FIG. 9.
Cell lines that constitutively express wt and mutant TBP resistant to cleavage by 3Cpro. (A) Detection of mutant TBP in various cell lines. Five clonally selected HeLa cell lines that constitutively express His-TBP were isolated as described in Materials and Methods. Each line was examined for expression of recombinant TBP by Western analysis using an antibody to the His tag. Two of these lines (GG1 and GG2) contained His-TBP in which the two alanines at positions 100 and 101 were replaced by glycine. The two alanine residues in three other lines (LL1, LL2, and LL3) were replaced by L. In both mutants the 18Q-19G and the 34Y-35G bonds were mutated to 18A-19A and 34A-35A, respectively. The wild-type line expresses wt His-TBP. In lane 1, HeLa cell extracts containing the endogenous TBP, not detected by anti-His antibody, was used. (B) GG1 rTBP is resistant to 3Cpro-mediated cleavage. Cell extracts from wild-type and GG1 rTBP (GG1) lines were digested with buffer (lanes 1 and 3) or purified 3Cpro (lanes 2 and 4) prior to Western analysis using anti-His antibody. (C) Five cellular mRNA levels as indicated were examined by RT-PCR from wt rTBP (odd-numbered lanes) and GG1 rTBP (even-numbered lanes) cells.