Cells, virus and antibodies
Vero cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 2 mM L-glutamine, nonessential amino acids, sodium pyruvate, 5% or 10% heat-inactivated fetal bovine serum (FBS), 100 U/ml penicillin and 100 μg/ml streptomycin (all reagents are purchased from Gibco Invitrogen). Cells were cultured at 37℃ in a humidified incubator with 5% CO2. PEDV strain CV777 stock with 1× 107/ml plaque forming units (PFU) is used in this paper. Anti PEDV-N (1:1000) mouse monoclonal antibody was obtained from Alpha Diagnostic International. Antibodies to actin (1:1000) and goat anti-mouse secondary antibodies conjugated to horseradish peroxidase (HRP, 1:10000) were obtained from Beyotime Biotechnology.
Design of shRNA plasmids
The shRNA expression vector pGPU6/GFP/Neo was selected to be the parental plasmid. The shRNA sequences were as follows (5'-3'; only the sense strand is shown). shRNA-N: GCAAAGACTGAACCCACTA; shRNA-M: CTGGAATTTCACATGGAAT. The shRNA-MN plasmid carrying two shRNAs was prepared based on the two intermediate plasmids shRNA-N and shRNA-M. Briefly, shRNA-MN DNA sense template was Bbs I- (shRNA-N) - hU6 promoter- (shRNA-M) - BamH I. Empty vector pGPU6/GFP/Neo and shRNA-MN sequence were digested with Bbs I and BamH I respectively. Then the shRNA-MN sense was ligased to the vector pGPU6/GFP/Neo by T4 DNA ligase. All of these nucleotides were chemically synthesized by Invitrogen.
Transfection of shRNA plasmids and virusinfection
For transfection experiments, 2x105 Vero cells were cultured in six-well plates to 50-70% confluence. Cells were transfected with plasmid using TransIntro EL according to the manufacturer's protocols. Briefly, for each well, 4 μg shRNA plasmid were incubated with 100 μl opti-MEM for 5 min, and 10 μl TransIntro ELwere incubated with 100 μl opti-MEM for 5 min. Subsequently, mixture of 10 μl TransIntro EL and 100 μl opti-MEM was added to the mixture of plasmid and opti-MEM. After incubation for 10-20 min at room temperature (RT), the mixture was added to the well. After incubation at 37 ℃ for 5 h, the medium containing the transfection mix was replaced with growth medium (DMEM-2%FBS). At 24 h post-transfection (h.p.t), cells were infected with PEDV at multiplicity of infection (MOI) of 0.1. Cultures were then incubated at 37 ℃, 5% CO2 in a humidified incubator for 1.5 h, at which point the medium was replaced with growth medium. After 48 h of incubation, cells were havested to detect virus titers and the levels of mRNA.
HHT and HCQ treatment and viral infection
For HCQ, Vero cells were treated with HCQ (25 mM and 50 mM ) at the time of PEDV infection (0.1MOI) for 1 h at 37°C, washed three times with PBS, and overlaid with DMEM-2% FBS containing HCQ with indicated concentrations. At 24 h.p.i., cells were harvested for WB assay and mRNA was extracted for RT-PCR. For HHT, Vero cells were infected with PEDV (0.1MOI) for 1 h at 37°C, washed three times with PBS, and overlaid with DMEM-2% FBS containing HHT with indicated concentrations. At 48 h.p.i., cells were harvested for WB assay and TCID50 assay. For combination of HCQ and HHT experiment, Vero cells were infected with PEDV (0.1MOI) in the absence or presence of HCQ (25 mM and 50 mM) for 1 h at 37°C, washed three times with PBS, and overlaid with DMEM-2% FBS in the absence or presence of HHT or HCQ with indicated concentrations. At 48 h.p.i., cells were harvested for TCID50 assay.
MTT assay
The MTT assay was conducted according to the manufacturer’s (Beyotime Biotechnology) protocol. Briefly, cells in 96-well plates were treated with shRNA at 37℃ and 5% CO2 for 24 h. Then, 10 µl of MTT solution was added to each well and the samples were incubated for 4 h. The medium was then removed and 100 µl of Formazan solution was added to each well. Optical density (OD) values were measured at a test wave length of 570 nm using a Mullikan Ascent Microplate Photometer (Thermo Fisher Scientific, Waltham, MA, USA).
Preparation of cell lysates and western blotting
The cells in 6-well plate were collected by centrifugation, washed three times with PBS and dissolved in 200 μl lysis buffer (pH7.5 20 mM Tris-HCl, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 2.5 mM Sodium pyrophosphate, 1 mM β-Glycerrophosphate, 1 mM NaVO4, 1 µg/ml Leupeptin) containing protease inhibitor cocktail (Roche) for 30min on ice. The cell suspension was then fractionated by centrifugation at 6000× g for 10 min at 4℃. Solubilized proteins were harvested, quantified by BCA assay, electrophoresed in denaturing polyacrylamide gels (with 40 μg/well ), electro blotted onto a polyvinylidene fluoride (PVDF) membrane, and the antibodies detected the viral protein PVDF. Protein bands were detected with secondary antibody conjugated to horseradish peroxidase (HRP) for 45 min at room temperature, and actin was used as a loading control.
