Cells and virus culture
Madin Darby canine kidney (MDCK, CCL–34), Vero E6 (CRL–1586), RD (CCL136), LLC- MK2 (CCL–7), A549 (CCL–185) cells obtained from ATCC (Manassas, VA, USA) were cultured in Dulbecco minimal essential medium (DMEM) or MEM supplemented with 10% fetal bovine serum (FBS), 100 IU ml−1 penicillin and 100 μg ml−1 streptomycin. The virus strains used in this study included 2019 new coronavirus (SARS-CoV–2)43, SARS-CoV, MERS-CoV (hCoV-EMC/2012), A/Hong Kong/415742/2009, A/Hong Kong/415742Md/2009 (H1N1) (a highly virulent mouse-adapted strain), A/Anhui/1/2013 (H7N9)13, rhinovirus44 and human parainfluenza 3 (ATCC-C243). For in vitro experiments, viruses were cultured in MDCK, Vero E6, RD and LLC-MK2 cells. For animal experiments, H1N1 virus was cultured in eggs as described previously45.
Design and synthesis of peptides
P9, P9R, PA1 and P9RS were designed as shown in Fig. 1a and synthesized by ChinaPeptide (Shanghai, China). The purity of all peptides was>95%. The purity and mass of each peptide were verified by HPLC and mass spectrometry.
Plaque reduction assay
Antiviral activity of peptides was measured using a plaque reduction assay as we described previously14. Briefly, peptides were dissolved in 30 mM phosphate buffer containing 24.6 mM Na2HPO4 and 5.6 mM KH2PO4 at a pH of 7.4. Peptides or bovine serum albumin (BSA, 0.4–50.0 μg ml−1) were premixed with 50 PFU of coronaviruses (SARS-CoV–2, MERS-CoV, and SARS-CoV), influenza viruses (H1N1 virus and H7N9 virus), rhinovirus, or parainfluenza 3 in phosphate buffer at room temperature. After 1 h of incubation, peptide-virus mixture was transferred to Vero E6 for coronaviruses, MDCK for influenza viruses, RD for rhinoviruses, or LLC-MK2 for parainfluenza virus. At 1 h post infection, infectious media were removed and 1% low melting agar was added to cells. Cells were fixed using 4% formalin at 2–4 day post infection. Crystal blue (0.1%) was added for staining, and the number of plaques was counted.
Antiviral multicycle growth assay
Coronaviruses (SARS-CoV–2, MERS-CoV, and SARS-CoV), influenza viruses (H1N1 and H7N9 virus) and rhinovirus (0.005 MOI) were premixed with P9R or BSA (50–100 μg ml–1) in phosphate buffer for 1 h. After incubation, coronaviruses were inoculated onto Vero E6. Influenza viruses were inoculated onto MDCK cells. Rhinovirus was inoculated onto RD cells. After 1h infection, infectious media were removed and fresh media with supplemented P9R or BSA (50–100 μg ml–1) were added to infected cells for virus and cell culture. At 24–30h post infection, the supernatants of cells were collected for detecting viral RNA copies.
Cytotoxicity of peptides was determined by the detection of 50% cytotoxic concentration (CC50) using a tetrazolium-based colorimetric MTT assay as we described previously13. Briefly, cells were seeded in 96-well cell culture plate at an initial density of 2 × 104 cells per well in MEM or DMEM supplemented with 10% FBS and incubated for overnight. Cell culture media were removed and then DMEM supplemented with various concentrations of peptides and 1% FBS were added to each well. After 24 h incubation at 37 °C, MTT solution (5 mg ml−1, 10 μl per well) was added to each well for incubation at 37 °C for 4 h. Then, 100 μl of 10% SDS in 0.01M HCl was added to each well. After further incubation at room temperature with shaking overnight, the plates were read at OD570 using VictorTM X3 Multilabel Reader (PerkinElmer, USA). Cell culture wells without peptides were used as the experiment control and medium only served as a blank control.
Peptide-virus binding assay
Peptides (0.1 μg per well) dissolved in H2O were coated onto ELISA plates and incubated at 4 °C overnight. Then, 2% BSA was used to block plates at 4°C overnight. For virus binding to peptides, viruses were diluted in phosphate buffer and then were added to ELISA plate for binding to the coated peptides at room temperature for 1h. After washing the unbinding viruses, the binding viruses were lysed by RLT buffer of RNeasy Mini Kit (Qiagen, Cat# 74106) for viral RNA extraction. Viral RNA copies of binding viruses were measured by RT-qPCR.
ELISA assay was done as described previously14. Peptides (0.1 μg per well) dissolved in H2O were coated onto ELISA plates and incubated at 4 °C overnight. Then, 2% BSA was used to block plates at 4 °C overnight. For HA and S binding, 150 ng HA1 or S in solution I buffer (Sino Biological Inc., Cat# 11055-V08H4) was incubated with peptides at 37 °C for 1 h. The binding abilities of peptides to HA1 or S proteins were determined by incubation with rabbit anti-His-HRP (Invitrogen, Cat# R93125, 1: 2,000) at room temperature for 30 min. The reaction was developed by adding 50 μl of TMB single solution (Life Technologies, Cat# 002023) for 15 min at 37 °C and stopped with 50 μl of 1 M H2SO4. Readings were obtained in an ELISA plate reader (Victor 1420 Multilabel Counter; PerkinElmer) at 450 nm.
