Virus propagation in mosquito cells and titration
HepG2 cells with passage number less than 60 were maintained in DMEM medium supplemented with 10% FBS and incubated at 37°C in 5% CO2. HepG2 cells were used to study the peptide cytotoxicity and antiviral activity. Dengue virus serotype-2 (DENV2) was first propagated in C6/36 cells. The DENV2-infected cells that showed cytopathic effects (CPE) were lysed with a freeze and thaw cycle. The culture medium was then centrifuged at 1800 rpm for 10 min to remove the cell debris, filtered (0.2 μm), portioned into aliquots and stored at -80°C until use. The viral titre of the DENV2 suspension was established by serial dilutions on Vero cells using a plaque assay.
The Ltc 1 peptides were manufactured chemically using standard solid-phase peptide synthesis with a Symphony parallel synthesiser (Protein Technologies, Tucson, AZ, USA) as previously described
. Briefly, the aqueous phase of the peptide synthesis was lyophilised to yield the crude peptide. The identity of the crude peptide was confirmed by LC-MS, and purification of the crude peptide was performed by RP-HPLC (Agilent 1200 series, USA). The identity of the 98% pure purified peptide was confirmed by LC-MS (Shimadzu LC/MS 2020, single quad, Japan). The purified peptide was then lyophilised using a Savant AES 2000 Automatic Environmental SpeedVac system. To prepare 2 mM pure peptide, 6.14 mg lyophilised peptide was dissolve into 1 ml filtered-deionised water for use as a stock solution.
The interaction between the Ltc 1 peptide and dengue NS2B-NS3pro was identified by protein-protein docking study. The Protein Data Bank (PDB) files of Ltc 1 (2PCO) and NS2BNS3pro (4M9F) were used in rigid global docking using an available online server (FireDoc, http://bioinfo3d.cs.tau.ac.il/FireDock/refs.html) as described previously
[23, 24]. The results of the protein-protein docking were further analysed using Discovery Studio software version 3.5.
ELISA binding of Ltc 1 to dengue NS2B-NS3pro
Enzyme-linked immunosorbent assay (ELISA) was used to examine the binding affinity of Ltc 1 to dengue NS2B-NS3pro. Increasing concentrations of purified dengue NS2B-NS3pro (0, 20, 30 and 50 nM/well) in carbonate/bicarbonate buffer (Sigma, USA) were bound to black 96-well plate with transparent bottom at 4°C overnight in triplicates. The wells were blocked with PBS containing 0.05% Tween 20 (PBS-T) plus 0.5% BSA for 1 h at room temperature and washed three times with PBS-T. Increasing concentrations of the Ltc 1 peptide labeled with FITC fluorescence dye (0, 0.1, 0.5, 1, 5, 10, 20, 30, 50 nM) were prepared in PBS-T plus BSA; 100 μl of each dilution of the Ltc 1 bound to plates for 3 h on ice in dark place. After the plates were washed, the fluorescence signals of bound Ltc 1 were detected using Tecan Infinite M200 Pro fluorescence spectrophotometer (Tecan Group Ltd., Switzerland).
Dengue NS2B-NS3 protease (NS2B-NS3pro) assay
The NS2B-NS3pro assay was performed to examine whether the Ltc 1 peptide inhibits the DENV2 serine protease
]. Briefly, a single chain NS2B (G4
) NS3pro was produced as a recombinant protein in E. coli
as previously described
]. The end point reaction mixture was performed in black 96-well plates, which contained 2 μM recombinant NS2B-NS3pro, 100 μM fluorogenic peptide substrate (Boc-Gly-Arg-Arg-AMC) and varying concentrations of the Ltc 1 peptide (0.1 to 40 μM) buffered at pH 8.5 with 200 mM Tris-HCl in a total volume of 200 μl. The reaction mixtures without peptide, substrate with the peptides, enzyme and different concentrations of the peptides were used as controls. Thereafter, all reaction mixtures were incubated at either 37°C or 40°C for 30 min, and the substrate was added to the specific reaction mixtures and incubated at the same temperatures for an additional 30 min. Measurements were performed in triplicate using a Tecan Infinite M200 Pro fluorescence spectrophotometer (Tecan Group Ltd., Switzerland). Substrate cleavage was normalised against the buffer only (control) at an emission wavelength of 440 nm with excitation at 350 nm. The fluorescence values obtained with the no-inhibitor control (0.0 μM peptide) were set at 100%, and those in the presence of peptide were calculated as a percentage of the control using non-linear regression in GraphPad Prism (version 5.01) software. The IC50
was calculated from nonlinear regression fitting of the signal vs.
