Phage cmv luc2 ires zsgreen

All the spike mutations were inserted into a codon-optimized version of the Wuhan-Hu-1 SARS-CoV-2 spike (GenBank accession no. QHD43416.1) cloned into a phCMV backbone (GenBank accession no. AJ318514). Mutations were introduced into the phCMV-Spike plasmids using mutagenic primers and the Q5 site-directed mutagenesis kit (New England Biolabs [NEB]). In addition, a truncation of 19 amino acids was introduced into the cytoplasmic tail of each spike studied (deletion at amino acids 1255 to 1273 in the Wuhan-Hu-1 spike) to promote spike incorporation into lentiviral particles, as described previously (67 (link), 68 (link)). All the mutant plasmids were sequenced prior to use. Primers used for mutagenesis and sequencing are reported in Table S1. The plasmid pQCXIP-empty was used as a negative control for spike expression (50 (link)). Plasmids used to produce GFP-expressing lentiviruses were the lentivector backbone pCDH-EF1α-GFP (System Biosciences), the packaging plasmid psPAXII (Addgene), and the pRev plasmid (a gift from P. Charneau). For the production of luciferase-expressing lentivirus, the plasmids used were pHAGE-CMV-Luc2-IRES-ZsGreen (NR-52516), pHDM-Hgpm2 (NR-52517), pHDM-tat1b (NR-52518), pRC-CMV-rev1b (NR-52519), all generated in J. Bloom’s laboratory (69 (link)) and obtained from BEI Resources (kit NR-53816).