FIG. 3.

In vitro cotranslation of NSP-ORF expression constructs to detect trans cleavage. Transcription-translation was carried out with the TNT Coupled Reticulocyte Lysate System in the presence of Trans ³⁵S-Label according to the manufacturer’s instructions, and the resulting proteins were resolved on sodium dodecyl sulfate–10% polyacrylamide gels. (A) Lysates were programmed with pTM1/nsRUB (NSP, lane 1), pTM1/nsRUB* (NSP*, a protease catalytic site mutant, lane 2), pTM1/nsRUB-S (NSP-S, a deletion mutant lacking the C-terminal 31 amino acids, lane 3), or pTM1/nsRUB* plus pTM1/nsRUB-S (lane 4). The positions of migration of the RUB NSPs as well as of the truncated P90 product (P90-S) are indicated. (B) Reactions programmed with pTM1/nsRUB* were carried out in the presence of radiolabel for 120 min. Reactions programmed with pTM1/P150 (a construct containing the P150 sequences), pTM1/nsRUB-S, or pTM1/nsRUB were carried out in the absence of radiolabel. RNase and cycloheximide were then added to final concentrations of 1 and 0.6 mg/ml, respectively, to stop translation. Aliquots of the pTM1/nsRUB* reaction then were mixed with aliquots of the cold, unlabeled pTM1/P150 reaction mixture (P150, lane 2), the pTM1/nsRUB-S reaction mixture (NSP-S, lane 3), or the pTM1/nsRUB reaction mixture (NSP) and incubated for another 120 min at 30°C. An aliquot of the pTM1/nsRUB* reaction mixture was also incubated alone (lane 1). (C) Transcription-translation reactions in the presence of radiolabel were programmed with the following dilutions of the standard amount of pTM1/nsRUB DNA: 1:30 (lane 1), 1:60 (lane 2), 1:100 (lane 3), 1:200 (lane 4), 1:400 (lane 5), or 1:800 (lane 6).