I1 hybridoma cells

Expression plasmids for vesicular stomatitis virus (VSV, serotype Indiana) glycoprotein (VSV-G) and SARS-CoV-2 spike variants B.1.1.7, B.1.351, B.1.617.1, and B.1.617.2 (codon-optimized; with a C-terminal truncation for increased pseudovirus packaging) have been described elsewhere.²⁴ (link)^,26 (link)^,27 (link) Transfection of cells was carried out by Transit LT-1 (Mirus). Rhabdoviral pseudotype particles were prepared as previously described.²⁸ (link) A replication-deficient VSV vector in which the genetic information for VSV-G was replaced by genes encoding two reporter proteins, enhanced green fluorescent protein and firefly luciferase (FLuc), VSV∗ΔG-FLuc²⁹ (link) (kindly provided by Gert Zimmer, Institute of Virology and Immunology, Mittelhäusern, Switzerland) was used for pseudotyping. One day after transfection of HEK293T cells to express the viral glycoprotein, they were inoculated with VSV∗ΔG-FLuc and incubated for 1-2 h at 37°C. Then the inoculum was removed, cells were washed with PBS and fresh medium added. After 16-18 h, the supernatant was collected and centrifuged (2,000 × g, 10 min, room temperature) to clear cellular debris. Cell culture medium containing anti-VSV-G antibody (I1-hybridoma cells; ATCC no. CRL-2700) was then added to block residual VSV-G-containing particles. Samples were then aliquoted and stored at -80°C.

Vero-TMPRSS2 cells were generously provided by Dr Benhur Lee (Icahn School of Medicine at Mount Sinai) and are previously described.^{55 (link)} Vero-TMPRSS2 cells were maintained in DMEM (Gibco 11965-092) supplemented with 10% fetal bovine serum (FBS; Gibco 10437-028) and penicillin–streptomycin (1:50; Sigma P4458) at 37°C in a 5% CO₂ atmosphere. I1 hybridoma cells (ATCC CRL-2700) were obtained from the ATCC and maintained at 37°C in 100% room air, in minimal essential media with Hank’s balanced salts (Gibco 11575032) supplemented with 2mM L-glutamine (Sigma G7513) and 20% FBS.

Baby hamster kidney (BHK-G43) cells were a kind gift from PD Dr. Gert Zimmer (Institute of Virology and Immunology, Mittelhäusern, Switzerland) and were kept in Glasgow’s Minimal Essential Medium (GMEM) supplemented with 5–10% fetal bovine serum (FBS). I1-Hybridoma cells (ATCC, CRL-2700) were kept in Dulbecco’s modified Eagle medium (DMEM-15% FBS) with 1% l-Glutamine, 100 IU/mL penicillin, and 0.1 mg/mL streptomycin. Human embryonic kidney (HEK293T) and African green monkey kidney (Vero E6) cells were maintained in DMEM-10 % FBS. All cells were incubated at 37 °C/5% CO₂.