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LitCovid-PMC-OGER-BB

LitCovid-PD-FMA-UBERON

LitCovid-PD-UBERON

{"project":"LitCovid-PD-UBERON","denotations":[{"id":"T3","span":{"begin":6245,"end":6254},"obj":"Body_part"}],"attributes":[{"id":"A3","pred":"uberon_id","subj":"T3","obj":"http://purl.obolibrary.org/obo/UBERON_0001982"}],"text":"Experimental Section\n\nChemicals\nThe compounds P1, P2, and P3 were purchased from Maybridge Chemical Company, TCI Chemicals, and Sigma-Aldrich Corporation, respectively. The reagents used in this study were purchased from Sigma Chemical Co. (St. Louis, MO). The purity of all compounds is higher than 95% and was used without further purification.\n\nCloning, Protein Expression, and Purification\nThe MERS-CoV N proteins were prepared according to previously described methods.46 In brief, the cDNA fragments of MERS-CoV N proteins were cloned into a pET-28a expression vector (Merck, Darmstadt, Germany) containing a histidine tag-encoding sequence. Vectors encoding the single mutants N39A, N39G, and W43A were generated using the QuikChange site-directed mutagenesis protocol with the primers listed in Table S3. The vectors were transformed into Escherichia coli BL21 (DE3) pLysS cells. The cells were grown to an optical density range of 0.6–0.8 at 600 nm at 37 °C and protein expression induced with 1.0 mM isopropyl β-d-1-thiogalactopyranoside (IPTG), followed by incubation at 10 °C for 24 h. The cells were harvested by centrifugation (6000g, 12 min, 4 °C) and resuspended in lysis buffer (150 mM NaCl, 50 mM Tris-HCl, 15 mM imidazole, and 1 mM PMSF; pH 7.5). The cells were lysed by sonication and centrifuged (10000g, 40 min, 4 °C) to remove debris. The supernatant was purified by injection into a Ni–NTA column (Merck, Darmstadt, Germany) and eluted with buffer containing imidazole at a gradient range of 15–300 mM. Pure protein fractions were collected, dialyzed with low-salt buffer, concentrated, and quantified by the Bradford method (BioShop Canada Inc., Burlington, ON, Canada).\n\nCrystallization and Data Collection\nMERS-CoV N-NTD crystals were grown as previously described:46 MERS-CoV N-NTD was crystallized at room temperature (∼25 °C) by the sitting-drop vapor-diffusion method. A protein solution (2 μL; 10 mg mL–1) was mixed with an equal volume of crystallization solution consisting of 75 mM ammonium sulfate, 2 mM NaBr, and 29% PEG 3350 (Sigma-Aldrich Corp., St. Louis, MO) and equilibrated against a 300 μL solution. MERS-CoV N-NTD:P3 co-crystals were obtained using a crystallization solution containing 2 mM P3. MERS-CoV N-NTD crystals in complex with P1 were obtained by soaking native MERS-CoV N-NTD crystals for 90 s at room temperature in a crystallization solution containing 2 mM P1. Diffraction datasets for MERS-CoV N-NTD alone and in complex with P1 were collected at beamline 13B1 of the Taiwan Light Source (TLS) of the National Synchrotron Research Center (NSRRC; Hsinchu City, Taiwan). Diffraction of the MERS-CoV N-NTD:P3 complex was performed at the beamline SP44XU of SPring-8 (Hyogo, Japan).\n\nStructural Determination and Refinement\nDiffraction data were processed and scaled with HKL-2000 software. The structures were solved by molecular replacement (MR) in Phenix47 using HCoV-OC43 N-NTD (PDB:4J3K) as the search model.24 The initial models were rebuilt and refined by Coot48 and Phenix. Structures were visualized using PyMOL (The PyMOL Molecular Graphics System, version 2.3.0).49\n\nChemical Cross-Link Assay\nProtein solutions containing 40 μM of wild-type or mutated MERS-CoV N-NTD were incubated with glutaraldehyde at a final concentration of 1% v/v. The reaction was conducted at room temperature for 10 min and quenched with the addition of 1 M Tris-HCl (pH 7.5). The samples were then stored on ice and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).\n\nDiscovery of Orthosteric PPI Stabilizers for MERS N Protein\nTo screen for compounds that induce hydrophobic PPI between MERS N-NTDs, a model of dimeric MERS-CoV N-NTD without the H37 and M38 residues was used in virtual drug screening. The Sigma-Aldrich, Acros Organics, and ZINC drug databases were screened with LIBDOCK molecular docking software to obtain compounds acting on the N protein. The N protein binding pocket was represented by a set of spheres. Each compound in the database was docked in a pocket comprising W43. The hydrophobic complementarity between ligands and receptors was calculated with PLATINUM.31 Compounds with higher docking scores are listed in Table S2.\n\nFluorescence Measurements\nFluorescence assays were performed in a buffer consisting of 50 mM Tris-HCl (pH 8.3) and 150 mM NaCl. One micromolar N protein was incubated either with the control buffer or each compound (10 μM) at 4 °C for 1 h. Tryptophan fluorescence was acquired with a Jasco FP-8300 fluorescence spectrometer (JASCO International Co. Ltd., Tokyo, Japan) at an excitation wavelength of 280 nm and an emission wavelength range of 300–400 nm.\n\nThermostability Measurements\nThermostability assays were conducted in a buffer consisting of 50 mM Tris-HCl (pH 7.5) and 150 mM NaCl and with a JASCO FP-8300 fluorescence spectrometer (JASCO International Co. Ltd., Tokyo, Japan). One micromolar N protein was incubated either with the control buffer or each compound (10 μM) at 4 °C for 2 h. UV absorbance vs temperature profiles were acquired by ramping the temperature from 4–95 °C at a 1 °C min–1 and recording the absorbance at 280 nm every 0.5 min.