coronaviruses assembly inhibitor is used to make sure the viral is stopped

Work with Live Coronaviruses at the KU Leuven Rega Institute: H&E-stained Left Lung lobes of Syrian Golden Hamsters Infected with SARS-CoV-2

rSARS-CoV-2 WT-USA-WA1, rSARS-CoV-2 WT-USA-WA1-3CLpro: L50F, rSARS-CoV-2 WT-USA-WA1- 3CLpro: T21I-D263G, rSARS-CoV-2 WT-USA-WA1-3CLpro: L50F- E166A L167V have been described previously30. The strain of the disease was obtained from the Centers for Disease Control and Prevention. The high-containment A3 and biosafety level 3 facilities of the KU Leuven Rega Institute have live work done on them. SBB 219 2018 0892 and AMV 23102017 SBB 219 20170589, according to institutional guidelines. The work with live coronaviruses was done in a level 3 laboratory at the Leiden University Medical Center.

In Fig. 3e, representative images are shown of H&E-stained left lung lobes of Syrian golden hamsters infected with SARS-CoV-2 and vehicle or JNJ-9676 treated. In this experiment, a full cross-section of the left lung of each of the five animals per group was assessed by a skilled pathologist.

Micrograph of the production of JNJ-9676 isolated from Western Blots as a control on the quality of the product and application to SARS-CoV-2 infection

In Extended Data Fig. 2g, uncut western blots are shown of purified M proteins. These blots were once used to control the quality of the product.

The data is extended in a fig. The micrograph from the data collection is shown. To obtain this representative image, 12,988 images were taken.

There are two methods for the synthesis of JNJ-9676, one in patent and one in supplementary methods. Molnupiravir was ordered at MedChemExpress (HY-135853) and nirmatrelvir was synthesized according to literature procedures59. JNJ-9676 had been dissolved in 100% dimethyl sulfoxide as a 5– 100 mM stock. For in vivo experiments, JNJ-9676 was dissolved in 100% polyethylene glycol 400 (PEG400) as stocks of 75, 25 or 8.33 mg ml−1, molnupiravir was dissolved in 100% PEG as a stock of 300 mg ml−1 and nirmatrelvir as a stock of 250 mg ml−1.

Previously described procedures52 were followed. Epithelix obtained human airway epithelial cells from aMucil Air pool of donors, using an air–liquid set-up. After they arrived, the inserts were washed with 1 PBS and kept in the Mucil Air medium for 4 days before use. After the experiment had taken place, the cultures were pretreated with a mixture of compounds at different concentrations for 1 h before they were exposed to the inoculum. 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. The medium in the basolateral side of the cultures was refreshed on day 2 after an illness. All incubations from start of the infection were done at 35 °C and 5% CO2.

The gene encoding SARS-CoV-2 M protein (1–222, UniProt: P0DTC5) was synthesized and cloned into a pcDNA3.4 vector, with an added C-terminal linker sequence, an ALFA-tag and a C-tag (SNSLEVLFQGP-SRGGSGAAAGSGSGSGSPSRLEEELRRRLTE-GS-EPEA).

Derived and isolated from infectious clones of Pg CoVGD, RsSHC014, and WIV-1 are recommencedviruses that can be used as working stocks.

VeroE6–GFP cells were seeded at a density of 25,000 cells per well in 96-well plates (Greiner Bio One, 655090) and pretreated with three-fold serial dilutions of the compounds overnight in the presence of the MDR1 inhibitor CP-100356 (final concentration, 0.5 μM). The next day (day 0), cells were infected with SARS-CoV-2 inoculum at a multiplicity of infection (MOI) of 0.001 median TCID50 per cell. The read-out of the number of GFP signals determined by high-content images was done after the 4th day. The percentage inhibition was calculated using the number of fluorescent cells in theinfecting control wells and the uninfecting control wells. The EC50 was determined by logarithmic interpolation. A similar protocol was used to determine the antiviral activity in A549ACE2+TMPRSS2 (InvivoGen) cells, but no MDR1 inhibitor CP-100356 was used and the cell viability was determined four days after infection using viability staining with 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS). The percentage of antiviral activity was calculated with the help of the background and normalizing control wells, and the EC50 was determined using a combination of these methods. The potential toxicity of compounds was assessed in a similar manner in both cell lines with the same set-up in the uninfecting culture where the metabolic activity was quantified. The 50% cytotoxic concentration (CC50, the concentration at which cell viability reduced to 50%) was calculated by logarithmic interpolation.

