SARS-CoV (String 200300592): A Disease Control and Prevention Study in a Biosafety Level 3 Laboratory at the Leiden University Medical Center
rSARS- CoV-2 WT-USA-WA1, rSCRS-CoV-2 WT-USA-WA3- 3CLpro: L50F. SARS-CoV, strain 200300592, was obtained from the Centers for Disease Control and Prevention. There were live work done in the bio safety level 3 and A3 high-containment facilities of the KU Leuven Rega Institute. SBB 2192018 0884 and AMV 2310/2017 are according to institutional guidelines. All work with live SARS-CoV (strain Frankfurt-1) and MERS-CoV (strain Jordan N3/2012) virus was done in a biosafety level 3 laboratory at the Leiden University Medical Center.
In Fig. The images are representative of H&E-stained left lung lobes of Syrian golden hamsters that have been treated for the disease. A full cross-section of each animal’s left lung was assessed in this experiment.
Micrograph of the SARS-CoV-2 M-FabB -JNJ-9676 Experiment. In vitro Synthesis and In Vitro Infections
In Extended Data Fig. 2g, uncut western blots are shown of purified M proteins. These blots were generated once as a quality control of the protein obtained.
In a data table. 6a, a micrograph from the SARS-CoV-2 M–FabB–JNJ-9676 data collection is shown. There were more than 12 thousand images taken to create this image.
The synthesis of JNJ-9676 is described in patent WO-2024/008909 and in the Supplementary Methods. Molnupiravir was ordered at MedChemExpress (HY-135853) and nirmatrelvir was synthesized according to literature procedures59. For in vitro experiments, JNJ-9676, molnupiravir or nirmatrelvir was dissolved in 100% dimethyl sulfoxide (DMSO) as a 5–100 mM stock. In a trial, JNJ-9676 was dissolved in 100% PEG as a stock of 75, 25 or 8.33 million units, and as a stock of 300 million units.
Previously described procedures52 were followed. In brief, human airway nasal epithelial cells (MucilAir pool of donors, product code EP02) were obtained from Epithelix in an air–liquid set-up. The inserts were washed with 1 PBS before they were placed in Muccliair medium, which was maintained for at least four days before use. On the day of the experiment, the cultures were pretreated with basal medium containing compounds at different concentrations for 1 h before infection with 100 μl SARS-CoV-2 inoculum (1,000 TCID50 per insert) at the apical side for 1.5 h, after which the viral inoculum was removed. Viral release from the cultures was measured by washing the apical sides with 250 µl Mucilair medium and determination of the viral load by RT–qPCR or titration. After a few days, the medium in the basolateral side of the cultures was refreshed. During the first few days of the infection, 35 C and 5% CO2 were used.
A mixture of SARS- CoV-2 M and FabB was placed into ice for one hour. The SARS-coV-2m–FabB complex was loaded into a Superose 6 Increase 10/300 GL column with buffer. NaCl, 0.001% LMNG (w/v), 0.0001% CHS (w/v), 0.00033% GDN (w/v)). The peak fractions containing the SARS-CoV-2 M–FabB complex were pooled, 100 µM JNJ-9676 was added and incubated for 1 h on ice. The sample was diluted to 0.2–0.8 mg ml−1 with size-exclusion chromatography (SEC) buffer containing 100 µM JNJ-9676 for cryo-EM.
The isolated, infectious clones of PgCoV and RsSHC014 were the source for theombinant viruses.
Toxicity assessment of the treated but uninfected cell using an MTS (3-Dimethylthiazol-2-Yl)-5-(3-Carboxymethoxyphenyl)-2-(4
Cytotoxicity was evaluated on day 5 in treated but uninfected cells using an MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt) reduction assay65.
The Toxicity assessment was done by exposing non-insurance inserts to the same concentration of JNJ-9676 as was done for antiviral treatment, in order to verify the integrity of the cell layer. Brefeldin (0.3 µM; internally synthesized) was used as a toxicity control.
