Distinct Roles for Sialoside and Protein Receptors in Coronavirus Infection

Qing, Enya; Hantak, Michael; Perlman, Stanley; Gallagher, Tom

Cells. HEK293T (ATCC), HeLa (ATCC), and HeLa-mCEACAM (77, 78) cells were maintained in DMEMϪ10% FBS medium (Dulbecco's modified Eagle medium [DMEM] containing 10 mM HEPES, 100 nM sodium pyruvate, 0.1 mM nonessential amino acids, 100 U/ml penicillin G, and 100 g/ml streptomycin, and supplemented with 10% fetal bovine serum [FBS] [Atlanta Biologicals]). BHK-21 cells (ATCC) were maintained in DMEMϪ5% FBS medium. LET-1 cells (BEI Resources) (79) were maintained in DMEMϪ10% FBS medium lacking HEPES, sodium pyruvate and nonessential amino acids. Calu3 cells (ATCC) were maintained in MEMϪ20% FBS medium (minimum essential medium [MEM] supplemented with 20% FBS, 100 U/ml penicillin G, and 100 g/ml streptomycin). DBT cells (80, 81) were maintained in MEMϪ5% FBS medium (MEM supplemented with 5% FBS, 10% tryptose-phosphate broth, 26.8 mM sodium bicarbonate, 2 mM L-glutamine, 100 U/ml penicillin G, and 100 g/ml streptomycin). All cell lines were cultured in a 5% CO 2 incubator at 37°C. Viruses. Recombinant MHV strains JHM.SD (82) and A59 (83) , both containing a firefly luciferase (Fluc) reporter between the viral E (envelope) and M (matrix) genes, were grown in DBT cells. Media were collected at 24 to 48 h postinfection. JHM HEϪ arose during laboratory passaging of JHM.SD.