The panel of plasmids expressing RBD-mutant SARS-CoV-2 spike proteins in the context of pSARS-CoV-2-SΔ19 has been described13 (link),25 (link),54 . Variant pseudoviruses resembling variants of interest/concern B.1.1.7 (first isolated in the UK), B.1.351 (first isolated in South Africa), B.1.526 (first isolated in New York), P.1 (first isolated in Brazil) and B.1.617.2 (first isolated in India) were generated by introduction of substitutions using synthetic gene fragments (IDT) or overlap extension PCR-mediated mutagenesis and Gibson assembly. Specifically, the variant-specific deletions and substitutions introduced were as follows: B.1.1.7: ΔH69/V70, ΔY144, N501Y, A470D, D614G, P681H, T761I, S982A, D118H; B.1.351: D80A, D215G, L242H, R246I, K417N, E484K, N501Y, D614G, A701V; B.1.526: L5F, T95I, D253G, E484K, D614G, A701V; P.1: L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, D614G, H655Y, T1027I, V1167F; B.1.617.2: T19R, Δ156–158, L452R, T478K, D614G, P681R, D950N.
The E484K, K417N/E484K/N501Y, L452R/E484Q and L452R/T478K substitutions, as well as the deletions/substitutions corresponding to the variants of concern listed above, were incorporated into a spike protein that also included the R683G substitution, which disrupts the furin cleavage site and increases particle infectivity. Neutralizing activity against mutant pseudoviruses was compared to that against a WT SARS-CoV-2 spike sequence (NC_045512 ), carrying R683G where appropriate.
SARS-CoV-2-pseudotyped particles were generated as previously described3 (link),8 . In brief, 293T (CRL-11268) and HT1080 (CCL-121) cells were obtained from ATCC. Cells were transfected with pNL4-3ΔEnv-nanoluc and pSARS-CoV-2-SΔ19 particles were collected 48 h after transfection, filtered and stored at –80 °C to propagate 293T/ACE2 and HT1080/ACE2.cl14 cells. Cell lines were checked for mycoplasma contamination by Hoeschst staining and confirmed negative.
The E484K, K417N/E484K/N501Y, L452R/E484Q and L452R/T478K substitutions, as well as the deletions/substitutions corresponding to the variants of concern listed above, were incorporated into a spike protein that also included the R683G substitution, which disrupts the furin cleavage site and increases particle infectivity. Neutralizing activity against mutant pseudoviruses was compared to that against a WT SARS-CoV-2 spike sequence (
SARS-CoV-2-pseudotyped particles were generated as previously described3 (link),8 . In brief, 293T (CRL-11268) and HT1080 (CCL-121) cells were obtained from ATCC. Cells were transfected with pNL4-3ΔEnv-nanoluc and pSARS-CoV-2-SΔ19 particles were collected 48 h after transfection, filtered and stored at –80 °C to propagate 293T/ACE2 and HT1080/ACE2.cl14 cells. Cell lines were checked for mycoplasma contamination by Hoeschst staining and confirmed negative.
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