Gaussian fitting (green line). The upper right image showed a single DiD-labeled DENV particle spot and the scale bar represents 1 μm. (d) The two-dimensional trajectories of DENV within Huh7.5 cells in the absence (top panel) or presence (bottom panel) of CPZ, an inhibitor of clathrin-mediated cytocytosis were shown. Both motion trajectories (right panel) and MSD analysis (left panel) graph showed corresponding colors for individual DiD-labeled DENV particles. The scale bars represent 1 μm.

Subsequently, we would like to investigate whether DENV particles interact with autophagosomes. If the interaction of DENV particle with autophagosomes was one of the early events after viral entrance, the intracellular trajectories of both should be overlapped during viral infection. The fluorescent signals of DiD-DENV particles and GFP-LC3 puncta were simultaneously recorded by a time-lapsed fluorescence microscopy. After 30-min incubation at 4°C for DENV attachment, Huh7.5 cells were placed on the object table in the culture chamber of time-lapsed fluorescence microscopy at 37°C and two-color fluorescence series images were recorded. In Fig. 5(a), the DiD-DENV particles were shown in red, GFP-LC3 puncta in green, and the vesicles containing both DiD-DENV and GFP-LC3 puncta in yellow. Clearly, these images revealed the co-localization of DENV and LC3 puncta [Fig. 5(a)]. The trajectories of both DiD-DENV and GFP-LC3 puncta were also shown to be almost identical [Fig. 5(b)], strongly suggesting that DENV particles traveled with LC-3 pancta. Therefore, we concluded that DENV particles interacted with autophagosomes during the early stage of viral infection.

Fig. 5

Co-transportation of DENV particles and autophagosomes. (a) The selected frames showed the endocytic trafficking behavior of a single DiD-labeled DENV particle co-localized with GFP-LC3 luncta. The GFP-LC3 puncta were displayed in green (top panel), the DiD-labeled DENV particles in red (middle panel), and the vesicles containing both DiD-DENV and GFP-LC3 puncta in yellow (bottom panel). (b) The overlapped trajectory profiles of DENV particle (in red) and LC3 puncta (in green) in Huh7.5 cell were determined by Polyparticletracker (Ref. 40). Black arrow indicates the start point of tracking.

3.5.

Inhibition of Autophagy Changes DENV Distribution in Host Cells

Previous studies have shown that DENV induces autophagy in various types of cells.^{21,22,24,25,41} In the present work, DENV infection was able to induce autophagic responses and caused the formation of autophagosomes that engulfed the DENV particles (Figs. 1 and 3). It was intriguing to investigate the influence of DENV-induced autophagy on the production of the virus. Extracellular virus titers from the culture supernatant of the infected cells in the presence of the autophagy suppressor 3-MA and the autophagy stimulator rapamycin, respectively, were determined by the plaque assay. Virus yield was reduced in 3-MA treated cells and increased in response to the rapamycin treatment [Fig. 6(a)]. Moreover, it would be interesting to examine whether the intracellular location of DENV particles was altered while Huh-7.5 cells were treated with either 3-MA or rapamycin. At the initial step of infection, DENV-induced formation of autophagosomes was suppressed dramatically by the treatment of 3-MA [Fig. 6(b)]. In addition, compared to the distribution of DENV particles in drug-free cells [Fig. 3(a)], the internalized virus particles were dispersed in the cytoplasm of 3-MA treated cells instead of in the perinuclear region 30-min postinfection [Fig. 6(b)]. These results revealed that the treatment of 3-MA not only suppressed autophagosome formation but also altered the distribution of DENV particles in the host cells. On the other hand, autophagosomes were stimulated dramatically in rapamycin-treated Huh7.5 cells 30 min after DENV infection [Fig. 6(c)]. As expected, DENV particles were engulfed by autophagosomes and distributed in the perinuclear region of cytoplasm [Fig. 6(c)]. In summary, the location of DENV particles is affected due to the alteration of autophagy status, resulting in the alteration of DENV replication.

Fig. 6

The effects of 3-methyladenine (3-MA) and rapamycin on the production of DENV and the distribution of DENV particles. Huh7.5 cells treated with either the autophagy suppress 3-MA or the autophagy stimulator rapamycin, respectively, were infected with DiD-labeled DENV at an MOI of 10 for the indicated time. (a) The amounts of newly produced virus isolated from the culture supernatant of the infected cells were determined by plaque assay at 12, 24, and 48-h postinfection. The data were derived from three independent replicates. (b and c) Using an Olympus FluoView 1000 confocal microscopy, fluorescence images of DENV particle (in red) and autophagosomes (in green) were acquired at 30 and 60 min in the presence of 3-MA (b) or rapamycin (c). GPF-LC3 puncta was shown in green. Nuclei were visualized by DAPI staining. The scale bar represents 10 μm.