FIG. 2.

Expression of L1 and L2 proteins and GFP and of the corresponding mRNAs from wt or codon-modified genes. Cos-1 cells were transfected with wt or modified L1 and L2 expression plasmids pCDNA/BPVL1wt plus pCDNA/HBL1 (row 1), pCDNA/BPVL2wt plus pCDNA/HBL2 (row 2), and pWt gfp plus P gfp (row 3). Cells were fixed after 36 h and incubated with rabbit anti-BPV1 L1 (row 1) or L2 (row 2) antiserum, followed by FITC-conjugated goat anti-rabbit IgG antibody, and protein was visualized by immunofluorescence. L1 and L2 proteins were visible in the nuclei of cells transfected with HBL1 (row 1, panel 2) and HBL2 (row 2, panel 2) plasmids. The cells transfected with wt L1 (row 1, panel 1) or L2 (row 2, panel 1) sequences failed to produce detectable protein. Cells transfected with humanized gfp (row 3, panel 2) produced GFP, whereas cells transfected with the Pgfp produced little GFP (row 3, panel 3). Immunoblotting of cell lysates of HBL1-transfected cells, and of cell transfected with purified BPV1 but not wt BPV1 L1 (row 1 panel 3), was positive for L1 protein. L2 protein appeared as a 78-kDa band in cells transfected with HBL2 and BPV1 but not in cells transfected with wt L2 (row 2, panel 3). mRNA extracted from the cytoplasm (Cyto) or whole-cell preparation (total) of cells transfected with control pCDNA3 (V), with pCDNA with wt or humanized (HB) L1 or L2 gene inserts, or with wt gfp or Pgfp was probed with ³²P-labelled probes made with wt L1 (row 1, panel 4) or L2 (row 2, panel 4) or gfp (row 3, panel 4) sequences. The total amount of mRNA in these samples was examined by hybridization of the mRNA sample with a GAPDH probe (panel 4, lower).