Figure 5.

Evidence for a second PL1pro cleavage site in theHCoVpp1a and pp1ab polyproteins.A, in vitro translation reactions of capped RNAs derived fromEcoRI-linearized plasmids pBST-111–103 and pBST-111–103_C1054A. The respective RNAs encode the pp1a/1ab amino acids 717–1285 (WT, lanes 1 and 2) or the same sequence with an active-site replacement of the catalytic Cys¹⁰⁵⁴ (C1054A, lanes 3 and 4). The translation reactions were done as described under “Experimental Procedures,” and the reaction products were either analyzed directly (lanes 1 and 3) or after further incubation for 120 min (lanes 2 and 4). The positions of full-length precursor proteins and cleavage products are indicated.B, a protein called pp717–1285_VM was translated in a reticulocyte lysate in the presence of [³⁵S]methionine. Except for three amino acid substitutions (V900M, V906M, and V908M), which had been introduced downstream to the presumed cleavage site, this protein contained the HCoV pp1a/1ab wild-type sequence from residues 717 to 1285. The translation reaction was incubated for 160 min at 30 °C, and the reaction products were separated on an SDS-12.5% polyacrylamide gel. After electrophoretic 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 pp717–1285_VM are shown in boldface type, and the newly identified PL1pro cleavage site is indicated by an arrow.