BHK-21, Vero-E6, LLC-MK2, DBT, 293FT, DPP4-expressing Huh7.5 cells, and 17Cl-1 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco) and incubated at 37°C in an atmosphere containing 5% CO2.
HCoV-OC43 (GenBank accession numberAY391777.1 ) expressing the Rluc gene (rOC43-ns2Del-Rluc) and derived from an infectious cDNA clone (17 (link)) was used for HTS in HBK-21 cells. HCoV-NL63 strain Amsterdam I was used to infect monolayers of LLC-MK2 cells at an MOI of 0.01. The MERS-CoV strain EMC was used to infect monolayers of Vero-E6 cells at an MOI of 0.01. The MHV strain A59 was propagated in 17Cl-1 cells, and a plaque assay was performed in monolayers of DBT cells infected with MHV at an MOI of 0.01.
A 2,000-compound library containing FDA-approved drugs and pharmacologically active compounds was purchased from MicroSource Discovery Systems, Inc. (Gaylordsville, CT, USA) (see Table S1 in the supplemental material).
The anti-HCoV-OC43 nucleocapsid protein mouse monoclonal antibody was made in-house. Anti-β-actin (13E5) rabbit monoclonal antibody was obtained from Cell Signaling Technology (Danvers, MA, USA). Infrared IRDye 800CW-labeled goat anti-mouse IgG and 680RD-labeled goat anti-rabbit IgG were purchased from LI-COR Biosciences (Lincoln, NE, USA).
HCoV-OC43 (GenBank accession number
A 2,000-compound library containing FDA-approved drugs and pharmacologically active compounds was purchased from MicroSource Discovery Systems, Inc. (Gaylordsville, CT, USA) (see Table S1 in the supplemental material).
The anti-HCoV-OC43 nucleocapsid protein mouse monoclonal antibody was made in-house. Anti-β-actin (13E5) rabbit monoclonal antibody was obtained from Cell Signaling Technology (Danvers, MA, USA). Infrared IRDye 800CW-labeled goat anti-mouse IgG and 680RD-labeled goat anti-rabbit IgG were purchased from LI-COR Biosciences (Lincoln, NE, USA).