\n\nDetermining CC50 and EC50 of Hit Compounds\nVero E6 cells were infected with MERS-CoV with M.O.I = 0.1 and treated with lead compounds for 48 h. Cell viability was determined by the neutral red uptake assay. CC50 and EC50 were determined by % cell viability. CC50 was determined for cells treated with drugs only. EC50 was determined for MERS-infected cells after drug treatments.\n\nSmall-Angle X-ray Scattering (SAXS) Experiments\nSAXS experiments were performed at the BL23A SAXS beamline at the TLS of NSRRC, using a monochromatic X-ray beam (λ = 0.828 Å), with an integrated HPLC system of an Agilent-Bio SEC-3 300 Å column (Agilent Technologies, Inc. Santa Clara, CA). Protein samples (44 μM MERS-CoV N and MERS-CoV N:P3 complex prepared by incubating the 44 μM native protein with 440 μM P3) were prepared in a buffer consisting of 50 mM Tris-HCl (pH 8.5) and 150 mM NaCl on ice for 1 h. Then, a 100 μL aliquot was injected into the column at a flow rate of 0.02 mL min–1. After passing through the column, the sample solution was directed into a quartz capillary (2 mm dia.) for subsequent buffer and sample SAXS measurement at 288 K. The sample-to-detector distance of 2.5 m used covered a scattering vector q range of 0.01–0.20 Å–1. Here, q is defined as q = (4π/λ) sin θ, with the scattering angle 2θ. Thirty-six frames were collected for each sample elution with an X-ray frame exposure time of 30 s. Frames of good data overlapping (namely, of low radiation damage effects) were merged for improved data statistics and analyzed to determine initial Rg using PRIMUS (version 3.1). The P(r) distance distribution and Dmax were calculated from the experimental scattering curve using GNOM (version 4.1). An ensemble optimization method (EOM) analysis was performed through the EMBL Hamburg web interface.50 Modeling of the rigid body crystal structure was calculated and generated using CRYSOL (ATSAS Program Suite v. 2.8.2).51 The crystal structures of MERS-CoV NTD (PDB ID: 4UD1)27 and MERS-CoV NTD:P3 (solved in this study) and the CTD domain of MERS-CoV N protein (PDB ID: 6G13)23 were used as rigid bodies in EOM analysis. With the EOM analysis, 1000 models were generated in the beginning as a structural pool. Selected from the SAXS profiles of the structural pool was an ensemble of models that could fit the experimental scattering curve with their linear combination. Tetrameric MERS-CoV NP conformations and 16-mer MERS-CoV:P3 conformations were selected because their ensemble generated curves fit best to the experimental SAXS results.\n\nViral Infection\nVero E6 cells (ATCC No: CRL-1586) were seeded onto culture plates with complete Dulbecco’s modified Eagle’s medium (DMEM) and incubated overnight prior to infection. MERS-CoV (HCoV-EMC/2012) at a multiplicity of infection (M.O.I.) of 0.1 was added to the cells and incubated at 37 °C for 1 h, followed by washing thrice with phosphate-buffered saline (PBS) to remove the unattached virus. Fresh complete culture medium was then added to the plates.\n\nPlaque Assay\nVero E6 cells were seeded in 12-well plates and incubated overnight before the assays. Samples containing MERS-CoV were serially diluted 10× with MEM, added to the wells, and incubated for 1 h with agitation every 15 min. After incubation, the inocula were removed and washed with PBS. An overlay medium comprising 2× MEM and 1.5% (w/v) agarose (1:1) was added to the wells followed by incubation at 37 °C and 5% CO2 for 3 days. The plates were fixed with 10% (v/v) formalin containing 0.2% (w/v) crystal violet, and the plaques were counted.\n\nRT-qPCR\nTotal RNA of infected Vero E6 cells was extracted with the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Reverse transcription and PCR amplification were performed with an iTaq Universal One-Step RT-qPCR Kit (Bio-Rad Laboratories, Hercules, CA). Real-time PCR was performed in a StepOnePlus Real-Time PCR System (Applied Biosystems, Foster City, CA). The primer pairs used to amplify the viral RNA were as follows: GAPDH-F: 5′-GAAGGTGAAGGTCGGAGTC-3′; GAPDH-R: 5′-GAAGATGGTGATGGGATTTC-3′;53 MERS-CoV-F: 5′-CCACTACTCCCATTTCGTCAG-3′; MERS-CoV-R: 5′-CAGTATGTGTAGTGCGCATATAAGCA-3′.54 The MERS RNA levels were normalized to that of GAPDH and compared between MERS-CoV groups at 24 h.p.i. and at 48 h.p.i.\n\nImmunofluorescence Assay\nVero E6 cells were seeded in eight-well chamber slides and incubated overnight prior to infection with MERS-CoV at M.O.I. = 0.1. The cells were fixed with 4% (v/v) paraformaldehyde for 20 min at 4 °C, followed by permeabilization in 0.1% (v/v) Triton X-100 for 10 min. Then, 7.5% (v/v) BSA was used as a blocking buffer for 30 min at 37 °C. Anti-MERS-CoV N primary antibody (1:500 dilution; Sino Biological Inc., Beijing, China) was used to stain the virus. The cells were incubated overnight, washed thrice with PBS, and incubated with Alex Fluor 568 anti-rabbit secondary antibody (1:1000 dilution; Thermo Fisher Scientific, Waltham, MA) for 1 h at room temperature. 4′,6-Diamidino-2-phenylindole (DAPI) was added during the PBS wash. MERS nucleocapsid expression was examined under a confocal microscope (LSM-700; Carl Zeiss AG, Oberkochen, Germany)."}

LitCovid-PD-MONDO

LitCovid-PD-CLO

LitCovid-PD-CHEBI

LitCovid-PD-GO-BP

