Figure 6.

Proteolytic activity of the PL2pro domain and identification of its cleavage site.A, the HCoV ORF1a-encoded amino acids 717–1910 were translated in rabbit reticulocyte lysates in the presence of [³⁵S]methionine. The proteins to be tested for proteolytic activity contained wild-type sequence (lanes 1 and 2) or the same sequence with active-site replacements in PL1pro (C1054A, lanes 3 and4) and both PL1pro and PL2pro (C1054A and W1702L), respectively. The proteins were translated at 30 °C for 40 min, and after the termination of translation, the reaction products were either analyzed directly (lanes 1, 3,and 5) or after further incubation for 120 min (lanes 2, 4,and6). The analysis was done by SDS-polyacrylamide gel electrophoresis in a 10–17% polyacrylamide gradient gel. The positions of full-length precursor proteins and cleavage products are indicated. B, a protein called pp717–1910_C1054A-VM was translated in a reticulocyte lysate in the presence of [³⁵S]methionine. Except for a PL1pro-inactivating amino acid replacement (C1054A) and three additional substitutions (V900M, V906M, and V908M), which had been introduced downstream to the predicted cleavage site, this protein contained the HCoV pp1a/1ab wild-type sequence from residues 717 to 1910. The translation reaction was incubated for 160 min at 30 °C, and the reaction products were separated on an SDS-10% polyacrylamide gel. After electro- phoretic transfer to PVDF membranes, the position of the C-terminal cleavage product was determined by autoradiography. The isolated protein was subjected to 16 cycles of Edman degradation, and the distribution of radiolabeled amino acids was determined by scintillation counting. The amino acid sequence of pp1a and pp1ab from positions 895 to 913 is shown. The amino acids Met⁹⁰⁰, Met⁹⁰⁶, and Met⁹⁰⁸ present in pp717–1910_C1054A-VM are shown in boldface type, and the deduced PL2pro cleavage site is indicated by an arrow.