Quantitative real-time PCR (qRT-PCR)
Cells were harvested, and total RNA was extracted with Trizol (Invitrogen). The RNA was reverse-transcribed into complementary DNA (cDNA). A two-step RT-PCR (SYBR Green I technology, Applied Roche) was performed using SYBR green super mix (Toyobo) according to the manufacturer's protocol to measure transcription levels for several genes of interest. The primers used were as follows: PEDV-N: 5’- CTG GGT TGC TAA AGA AGG CG -3’ (forward), 5’- CTG GGG AGC TGT TGA GAG AA -3’ (reverse). IL-1b: 5’- GAC CTG GAC CTC TGC CCT CTG-3’ (forward), 5’- AGG TAT TTT GTC ATT ACT TTC-3’ (reverse). IL-6: 5’- AAC TCC TTC TCC ACA AGC -3’ (forward), 5’- TGG ACT GCA GGA ACT CCT -3’ (reverse). GAPDH: 5’-GAT CAT CAG CAA TGC CTC CT -3’ (forward), 5’- TGA GTC CTT CCA CGA TAC CA -3’ (reverse).
Relative fold changes were automatically calculated by the Step One Plus real-time PCR system software (Applied Bio systems), following the 2-∆∆CT method. GAPDH as a control.
Determination of 50% tissue culture infectious dose (TCID50)
Vero cells in 6-well plates were cultured overnight with 80% confluency. Serial dilutions of the PEDV were added to the cells and let them infect of 90 min, and then the culture supernate was replaced with 2% FBS of DMEM and incubated for 48 h. Virus titration was quantified. Ten-fold serial dilutions were prepared for each sample and 100 ml/well of each dilution were added to the cells in 96-well plates in quadruplicates. Wells with cytopathic effect were scored as positive for virus growth and TCID50 was determined by the method of Reed and Muench.
Prediction and construction of HR1 and tHR2
The software packages LearnCoil-VMF (http://nightingale.lcs.mit.edu) and ExPASy-Coils (http://www.ch.embnet.org/software/COILS) were used to study the coiled coils. HR1 and HR2 domains of spike glycoprotein (gS) from the PEDV (GeneBank Accession No. ALS35469.1), derived from amino acids 978 to 1118 and 1263 to 1314 (52Aa), respectively, were predicted. In addition, an optimised Lupas algorithm was used with window widths of 14, 21 and 28 and the MTIDK matrix. The predicted probability of forming coiled coils was 0.9. Optimal focused HR1 and HR2 domains were predicted as follows: NSAIGNITSA FESVKEA ISQTSKGL NTVAH ALTKVQEVVN SQGSALNQLT VQLQHNFQAI, and LTGE IADLEQR SESLRNT TEELRSL INNINNT LVDLEWL (39Aa), respectively. The scramble peptide is LTGE IADVEQR SESVRNT TEEVRSL INNVNNT LVDVEWL, which is also derived from the PEDV HR2 domain, as negative control (NC). All of these peptides were chemically synthesized by China Peptides (http://www.chinapeptides.com/english/index.aspx).
Gel-filtration analysis
The highly purified HR1 and tHR2 were loaded onto the Superdex G75 column in a solution buffer of 20 mM Tris-HCl, pH 8.0. The peak MW was estimated by comparing the substrate with protein standards running on the same column. The analytical column was calibrated with a series of individual runs of standard molecular mass proteins as markers, including bovine serum albumin (68 kDa), egg white albumin (43 kDa), ribose nucleotides (13.7 kDa), and antimicrobial peptides (5 kDa).
Circular dichroism (CD) spectra analysis
Peptides were dissolved in PBS (pH 7.4) at a concentration of 30 µM. The wavelength-dependence of molar ellipticity [q] was monitored at 25 °C using an average of eight scans in a spectropolarimeter (Model J-710, JASCO) with 0.2-cm path length cuvettes from 195-245 nm, equipped with a thermoelectric temperature controller. The buffers were filtered in a vacuum pump system with 0.2-μm pore membrane filters. Routine calibration of the machine was performed with d-10 camphor sulphonic acid (60 mg 100 ml-1) according to the equation [Q]=100 Q cnl-1, where Q is the ellipticity (mdeg), c is the peptide concentration (mM), n is the number of residues, and l is the path length (cm). Data analyses and acquisition were performed using the spectra manager software provided by the equipment manufacturer. On average, three scans were taken with a scanning rate of 200 nm min-1. The results were expressed as the mean residue ellipticity [Q] (10-3 degree cm2 dmol-1). A CD spectra analysis was performed to study the secondary structural changes in single peptides and any combination of two peptides (peptides mixtures) at an equimolar concentration in PBS. In these studies, single peptides were prepared at a concentration of 30 µM, and peptide mixtures at an equimolar concentration in a constant volume (for example, for each peptide mixture, the final concentration of each peptide was 15 µM).
Immunofluorescence assay
HCQ treatment of PEDV-infected Vero cells (Fig. 3C) growing on glass coverslips in 24-well plates were fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.1% Triton X-100 for 15 min,and blocked with phosphate-buffered saline (PBS) containing 10% horse serum plus 1% bovine serum albumin (BSA) (purchase from Solarbio life sciences) for 2 h. The cells were washed three times with PBS and incubated with diluted primary antibodies overnight at 4℃. Cells were washed with PBS three times at room temperature (RT), then incubated with fluorochrome-conjugated secondary antibodies in the dark for 1h at RT. The cells were then rinsed by PBS, mounted on glass slide and examined; images were captured using an microscope Olympus FluoViewÔ FV1000. The primary antibody to PEDV-N (1:1000) and the goat anti-mouse IgG (H+L) secondary antibody conjugated to Alexa Flour 488 (Invitrogen).
Statistics
All results were expressed as means and standard deviations (SD). Statistical analyses were performed using Prism 5.01 (GraphPad Software). Significance was determined by one-way analysis of variance (ANOVA) with Dennett’s multiple-comparison test.