Viral RNA extraction and RT-qPCR
Viral RNA was extracted by Viral RNA Mini Kit (QIAGEN, Cat# 52906, USA) according to the manufacturer’s instructions. Real-time RT-qPCR was performed as we described previously14. Extracted RNA was reverse transcribed to cDNA using PrimeScript II 1st Strand cDNA synthesis Kit (Takara, Cat# 6210A) using GeneAmp® PCR system 9700 (Applied Biosystems, USA). The cDNA was then amplified using specific primers (Table S1) for detecting SARS-CoV–2, MERS-CoV, SARS- CoV, H1N1, H7N9, and rhinovirus using LightCycle® 480 SYBR Green I Master (Roach, USA). For quantitation, 10-fold serial dilutions of standard plasmid equivalent to 101 to 106 copies per reaction were prepared to generate the calibration curve. Real-time qPCR experiments were performed using LightCycler® 96 system (Roche, USA).
Endosomal acidification assay
Endosomal acidification was detected with a pH-sensitive dye (pHrodo Red dextran, Invitrogen, Cat#P10361) according to the manufacturer’s instructions as previously described but with slight modification14. First, MDCK cells were treated with BSA (25.0 μg ml−1), P9 (25.0 μg ml−1), P9R (25.0 μg ml−1), PA1 (25.0 μg ml−1), or P9RS (25.0 μgml−1) at 4 °C for 15 min. Second, MDCK cells were added with 100 μg ml−1 of pH-sensitive dye and DAPI and then incubated at 4 °C for 15 min. Before taking images, cells were further incubated at 37 °C for 15 min and then cells were washed twice with PBS. Finally, PBS was added to cells and images were taken immediately with confocal microscope (Carl Zeiss LSM 700, Germany).
Colocalization assay of peptide binding to virus in cells
H1N1 virus was labeled by green Dio dye (Invitrogen, Cat#3898) according to the manufacture introduction. DIO-labeled virus was treated by TAMRA-labeled P9R and TAMRA-labeled P9RS for 1h at room temperature. Pre-cool MDCK cells were infected by the peptide-treated virus on ice for 15 min and then moved to 37 for incubation for 15 min. Cells were washed twice by PBS and then fixed by 4% formalin for 1h. Nuclei were stained by DAPI for taking images by confocal microscope (Carl Zeiss LSM 700, Germany).
Nucleoprotein (NP) immunofluorescence assay.
NP staining was carried out as described previously14. MDCK cells were seeded on cell culture slides and were infected with A(H1N1)pdm09 virus at 1 MOI pretreated with BSA (25.0 μg ml−1), bafilomycin A1 (50.0 nM) or P9R (25.0 μg ml−1). After 3.5 h post infection, cells were fixed with 4% formalin for 1 h and then permeabilized with 0.2 % Triton X–100 in PBS for 5 min. Cells were washed by PBS and then blocked by 5% BSA at room temperature for 1 h. Cells were incubated with mouse IgG anti-NP (Millipore, Cat# 2817019, 1:600) at room temperature for 1 h and then washed by PBS for next incubation with goat anti-mouse IgG Alexa–488 (Life Technologies, Cat# 1752514, 1:600) at room temperature for 1 h. Finally, cells were washed by PBS and stained with DAPI. Images were taken by confocal microscope (Carl Zeiss LSM 700, Germany).
NMR Structure analysis of P9R
Freshly prepared 1 mg ml−1 (0.29 mM) of P9R in 0.5 ml solvent was used for the NMR study. Data were collected in H2O/D2O (19:1 v/v), as well as 99.996% D2O, with the internal reference trimethylsilylpropanoic acid. All NMR spectra were acquired on either a Bruker AVANCE III 600 MHz spectrometer (Bruker BioSpin, Germany) or a Bruker AVANCE III 700 MHz spectrometer at 25˚C. 2D 1H–1H correlation spectroscopy (COSY), total correlated spectroscopy (TOCSY) and nuclear Overhauser effect spectroscopy (NOESY) spectra were recorded for resonance assignments. Inter- proton distance restraints were derived from 2D NOESY spectrum with mixing times of 300 ms and 500 ms using automated NOE assignment strategy followed by a manual check. NOE intensities and chemical shifts were extracted using CCPNMR Analysis 2.4.246 and served as inputs for the Aria program. Dihedral angle is predicted from the chemical shifts using the program DANGLE47. The NMR solution structure of P9R was calculated iteratively using Aria 2.3 program48. One hundred random conformers were annealed using distance restraints in each of the eight iteratively cycles of the combined automated NOE assignments and structure calculation algorithm. The final upper limit distance constraints output from the last iteration cycle were subjected to a thorough manual cross- checking and final water solvent structural refinement cycle. The 10 lowest energy conformers were retained from these refined 100 structures for statistical analysis. The convergence of the calculated structures was evaluated using root-mean-square deviations (RMSDs) analyses. The distributions of the backbone dihedral angles (φ, ψ) of the final converged structures were evaluated by representation of the Ramachandran dihedral pattern using PROCHECK-NMR49. Visualization of three-dimensional structures and electrostatic surface potential of P9R were achieved using UCSF Chimera 1.13.150.
Antiviral analysis of P9R in mice
BALB/c female mice, 10–12 weeks old, were kept in biosafety level 2 laboratory and given access to standard pellet feed and water ad libitum. All experimental protocols followed the standard operating procedures of the approved biosafety level 2 animal facilities and were approved by the Committee on the Use of Live Animals in Teaching and Research of the University of Hong Kong45. The mouse adapted H1N1 virus was used for lethal challenge of mice. To evaluate the therapeutic effect, mice were challenged with 3 LD50 of the virus and then intranasally inoculated with PBS, P9, P9R, PA1 or zanamivir at six hours after the viral inoculation. Two more doses were given to H1N1- challenged mice at the following one day. Survival and general conditions were monitored for 16 days or until death.
Survival of mice and the statistical significance were analyzed by GraphPad Prism 5. The statistical significance of the other results was calculated by the two-tailed Student t test using Stata statistical software. Results were considered significant at P < 0.05.