Purified SARS-CoV-2 nsp14 (N7-MTase) and nsp10/nsp16 (2′O-MTase) were incubated in a reaction mixture containing 40 mM Tris-hcl has one mM DTT, 1 mM MgCl2, 2 M SAM and 0.1 M. 3H-SAM (Perkin Elmer), in the presence of either 0.7 μM GpppAC4 or 7m GpppAC4 synthetic RNA79. The compounds were suspended in less than 3% of their final concentration. Reactions were stopped after 30 minutes of dilution in ice-cold water. Samples were transferred to a diethylaminoethyl filter mat (Perkin Elmer) using a Filtermat Harvester (Packard Instruments). The mats were washed with water, water, water and a small amount of formate, 10 mM. They were soaked with scintillation fluid, and the 3H-methyl transfer was determined using a wallac MicroBeta TriLux liquid scintivity counter.
pcDNA3.4 vectors containing FabB heavy chain and light chain were co-transfected into Expi293F cells (Invitrogen) according to the manufacturer’s protocol and incubated for 96 h at 37 °C with 8% CO2.
Conditioned medium was loaded onto a 10 ml HisTrap excel column (Cytvia) at a flow rate of 8 ml min−1. The column was washed with 6 CV of wash buffer (20 mM sodium phosphate pH 6.5, 150 mM A 39.2–500 mM imidazole gradient prepared in a buffer is used for eluted with over 5 CV. 500 mM imidazole is NaCl. The peak fractions of FabB were then put onto a buffer of 20 mM with the help of a HiLoad 16/600 Superdex 75 pg column. It’s NaCl.
Experiments were performed in a total volume of 10 µl. The Prometheus NT.Plex instrument was used to measure the melting temperatures. The samples were prepared in a 384-well plate with 0.5 mg ml−1 purified recombinant SARS-CoV-2 M and 100 µM of JNJ-9676 in 20 mM HEPES pH 7.5, 150 mM NaCl, 0.001% LMNG (w/v), 0.0001% CHS (w/v), 0.00033% GDN (w/v) and 1% DMSO (v/v). The samples were loaded into the glass capillaries with a temperature range of 25–95 C, and the temperature was changed to 1 C for each sample. The data were analysed using PR.ThermControl v.2.1.6 (NanoTemper Technologies) (technical replicates ≥ 3).
The offline ASMS experiment consisted of the preparation of three sample types: compound QC, protein target (M protein) and no-protein control (breakthrough).
For the preparation of SEC filter plates for offline ASMS, 130 µl of pre swollen Bio-Gel P10 resin slurry was added to each well of a low-protein-binding Millipore HTS 384 HV filter plate (hereafter, size-exclusion plate) with a 0.45 µm Durapore (PVDF) membrane (MZHCN0W10). The size-exclusion plate was placed into a 4 °C refrigerated centrifuge, centrifuged at 1,000g for 2 min and the flowthrough was discarded. After washing each single one, the cartridge was put into a 50 l buffer and then washed four more times. NaCl, 0.001% LMNG, 0.0001% CHS, 0.00033% GDN and 2% DMSO, whereby the flowthrough from each wash was discarded after centrifugation at 1,000g for 2 min. The ASMS plate was prepared using an acoustic liquid handler and a maximum of 20 lm of 5 mM of compound was transferred from the source plate to four separate wells. An aliquot of purified recombinant M protein stock solution was thawed on ice, then diluted using assay buffer to a working concentration of 5 µM and 2% DMSO. The compound was put into three wells which yielded a final concentration of 5 M. To control for compound breakthrough of the SEC resin, either in-solution or through micelle partitioning, a separate working stock was prepared without protein and dispensed as a 20 µl aliquot into the remaining compound well. The plate was put into a solution and put into a petri dish for 30 min at 25 C.
All of the samples were transferred to the size-exclusion plate, which was quickly centrifuged at 1,000g for 2 min at 4 °C to minimize compound breakthrough. The flowthrough was partially immersed with 15 lms-level water (Honeywell) in order to reduce the detergent concentration and then completely immersed at 2000g per minute for 5 min to collect any insoluble particles.
A 5% compound was transferred from the source plate into a 384-well plate and 25 l of acetonitrile, 2% DMSO was added to the sample.
