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.