Virus and cells
African green monkey kidney (Vero) cells (ATCC number: CCL-81; RRID:CVCL_0059) were maintained at 37 °C under 5% CO2 in standard medium [Dulbecco-modified Eagle’s medium (DMEM, Thermofisher Scientific, Poland, Poland) supplemented with 3% heat-inactivated fetal bovine serum (FBS, Thermofisher Scientific, Poland), 100 U/ml penicillin and 100 μg/ml streptomycin (Immuniq, Poland)] with addition of 5 μg/ml ciprofloxacin.
ZIKV H/PF/2013 strain acquired from European Virus Archive [29] was propagated in Vero cells under standard medium. After 3 days of infection at 37 °C, virus-containing medium was collected and titrated. As a control, mock-infected Vero cells were subjected to the same procedure. Virus and mock aliquots were stored at − 80 °C. Virus titration was performed on confluent Vero cells on a 96-well plate, according to the Reed–Muench method [30].
Inhibitors
Amantadine (1-adamantylaminean, AMTD; Sigma Aldrich, Poland) was used at 400 μM as clathrin-coated pit stabilizing agent [31]. Bafilomycin A1 [(3Z,5E,7R,8S,9S,11E,13E,15S,16R)-8-hydroxy-16-[(1S,2R,3S)-2-hydroxy-1-methyl-3-[(2R,4R,5S,6R)-tetrahydro-2,4-dihydroxy-5-methyl-6-(1-methylethyl)-2H-pyran-2-yl]butyl]-3,15-dimethoxy-5,7,9,11-tetramethyloxacyclohexadeca-3,5,11,13-tetraen-2-one, Baf A1; Sigma Aldrich, Poland] was used at 100 nM as a vacuolar type H+-ATPase inhibitor that hampers endosome acidification [32]. Camostat [4-[[4-[(aminoiminomethyl)amino]benzoyl]oxy] benzeneacetic acid 2-(dimethylamino)-2-oxoethyl ester methanesulfonate, cam; Sigma Aldrich, Poland] was used at 100 μM as a serine protease inhibitor [33]. Decanoyl-arg-val-lys-arg-chloromethylketone (CMK; Sigma Aldrich, Poland) was used at 50 μM as furin inhibitor [34]. Dynasore [3-hydroxynaphthalene-2-carboxylic acid (3,4-dihydroxybenzylidene) hydrazide, Dyn; Abcam, UK] was used at 100 μM as an inhibitor of the GTPase activity of dynamin [35]. Trans-epoxysuccinyl-L-leucylamido (4-guanidino) butane, L-trans-3-Carboxyoxiran-2-carbonyl-L-leucylagmatine, N-(trans-epoxysuccinyl)-L-leucine 4-guanidinobutylamide (E64; Sigma Aldrich, Poland) was used at 100 μM as a cysteine protease inhibitor [33]. MitMAB (tetradecyltrimethylammonium bromide, MM; Abcam, UK) was used at 20 μM as an inhibitor that targets dynamin-phospholipid interaction [36]. NH4Cl (Bioshop, Poland) was used at 50 mM as an intracellular alkalizing agent [37, 38]. PitStop 2 [N-[5-[4-bromobenzylidene]-4-oxo-4,5-dihydro-1,3-thiazol-2-yl] naphthalene-1-sulfonamide, PtS; Abcam, UK] was used at 50 μM as an inhibitor of ligand association with clathrin’s amino terminal domain [39].