The toxicity was assessed by exposing non-infecting inserts to the same concentrations of JNJ-9676 as for anti-parasitivity treatment using anEVOM3 to represent the health of the cell layer. Brefeldin (0.3 µM; internally synthesized) was used as a toxicity control.

VeroE6 cells were seeded in 12-well plates the day before infection. Cells were infected with an MOI of 1 TCID50 per cell (SARS-CoV-2 GHB) and compounds were added 0, 3, 5 and 7 h after infection. The supernatant was removed at 10 h and the cells were either lysed or collected for the purpose of detection of the infectious virus. The VeroE6–mCherry cells were put in 96-well plates the day before the test. Cells were infected with SARS-CoV-2–mNeonGreen virus (at a final MOI of 0.1 TCID50 per cell) and compounds were added −0.5, 0, 2, 4, 6 and 8 h after infection. At 10 h after infection, cells were imaged using high-content imaging and the number of infected cells was calculated, after which the percentage inhibition relative to the DMSO-treated control was calculated.

The Expi23F cells were grown in a lab with 8% CO2 at 37 C after being co-delivered into the FabB heavy chain and light chain.

There was a loaded medium. 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 buffer of 20 mM sodiumphosphate and 50 mM imidazole was prepared and then eluted with over 5 CV. NaCl, 500 mM imidazole). Peak fractions of FabB were subsequently purified on to a HiLoad 16/600 Superdex 75 pg column (Cytvia) in buffer (20 mM sodium phosphate pH 6.5, 150 mM Something called NaCl.

Experiments were performed in a total volume of 10 µl. A Prometheus NT.Plex instrument (NanoTemper Technologies) 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 0.00033% GDN, 0.0001% CHS and 1% DMSO are all levels of NaCl. The samples loaded into standard‐grade glass capillaries were measured under a temperature range of 25–95 °C with a temperature gradient of 1 °C min−1, and the intrinsic protein fluorescence at 330 and 350 nm was recorded. The data were analysed using PR.ThermControl v.2.1.6 (NanoTemper Technologies) (technical replicates ≥ 3).

The preparation of three sample types was the focus of the offline ASMS experiment.

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 flowthrough was discarded after the size-exclusion plate was placed into a 4 C refrigeratedcentrifugation for 2 minutes at 1,000g. A buffer containing 20 mM HePES and a pH 7.5 was used for the four washings. 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 source plate was prepared with an acoustic liquid handler and transferred into four wells of a natural V bottom plate, with 20 mM of compound dissolved in 100% DMSO. A fraction of a stock solution was melted on ice and then dripped with reagent into the sample to make it work. Then, 20 µl of the resulting working protein stock was dispensed into three wells containing compound to yield a final concentration of 5 µM (3 technical replicates). 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. After 1000g was added to the plate, it was put into a dish at 25C for 30 minutes.

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 detergent concentration was reduced and the flowthrough was altered to make it harder to collect insoluble precipitate with the added help of 15 lMS-grade water.

5 mM compound was transferred from the source plate into a 960-wellplate and together they had 25% acetonitrile and 2% DMSO solution.