The LC–MS analyses were performed using an adler 1290 Infinity II and an accuracy 6556XT qTOF combined with the adler MassHunter software. A 4 µl sample injection was loaded with water as a loading solvent onto the reversed-phase column (2.1 × 35 mm ACQUITY UPLC BEH C18 column, 130 Å, 1.7 µm), heated to 40 °C. The mobile phases consisted of water and acetonitrile and each contained 0.4% formic acid. The LC method used a constant flow rate of 0.1 ml min−1 and consisted of a 1 min wash with 5% solvent B, a steep gradient from 5% to 20% B over 0.1 min, a subsequent shallow gradient from 5% to 95% B over 1.9 min, followed by a hold for 1 min and a return to 5% B in 0.1 min with a 0.9 min hold. The instrument was operated in positive polarity mode with centroided data acquisition and had a source set to 350 C drying gas temperature and 13 min1 drying gas flow rate. A reference mass solution consisting of purine and HP-0921 (Agilent, G1969-85001) was prepared according to the manufacturer’s instructions and infused to apply automatic mass correction to all spectra acquired from 110 to 1,100 m/z at a rate of 1 spectrum per second.
The data were processed using a mass error tolerance window of 3 parts per million.
The cryo-EM cleaning of the PELCO easiGlow discharge grids using a Vitrobot (Thermo Fisher Mark IV)
The PELCO easiGlow Discharge Cleaning System was used to remove glow discharge from the mesh grids. A total of 3 µl recombinant M protein sample (0.8 mg ml−1), prepared as described above, was applied to the EM grids, which were vitrified with a Vitrobot (Thermo Fisher Mark IV) using the following settings: blot time 4 s, blot force 0, wait time 0 s, inner chamber temperature 4 °C, and 100% relative humidity. Liquid nitrogen was used for the freezing of liquid ethane. Cryo-EM data collection was automated on the 200 kV Thermo Scientific Glacios microscope controlled by EPU software. At 105,000 magnification, the microscope was taken using a Gatan detector. Each 6 s exposure recorded 40 frames with a total dose of 40 e− Å−2. The digital micrographs had a scuple size of 0.910.
Cryo-EM data collection and image quality were monitored using cryoSPARC Live v.3.2. The image preprocessing steps were performed at the same time. A total of 12,988 raw micrographs was recorded during a 4-day data collection session using the Glacios microscope. Acceptable 2D classes served as templates for particle repicking. About 1.2 million good particle images were produced by a single round of live 2D image classification. These particles were used for 3D reconstruction. The first round of five starting 3D models were calculated, resulting in one major 3D class, followed by a second round of four 3D classes. One major class underwent non-uniform 3D refining using 484,610 particles and has an average resolution of 3.06 on a 3D EM map.
Resolutions were estimated by applying a soft mask around the protein complex density using the gold-standard (two halves of data refined independently) The criterion is 0.143. Before visualization, all density maps were sharpened by applying different negative temperature factors along with the half maps and used for model building. Local resolution was determined using ResMap. Detailed statistics about the cryo-EM data processing can be found in Extended Data Fig. 6a–f.
Initially, a previously determined SARS-CoV-2 M structure (PDB: 8CTK; ref. 34) and an Alphafold2 model62 of Fab–B were rigid-body fitted into the M–Fab–B-CIM map using the UCSF Chimera Fit in map tool63. The initial round of flexible fitting was done using the combined model and then manually adjusted in Coot65. The ligands builder tool was used to add the compound during the last step. When selecting the pose of CIM-834, the following were taken into account. The density’s phemetres are first. The piperidine and pyrimidine rings ofCIM-734 were similar to the flat plate-like morphology of the density closer to the M two-fold symmetry axis. The density for the opposing end of the density was thinner and less well resolved (Extended Data Fig. 6), consistent with the increased flexibility of the molecule beyond the amide position. There is a position of hydrogen-bonding partners. The selected orientation of CIM-834 positions the pyridazine ring in proximity to S99 and N117, allowing for potential direct or water-mediated hydrogen bonds. iterative cycles of manual model building using Coot65, real-space refinement, and eLBOW were used to refined the model. The model validation was done with the help of a program called Molprobity68.
Blind scoring of lung tissue sections with Eosin and haematoxylin for lung histopathology using GraphPad Prism
After staining the fixed lung tissue sections with haematoxylin and Eosin, they were scored blindly for lung damage by an expert pathologist. The scored parameters, (cumulative score, 1 to 3), were as follows: congestion, intra-alveolar haemorrhagic, apoptotic bodies in the bronchus wall, necrotizing bronchiolitis, perivascular oedema, bronchopneumonia, perivascular inflammation, peribronchial inflammation and vasculitis.