{"project":"LitCovid-PD-GO-BP","denotations":[{"id":"T23","span":{"begin":1182,"end":1187},"obj":"http://purl.obolibrary.org/obo/GO_0019835"},{"id":"T24","span":{"begin":5381,"end":5387},"obj":"http://purl.obolibrary.org/obo/GO_0098739"},{"id":"T25","span":{"begin":5381,"end":5387},"obj":"http://purl.obolibrary.org/obo/GO_0098657"},{"id":"T26","span":{"begin":7744,"end":7759},"obj":"http://purl.obolibrary.org/obo/GO_0016032"},{"id":"T27","span":{"begin":8922,"end":8943},"obj":"http://purl.obolibrary.org/obo/GO_0001171"},{"id":"T28","span":{"begin":8930,"end":8943},"obj":"http://purl.obolibrary.org/obo/GO_0006351"}],"text":"Experimental Section\n\nChemicals\nThe compounds P1, P2, and P3 were purchased from Maybridge Chemical Company, TCI Chemicals, and Sigma-Aldrich Corporation, respectively. The reagents used in this study were purchased from Sigma Chemical Co. (St. Louis, MO). The purity of all compounds is higher than 95% and was used without further purification.\n\nCloning, Protein Expression, and Purification\nThe MERS-CoV N proteins were prepared according to previously described methods.46 In brief, the cDNA fragments of MERS-CoV N proteins were cloned into a pET-28a expression vector (Merck, Darmstadt, Germany) containing a histidine tag-encoding sequence. Vectors encoding the single mutants N39A, N39G, and W43A were generated using the QuikChange site-directed mutagenesis protocol with the primers listed in Table S3. The vectors were transformed into Escherichia coli BL21 (DE3) pLysS cells. The cells were grown to an optical density range of 0.6–0.8 at 600 nm at 37 °C and protein expression induced with 1.0 mM isopropyl β-d-1-thiogalactopyranoside (IPTG), followed by incubation at 10 °C for 24 h. The cells were harvested by centrifugation (6000g, 12 min, 4 °C) and resuspended in lysis buffer (150 mM NaCl, 50 mM Tris-HCl, 15 mM imidazole, and 1 mM PMSF; pH 7.5). The cells were lysed by sonication and centrifuged (10000g, 40 min, 4 °C) to remove debris. The supernatant was purified by injection into a Ni–NTA column (Merck, Darmstadt, Germany) and eluted with buffer containing imidazole at a gradient range of 15–300 mM. Pure protein fractions were collected, dialyzed with low-salt buffer, concentrated, and quantified by the Bradford method (BioShop Canada Inc., Burlington, ON, Canada).\n\nCrystallization and Data Collection\nMERS-CoV N-NTD crystals were grown as previously described:46 MERS-CoV N-NTD was crystallized at room temperature (∼25 °C) by the sitting-drop vapor-diffusion method. A protein solution (2 μL; 10 mg mL–1) was mixed with an equal volume of crystallization solution consisting of 75 mM ammonium sulfate, 2 mM NaBr, and 29% PEG 3350 (Sigma-Aldrich Corp., St. Louis, MO) and equilibrated against a 300 μL solution. MERS-CoV N-NTD:P3 co-crystals were obtained using a crystallization solution containing 2 mM P3. MERS-CoV N-NTD crystals in complex with P1 were obtained by soaking native MERS-CoV N-NTD crystals for 90 s at room temperature in a crystallization solution containing 2 mM P1. Diffraction datasets for MERS-CoV N-NTD alone and in complex with P1 were collected at beamline 13B1 of the Taiwan Light Source (TLS) of the National Synchrotron Research Center (NSRRC; Hsinchu City, Taiwan). Diffraction of the MERS-CoV N-NTD:P3 complex was performed at the beamline SP44XU of SPring-8 (Hyogo, Japan).\n\nStructural Determination and Refinement\nDiffraction data were processed and scaled with HKL-2000 software. The structures were solved by molecular replacement (MR) in Phenix47 using HCoV-OC43 N-NTD (PDB:4J3K) as the search model.24 The initial models were rebuilt and refined by Coot48 and Phenix. Structures were visualized using PyMOL (The PyMOL Molecular Graphics System, version 2.3.0).49\n\nChemical Cross-Link Assay\nProtein solutions containing 40 μM of wild-type or mutated MERS-CoV N-NTD were incubated with glutaraldehyde at a final concentration of 1% v/v. The reaction was conducted at room temperature for 10 min and quenched with the addition of 1 M Tris-HCl (pH 7.5). The samples were then stored on ice and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).\n\nDiscovery of Orthosteric PPI Stabilizers for MERS N Protein\nTo screen for compounds that induce hydrophobic PPI between MERS N-NTDs, a model of dimeric MERS-CoV N-NTD without the H37 and M38 residues was used in virtual drug screening. The Sigma-Aldrich, Acros Organics, and ZINC drug databases were screened with LIBDOCK molecular docking software to obtain compounds acting on the N protein. The N protein binding pocket was represented by a set of spheres. Each compound in the database was docked in a pocket comprising W43. The hydrophobic complementarity between ligands and receptors was calculated with PLATINUM.31 Compounds with higher docking scores are listed in Table S2.\n\nFluorescence Measurements\nFluorescence assays were performed in a buffer consisting of 50 mM Tris-HCl (pH 8.3) and 150 mM NaCl. One micromolar N protein was incubated either with the control buffer or each compound (10 μM) at 4 °C for 1 h. Tryptophan fluorescence was acquired with a Jasco FP-8300 fluorescence spectrometer (JASCO International Co. Ltd., Tokyo, Japan) at an excitation wavelength of 280 nm and an emission wavelength range of 300–400 nm.\n\nThermostability Measurements\nThermostability assays were conducted in a buffer consisting of 50 mM Tris-HCl (pH 7.5) and 150 mM NaCl and with a JASCO FP-8300 fluorescence spectrometer (JASCO International Co. Ltd., Tokyo, Japan). One micromolar N protein was incubated either with the control buffer or each compound (10 μM) at 4 °C for 2 h. UV absorbance vs temperature profiles were acquired by ramping the temperature from 4–95 °C at a 1 °C min–1 and recording the absorbance at 280 nm every 0.5 min.