SARS-CoV-2 spike-protein D614G mutation increases virion spike density and infectivity

Abstract

SARS-CoV-2 variants with spike (S)-protein D614G mutations now predominate globally. We therefore compare the properties of the mutated S protein (S^G614) with the original (S^D614). We report here pseudoviruses carrying S^G614 enter ACE2-expressing cells more efficiently than those with S^D614. This increased entry correlates with less S1-domain shedding and higher S-protein incorporation into the virion. Similar results are obtained with virus-like particles produced with SARS-CoV-2 M, N, E, and S proteins. However, D614G does not alter S-protein binding to ACE2 or neutralization sensitivity of pseudoviruses. Thus, D614G may increase infectivity by assembling more functional S protein into the virion.

Fig. 1. The D614G mutation is associated with enhanced infectivity.

a Cryo-EM structure of S1 (gray) and S2 (orange) heterodimer (PBD entry 6VXX). Residues 581-676, a C-terminal region of the S1 domain involved in S2 interaction, are shown in green. The black square area is magnified on the right. Residues within 5.5 Å of D614 are shown in a ball-and-stick representation. Aspartic acid 614 is shown in bright green. b A representation of the SARS-CoV-2 S protein (top) and D/G variation at residue 614 presented in logo plots at different time points between January 1^st and May 30^th, 2020 (bottom). c–e Mock- and hACE2-293T cells (c, d) or the same cells transfected to express hTMPRSS2 (e) were infected with the indicated PV. See Supplementary Figs. 1a–c and 2 for PV yields, TMPRSS2 expression in transfected HEK293T cells, and the sequences for various FKO mutations, respectively. f NCI-H1975 cells transduced to express hACE2 were infected with PVs. See Supplementary Fig. 3a for PV yields. The S proteins used in c–f have the FLAG tag at C-terminus. Mean values ± SEM of n = 5 (d), n = 3 (e), or n = 4 (f). Experiments conducted using two or three biologically independent PV batches are shown. The p values by two-way ANOVA (analysis of variance) with Sidak multiple comparisons test are indicated (d, e, f). NTD N-terminal domain, RBD receptor-binding domain, FP fusion peptide, HR1 and HR2 heptad-repeat regions 1 and 2, respectively, TM transmembrane region, CT cytoplasmic tail, GFP green fluorescent protein, FKO furin-cleavage knockout mutant, PV pseudovirus, M.F.I. mean fluorescence intensity.

Fig. 2. Superior infectivity of PV^G614 is associated with decreased S1 shedding and higher level of S protein in the virion.

a–f Indicated MLV PVs produced with the S protein containing the FLAG tag at both the N- and C-termini were purified by pelleting through a 20% sucrose layer. PV yields were assessed by RT-qPCR (a). The same symbols in different PV groups indicate they are from the same batch. The same PVs were assessed for their infectivity in Mock and hACE2-293T cells (b). Mean ± SEM of n = 3 biologically independent PV batches (a) and n = 4 experiments using those three PV batches (b) are shown. The same amount (1 × 10¹⁰ vg per lane) (c, d) or different amounts to more accurately compare the S1 and S2 ratio (e, f) of the purified PVs were analyzed by WB (western blot) using the anti-FLAG M2 antibody or anti-p30 MLV gag antibody. The same PVs visualized by silver stain are shown in Supplementary Fig. 5. Total virion S protein (d) and the S1:S2 ratio (f) of PV^D614 and PV^G614 were calculated from n = 4 (d) or n = 5 (f) WBs performed with three independently prepared PV batches and presented as mean ± SEM. The p values by one-way ANOVA (a), two-way ANOVA with Sidak multiple comparison tests of log-transformed data (b), or two-sided unpaired Student’s t-test (d, f) are indicated. GFP green fluorescent protein, MLV Maloney murine leukemia virus, PV pseudovirus, FKO furin-cleavage knockout mutant, M.F.I. mean fluorescence intensity.

Fig. 3. SARS-CoV-2 VLP^G614 and lung epithelial cell-produced PV^G614 also exhibit decreased S1 shedding and increased total virion S protein.

a–f PVs produced from NCI-H1299, a lung epithelial cell line (a–c), and VLPs (virus-like particles) produced from HEK293T cells by transfecting M, N, E, and S proteins of SARS-CoV-2 (d–f) are analyzed. The S protein is FLAG tagged at both the N- and C-termini. PVs were harvested at 48 h and VLPs at 43 h and purified by pelleting through a 20% sucrose layer. The S protein bands were visualized using the anti-FLAG tag M2 antibody (a, d), p30 band with an anti-p30 antibody (a), and the N protein band using pooled convalescent plasma (d). Representative images of WBs (western blot) performed with n = 4 independently prepared PV (a) or VLP (d) batches are shown. The S1:S2 ratio and the difference in total virion S protein incorporation are presented as mean ± SEM, which were calculated from those four PV WBs (b,c) or four VLP WBs (e,f). The p values by two-sided unpaired Student’s t-test are indicated (b, c, e, f). MLV Maloney murine leukemia virus, PV pseudovirus, FKO furin-cleavage knockout mutant.

Fig. 4. The D614G mutation neither increases S protein affinity for ACE2 nor makes PV more resistant to neutralization.

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-Ctag 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 (k_on) and dissociation (k_off) 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 k_on, k_off, and K_D 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.