All statistical analyses were performed in GraphPad Prism v.9.5.0 and validated using R (v.3.6.1). A log10 transformation was applied to the lung viral-load data (RNA and infectious virus) to approximate normality. The mean differences between the treatment groups and the vehicle group were estimated using the one-way analysis of variance with Šídák’s multiplicity correction to account for multiple testing.
In the case that normality could not be assumed for the outcome variable or in case of lung histopathology, the nonparametric Kruskal–Wallis test by ranks was applied. After the post hoc Dunn’s test with the Benjamini–Hochberg’s correction was applied, multiple testing was taken into account. The significance level was used.
The graphs were prepared using GraphPad Prism. Figures and schemes were created using BioRender.com and Adobe Illustrator 28.1 (Windows).
All statistical comparisons in the study were done in GraphPad Prism (v.6.07, v.8.3.0, v.9.2.0, v.10.1.2 and v.10.2.3) and the statistical tests used are indicated in the figure legends. The 3 independent preparations which included the 2DEMs for the quantification of the genes were used to acquire them. Tomography was performed on a number of technical samples previously characterized in 2D, but only the ones that were prepared in a single preparation were used.
Determination of the microsomal stability and active enzyme activity of a protein-containing mixture using a biomek I5 automate
Compound microsomal stability was determined using mouse microsomal fractions (Gibco; final protein concentration, 0.5 mg ml−1), with hamster microsomal fractions (Xenotech; final protein concentration, 0.5 mg ml−1) and with human microsomal fractions (Xenotech; final protein concentration, 0.5 mg ml−1) at a substrate concentration of 1 μM with or without NADPH (final concentration, 1 mM). Incubations were done for 120 minutes at 37 C. At various time points (5, 15, 30, 60 and 120 min), 25 μl of the reaction mixture was sampled and quenched in 300 μl of acetonitrile containing internal standard. The suspension was whirled and then sucks the water out of the supernatant. The resulting solution was analysed by liquid chromatography with tandem mass spectrometry to determine the half-life of the compound.
The IC50 is determined by the percentage of active enzymes and the amount of activity that is reflected in the light. The IC50 was determined from curve fitting using Prism software. For each measurement, results were obtained in triplicate.
The reaction assays had to be stopped by 20 l EDTA. 5% DMSO or 100 mM was used in the reaction mix for the positive and negative controls. Reaction mixes were transferred to a Greiner plate using a Biomek I5 automate. 60 l of PicoGreen fluorescent reagent was distributed to the Greiner plates according to the manufacturer’s instructions. The plate was incubated for 5 min in the dark at room temperature and the fluorescence signal was then read at 480 nm (excitation) and 530 nm (emission) using a Tecan Safire2 and/or a ClarioStar.
Reactions were done in a volume of 40 µl on a 96-well Nunc plate. The experiments were robotized with the help of the BioMek I5 automate. Then, 2 µl of each diluted compound in 100% DMSO was added in wells to the chosen concentration (5% DMSO final concentration). For each assay, the (nsp8L7 + nsp8) mix was distributed in wells after an 8-min incubation at room temperature to pre-form the active complex. After 8 min., Nsp12 was added to wells. Reactions were started by adding the UTP + poly(A) template mix and were incubated at 30 °C for 20 min, using 350 nM of poly(A) template and 750 µM of UTP final concentration.
The compound concentrations leading to 50% inhibition of polymerase-mediated RNA synthesis was determined in IC50 buffer (50 mM HEPES, pH 8.0, 10 mM KCl, 2 mM MnCl2, 2 mM MgCl2 and 10 mM DTT) containing seven increasing concentrations of compound (from 1 µM to 100 µM) and 150 nM of nsp12 (ref. The complex has 450 nM nsp8 and 450 nM nsp8L7.
