FIG. 5.

Analysis of products expressed from SARS coronavirus pPLpro-HD, pPLpro, and pNSP*3-4 constructs alone and during cotransfection in a trans-cleavage assay. (A) Schematic diagram of the predicted processing of SARS coronavirus ORF1a, the constructs expressing PLpro, PLpro-HD, and the NSP*3-4 substrate. (B) Detection of processing by PLpro-HD at the nsp3/4 cleavage site of ORF1a. The trans-cleavage assay was performed as described for Fig. 4 and in Materials and Methods. (C) Effect of amino acid substitutions on PLpro activity. Predicted catalytic and control residues (indicated above the lanes) were changed to alanine, plasmid DNA was cotransfected with pNSP*3-4, and products were analyzed by SDS-PAGE (lanes 1 to 4). The effect of changing glycine to alanine in the P1 position of the nsp3/4 cleavage site is shown in lane 6. The positions of molecular size markers are shown on the left of each gel (in kilodaltons).