Fig. 4: The D614G mutation neither increases S protein affinity for ACE2 nor makes PV more resistant to neutralization.
From: SARS-CoV-2 spike-protein D614G mutation increases virion spike density and infectivity
a The S protein containing C-terminal FLAG tag is transfected into HEK293T cells and assessed for hACE2-NN-Ig binding. Total S protein was measured by detecting the FLAG tag in the permeabilized cells. The ratio of hACE2-NN-Ig binding to FLAG-tag staining is shown. b Experiments similar to those in a except that the S protein contains N-Myc and C-FLAG tags, and S1 level was assessed using an anti-Myc antibody. The data in a,b before normalization are presented in Supplementary Fig. 9a, b. Mean ± SEM of n = 3 independent experiments are shown. The p values by one-way ANOVA and Sidak multiple comparisons test are indicated. c Surface plasmon resonance assay design (left) and sensorgrams (middle and right). S1-Fc was immobilized and monomeric hACE2-NN-Ctag40 was injected at 500, 250, 125, 62.5, and 31.25 nM. Colored lines are the experimental traces and black lines are the best global fits (1:1 Langmuir binding model) used to calculate the association (kon) and dissociation (koff) rate constants. Representative sensorgrams of three independent experiments with nearly identical results are shown. Supplementary Fig. 9c shows the proteins used in these assays and Fig. 9d presents kon, koff, and KD values derived from n = 3 independent experiments. d MLV PVs pseudotyped with the indicated S protein (C-term FLAG) or VSV G protein were preincubated without (presented at x = −6) or with serially diluted convalescent or control plasmas. hACE2-293T cells were incubated with these preincubated mixes and analyzed 24 h later by measuring luciferase activity. Mean ± SEM of n = 3 independent experiments are presented. FKO furin-cleavage knockout mutant.