The FRET was performed in black 384-well HiBase Non-binding Plates and they were used for activity and inhibition. In brief, increasing concentrations of inhibitor were incubated with purified PLpro protein (55 nM) in the presence of 5 μM of a fluorescent synthetic peptide (Dabcyl-FTLKGG↓APTK-Edans, Genscript) in HEPES buffer (20 mM, pH 6.5) containing 120 mM NaCl, 0.4 mM 4% DTT, 4% EDTA, and 10% glycerol. The final concentration was raised to a bit over 1%. Cleavage of the fluorogenic peptide separates the Edans/Dabcyl fluorophore–quencher pair. The 40 min follow up of the enzymatic reaction was done by using a TeCan Safire2 fluorimeter which would show the increase in emission at 335 nm. Enzymatic activities were estimated by calculating the slope of the linear part of the reaction curve and were normalized with respect to the activity measured in the absence of inhibitor.
A fluorescent substrate comprising the cleavage site of SARS-CoV-2 Mpro (Dabcyl-KTSAVLQ↓SGFRKM-E(Edans)-NH2) and buffer composed of 20 mM HEPES, 120 mM NaCl, 0.4 mM EDTA, 4 mM DTT, 20% glycerol, pH 7.0, was used for the inhibition assay. The Edans generated as a result of the Mpro cleavage was observed at an emission wavelength of 465 nm using a Tecan Spark multimode microplate. The compound was diluted with 100% DMSO to prepare a stock solution. The IC50 was determined after 10 minutes in reaction buffer at 37 C with concentrations from 0 to 500 M. The final concentration of FRET was added to each well and the reaction began at a final amount of 100 l. The IC50 was calculated using GraphPad Prism 9.2.0 software. Inhibitory activity of the compound was measured in triplicates and data are presented as mean ± s.d. Two positive control tests were also done against Mpro.
The coding sequences for SARS-CoV-2 Wuhan-Hu-1 (GenBank: NC_045512.2) were used for recombinant protein expression. Nsp3 (PLpro domain), nsp15 (Mpro domain), nsp14 (N7-MTase), nsp10 and nsp16 (2′O-MTase) were cloned in fusion with an N-terminus hexa-histidine tag, as previously described75. The proteins were expressed in E. coli and purified by affinity using IMAC cobalt bead. In brief, after cell sonication in a buffer containing 50 mM Tris has a pH of 7.8. NaCl, 10 mM imidazole, 5 mM MgCl2 and 1 mM BME, supplemented with 0.25 mg ml−1 lysozyme, 10 μg ml−1 DNase and 1 mM PMSF, the proteins were purified through affinity chromatography with HisPur Cobalt resin 480 (Thermo Scientific), washed with an increased concentration of salt (1 M NaCl) and imidazole (20 mM), before elution in a buffer supplemented with 250 mM imidazole. The final buffer of 50 mM was used to prepare the MTses for purification. The tris is 300 mM. 5 mM NaCl, 1 mM BME. The proteins were concentrated using Vivaspin 20 centrifugal concentrators with a 10 kDa MWCO (GE Healthcare, VS2001), dialysed against the elution buffer in the absence of imidazole, and stored at −20 °C in a buffer containing 50% glycerol. The complex is endowed with RdRp activity, according to the ref. There were variations described in the ref. There is a recommendation for 77.
Huh-7 cells53 were seeded in 100mm dishes at a concentration of 2106 cells per dish. On the next day, the medium for the cell-culture was changed so that it contained different concentrations of CIM-834 or DMSO. The M(P132S) and E-HSV-encoding plasmids were transfected with 2 g and 1 g of cells, respectively. Then, 48 h after transfection, VLPs and cell lysates were collected and stored as described previously54 for MERS-CoV VLPs. VLP and lysate samples were resuspended in reducing Laemmli loading buffer and separated on a 12% polyacrylamide gel by SDS–PAGE. The primary anti-V5 antibodies and secondary HRP-labelled goat anti-mouse antibody were used to create a clone of the MProteins. Tubulin was used as a loading control and stained using a mouse anti-β-tubulin IgG1 antibody (Sigma-Aldrich, T5201, clone TUB 2.1, 1:2,000). The band quantification function was used to quantify the western blotting bands. Supplementary fig. 1 is for blots that are not boiled down.
Interactions between M and CIM-834 were calculated using LigPlot71 and UCSF ChimeraX72. The UCSF Chimera MatchMaker tool was used to obtain root mean square deviation values using default settings. The figures were generated using the UCSF computer system and the structural-biology applications used in the project were compiled and configured.