We put the 5 M Mprotein in a buffer only and put it at 25 C for 30 minutes. NaCl, 0.001% LMNG, 0.0001% CHS, 0.00033% GDN, supplemented with 2% DMSO) or in buffer with CIM-834 (in DMSO, 2% final concentration). The unbound compound was removed by the Spin Desalting columns from the bound compound. The experiments were done with triplicate. Mass-spectrometry was done on a time-of-flight for liquid chromatography with a tandem mass spectrometry instrument. For data analysis, the Agilent Mass Hunter Qualitive Analysis software (target screening workflow) and the Find by Formula method for compound identification by mass/isotope pattern matching was used. An EIC peak area of [M + H]+, [M+Na]+ and [M + K]+ masses was extracted using a mass error tolerance window of 10 ppm.

MS data processing was performed using Agilent MassHunter Qualitative Analysis (v.10.0), where the [M+H]+, [M+Na]+, and [M+K]+ masses were extracted and merged using a mass error tolerance window of 3 ppm.

The PELCO easiGlow Discharge Cleaning System for the Fab-Fab complexes: Sample collection, processing, and characterization

The PELCO easiGlow Discharge Cleaning System was used to clean the grids. The three l sample was applied to the EM grids with settings like Blot Time 4 s, Blot Force 0, as well as a ready-made cell culture medium with the following instructions. Flash-freezing in liquid ethane cooled by liquid nitrogen was performed. The data collection was done on the Glacios microscope by the automated system. Micrographs were taken at ×105,000 magnification using a Facon4 detector (Gatan) in counting mode. The frames were recorded in 40 frames and a dose of 40 e 2. The calibrated physical pixel size for all digital micrographs was 0.910 Å. All details corresponding to individual datasets are summarized in Extended Data Table 6.

patch motion correction was performed for the Fab–E and Fab–B complexes. Out of the 5,048 images left for processing, less than 10 were discarded, because of their estimated resolution of 10. The blob picker tool was then used to select 4,327,548 and 4,497,785 particles, respectively, which were then extracted in an 80-pixel box (Fourier binned 4.5 × 4.5). For the Fab–E complex, two rounds of 2D classification were done, resulting in 270,970 particles being selected for further processing. The Fab-E complex was reconstructed with Ab initio reconstruction. Particles belonging to this class were then re-extracted in a 300-pixel box. After the particles were obtained, a final reconstruction was done with a global resolution of 3.3. One well-defined reconstruction of the Fab–B complex was generated by Ab initio reconstruction. For five rounds of heterogeneous refining, the Fab–B complex was used as a reference volume for high, using the particles picked by the blob picking tool. The last two rounds of ab initio were done to further separate the best-quality particles. The remaining 73,185 particles were then re-extracted in a 360-pixel box. The final size of particles was 0.836, after they were binned by a non-integer value. These particles were then subjected to a final non-uniform refinement, with C2 symmetry imposed60, yielding a final reconstruction with a global resolution of 3.2 Å. The gold standard Fourier shell correlation (FSC) criterion (FSC = 0.143) was used for calculating all resolution estimates, and 3D-FSC plots were generated in cryoSPARC61. To enable model building, globally refined maps were filtered by local resolution in cryoSPARC.

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. ResMap was used to determine local resolution. Detailed statistics about the cryo-EM data processing can be found in Extended Data Fig. 6a–f.

The M–Fab– B-CIM map used the Alphafold2 model62 and a previously determinedSRS-CoV-2 M structure in it’s design. An initial round of molecular-dynamics flexible fitting was then done on the combined model using Namdinator64 and then manually adjusted in Coot65. The compound was added using a tool known as the ligand builder. The following were taken into account when choosing the pose. First, the morphology of the density. The flat plate-like morphology of the density orientated away from the M two-fold symmetry axis was consistent with the piperidine and pyrimidine rings of CIM-834. 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. The hydrogen-bonding partners are placed in the second position. The pyridazine ring is positioned close to S99 and N117 with the potential for water- or hydrogen-based bonds. The model was refined using iterative cycles of manual model building, Phenix 66, real-space refinement, and eLBOW. Model validation was done with the aid of Molprobity68.