\n\nDetermining CC50 and EC50 of Hit Compounds\nVero E6 cells were infected with MERS-CoV with M.O.I = 0.1 and treated with lead compounds for 48 h. Cell viability was determined by the neutral red uptake assay. CC50 and EC50 were determined by % cell viability. CC50 was determined for cells treated with drugs only. EC50 was determined for MERS-infected cells after drug treatments.\n\nSmall-Angle X-ray Scattering (SAXS) Experiments\nSAXS experiments were performed at the BL23A SAXS beamline at the TLS of NSRRC, using a monochromatic X-ray beam (λ = 0.828 Å), with an integrated HPLC system of an Agilent-Bio SEC-3 300 Å column (Agilent Technologies, Inc. Santa Clara, CA). Protein samples (44 μM MERS-CoV N and MERS-CoV N:P3 complex prepared by incubating the 44 μM native protein with 440 μM P3) were prepared in a buffer consisting of 50 mM Tris-HCl (pH 8.5) and 150 mM NaCl on ice for 1 h. Then, a 100 μL aliquot was injected into the column at a flow rate of 0.02 mL min–1. After passing through the column, the sample solution was directed into a quartz capillary (2 mm dia.) for subsequent buffer and sample SAXS measurement at 288 K. The sample-to-detector distance of 2.5 m used covered a scattering vector q range of 0.01–0.20 Å–1. Here, q is defined as q = (4π/λ) sin θ, with the scattering angle 2θ. Thirty-six frames were collected for each sample elution with an X-ray frame exposure time of 30 s. Frames of good data overlapping (namely, of low radiation damage effects) were merged for improved data statistics and analyzed to determine initial Rg using PRIMUS (version 3.1). The P(r) distance distribution and Dmax were calculated from the experimental scattering curve using GNOM (version 4.1). An ensemble optimization method (EOM) analysis was performed through the EMBL Hamburg web interface.50 Modeling of the rigid body crystal structure was calculated and generated using CRYSOL (ATSAS Program Suite v. 2.8.2).51 The crystal structures of MERS-CoV NTD (PDB ID: 4UD1)27 and MERS-CoV NTD:P3 (solved in this study) and the CTD domain of MERS-CoV N protein (PDB ID: 6G13)23 were used as rigid bodies in EOM analysis. With the EOM analysis, 1000 models were generated in the beginning as a structural pool. Selected from the SAXS profiles of the structural pool was an ensemble of models that could fit the experimental scattering curve with their linear combination. Tetrameric MERS-CoV NP conformations and 16-mer MERS-CoV:P3 conformations were selected because their ensemble generated curves fit best to the experimental SAXS results.\n\nViral Infection\nVero E6 cells (ATCC No: CRL-1586) were seeded onto culture plates with complete Dulbecco’s modified Eagle’s medium (DMEM) and incubated overnight prior to infection. MERS-CoV (HCoV-EMC/2012) at a multiplicity of infection (M.O.I.) of 0.1 was added to the cells and incubated at 37 °C for 1 h, followed by washing thrice with phosphate-buffered saline (PBS) to remove the unattached virus. Fresh complete culture medium was then added to the plates.\n\nPlaque Assay\nVero E6 cells were seeded in 12-well plates and incubated overnight before the assays. Samples containing MERS-CoV were serially diluted 10× with MEM, added to the wells, and incubated for 1 h with agitation every 15 min. After incubation, the inocula were removed and washed with PBS. An overlay medium comprising 2× MEM and 1.5% (w/v) agarose (1:1) was added to the wells followed by incubation at 37 °C and 5% CO2 for 3 days. The plates were fixed with 10% (v/v) formalin containing 0.2% (w/v) crystal violet, and the plaques were counted.\n\nRT-qPCR\nTotal RNA of infected Vero E6 cells was extracted with the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Reverse transcription and PCR amplification were performed with an iTaq Universal One-Step RT-qPCR Kit (Bio-Rad Laboratories, Hercules, CA). Real-time PCR was performed in a StepOnePlus Real-Time PCR System (Applied Biosystems, Foster City, CA). The primer pairs used to amplify the viral RNA were as follows: GAPDH-F: 5′-GAAGGTGAAGGTCGGAGTC-3′; GAPDH-R: 5′-GAAGATGGTGATGGGATTTC-3′;53 MERS-CoV-F: 5′-CCACTACTCCCATTTCGTCAG-3′; MERS-CoV-R: 5′-CAGTATGTGTAGTGCGCATATAAGCA-3′.54 The MERS RNA levels were normalized to that of GAPDH and compared between MERS-CoV groups at 24 h.p.i. and at 48 h.p.i.\n\nImmunofluorescence Assay\nVero E6 cells were seeded in eight-well chamber slides and incubated overnight prior to infection with MERS-CoV at M.O.I. = 0.1. The cells were fixed with 4% (v/v) paraformaldehyde for 20 min at 4 °C, followed by permeabilization in 0.1% (v/v) Triton X-100 for 10 min. Then, 7.5% (v/v) BSA was used as a blocking buffer for 30 min at 37 °C. Anti-MERS-CoV N primary antibody (1:500 dilution; Sino Biological Inc., Beijing, China) was used to stain the virus. The cells were incubated overnight, washed thrice with PBS, and incubated with Alex Fluor 568 anti-rabbit secondary antibody (1:1000 dilution; Thermo Fisher Scientific, Waltham, MA) for 1 h at room temperature. 4′,6-Diamidino-2-phenylindole (DAPI) was added during the PBS wash. MERS nucleocapsid expression was examined under a confocal microscope (LSM-700; Carl Zeiss AG, Oberkochen, Germany)."}

LitCovid-PD-HP