Immunostaining
Vero cells were seeded on glass slides in a cell culture plate and cultured in standard medium for two days at 37 °C. Upon experimental procedure, the cells were fixed with ice-cold 4% formaldehyde in PBS for 20 min at room temperature, washed with PBS and permeabilized with 0.5% TWEEN-20 for 10 min at room temperature. Afterwards, non-specific binding sites were blocked overnight at 4 °C with 5% BSA and slides were incubated for 2 h at room temperature with primary anti-ZIKV antibodies (specific to envelope protein (Merck Millipore, Poland) or capsid protein (kind gift from prof. Jassoy, Institut für Virologie, Leipzig, Germany) diluted 1:100 in 3% BSA in PBS. To visualize host cell proteins, slides were incubated with primary antibodies against clathrin, EEA1, Rab7, LAMP1, Rab11, Rab27 and Rab35 [goat anti-clathrin HC (RRID:AB_2083170), rabbit anti-EEA1 (RRID:AB_2277714) and rabbit anti-Rab7 (RRID:AB_2175483) polyclonal antibodies from Santa Cruz Biotechnology, Poland, rabbit anti-Rab11A (RRID:AB_2173458) polyclonal antibody from Proteintech, UK, rabbit anti-Rab27A monoclonal antibodies from Cell Signaling Technology, Poland, rabbit anti-Rab35 polyclonal antibody from Novus Biologicals, Poland, rabbit anti-LAMP1 (RRID:AB_2134611) polyclonal antibody from Thermofisher Scientific, Poland] diluted 1:100 in 3% BSA in PBS, together with anti-ZIKV antibodies. Next, the cells were incubated for another 1 h with Alexa Fluor 488-labeled goat anti-mouse IgG (RRID:AB_2534069, Thermofisher Scientific, Poland) or Alexa Fluor 488-labeled rabbit anti-mouse IgG (RRID:AB_2534106, Thermofisher Scientific, Poland) diluted 1:200 in 3% BSA in PBS. For staining of host cell proteins also Alexa Fluor 546 goat anti-rabbit secondary antibodies (RRID:AB_2534077, Thermofisher Scientific, Poland) or Alexa Fluor 546 donkey anti-goat secondary antibodies (RRID:AB_2534103, Thermofisher Scientific, Poland) diluted 1:200 were added to the mix. In experiments focused on siRNA silencing and inhibitors’ influence on virus internalization, the cell surface was labelled by Atto 633-phalloidin (Thermofisher Scientific, Poland) diluted 1:50 in PBS for 1 h at room temperature. Nuclei were stained with DAPI (RRID:AB_2629482, Thermofisher Scientific, Poland) diluted 1:10000 in PBS. At the end of immunostaining procedure slides were thoroughly washed with 0.5% TWEEN-20 in PBS. Stained slides were mounted in ProLong Diamond antifade medium (Thermofisher Scientific, Poland) and stored at 4 °C.
Staining of living cells
Vero cells were seeded on 35 mm glass bottom dishes and cultured in standard medium for two days at 37 °C. Afterwards, the cells were washed with PBS and incubated in standard medium containing 50 nM LysoTracker™ Red DND-99 (Thermofisher Scientific, Poland) for 90 min at 37 °C to visualize acidic organelles. Next, the medium was discarded, and cells were overlaid with BafA1/NH4Cl-containing or control standard medium and observed for 30 min with a fluorescence microscope. The first images (“0 min”) were acquired in < 1 min upon treatment of the cells with the two above mentioned agents.
Fluorescence and confocal microscopy
Images of living cells were acquired using EVOS FL Imaging System (Thermofisher Scientific, Poland) with 60× oil immersion lens. Images of fixed cells were taken under a ZEISS LSM 710 (version 8.1) confocal microscope with 40× oil immersion lens and ZEN 2012 SP1 (black edition, version 8.1.0.484). Image processing was performed with ImageJ FIJI (RRID:SCR_002285, National Institutes of Health, Bethesda, Maryland, USA). Co-localization parameters (Pearson’s and Manders’ coefficients) were calculated using JaCop plugin [40].
Flow cytometry
Vero cells were seeded in a 6-well cell culture plate and cultured in standard medium for two days at 37 °C. Upon experiment, the cells were fixed, permeabilized, blocked and immunostained with primary antibodies specific to viral envelope protein (Merck Millipore, Poland) and secondary rabbit anti-mouse antibodies labeled with Alexa Fluor 488 (RRID:AB_2534106, Thermofisher Scientific, Poland), as indicated in Immunostaining section. Proportion of ZIKV-infected cells (corresponding to the median fluorescence of the analyzed cells population) was evaluated with flow cytometry using FACSCalibur (RRID:SCR_000401, Becton Dickinson, Poland). Cell Quest software (RRID:SCR_014489, Becton Dickinson, Poland) was used for data processing and analysis.
