Fig 3.

(A) Internal cleavage of a CSFV NS4A3 construct. (B) NS4A3 immediately after purification by IMAC (0 h NS4A3) or after incubation for 0.5 h or 2 h at 37°C (0.5 h or 2 h NS4A3) was solved by SDS-PAGE and visualized by Coomassie staining. (C) Degradation products were specified using antibodies against the N and C termini of NS3 (the applied antibodies are indicated below the figure panel). The dominant protein bands were attributed to NS3 (75 kDa), two C-terminal fragments (58 and 55 kDa), and one short-lived N-terminal fragment (35 kDa). To inactivate the NS4A3 protease, a part of the NS4A cofactor peptide was deleted (NS4A_del3) or the essential Ser₁₇₅₂ was mutated (NS4A3_S1752A). Inactivation of the NS4A3 protease stabilized the construct (shown in panel D). The positions of NS4A3 and processing products are indicated at the right. A weak 58-to-60-kDa band visible in all preparations (marked with stars in panels B and D) is most likely a copurified bacterial protein that is not detected by NS3-specific antibodies (C). Molecular mass standards (kDa) are shown on the left.