{"project":"LitCovid-PD-HP","denotations":[{"id":"T5","span":{"begin":8421,"end":8430},"obj":"Phenotype"}],"attributes":[{"id":"A5","pred":"hp_id","subj":"T5","obj":"http://purl.obolibrary.org/obo/HP_0000713"}],"text":"Experimental Section\n\nChemicals\nThe compounds P1, P2, and P3 were purchased from Maybridge Chemical Company, TCI Chemicals, and Sigma-Aldrich Corporation, respectively. The reagents used in this study were purchased from Sigma Chemical Co. (St. Louis, MO). The purity of all compounds is higher than 95% and was used without further purification.\n\nCloning, Protein Expression, and Purification\nThe MERS-CoV N proteins were prepared according to previously described methods.46 In brief, the cDNA fragments of MERS-CoV N proteins were cloned into a pET-28a expression vector (Merck, Darmstadt, Germany) containing a histidine tag-encoding sequence. Vectors encoding the single mutants N39A, N39G, and W43A were generated using the QuikChange site-directed mutagenesis protocol with the primers listed in Table S3. The vectors were transformed into Escherichia coli BL21 (DE3) pLysS cells. The cells were grown to an optical density range of 0.6–0.8 at 600 nm at 37 °C and protein expression induced with 1.0 mM isopropyl β-d-1-thiogalactopyranoside (IPTG), followed by incubation at 10 °C for 24 h. The cells were harvested by centrifugation (6000g, 12 min, 4 °C) and resuspended in lysis buffer (150 mM NaCl, 50 mM Tris-HCl, 15 mM imidazole, and 1 mM PMSF; pH 7.5). The cells were lysed by sonication and centrifuged (10000g, 40 min, 4 °C) to remove debris. The supernatant was purified by injection into a Ni–NTA column (Merck, Darmstadt, Germany) and eluted with buffer containing imidazole at a gradient range of 15–300 mM. Pure protein fractions were collected, dialyzed with low-salt buffer, concentrated, and quantified by the Bradford method (BioShop Canada Inc., Burlington, ON, Canada).\n\nCrystallization and Data Collection\nMERS-CoV N-NTD crystals were grown as previously described:46 MERS-CoV N-NTD was crystallized at room temperature (∼25 °C) by the sitting-drop vapor-diffusion method. A protein solution (2 μL; 10 mg mL–1) was mixed with an equal volume of crystallization solution consisting of 75 mM ammonium sulfate, 2 mM NaBr, and 29% PEG 3350 (Sigma-Aldrich Corp., St. Louis, MO) and equilibrated against a 300 μL solution. MERS-CoV N-NTD:P3 co-crystals were obtained using a crystallization solution containing 2 mM P3. MERS-CoV N-NTD crystals in complex with P1 were obtained by soaking native MERS-CoV N-NTD crystals for 90 s at room temperature in a crystallization solution containing 2 mM P1. Diffraction datasets for MERS-CoV N-NTD alone and in complex with P1 were collected at beamline 13B1 of the Taiwan Light Source (TLS) of the National Synchrotron Research Center (NSRRC; Hsinchu City, Taiwan). Diffraction of the MERS-CoV N-NTD:P3 complex was performed at the beamline SP44XU of SPring-8 (Hyogo, Japan).\n\nStructural Determination and Refinement\nDiffraction data were processed and scaled with HKL-2000 software. The structures were solved by molecular replacement (MR) in Phenix47 using HCoV-OC43 N-NTD (PDB:4J3K) as the search model.24 The initial models were rebuilt and refined by Coot48 and Phenix. Structures were visualized using PyMOL (The PyMOL Molecular Graphics System, version 2.3.0).49\n\nChemical Cross-Link Assay\nProtein solutions containing 40 μM of wild-type or mutated MERS-CoV N-NTD were incubated with glutaraldehyde at a final concentration of 1% v/v. The reaction was conducted at room temperature for 10 min and quenched with the addition of 1 M Tris-HCl (pH 7.5). The samples were then stored on ice and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).\n\nDiscovery of Orthosteric PPI Stabilizers for MERS N Protein\nTo screen for compounds that induce hydrophobic PPI between MERS N-NTDs, a model of dimeric MERS-CoV N-NTD without the H37 and M38 residues was used in virtual drug screening. The Sigma-Aldrich, Acros Organics, and ZINC drug databases were screened with LIBDOCK molecular docking software to obtain compounds acting on the N protein. The N protein binding pocket was represented by a set of spheres. Each compound in the database was docked in a pocket comprising W43. The hydrophobic complementarity between ligands and receptors was calculated with PLATINUM.31 Compounds with higher docking scores are listed in Table S2.\n\nFluorescence Measurements\nFluorescence assays were performed in a buffer consisting of 50 mM Tris-HCl (pH 8.3) and 150 mM NaCl. One micromolar N protein was incubated either with the control buffer or each compound (10 μM) at 4 °C for 1 h. Tryptophan fluorescence was acquired with a Jasco FP-8300 fluorescence spectrometer (JASCO International Co. Ltd., Tokyo, Japan) at an excitation wavelength of 280 nm and an emission wavelength range of 300–400 nm.\n\nThermostability Measurements\nThermostability assays were conducted in a buffer consisting of 50 mM Tris-HCl (pH 7.5) and 150 mM NaCl and with a JASCO FP-8300 fluorescence spectrometer (JASCO International Co. Ltd., Tokyo, Japan). One micromolar N protein was incubated either with the control buffer or each compound (10 μM) at 4 °C for 2 h. UV absorbance vs temperature profiles were acquired by ramping the temperature from 4–95 °C at a 1 °C min–1 and recording the absorbance at 280 nm every 0.5 min.