FIG. 4.
Analysis of products expressed from SARS coronavirus pPLpro and pNSP1-3* constructs alone and during coexpression in a trans-cleavage assay. (A) Schematic diagram of the predicted processing of SARS coronavirus ORF1a, the constructs expressing the NSP1-3* substrate and PLpro, and the expected final products. (B) Detection of processing by PLpro of the nsp1/2 and nsp2/3 cleavage sites in ORF1a. HeLa-MHVR cells were infected with vTF7.3 and transfected with plasmid DNAs as indicated above each lane. Newly synthesized proteins were labeled with Trans35S label from 5.5 to 10.5 h postinfection. Lysates were prepared and subjected to immunoprecipitation with anti-R1, anti-R2, or anti-V5 antibody. Immunoprecipitated proteins were analyzed by electrophoresis on an SDS-5.0 to 12.5% polyacrylamide gel, processed, and subjected to autoradiography. Products were immunoprecipitated with anti-R1, anti-R2, or anti-V5 epitope tag antibody from cells transfected with DNA encoding the SARS coronavirus NSP1-3* substrate region alone (lanes 1, 4, and 7), the SARS coronavirus PLpro alone (lanes 2, 5, and 8), or cotransfected with both constructs (lanes 3, 6, and 9) or an inactive mutant of PLpro (lane 12). The positions of molecular size markers are shown on the left of each gel (in kilodaltons).