Lung histopathology and Tomography using the GraphPad Prism (v.6.07) and v.10.2 (version.8.0)

For histological examination, the fixed lung tissue sections (5 μm) were analysed after staining with haematoxylin and eosin and 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.

The statistical analyses were performed using GraphPad Prism. A log10 transformation was applied to the lung viral-load data. 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. The post hoc Dunn’s test with the Benjamini–Hochberg’s multiplicity correction was applied to account for multiple testing. A significance level of 0.05 was used.

All graphs were prepared using GraphPad Prism (v.6.07, v.8.3.0, v.9.2.0, v.10.1.2 and v.10.2.3). 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 2D EMs for quantification of DMVs were acquired from three independent preparations. Tomography was acquired on a selection of technical duplicates of the samples previously characterized in 2D, with the exception of the HPF samples that were prepared in duplicates in one preparation.

Nanoscale microstability and IC50 determinations using a biomek I5 automate for the automation of reaction assays on Nunc plates

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). Incubation took place at 37 C for 120 min. 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 supernatant was immersed in water with two-fold. The resulting solution was analysed by liquid chromatography with tandem mass spectrometry to determine the half-life of the compound.

The IC50 was determined using this equation: percentage of active enzyme = 100/(1 + I2/IC50), where I is the concentration of inhibitor and 100% of activity is the fluorescence intensity without the inhibitor. The IC50 was determined from curve fitting using Prism software. For each measurement, results were obtained in triplicate.

Reaction assays were stopped by adding 20 µl EDTA (100 mM). Positive and negative controls consisted of a reaction mix with 5% DMSO (final concentration) or EDTA (100 mM) instead of compounds, respectively. The reactions were transferred to a plate using aBiok I5 automate. 60 l of reagent was distributed into 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.

The reactions were done on a 96-well Nunc plate. The experiments were robotized using the BioMek I5 automate. 2 l of each compound in 100% DMSO wasadded to wells of the chosen concentration. The (nsp8l7 + nsp8) mix was spread out in the wells after 8 minutes at a room temperature, when it was ready to form the active complex. Nsp12 was then added to wells and incubated for 8 min. The reactions were started with a UTP + poly(A) template mix, and kept at 30 C for 20 min.

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. 76) in complex with 450 nM nsp8 and 450 nM nsp8L7 (ref. 77).

Activity and inhibition were determined by FRET, performed in black 384-well HiBase non-binding plates (Greiner Bio One, 784900), as previously described75. The HEPES buffer was prepared with 5 M of a fluorescent synthetic peptide in the presence of 55 nM of PLproprotein, which was then acclimated with inhibitor. EDTA, 4 mM DTT and 10% glycerol. The final concentration of the drug was adjusted. The Edans/Dabcyl Fluorophore–quencher pair are separated by the fluorogenic peptide. The time courses were followed for 40 minutes by using a Tecan Safire2 fluorimeter to check the increase in emission at 493 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. Edans generated by Mpro’s caspase activity was seen at a wavelength of 490 nm with a temperature of 400 degrees Centigrade, using a Tecan Spark multimode microplate. The compound was prepared with a stock solution. The IC50 of CIM-834 was determined after incubation of 0.5 µM of SARS-CoV-2 Mpro and CIM-834 at various concentrations from 0 to 500 µM in reaction buffer at 37 °C for 10 min. At the end, 50 M was added to each well and a final total volume of 100 l was added to initiate the reaction. The IC50 value was calculated with the help of the GraphPad Prism 9.2.0 software. The data for the Inhibitory activity of the compound is presented as a mean. There were two positive control tests 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 separated by affinity using the IMAC bead. In a buffer containing 50 mM, after cell sonication. 300 mM of tris. The results of the purification were obtained by using 0.25 g ml1 lysozyme, 10 g colloid, 1 mM B ME, and 1 mM PMSF. The MTases were subsequently purified by size-exclusion chromatography (GE Superdex S200) in a final buffer of 50 mM 300 mM of tris. NaCl, 5 mM MgCl2 and 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 endowed with RdRp activity was assembled. Variations were described in the ref. There is a score of 77.