A final concentration of 44.34 M Mprotein, 100 M CIM-834 and Fab–E/B was achieved for both the Fab–E and Fab–B complexes. In the buffer solution, 20mM HePES-NaOH was used to conjugate all components. NaCl, 0.0025% LMNG and 0.00025% CHS. The samples were mixed and put on ice. The sample was pipetted onto glow-discharged R1.2/1.3 200 mesh holey copper carbon grids and then plunge-frozen in liquid ethane using a Vitrobot mark IV. Both datasets were collected at the Netherlands Center for Electron Nanoscopy. Grids were loaded into a Titan Krios electron microscope (Thermo Fisher Scientific) operating at 300 kV, equipped with a K3 direct electron detector and Bioquantum energy filter (Gatan). The slit width of the energy filter was set to 20 eV. Imaging was done at a nominal magnification of ×81,000 and ×105,000 for Fab–E and Fab–B, respectively, in super-resolution mode using EPU software (Thermo Fisher Scientific). A total of 5,058 and 5,037 movies were recorded for the Fab–E and Fab–B complexes, respectively. Detailed data-acquisition parameters are summarized in Extended Data Table 2.
For tomography, semithin sections of 200 nm or 300 nm were screened and imaged using a Tecnai F30 microscope (Thermo Fisher Scientific) equipped with a Gatan OneView camera. At 15,500 magnification, the positions were manually selected and acquired by single-axis tomography. The tilt series were reconstructed using IMOD56,57,58. Volume rendering and animation were done with the same software, and also using a brush segmenting tool.
Cells prepared on sapphires for high-pressure freezing were prefixed and exported from BSL3 in 6% PFA, similarly to the samples on coverslips. The samples were immersed in 100 mM. PHEM with 15% BSA as a cryo-protectant and high-pressure frozen using a BalTec HPM-010 with carriers forming a 40 µm-deep cavity (3-mm aluminium carriers, type B 0/0.3 mm and type 748 0.04/0.020 mm, Engineering Office M. Wohlwend). The high-pressure frozen samples were subjected to freeze substitution using cryo-tubes and rubber sealing rings, with a fixative cocktail of 2% OsO4, 1% uranyl acetate and 5% acetone. The AFS chamber temperature was increased over the course of 24 h as follows: 1 h at −90 °C; 8 h at −90 °C to −80 °C; 8 h at −80 °C to −50 °C; 2 h at −50 °C to −20 °C; 2 h at −20 °C to 0 °C. After rinsing the samples with dry acetone on ice they were further processed using a microwave and with increasing concentrations of Epon 822 in the sample. At the last 100% step in Epon 812, sapphires were transferred to AFS plastic moulds and polymerized at 60 °C for 48–72 h. For both chemically fixed and high-pressure frozen samples, ultrathin sections of 70 nm and 300 nm were collected using a 30 diamond knife and a UC7 Leica Ultra Microscoptor. Grids were post-stained for 5 min with 3% uranyl acetate in 70% methanol and 2 min with lead citrate. To locate the infected cells, the Serial-EM Navigator functionality and a procedure adapted from ref. 55 were used to map the central portion of the ribbon of 5 that was mounted on a JEOL 1400. A Matataki sCMOS camera was attached to a JEOL 1800+ that was used to acquire images of the whole perinuclear region of the selected cells at a magnification of.
Source: A coronavirus assembly inhibitor that targets the viral membrane protein
Animal experimentation of GFP and TMPRSS2 cell lines for antiviral studies: Application to the African monkey kidney cell line A549ACE2+TM PRSS2 cells
Animal housing conditions and experimental procedures were approved by the ethics committee of animal experimentation of KU Leuven (licence P001/2021).
GS-441524 was obtained from MedChem Express (HY-103586). Cell Signaling Technology bought Hydroxychloroquine. It was from the city of Wuxi.
