Figure 7.

Relationship between PL1pro and PL2pro proteolytic activities.A, in vitrotranslation reactions of capped RNAs encoding the pp1a/1ab amino acids 717–1285 (lanes 1 and 2), amino acids 717–1436 (lanes 3 and 4), amino acids 717–1910 (lanes 5 and 6), and amino acids 759–1910 (lanes 7 and 8). The proteins to be tested for proteolytic activity were translated in rabbit reticulocyte lysates in the presence of [³⁵S]methionine at 30 °C for 40 min. After the termination of translation, the reaction products were either analyzed directly (lanes 1, 3, 5, and 7) or after further incubation for 120 min (lanes 2, 4, 6, and8). The analysis was done by SDS-polyacrylamide gel electrophoresis in a 10–17% polyacrylamide gradient gel. Full-length precursor proteins and major processing products are indicated (*, precursor protein; ●, processing product). Also, the calculated cleavage activities of the full-length precursor proteins are given (see “Experimental Procedures” for details). B,proteolytic activities of pp717–1910-derived proteins carrying active-site mutations in the two HCoV PLpro domains. The proteins to be tested for proteolytic activity were translated in rabbit reticulocyte lysates in the presence of [³⁵S]methionine at 30 °C for 40 min. After termination of translation, the reaction products were separated by SDS-polyacrylamide gel electrophoresis. They were either analyzed directly (lanes 1, 3, 5, 7, and9) or after further incubation for 120 min (lanes 2, 4, 6, 8, and 10). The proteins, which all encompassed the HCoV pp1a/pp1ab amino acids 717–1910, contained Cys-to-Ala replacements of the putative nucleophilic residues of PL1pro (C1054A,lanes 1 and 2) and PL2pro (C1701A, lanes 5 and 6), 8-amino acid deletions including the putative nucleophilic residues of PL1pro (Δ1054–1061, lanes 3 and4) and PL2pro (Δ1701–1708, lanes 7 and8) or wild-type sequence (lanes 9and10). The positions of full-length precursor proteins and cleavage products are indicated. Also the calculated cleavage activities of the full-length precursor proteins are given.