Figure 5.

The Engineered S-HRV3C/D614G Ectodomain Is More Susceptible to S1/S2 Cleavage by the HRV3C Protease Than S-HRV3C

(A) SEC elution profile on a Superose 6 10/300 column of the S-HRV3C (red) and S-HRV3C/D614G (blue) ectodomains. Fractions isolated for further characterization are indicated by vertical red dotted lines. Elution volumes of standards at 669 and 44 kDa are labeled for reference.

(B) SDS-PAGE of the SEC purified ectodomains.

(C and D) Representative NSEM micrograph of (C) S-HRV3C and (D) S-HRV3C/D614G ectodomains and 2D class averages (related to Data S4).

(E and F) SDS-PAGE of an HRV3C digestion of the (E) S-HRV3C and (F) S-HRV3C/D614G engineered ectodomains at 25°C for 24 h in the presence of 0.03 U of enzyme per microgram of ectodomain. Aliquots corresponding to 1 μg protein at the time points before HRV3C addition, at addition (0 min) and 10 min, 30 min, 60 min, 240 min, and 24 h following HRV3C addition are presented. After 24 h, 0.03 supplementary unit of the HRV3C enzyme per microgram of ectodomain was added, and aliquots were analyzed after 4 additional hours of incubation aiming at completion of the digestion (labeled Suppl.).

(G) Quantification of S protomer (200 kDa) band intensity on SDS-PAGE at the time points presented on (E) and (F) (S-HRV3C in red, S-HRV3C/D614G in blue).

NR, non-reduced sample; R, reduced sample.