Fig. 4.

Further characterization of a select panel of mutants. (a) E7 mutants with decreased transformation potential. The M84S and QKP96-98EEA mutants, as well as the CR2 del21-24 deletion mutant, which targets the LXCXE motif, were assessed for the ability to target pRb for degradation. Saos2 cells were transfected with a pRb expression plasmid and vectors expressing the indicated E7 mutant; pRb levels were determined by Western blotting at 48 h posttransfection. (b) The ability of E7 mutants to bind pRb was also assessed in a yeast two-hybrid assay. pRb was expressed as bait, with the indicated E7 mutant as prey. β-Galactosidase activity was measured and reported relative to that of wild-type E7. (c) The L67R mutant, with a mutation in the hydrophobic core of E7 which leads to decreased dimerization, and the QKP96-98EEA mutant, which dimerizes but fails to transform cells, were transfected into HEK293 cells together with a GFP expression plasmid. At 24 h posttransfection, cells were treated with cycloheximide for the indicated times and the levels of E7 determined by Western blotting. (d) Cellular localization of M84S and QKP96-98EEA mutants was assessed by immunofluorescence in U2OS cells.