concentration data points to the standard dose–response equation Y
)/(1 + 10^
In this equation, X
is the log of the compound concentration, Y
is the response signal, and the bottom and top refer to the plateaus of the sigmoid response curve. All assays were performed in triplicate and repeated twice. The inhibition percentage was calculated using the following formula:
Ltc 1 peptide cytotoxicity
The cytotoxicity of the Ltc 1 peptides was evaluated by determining the maximal non-toxic dose (MNTD) and the 50% cytotoxic concentration (CC50) of the cells using the Non-Radioactive Cell Proliferation assay (Promega, USA) according to the manufacturer’s instructions. The peptide concentration of 25 μM showed 80% cell viability and was considered the MNTD value, assuming that approximately 80% of the cells were healthy. Vero cells were seeded at 1×104 cells/well in triplicate under optimal conditions (37°C, 5% CO2 in a humidified incubator) in 96-well plates with blank controls (media only) and cell controls (cells only). After an overnight incubation, the cells were treated with increasing concentrations of Ltc 1 peptide (0, 4, 8, 16, 32, 64 and 120 μM) with DMEM medium supplemented with 2% FBS and the cell culture was analysed after 72 h. The percentage of cell viability was calculated as follows: 100 - (absorbance of treated cells/absorbance of untreated cells) × 100. The MNTD and CC50 values were calculated from the dose-response curves.
Real Time Cell Proliferation Assay (RTCA assay)
This assay was performed to test the real time effects of the Ltc 1 peptide on cell viability. Cell proliferation was measured using the xCELLigence Real-Time Cellular Analysis (RTCA) system (Roche, Germany) as described previously
. Cell viability and growth were monitored continuously after applying increasing concentrations of the Ltc 1 peptide (0, 12.5, 25, 50, 100, 150, 200, 250 μM). Briefly, the background measurements were recorded after adding 100 μl culture medium to the wells. Next, the cells were seeded at a density of 1 × 104 cell/well in a 16-well plate with electrodes for 18 h to allow the cells to grow to log phase. The cells were treated with different concentrations of peptide dissolved in cell culture medium and continuously monitored for up to 100 h. The cell sensor impedance was expressed as an arbitrary unit named the cell index. The cell index was recorded every 5 minutes using a RTCA analyser. To eliminate variation between the wells, the cell index values were normalised to the value at the beginning of the treatment.
Treatment of DENV-infected cells with the Ltc 1 peptide
To infect the HepG2 cells with DENV2, the cells were cultured in 24-well plates (1.5 × 105 cells/well) for 24 h at 37°C and 5% CO2. The virus supernatant was added to the cells at a MOI of 2, followed by incubation for 1 h with gentle shaking every 15 min for optimal virus to cell contact. The cells were washed twice with fresh serum-free DMEM after removal of the virus supernatant. Then, fresh complete DMEM containing 25 μM Ltc 1 peptide was added to the cultures and incubated for 72 h. The HepG2 cells were then collected, and the virus particles and expression level of the viral NS1 protein were examined using immunostaining and western immunoblotting.
This assay was performed to identify the mode of antiviral activity of the Ltc 1 peptide against DENV2 entry, replication and release from the infected cells. Three independent experiments were performed in triplicate for pre-, simultaneous and post-infection treatments. HepG2 cells were grown in a 24-well tissue culture plate (1.5 × 105 cells/well), incubated 24 h under optimal conditions and infected with DENV2 at an MOI of 2. For pre-treatment infection, 25 μM peptide was added to the cells before virus inoculation and incubated for 24 h. After removal of the old medium containing the peptide, the DENV2 supernatant was added, followed by incubation for 1 h with gentle shaking every 10 min for optimal virus to cell contact. The virus supernatant was removed and the cells were washed twice with fresh serum-free DMEM medium to remove the residual virus. Fresh complete DMEM medium was added and the cultures were incubated for 72 h at 37°C, supplemented with 5% CO2. Identical applications were performed for the simultaneous treatment, except the peptide was mixed with the virus supernatant and incubated at 37°C for 1 h, and then inoculated onto the HepG2 cells. The post-treatment infection was performed after inoculation of the HepG2 cells with DENV2, and complete DMEM medium with the Ltc 1 peptide was then added. The cultures including the peptide were incubated for 72 h at 37°C and 5% CO2, and three wells of infected cells in each experiment were maintained without treatment as controls. The cell supernatants were collected and stored at -80°C for viral load determination using a plaque formation assay.