\n\nDetermining CC50 and EC50 of Hit Compounds\nVero E6 cells were infected with MERS-CoV with M.O.I = 0.1 and treated with lead compounds for 48 h. Cell viability was determined by the neutral red uptake assay. CC50 and EC50 were determined by % cell viability. CC50 was determined for cells treated with drugs only. EC50 was determined for MERS-infected cells after drug treatments.\n\nSmall-Angle X-ray Scattering (SAXS) Experiments\nSAXS experiments were performed at the BL23A SAXS beamline at the TLS of NSRRC, using a monochromatic X-ray beam (λ = 0.828 Å), with an integrated HPLC system of an Agilent-Bio SEC-3 300 Å column (Agilent Technologies, Inc. Santa Clara, CA). Protein samples (44 μM MERS-CoV N and MERS-CoV N:P3 complex prepared by incubating the 44 μM native protein with 440 μM P3) were prepared in a buffer consisting of 50 mM Tris-HCl (pH 8.5) and 150 mM NaCl on ice for 1 h. Then, a 100 μL aliquot was injected into the column at a flow rate of 0.02 mL min–1. After passing through the column, the sample solution was directed into a quartz capillary (2 mm dia.) for subsequent buffer and sample SAXS measurement at 288 K. The sample-to-detector distance of 2.5 m used covered a scattering vector q range of 0.01–0.20 Å–1. Here, q is defined as q = (4π/λ) sin θ, with the scattering angle 2θ. Thirty-six frames were collected for each sample elution with an X-ray frame exposure time of 30 s. Frames of good data overlapping (namely, of low radiation damage effects) were merged for improved data statistics and analyzed to determine initial Rg using PRIMUS (version 3.1). The P(r) distance distribution and Dmax were calculated from the experimental scattering curve using GNOM (version 4.1). An ensemble optimization method (EOM) analysis was performed through the EMBL Hamburg web interface.50 Modeling of the rigid body crystal structure was calculated and generated using CRYSOL (ATSAS Program Suite v. 2.8.2).51 The crystal structures of MERS-CoV NTD (PDB ID: 4UD1)27 and MERS-CoV NTD:P3 (solved in this study) and the CTD domain of MERS-CoV N protein (PDB ID: 6G13)23 were used as rigid bodies in EOM analysis. With the EOM analysis, 1000 models were generated in the beginning as a structural pool. Selected from the SAXS profiles of the structural pool was an ensemble of models that could fit the experimental scattering curve with their linear combination. Tetrameric MERS-CoV NP conformations and 16-mer MERS-CoV:P3 conformations were selected because their ensemble generated curves fit best to the experimental SAXS results.\n\nViral Infection\nVero E6 cells (ATCC No: CRL-1586) were seeded onto culture plates with complete Dulbecco’s modified Eagle’s medium (DMEM) and incubated overnight prior to infection. MERS-CoV (HCoV-EMC/2012) at a multiplicity of infection (M.O.I.) of 0.1 was added to the cells and incubated at 37 °C for 1 h, followed by washing thrice with phosphate-buffered saline (PBS) to remove the unattached virus. Fresh complete culture medium was then added to the plates.\n\nPlaque Assay\nVero E6 cells were seeded in 12-well plates and incubated overnight before the assays. Samples containing MERS-CoV were serially diluted 10× with MEM, added to the wells, and incubated for 1 h with agitation every 15 min. After incubation, the inocula were removed and washed with PBS. An overlay medium comprising 2× MEM and 1.5% (w/v) agarose (1:1) was added to the wells followed by incubation at 37 °C and 5% CO2 for 3 days. The plates were fixed with 10% (v/v) formalin containing 0.2% (w/v) crystal violet, and the plaques were counted.\n\nRT-qPCR\nTotal RNA of infected Vero E6 cells was extracted with the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Reverse transcription and PCR amplification were performed with an iTaq Universal One-Step RT-qPCR Kit (Bio-Rad Laboratories, Hercules, CA). Real-time PCR was performed in a StepOnePlus Real-Time PCR System (Applied Biosystems, Foster City, CA). The primer pairs used to amplify the viral RNA were as follows: GAPDH-F: 5′-GAAGGTGAAGGTCGGAGTC-3′; GAPDH-R: 5′-GAAGATGGTGATGGGATTTC-3′;53 MERS-CoV-F: 5′-CCACTACTCCCATTTCGTCAG-3′; MERS-CoV-R: 5′-CAGTATGTGTAGTGCGCATATAAGCA-3′.54 The MERS RNA levels were normalized to that of GAPDH and compared between MERS-CoV groups at 24 h.p.i. and at 48 h.p.i.\n\nImmunofluorescence Assay\nVero E6 cells were seeded in eight-well chamber slides and incubated overnight prior to infection with MERS-CoV at M.O.I. = 0.1. The cells were fixed with 4% (v/v) paraformaldehyde for 20 min at 4 °C, followed by permeabilization in 0.1% (v/v) Triton X-100 for 10 min. Then, 7.5% (v/v) BSA was used as a blocking buffer for 30 min at 37 °C. Anti-MERS-CoV N primary antibody (1:500 dilution; Sino Biological Inc., Beijing, China) was used to stain the virus. The cells were incubated overnight, washed thrice with PBS, and incubated with Alex Fluor 568 anti-rabbit secondary antibody (1:1000 dilution; Thermo Fisher Scientific, Waltham, MA) for 1 h at room temperature. 4′,6-Diamidino-2-phenylindole (DAPI) was added during the PBS wash. MERS nucleocapsid expression was examined under a confocal microscope (LSM-700; Carl Zeiss AG, Oberkochen, Germany)."}

