FIG 9.

The C terminus of HPV11 E1 can be partially replaced by the analogous domain from BPV1 E1. (A) Depiction and amino acid sequence of the C termini of the two HPV11/BPV1 E1 chimeras created in this study. The regions highlighted in black are from HPV11 E1, while those in gray are from BPV1 E1. Chimera 617 (Ch617) is comprised of residues 1 to 617 of HPV11 E1 fused to amino acids 572 to 605 of BPV1 E1. Ch620 contains residues 1 to 120 of HPV11 E1 fused to amino acids 575 to 605 of BPV1 E1. Mutant derivatives of both chimeras were also created by replacing the two conserved C-tail residues F594 and C596 (boxed) with alanines. (B) DNA replication levels supported by the Ch617 and Ch620 chimeras and mutant derivatives were measured using the HPV11 DNA replication assay essentially as described in the legend to Fig. 3B but using 2.5, 1.25, and 0.625 ng of E1 expression vector. DNA replication activities were measured 72 h posttransfection and are reported as percentages of the levels obtained with 2.5 ng of wild-type HPV11 E1 expression plasmid [E1(1–649)]. Also presented are the levels of DNA replication supported by HPV11 truncated E1 proteins ending at residues 617 and 620 to depict the “baseline” DNA replication value for each chimera in the absence of the BPV1 E1 C terminus. Statistical significance was assessed by comparing the DNA replication activity of each chimera to that of its parental truncated protein (white bars) using one-way ANOVA followed by Dunnett's post hoc analysis. Significant differences are indicated (***, P ≤ 0.001; ****, P ≤ 0.0001). (C) Expression of the indicated E1 proteins and chimeras. Extracts from transfected cells were separated on an SDS-10% PAGE gel prior to immunoblotting with an anti-GFP antibody. Tubulin was used as a loading control.