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. 2010 May 5;84(14):7325–7336. doi: 10.1128/JVI.02490-09

FIG. 2.

FIG. 2.

Proteolytic processing of IBV polyproteins by IBV and SARS-CoV 3CLpros. (A) Diagram showing the constructs containing sequences flanking IBV cleavage sites and the 3CLpro-mediated processing in the present study. Uncleaved (U) and cleaved (C) products are represented with straight lines. The predicted sizes of the Flag-tagged (black box) uncleaved and cleaved products are indicated. Western blot analyses of the 3CLpro-mediated processing of wild-type and mutant substrates flanking nsp4-5/Q2779 (B), nsp5-6/Q3086 (B), nsp6-7/Q3379 (B), nsp7-8/Q3462 (C), nsp8-9/Q3672 (C), nsp9-10/Q3783 (C), and nsp12-13/Q4868 (C) and nsp13-14/Q5468 (D), nsp14-15/Q5989 (D), and nsp15-16/Q6327 (D) in IBV 1a and 1ab polyproteins, respectively. The Flag-tagged wild-type and P1-mutant substrates were coexpressed with IBV (I-3C) or SARS-CoV (S-3C) 3CLpro in H1299 cells using the vaccinia virus-T7 system. The transfected cell lysates were resolved on an SDS-15 to 20% polyacrylamide gel and subjected to Western blotting with anti-Flag antibodies. Q, E, H, N, and P represent constructs containing the corresponding amino acid residue at the P1 position. As a control, the amount of expressed 3CLpro was probed with IBV or SARS-CoV anti-3CLpro antibodies. The uncleaved (U) and cleaved (C) products and the relative cleavage efficiency (RCE) are indicated.