FIG. 4.
Formation of newly synthesized Vp1 folding intermediates in cells transfected with mutant DNAs. (A to D) Profiles of Vp1 intermediates in cytoplasmic fractions. The Sol (A and B) or Csk (C and D) fraction was prepared from TC7 cells 72 h posttransfection of wild-type (Wt), C87A-C207S mutant (87/207), or C49A-C87A mutant (49/87) NO-SV40 DNA, following 5 min of [35S]methionine pulse labeling with no chase (lanes 1, 4, and 7), with 10 min of chase (lanes 2, 5, and 8), or with 40 min of chase (lanes 3, 6, and 9). Aliquots of the Sol or Csk fraction (equivalent to 6 × 105 cells) were immunoprecipitated with affinity-purified anti-Vp1 IgG, and the IPs were denatured under nonreducing (A and C) or reducing (B and D) conditions, resolved by SDS-PAGE, and processed for fluorography. Bands corresponding to monomeric, dimeric, trimeric, and higher oligomeric forms of Vp1 are denoted 1, 2, 3, and 4+, respectively. A single asterisk marks a 73- to 75-kDa doublet present prominently in C49A-C87A samples. (E) Steady-state intracellular Vp1 species. Samples of 4 × 105 whole cells transfected with wild-type, C87A-C208S, or C49A-C87A NO-SV40 DNA were separated by nonreducing or reducing SDS-PAGE and processed for anti-Vp1 Western blots. Lane 4 represents a longer exposure of the image in lane 3, and lanes 8 to 10 represent longer exposures of the images in lanes 5 to 7, respectively.