Distinct Roles for Sialoside and Protein Receptors in Coronavirus Infection

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

Virus and pseudovirus particle entry assays. Virus particles were incubated with cells, either with or without prior neuraminidase treatments, for 2 h at 4°C. Cells were then rinsed three times with PBS and replenished with FBS-containing DMEM/MEM. After 16 h at 37°C, cells were dissolved in lysis buffer (25 mM Cell-cell fusion assay. Effector and target cells were prepared by introducing expression plasmids, using the PEI transfection method. Effector cells were cotransfected with pDSP 1-7 and the indicated S-expressing plasmids. S-expressing plasmids included pcDNA3.1-229E S C9 (GenBank accession no. AB691763.1, obtained from Fang Li, University of Minnesota). Control effector cells received pDSP 1-7 and empty vector plasmids. Target cells were cotransfected with pDSP [8] [9] [10] [11] and the indicated S receptorexpressing plasmids. These plasmids included pCMV6-Entry-hDPP4 FLAG (GenBank accession no. NM_001935; purchased from OriGene) and pcDNA3.1-hAPN (GenBank accession no. M22324; obtained from Fang Li). Indicated experiments included additional cotransfection of target cells with pCAGGS-TMPRSS2 FLAG (44) .