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{"project":"2_test","denotations":[{"id":"32105468-26249685-61929524","span":{"begin":474,"end":476},"obj":"26249685"},{"id":"32105468-26249685-61929525","span":{"begin":1792,"end":1794},"obj":"26249685"},{"id":"32105468-12393927-61929526","span":{"begin":2912,"end":2914},"obj":"12393927"},{"id":"32105468-15572765-61929527","span":{"begin":3022,"end":3024},"obj":"15572765"},{"id":"32105468-19244385-61929528","span":{"begin":4162,"end":4164},"obj":"19244385"},{"id":"32105468-25484842-61929529","span":{"begin":6998,"end":7000},"obj":"25484842"},{"id":"32105468-26894667-61929530","span":{"begin":7175,"end":7177},"obj":"26894667"},{"id":"32105468-30644840-61929531","span":{"begin":7276,"end":7278},"obj":"30644840"},{"id":"32105468-22026976-61929532","span":{"begin":9304,"end":9306},"obj":"22026976"},{"id":"32105468-26432410-61929533","span":{"begin":9393,"end":9395},"obj":"26432410"}],"text":"Experimental Section\n\nChemicals\nThe compounds P1, P2, and P3 were purchased from Maybridge Chemical Company, TCI Chemicals, and Sigma-Aldrich Corporation, respectively. The reagents used in this study were purchased from Sigma Chemical Co. (St. Louis, MO). The purity of all compounds is higher than 95% and was used without further purification.\n\nCloning, Protein Expression, and Purification\nThe MERS-CoV N proteins were prepared according to previously described methods.46 In brief, the cDNA fragments of MERS-CoV N proteins were cloned into a pET-28a expression vector (Merck, Darmstadt, Germany) containing a histidine tag-encoding sequence. Vectors encoding the single mutants N39A, N39G, and W43A were generated using the QuikChange site-directed mutagenesis protocol with the primers listed in Table S3. The vectors were transformed into Escherichia coli BL21 (DE3) pLysS cells. The cells were grown to an optical density range of 0.6–0.8 at 600 nm at 37 °C and protein expression induced with 1.0 mM isopropyl β-d-1-thiogalactopyranoside (IPTG), followed by incubation at 10 °C for 24 h. The cells were harvested by centrifugation (6000g, 12 min, 4 °C) and resuspended in lysis buffer (150 mM NaCl, 50 mM Tris-HCl, 15 mM imidazole, and 1 mM PMSF; pH 7.5). The cells were lysed by sonication and centrifuged (10000g, 40 min, 4 °C) to remove debris. The supernatant was purified by injection into a Ni–NTA column (Merck, Darmstadt, Germany) and eluted with buffer containing imidazole at a gradient range of 15–300 mM. Pure protein fractions were collected, dialyzed with low-salt buffer, concentrated, and quantified by the Bradford method (BioShop Canada Inc., Burlington, ON, Canada).\n\nCrystallization and Data Collection\nMERS-CoV N-NTD crystals were grown as previously described:46 MERS-CoV N-NTD was crystallized at room temperature (∼25 °C) by the sitting-drop vapor-diffusion method. A protein solution (2 μL; 10 mg mL–1) was mixed with an equal volume of crystallization solution consisting of 75 mM ammonium sulfate, 2 mM NaBr, and 29% PEG 3350 (Sigma-Aldrich Corp., St. Louis, MO) and equilibrated against a 300 μL solution. MERS-CoV N-NTD:P3 co-crystals were obtained using a crystallization solution containing 2 mM P3. MERS-CoV N-NTD crystals in complex with P1 were obtained by soaking native MERS-CoV N-NTD crystals for 90 s at room temperature in a crystallization solution containing 2 mM P1. Diffraction datasets for MERS-CoV N-NTD alone and in complex with P1 were collected at beamline 13B1 of the Taiwan Light Source (TLS) of the National Synchrotron Research Center (NSRRC; Hsinchu City, Taiwan). Diffraction of the MERS-CoV N-NTD:P3 complex was performed at the beamline SP44XU of SPring-8 (Hyogo, Japan).\n\nStructural Determination and Refinement\nDiffraction data were processed and scaled with HKL-2000 software. The structures were solved by molecular replacement (MR) in Phenix47 using HCoV-OC43 N-NTD (PDB:4J3K) as the search model.24 The initial models were rebuilt and refined by Coot48 and Phenix. Structures were visualized using PyMOL (The PyMOL Molecular Graphics System, version 2.3.0).49\n\nChemical Cross-Link Assay\nProtein solutions containing 40 μM of wild-type or mutated MERS-CoV N-NTD were incubated with glutaraldehyde at a final concentration of 1% v/v. The reaction was conducted at room temperature for 10 min and quenched with the addition of 1 M Tris-HCl (pH 7.5). The samples were then stored on ice and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).\n\nDiscovery of Orthosteric PPI Stabilizers for MERS N Protein\nTo screen for compounds that induce hydrophobic PPI between MERS N-NTDs, a model of dimeric MERS-CoV N-NTD without the H37 and M38 residues was used in virtual drug screening. The Sigma-Aldrich, Acros Organics, and ZINC drug databases were screened with LIBDOCK molecular docking software to obtain compounds acting on the N protein. The N protein binding pocket was represented by a set of spheres. Each compound in the database was docked in a pocket comprising W43. The hydrophobic complementarity between ligands and receptors was calculated with PLATINUM.31 Compounds with higher docking scores are listed in Table S2.\n\nFluorescence Measurements\nFluorescence assays were performed in a buffer consisting of 50 mM Tris-HCl (pH 8.3) and 150 mM NaCl. One micromolar N protein was incubated either with the control buffer or each compound (10 μM) at 4 °C for 1 h. Tryptophan fluorescence was acquired with a Jasco FP-8300 fluorescence spectrometer (JASCO International Co. Ltd., Tokyo, Japan) at an excitation wavelength of 280 nm and an emission wavelength range of 300–400 nm.\n\nThermostability Measurements\nThermostability assays were conducted in a buffer consisting of 50 mM Tris-HCl (pH 7.5) and 150 mM NaCl and with a JASCO FP-8300 fluorescence spectrometer (JASCO International Co. Ltd., Tokyo, Japan). One micromolar N protein was incubated either with the control buffer or each compound (10 μM) at 4 °C for 2 h. UV absorbance vs temperature profiles were acquired by ramping the temperature from 4–95 °C at a 1 °C min–1 and recording the absorbance at 280 nm every 0.5 min.