Spike plasmid cloning and lentiviral production

To express the D614 Spike, we used an existing cytomegalovirus (CMV)-driven SARS-CoV-2 plasmid (pcDNA3.1-SARS2-Spike, Addgene 145032) (Shang et al., 2020). To express the G614 Spike, we cloned pcDNA3.1-SARS2-SpikeD614G using the Q5 site-directed mutagenesis kit (NEB E0554S) and the following primers: 5′-CTGTACCAGGgCGTGAATTGCAC-3′ and 5′-CACGGCCACCTGGTTGCT-3′.

To make Spike-pseudotyped lentivirus, we co-transfected a d2EGFP-containing transfer plasmid (Addgene 138152) with accessory plasmid psPAX2 (Addgene 12260) and the pseudotyping plasmid (or omitted the pseudotyping plasmid to produce no-pseudotype lentivirus). Briefly, for each virus, a T-225 flask of 80% confluent HEK293T cells (Thermo) was transfected in OptiMEM (Thermo) using 25 μg of the transfer plasmid, 20 μg psPAX2, 22 μg Spike plasmid, and 175 μL of linear polyethylenimine (1 mg/mL) (Polysciences). Six hours post-transfection media was replaced with fresh D10 media, DMEM (Caisson Labs) with 10% Serum Plus II Medium Supplement (Sigma–Aldrich), with 1% bovine serum albumin (Sigma) added to improve virus stability. After 60 hr, viral supernatants were harvested and centrifuged at 3,000 rpm at 4°C for 10 min to pellet cell debris and filtered using 45 μm PVDF filters (CellTreat). The supernatant was then ultracentrifuged for 2 hr at 100,000 g (Sorvall Lynx 6000), and the pellet was resuspended overnight at 4°C in phosphate-buffered saline (PBS) with 1% bovine serum albumin (BSA).