Pcdna3 3 cov2 b 1

psPAX2 (Addgene, no.12260) was a gift from Didier Trono. pCDNA3.3_CoV2_B.1.1.7 (Addgene, no.170451) for Alpha-S and pcDNA3.3-SARS2-B.1.617.2 (Addgene, no.172320) for Delta-S proteins, were gifts from David Nemazee^{36 (link)}. pTwist-SARS-CoV-2 Δ18 B.1.351v1 (Addgene, no.169462) for Beta-S protein was a gift from Alejandro Balazs^{37 (link)}. Lentiviral vector, pWPI-ffLuc-P2A-EGFP for luciferase reporter assay and pTRC2puro-ACE2-P2A-TMPRSS2 for the generation of 293T cell line susceptible to SARS-CoV-2 infection was created from pWPI-IRES-Puro-Ak-ACE2-TMPRSS2, a gift from Sonja Best (Addgene, no.154987) by In-Fusion® technology (Takara Bio). pcDNA3.4 expression plasmids encoding SARS-CoV-2 S proteins with human codon optimization and 19 a.a deletion of C-terminus (C-del19) from Wuhan, D614G, and Omicron were generated by assembly of PCR products, annealed oligonucleotides, or artificial synthetic gene fragments (Integrated DNA Technologies, IDT) using In-Fusion® technology. For Delta plus, Kappa and Lambda variants, S proteins with only RBD, D614, and P681 mutations were created from pcDNA3.4 encoding human codon-optimized Wuhan S protein (C-del19). LentiX-293T cells (Takara Bio) and 293T cells were maintained in culture with Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% fetal bovine serum (FBS), penicillin-streptomycin (Nacalai tesque), and 25 mM HEPES (Nacalai tesque).

Construction of pseudovirus that carrying the spike protein of SARS-CoV-2 was performed as previously described (29 (link)). In brief, 60 μL Lipofectamine 3000 transfection reagents (Thermo Fisher) was mixed with 500 μL serum-free DMEM, held at room temperature for 5 min, and then mixed with the following plasmids that were diluted in 500 μL serum-free DMEM for another 20 min: pLAS3w-FLuc-Ppuro (9.5 μg) and pCMV-Δ8.91 (Gag-Pol provider, 6.5 μg), the spike plasmids (4.5 μg) pcDNA3.3_CoV2_B.1.1.7 (Addgene no. 170451) and pcDNA3.3-SARS2-B.1.617.2 (Addgene no. 172320), and the SARS-CoV-2 Omicron Strain S gene Human codon_pcDNA3.1(+) plasmid (B.1.1.529/BA.1) (GenScript no. MC_0101274). This DNA-Lipofectamine mixture was used for cotransfection of HEK-293T cells (4 × 10⁶ cells per 10-cm dish), and the cells were incubated at 37°C in a 5% CO₂ incubator. After overnight incubation for 16 h, the transfected cells were replenished with fresh medium for subculture. At 48 h posttransfection, the pseudovirus-containing culture medium was collected by centrifugation at 1,000 × g for 10 min to remove unwanted cells or large debris, followed by passing the clarified medium through a 0.45-μm-pore filter (Millipore Corporation. Billerica, MA, USA). The virus can be stored at 4°C for immediate use or frozen at –80°C. Pseudovirus titers were determined using the p24 ELISA kit. (TaKaRa Bio).

Construction of pseudovirus that carrying the spike protein of SARS-CoV-2 was performed as previously described (29 (link)). In brief, 60 μL Lipofectamine 3000 transfection reagents (Thermo Fisher) was mixed with 500 μL serum-free DMEM, held at room temperature for 5 min, and then mixed with the following plasmids that were diluted in 500 μL serum-free DMEM for another 20 min: pLAS3w-FLuc-Ppuro (9.5 μg) and pCMV-Δ8.91 (Gag-Pol provider, 6.5 μg), the spike plasmids (4.5 μg) pcDNA3.3_CoV2_B.1.1.7 (Addgene no. 170451) and pcDNA3.3-SARS2-B.1.617.2 (Addgene no. 172320), and the SARS-CoV-2 Omicron Strain S gene Human codon_pcDNA3.1(+) plasmid (B.1.1.529/BA.1) (GenScript no. MC_0101274). This DNA-Lipofectamine mixture was used for cotransfection of HEK-293T cells (4 × 10⁶ cells per 10-cm dish), and the cells were incubated at 37°C in a 5% CO₂ incubator. After overnight incubation for 16 h, the transfected cells were replenished with fresh medium for subculture. At 48 h posttransfection, the pseudovirus-containing culture medium was collected by centrifugation at 1,000 × g for 10 min to remove unwanted cells or large debris, followed by passing the clarified medium through a 0.45-μm-pore filter (Millipore Corporation. Billerica, MA, USA). The virus can be stored at 4°C for immediate use or frozen at –80°C. Pseudovirus titers were determined using the p24 ELISA kit. (TaKaRa Bio).