This assay was performed to evaluate the 50% effective concentration (EC50) of the Ltc 1 peptide against DENV2. HepG2 cells were grown in six-well microplates (1.5 × 106 cells/well) for 24 h in quadruplicate experiments. The cell culture media were removed and the cells were washed three times with PBS. Then, fresh medium containing the virus supernatant was added at MOI of 2, followed by incubation for 1 h with gentle shaking every 15 min. The viral residues were removed by washing with PBS, and serial dilutions of the Ltc 1 peptide (0, 2.5, 5, 10, 20, 40, 80 μM) were added. The cultures including the peptide were incubated for 72 h at 37°C and 5% CO2. The cell supernatants were collected and stored at -80°C for viral load determination using viral RNA and were quantified using one step qReal time-PCR.
Virus quantification by plaque formation assay
To determine the virus yield after treatment with different concentrations of peptide, the culture supernatants were collected and serially diluted to reduce the effects of the drug residues. A 10-fold serial dilution of medium supernatant was added to new Vero cells grown in 24-well plates (1.5 × 105
cells) and incubated for 1 hr at 37°C. The cells were then overlaid with DMEM medium containing 1.1% methylcellulose. The viral plaques were stained with crystal violet dye after a five-day incubation. The virus titres were calculated according to the following formula:
Cells lysates were prepared for immunoblotting against dengue viral antigen using ice-cold lysis buffer. The amount of protein in the cell lysates was quantified to ensure equal loading (20 μg) of the western blot gels using the 2-D Quant Kit (GE Healthcare Bio-Sciences, USA) according to the manufacturer’s instructions. The separated proteins were transferred onto nitrocellulose membranes and then blocked with blocking buffer. The membrane was incubated overnight with anti-DENV2 antibody specific to the viral NS1 protein (Abcam, UK, Cat. no. ab41616) and an anti-beta actin antibody (Abcam, UK, Cat. no. ab8226). After washing three times, the membranes were incubated with anti-mouse IgG conjugated to horseradish peroxidase (Dako, Denmark) at 1:1,000 for two h. Horseradish peroxidase substrate was added to for colour development.
To examine the efficacy of the Ltc 1 peptide for reducing viral particles, HepG2 cells were grown on cover slips in 6-well plates and infected with DENV2 at an MOI of 2. The DENV2-infected cells were then treated with 25 μM peptide for 24 h. The cells were washed three times with PBS to remove the peptide residues and then fixed with ice-cold methanol for 15 min at -20°C. After washing, the cells were incubated with coating buffer for 1 h at room temperature. A mouse antibody specific to the dengue envelop glycoprotein (Abcam, UK, Cat. no. ab41349) was added, and the cells were incubated overnight at 4°C. The cells were washed three times with PBS and incubated for 30 min with an anti-mouse IgG labelled with FITC fluorescent dye (Invitrogen, USA, Cat. no. 62-6511). To stain the cell nuclei, Hoechst dye was added (Invitrogen, USA, Cat. no. H1399) for the last 15 min of the incubation.
Viral RNA quantification
The DENV2 copy number was quantified in the culture supernatants using one-step quantitative real-time PCR. Known copies of the viral RNA were 10-fold serially diluted to generate a standard curve. The viral RNA was extracted using the QIAmp viral RNAmini kit (QIAGEN, Germany), and the qRT-PCR was performed using a SYBR Green Master Kit (Qiagen, Germany). Triplicate reactions were performed for each sample, and a no template control was included as a negative control. Absolute quantification was performed using an ABI7500 machine (Applied Biosystems, Foster City, CA). The results were analysed using Sequence Detection Software Version 1.3 (Applied Biosystems, Foster City, CA). The percentage of viral inhibition (%) was calculated as follows: 100 – (viral copy number of treated cells/viral copy number of untreated cells) × 100.
All the assays were performed in triplicate, and the statistical analyses were performed using GraphPad Prism version 5.01 (GraphPad Software, San Diego, CA). P values <0.05 were considered significant. The error bars are expressed as ± SD.