Cell viability
Cells were seeded on 96-well plates and cultured in standard medium for two days at 37 °C. Afterwards, the cells were washed with PBS, overlaid with standard medium supplemented with inhibitor or control and further incubated for 3 days at 37 °C. Cell viability was examined using XTT Cell Viability Assay (Biological Industries, Poland), according to the manufacturer’s protocol. Briefly, the medium was discarded and 50 μl of fresh standard medium with 50 μl of the activated XTT solution was added to each well. After 2 h incubation at 37 °C, the supernatant was transferred onto a new, transparent 96-well plate and signal from formazan derivative of tetrazolium dye was read at λ = 490 nm using colorimeter (Tecan i-control Infinite 200 Microplate Reader, 1.5.14.0). The obtained results were further normalized to the control, where cell viability was set to 100%.
Virus yield
Virus detection and quantification was performed using reverse transcription (RT) followed by quantitative real-time PCR (qPCR). Viral RNA was isolated from cell culture supernatant 3 days post-infection (p.i.) using Viral DNA / RNA Kit (A&A Biotechnology, Poland), while reverse transcription was carried out with High Capacity cDNA Reverse Transcription Kit (Thermofisher Scientific, Poland), according to manufacturers’ protocols. To assess virus yield, DNA standards were subjected to qPCR along with the cDNA acquired from the isolated samples. qPCR was performed using KAPA PROBE FAST qPCR Master Mix (Kapa Biosystem, Poland), ZIKV-specific primers (5′-TTG GTC ATG ATA CTG CTG ATT GC-3′ and 5′-CCT TCC ACA AAG TCC CTA TTG C-3′) and probe (5′-CGG CAT ACA GCA TCA GGT GCA TAG GAG-3′) labelled with FAM (6-carboxyfluorescein) and TAMRA (6-carboxytetramethylrhodamine). Rox was used as a reference dye. The signal was recorded and analysed using 7500 Fast Real-Time PCR System (Thermofisher Scientific, Poland).
siRNA silencing
Control (scrambled) siRNA (sc-44,237) and pooled siRNAs targeting heavy chain of clathrin (sc-35,067) were obtained from Santa Cruz Biotechnology, Poland. siRNA specific to simian Rab35 mRNA (GenBank sequence ID: XM008004920.1) was designed and synthesized by Thermofisher Scientific, Poland.
Vero cells cultured for 1 day in antibiotic- and serum-depleted standard medium on a 6-well plate were transfected with appropriate siRNAs using Lipofectamine RNAiMAX (Thermofisher Scientific, Poland). The procedure was performed according to the manufacturer’s instructions and repeated 24 h later to enhance the silencing effect. The efficiency of the procedure was assessed by Western blotting 24 h later (at the same time as microscopic studies on virus subcellular localization).
Western blot analysis
Cells were harvested in RIPA buffer (1 h, 4 °C; Thermofisher Scientific, Poland) supplemented with 0.5 M EDTA and protease inhibitors cocktail (cOmplete Tablets, Roche, Poland)]. Protein concentration was assessed with Pierce BCA Protein Assay Kit (Thermofisher Scientific, Poland), according to manufacturer’s protocol. Samples containing equal amounts of proteins were mixed with SDS-PAGE Sample Buffer (0.5 M Tris, pH 6.8, 10% SDS, 50 mg/ml DTT), denatured for 10 min at 95 °C and separated by SDS-PAGE electrophoresis. Subsequently, proteins were electrotransferred onto a PVDF membrane (1.5 h, 100 V; Amersham, Poland). The non-specific binding sites on the membrane were blocked for 1 h at room temperature with 10% milk (Bioshop) in Tris-buffered saline supplemented with 0.25% TWEEN-20 (Bioshop, Poland) (TBST) and incubated with primary antibodies specific to clathrin or Rab35 [rabbit anti-clathrin heavy chain polyclonal antibody, RRID:AB_10695306, Cell Signaling Technology, Poland; rabbit anti-Rab35 polyclonal antibody, Novus Biologicals, Poland] diluted 1:500 or 1:1000 (for clathrin and Rab35, respectively) in 3% BSA in TBST overnight at 4 °C and additionally for 1 h at room temperature; or with primary antibodies specific to GAPDH (rabbit anti-GAPDH antibodies, RRID:AB_561053, Cell Signaling Technology, Poland) diluted 1:5000 in 3% BSA in TBST for 1 h at room temperature. After being washed in TBST, the membrane was incubated with HRP-labelled anti-rabbit IgG antibody (RRID:AB_257896, Sigma Aldrich, Poland) diluted 1:20,000 in 3% BSA in TBST for 1 h at room temperature. Finally, the proteins were visualized with chemiluminescence, using the ECL system (Amersham, Poland).