Huh-7 cells were seeded on coverslips in 24-well plates at a concentration of 8 × 104 cells per well. The next day, 1 µM CIM-834 or DMSO was added to the medium and cells were transfected with a total of 250 ng plasmid encoding for V5-tagged WT-M using the TransIT-LT1 transfection reagent (Mirus Bio). Cells were fixed with 4% paraformaldehyde in PBS for 20 minutes at room temperature, followed by 1% Triton-X100 in PBS for 3 minutes. After blocking a 10% normal horse serum for fifteen minutes at room temperature, the V5-tags were visualized by an anti-V5 antibodies being injected into the 10% normal horse serum. The Trans-Golgi network was visualized with polyclonal sheep anti-human TGN46 and Cy 3-conjugated donkey anti-sheep IgG. Nuclei were visualized using 1 µg ml−1 of 4′,6-diamidino-2-phenylindole (DAPI), and coverslips were mounted in Mowiol mounting medium. Images were obtained with a confocal laser scanning microscope using an oilimmersion objective lens. The extent of colocalization between M and TGN46 was quantified by calculating the Pearson’s correlation coefficient using the JACoP plug-in of ImageJ. For both treated (1 µM CIM-834) and untreated (DMSO) conditions, 25 cells were analysed.

Interactions between M and CIM-834 were calculated using LigPlot71 and UCSF ChimeraX72. The tool used to get root mean square deviation values was used by the University of California, San Francisco. Figures were generated using UCSF ChimeraX72, and structural-biology applications used in this project were compiled and configured by SBGrid73.

For both the Fab–E and Fab–B complexes, M protein was combined with CIM-834 and either Fab–E or Fab–B, resulting in a final concentration of 44.44 µM M protein, 100 µM CIM-834 and Fab–E/B in a 2.2 molar excess of M protein. All components were diluted in a buffer solution comprising 20 mM HEPES-NaOH at pH 7.7, 150 mM NaCl, 0.0025% LMNG and 0.00025% CHS. Samples were mixed and incubated for 15 min on ice before vitrification. A sample of 3.5 l was pipetted onto glow-discharged R1.2/1.3200 mesh holey copper carbon grids and then plunge-frozen in liquid ethane using aVitrobot mark IV. The data were collected at the center. The grids were loaded into the Titan Krios electron microscope which was operating at 300 kv and had a K3 direct electron detector. Fab–E and Fab–B had their magnetic resonance images done at a magnification of 81,000 and 105,000, respectively, which was done in super-resolution mode. There were more than 5,000 films recorded for the Fab–B and Fab–E complexes. 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. Target positions were manually selected and acquired at ×15,500 magnification (−60° to +60° per axis; increment, 1°) by single-axis tomography (0.78 nm per pixel). The series was reconstructed using IMOD56,57,58. Segmentation of selected tomograms was done manually using the brush segmentation tool in Amira-Avizo software v.2020.1 (ThermoFisher), Volume renderings and animations were also computed with the same software.