The GFP cells of the African monkey kidneys cell line were kept in a modified Eagle medium supplemented with 10% V/V heat-inactivated fetal bovine serum. + 0.5 mg ml−1 geneticin. The cells were created as described in the ref. 41) were maintained in DMEM, supplemented with 10% (v/v) heat-inactivated FBS and 10 μg ml−1 blasticidin. The A549ACE2+TMPRSS2 cells (a human lung carcinoma cell line overexpressing human ACE2 and human TMPRSS2 receptors), used for antiviral studies, were from InvivoGen (a549d-cov2r, A549-Dual hACE2-TMPRSS2 cells) and were cultured in DMEM supplemented with 10% v/v heat-inactivated FBS, 300 μg ml−1 hygromycin, 0.5 μg ml−1 puromycin and 10 μg ml−1 blasticidin. A549ACE2+TMPRSS2 cells, used for subcellular studies, were generated in-house from A549 obtained from ATCC (CCL-185), using lentiviral transduction with pWPI vectors encoding ACE2 (selected using 500 μg ml−1 geneticin) and TMPRSS2 (selected using 1 μg ml−1 puromycin) under EF1alpha promotor control. These cells were cultivated in DMEM supplemented with 10% (v/v) FBS, 100 U ml−1 penicillin, 10 µg ml−1 streptomycin and 1% non-essential amino acids. The cell growth Medium used for the various studies using VeroE6– GFP, VeroE 6–mCherry, and A548ACE2+TMPRSS2, was 2% instead of 10%. All cell cultures were done at 37 °C and 5% CO2. BHK-21 cells (Baby hamster kidney cell line obtained from ATCC, CCL10) were maintained in Glasgow MEM (Invitrogen) supplemented with 5% v/v FBS, 10% tryptose phosphate broth, 100 U ml−1 penicillin, 100 µg ml−1 streptomycin and 10 mM HEPES, pH 7.4. Cells were kept in Eagle’s minimal essential medium for transfection experiments. Huh7 cells were cultured in DMEM supplemented with 10% FBS and 1 mM Glutamax.
In a vehicle containing 27% propylene glycol and 43% absolute ethanol, the product was used to make irrmatrelvir, which is derived from Excenen, batchesEXA5024). The formulated material was composed of 20% SYNTHETIC GAMBLE, 1% TREAD, and 85% PAIN 5 TICACE buffer. To evaluate in vivo efficacy, male SCID mice (CB-17/Icr-Prkdcscid/scid/Rj; Janvier Laboratories) 7–9 weeks old were treated by oral gavage with either the vehicle (n = 12, twice a day) or CIM-834 at 100 mg per kg (n = 12 twice a day and n = 12 once a day) or nirmatrelvir at 300 mg per kg (n = 12, twice a day) or 100 mg per kg (n = 6, twice a day), starting from day 0, just before infection with the beta variant B.1.351 (hCoV-19/Belgium/rega-1920/2021; EPI_ISL_896474, 2021-01-11). Animals were anesthetized with isoflurane and had 40 l containing 105 TCID50 injected into them. SARS-CoV-2 beta variant (day 0). In the therapeutic set-up, animals were infected on day 0 and treatment with CIM-834 (100 mg per kg, twice a day) was initiated 24 h, 30 h or 48 h after infection. Mice were housed in individually ventilated cages with three mice per cage and monitored daily for weight changes and any clinical signs. Animals were euthanized by injecting 100 l Dolethal on day 3, along with the lungs to be collected. Infectious viral lung loads were quantified by end-point virus titration. To prevent carry-over of the compound during determination of infectious virus titres, the cells were washed and given fresh medium, immediately after incubation for 2 h with lung homogenates. Cells were subsequently incubated at 37 °C for three days before TCID50 read-out.
All plasmids were validated by Sanger sequencing (Macrogen). Preparation of viral DNA, in vitro transcription and electroporation of BHK-21 cells was carried out as previously described51, except for the use of an ECM 830 Square Wave electroporation system (850 V, 3 pulses of 0.30 ms, a 3 s interval, BTX). The cells were added in medium with 10% FCS. The medium was replaced by the small FCS after 6 h at 37 C. Four days later, the virus stocks were collected, passaged once on A549ACE2+TMPRSS2 (InvivoGen) and subjected to whole-genome sequencing (Oxford Nanopore Technologies, by B. Vanmechelen, Rega Institute) to verify the desired sequence.
5-fold dilutions of the cell lysate supernatant were added to VeroE6 cell monolayers in 12-well plates with a 37 C incubated time. The mix was replaced with 0.8% methylcellulose in DMEM supplemented with 2% FBS. After three days of incubation at 37 °C, the overlays were removed, the cells were fixed with 3.7% PFA and stained with 0.5% crystal violet, and plaques were counted visually.