Fig. 1. The effect of the C39S and W32S E5 mutants in HDFs. (A) The amino acid sequence of the BPV E5 protein showing the positions of the amino acid substitutions present in the W32S and C39S mutants. Boxed region indicates the predicted transmembrane (TM) domain. (B) Extracts of HDFs stably expressing wild-type (wt) E5, W32S, C39S or no viral oncogene (control) were immunoprecipitated (IP) with an anti-E5 or anti-PDGFβR (PR) antiserum. Immunoprecipitates then were subjected to immunoblotting for PDGFβR (PR), E5 or phosphotyrosine (PY) as indicated. Mature (m) and precursor (p) forms of the PDGFβR (PR) are indicated by the arrows on the left. (C) HDFs expressing wild-type (wt) E5, the W32S or C39S mutant, or no viral oncogene (control) were plated at 1.6x10⁵ cells per 60 mm dish and counted in triplicate at various times thereafter. The mean number of cells with the standard error is plotted. (D,E) A focus-forming assay was performed by infecting HDFs with recombinant retroviruses expressing wild-type (wt) E5, C39S, W32S or no viral oncogene (control) and then maintaining cells at confluence as described in the Materials and Methods. In D, Crystal Violet-stained monolayers are shown for visualization of foci. In E, the number of foci induced by E5, C39S and W32S were counted, averaged, corrected for virus titer and expressed as the mean percent (with standard error) relative to the number of foci induced by wild-type E5.