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 after being washed six times. The Engineering Office has a cryo-protectant with 15% BSA as a high-pressure frozen using a BalTec HPM-012 with carriers that form a 40 m deep Cavity. High-pressure frozen samples were then subjected to freeze substitution in a Leica AFS II using cryo-tubes with screw caps and rubber sealing rings, containing 1 ml of fixative cocktail composed of 0.2% OsO4, 1% uranyl acetate and 5% water in 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 5 minutes of ice and dry acetone on ice, the samples were washed twice with a small amount of ethanol and then processed using a microwave with increasing concentrations of Epon 902 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. Ultrathin and semithin sections of 70 nm or 300 nm, respectively, were generated for both chemically fixed and high-pressure frozen samples using a UC7 Leica ultramicrotome and a 30° diamond knife (Diatome) and collected on Pioloform-coated slot grids. The grids were stained with 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 section of the ribbon of 5 on the JEOL 1400, equipped with a camera. Cells displayed in the map were identified and stored in the map, navigator maps and grids were transferred to a JEOL 2100+ that was equipped with a Matataki sCMOS camera to acquire photos of the entire perinuclear region of the selected cells.

Source: A coronavirus assembly inhibitor that targets the viral membrane protein

Animal Housing and Experimental Procedures for Antiviral Studies Applied to Human Lung Carcinoma (HACE2+TMPRSS2 Cells)

The animal housing conditions and experimental procedures were approved by the ethics committee.

GS-441524 was obtained from MedChem Express (HY-103586). Hydroxychloroquine was purchased from Cell Signaling Technology (85523S). Nirmatrelvir (PF-07321332) was from Wuxi.

The VeroE6–GFP cells were kept in a modified Eagle medium supplemented with 10% v/v heat-inactivated fetal bovine Serum, provided by M. van loock. + 0.5 mg ml−1 geneticin. VeroE6–mCherry cells were created as described in ref. The additions were made with 10% heat-inactivated FBS and 10 g ml1 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. The progenitor cells were created in-house using lentiviral transduction and a 500 g geneticin. 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 containing 2% and not 10% was used for all of the VeroE6–GFP and VeroE6–mCherry tests. At 37 C and 5% CO2 all cell cultures were done. BHK-21 cells were maintained in Glasgow MEM with 5% v/v FBS, 10% tryptose phosphate chondroitin and 100 U grams penicillin. For transfection experiments, cells were maintained in Eagle’s minimal essential medium (EMEM), as described42. Huh7 cells were cultured in DMEM supplemented with 10% FBS and 1 mM Glutamax.

Nirmatrelvir (from Excenen, batch EXA5024) was formulated as a 100 mg ml−1 and 33.3 mg ml−1 stock (for 300 mg per kg and 100 mg per kg dosing, respectively) in a vehicle containing 43% absolute ethanol and 27% propylene glycol (Sigma) in sterile distilled water. The buffer was composed of 20 million liters of 14 percent propylene glycol (Sigma), 1% Tween 80 (Sigma), and 85% pH 5 citrate. 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). For virus infection, animals were anesthetized with isoflurane and inoculated intranasally with 40 µl containing 105 TCID50 On day 0, there is a variant of the SRES-CoV-2. Animals were initiated with the treatment on day 0 after they were exposed to the infectious disease, and it takes up to 48 hours to cure the disease. The mice were kept in individuallyventilated cages and monitored daily for their weight and clinical signs. At day 3 after infection, animals were euthanized by intraperitoneal injection of 100 µl Dolethal (200 mg ml−1 sodium pentobarbital, Vétoquinol SA), and the lungs were collected. Infectious viral lung loads were quantified by end-point virus titration. To prevent carry-over of the compound during determination of infectious virus, the cells were washed and gave fresh medium after 2h with lung lygicates. The cells were placed in 37 C for three days.

The plasmids were checked bygenomics. 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 to the medium with 10% FCS. After 6 h at 37 C, the medium was removed and replaced with a small amount of FCS. The stock of the virus was collected four days later and subjected to whole-genomic sequencing to verify the desired sequence.

For this, 5-fold dilutions of the cell lysate supernatant were added to VeroE6 cell monolayers in 12-well plates and incubated at 37 °C for 1 h. Subsequently, the inoculum mixture was replaced with 0.8% (w/v) methylcellulose in DMEM supplemented with 2% FBS. After three days of being at 37 C, the cells were removed, and stained with Crystal violet, and plaques were counted.