\n\nDetermining CC50 and EC50 of Hit Compounds\nVero E6 cells were infected with MERS-CoV with M.O.I = 0.1 and treated with lead compounds for 48 h. Cell viability was determined by the neutral red uptake assay. CC50 and EC50 were determined by % cell viability. CC50 was determined for cells treated with drugs only. EC50 was determined for MERS-infected cells after drug treatments.\n\nSmall-Angle X-ray Scattering (SAXS) Experiments\nSAXS experiments were performed at the BL23A SAXS beamline at the TLS of NSRRC, using a monochromatic X-ray beam (λ = 0.828 Å), with an integrated HPLC system of an Agilent-Bio SEC-3 300 Å column (Agilent Technologies, Inc. Santa Clara, CA). Protein samples (44 μM MERS-CoV N and MERS-CoV N:P3 complex prepared by incubating the 44 μM native protein with 440 μM P3) were prepared in a buffer consisting of 50 mM Tris-HCl (pH 8.5) and 150 mM NaCl on ice for 1 h. Then, a 100 μL aliquot was injected into the column at a flow rate of 0.02 mL min–1. After passing through the column, the sample solution was directed into a quartz capillary (2 mm dia.) for subsequent buffer and sample SAXS measurement at 288 K. The sample-to-detector distance of 2.5 m used covered a scattering vector q range of 0.01–0.20 Å–1. Here, q is defined as q = (4π/λ) sin θ, with the scattering angle 2θ. Thirty-six frames were collected for each sample elution with an X-ray frame exposure time of 30 s. Frames of good data overlapping (namely, of low radiation damage effects) were merged for improved data statistics and analyzed to determine initial Rg using PRIMUS (version 3.1). The P(r) distance distribution and Dmax were calculated from the experimental scattering curve using GNOM (version 4.1). An ensemble optimization method (EOM) analysis was performed through the EMBL Hamburg web interface.50 Modeling of the rigid body crystal structure was calculated and generated using CRYSOL (ATSAS Program Suite v. 2.8.2).51 The crystal structures of MERS-CoV NTD (PDB ID: 4UD1)27 and MERS-CoV NTD:P3 (solved in this study) and the CTD domain of MERS-CoV N protein (PDB ID: 6G13)23 were used as rigid bodies in EOM analysis. With the EOM analysis, 1000 models were generated in the beginning as a structural pool. Selected from the SAXS profiles of the structural pool was an ensemble of models that could fit the experimental scattering curve with their linear combination. Tetrameric MERS-CoV NP conformations and 16-mer MERS-CoV:P3 conformations were selected because their ensemble generated curves fit best to the experimental SAXS results.\n\nViral Infection\nVero E6 cells (ATCC No: CRL-1586) were seeded onto culture plates with complete Dulbecco’s modified Eagle’s medium (DMEM) and incubated overnight prior to infection. MERS-CoV (HCoV-EMC/2012) at a multiplicity of infection (M.O.I.) of 0.1 was added to the cells and incubated at 37 °C for 1 h, followed by washing thrice with phosphate-buffered saline (PBS) to remove the unattached virus. Fresh complete culture medium was then added to the plates.\n\nPlaque Assay\nVero E6 cells were seeded in 12-well plates and incubated overnight before the assays. Samples containing MERS-CoV were serially diluted 10× with MEM, added to the wells, and incubated for 1 h with agitation every 15 min. After incubation, the inocula were removed and washed with PBS. An overlay medium comprising 2× MEM and 1.5% (w/v) agarose (1:1) was added to the wells followed by incubation at 37 °C and 5% CO2 for 3 days. The plates were fixed with 10% (v/v) formalin containing 0.2% (w/v) crystal violet, and the plaques were counted.\n\nRT-qPCR\nTotal RNA of infected Vero E6 cells was extracted with the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Reverse transcription and PCR amplification were performed with an iTaq Universal One-Step RT-qPCR Kit (Bio-Rad Laboratories, Hercules, CA). Real-time PCR was performed in a StepOnePlus Real-Time PCR System (Applied Biosystems, Foster City, CA). The primer pairs used to amplify the viral RNA were as follows: GAPDH-F: 5′-GAAGGTGAAGGTCGGAGTC-3′; GAPDH-R: 5′-GAAGATGGTGATGGGATTTC-3′;53 MERS-CoV-F: 5′-CCACTACTCCCATTTCGTCAG-3′; MERS-CoV-R: 5′-CAGTATGTGTAGTGCGCATATAAGCA-3′.54 The MERS RNA levels were normalized to that of GAPDH and compared between MERS-CoV groups at 24 h.p.i. and at 48 h.p.i.\n\nImmunofluorescence Assay\nVero E6 cells were seeded in eight-well chamber slides and incubated overnight prior to infection with MERS-CoV at M.O.I. = 0.1. The cells were fixed with 4% (v/v) paraformaldehyde for 20 min at 4 °C, followed by permeabilization in 0.1% (v/v) Triton X-100 for 10 min. Then, 7.5% (v/v) BSA was used as a blocking buffer for 30 min at 37 °C. Anti-MERS-CoV N primary antibody (1:500 dilution; Sino Biological Inc., Beijing, China) was used to stain the virus. The cells were incubated overnight, washed thrice with PBS, and incubated with Alex Fluor 568 anti-rabbit secondary antibody (1:1000 dilution; Thermo Fisher Scientific, Waltham, MA) for 1 h at room temperature. 4′,6-Diamidino-2-phenylindole (DAPI) was added during the PBS wash. MERS nucleocapsid expression was examined under a confocal microscope (LSM-700; Carl Zeiss AG, Oberkochen, Germany)."}

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