Co-localization assay
Vero cells were seeded in standard medium on glass slides in 12-well plates. After 2 days, 2 h prior to infection, cell culture medium was replaced with serum-depleted standard medium. Next, cells were cooled down to 4 °C, overlaid with 100 μl of non-diluted ZIKV stock (TCID50 ranging from 1000,000 to 10,000,000/ml, which approximately corresponds to MOI = 1.75–17.5 and 7 × 105–7 × 106 PFU/ml) and incubated for 30 min at 4 °C to synchronize cargo particles entry from the cell surface. Subsequently, after incubation at 37 °C (exact times indicated for each experiment) the cells were fixed, permeabilized, blocked and immunostained for viral and cellular proteins, as indicated in Immunostaining section.
Virus inhibition assays
For visualization of virus subcellular localization during experiments with agents interfering with virus trafficking, Vero cells were cultured in standard medium on glass slides in 12-well plates for 2 days and pre-treated with a particular inhibitor 1 h prior to infection. Afterwards, the cells were cooled down to 4 °C, overlaid with 100 μl of non-diluted ZIKV stock (TCID50 ranging from 1000,000 to 10,000,000/ml, which approximately corresponds to MOI = 1.75–17.5 and 7 × 105–7 × 106 PFU/ml) in the presence of inhibitory agents and incubated for another 30 min at 4 °C to synchronize cargo internalization. Subsequently, the virus-overlaid cells were warmed up to 37 °C. At indicated for each experiment time points, cells were washed with PBS, fixed, permeabilized, blocked and immunostained for viral and actin cytoskeleton proteins as indicated in Immunostaining section. Identical procedure was carried out for visualization of virus particles in cells depleted of certain proteins with siRNAs.
To test the influence of compounds on virus adhesion, Vero cells cultured in standard medium in a 6-well plate for 2 days were cooled down to 4 °C, overlaid with 100 μl of ice-cold non-diluted ZIKV stock (TCID50 ranging from 1000,000 to 10,000,000/ml, which approximately corresponds to MOI = 1.75–17.5 and 7 × 105–7 × 106 PFU/ml) and incubated for another 2 h at 4 °C. Further, cells were rinsed twice with ice-cold PBS. The cells were fixed, permeabilized, blocked, immunostained and analyzed with flow cytometry, as described in Flow cytometry section.
For assessment of the inhibitors’ influence on viral replication, Vero cells were cultured in standard medium in 96-well plates for 2 days and pre-treated with a selected agent 1 h prior to infection. Virus at TCID50 of 800/ml (which approximately corresponds to MOI = 0.0014 and 550 PFU/ml) was overlaid on the cells in the presence of inhibitors and samples were incubated for 2 h at 37 °C. Wells were washed thrice with PBS and incubated at 37 °C in standard medium supplemented with inhibitors. 3 days p.i. culture supernatants were collected, viral RNA was isolated and its yield was quantified with RT-qPCR. Whether the procedure was modified, it is described in the results section.
Statistical analyses
Each experiment was performed at least twice in triplicate. Chart bars represent mean ± SD. The significance of differences between compared groups was determined by Single-Factor Analysis of Variance (ANOVA); p values < 0.05 were considered significant.