SARS-CoV-2 spike protein pseudotyped VSIVs

Using a replication-incompetent VSIV containing the green fluorescent protein (GFP) instead of the receptor-binding VSIV glycoprotein (G) gene (VSVΔG*-G), VSIVs pseudotyped with S proteins of SARS CoVs (VSVΔG*-SCoV and VSVΔG*-SCoV-2) were generated as described previously (41 (link), 42 (link)). Briefly, 24 h after transfection of HEK293T cells with pCAGGS expressing the S protein of SARS-CoV (Tor2 strain; GenBank accession number NC_004718.3) or SARS-CoV-2 (strain WHU01; GenBank accession number MN988668.1), the cells were incubated with VSVΔG*-G for 60 min at 37°C. After three washes with DMEM, the medium was replaced by DMEM with 10% FCS. After 24 h, the supernatants were harvested and stored at −80°C until use. Virus IUs in HEK293T cells were determined by counting the number of GFP-positive cells with an IN Cell Analyzer 2500HS (GE Healthcare). To produce VSIVs pseudotyped with the S proteins having the substitutions in RBD derived from the Alpha (N501Y), Beta (K417N, E484K, and N501Y), Gamma (K417T, E484K, and N501Y), and Delta (L452R and T478K) variants, the mutant S protein genes were constructed by site-directed mutagenesis with KOD One (Toyobo) based on the S protein gene of SARS-CoV-2 WHU01, which is an early isolate from Wuhan. Each mutation was confirmed by Sanger sequencing.