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. 2016 Feb 26;90(6):3198–3211. doi: 10.1128/JVI.03127-15

FIG 8.

FIG 8

Replacement of the highly conserved F594 and C596 residues in BPV1 E1 indicates an essential role for the C-tail in BPV1 DNA replication. (A) Depiction and amino acid sequence of the BPV1 E1 C terminus highlighting the two most highly conserved amino acid residues in the C-tail, F594 and C596, analogous to F638 and C640 in HPV11 E1 (Fig. 7). (B) The DNA replication activities of mutant BPV1 E1 proteins carrying the F594A and C596A substitutions were tested for the ability to support BPV1 DNA replication using a luciferase-based assay (25) similar to the one described in this study for HPV11 and using three amounts of BPV1 E1 expression vector (2.5, 5, and 10 ng) 72 h posttransfection. DNA replication levels are reported as percentages of the activity obtained with 10 ng of wild-type BPV1 E1 expression plasmid (WT). Note the more profound effect of the two C-tail substitutions on the activity of BPV1 E1 compared to HPV11 (Fig. 7B). Statistical significance was assessed by comparing the DNA replication activity of each BPV1 E1 mutant protein to that of wild-type E1 (white bars) using one-way ANOVA followed by Dunnett's post hoc analysis. Significant differences are indicated (****, P ≤ 0.0001). (C) Dominant-negative inhibition of BPV1 DNA replication by E1 F638A and E1 C640A. BPV1 DNA replication was performed using 10 ng of wild-type E1 expression plasmid and increasing amounts of expression vector (0, 3.13, 6.26, 12.5, 25, 50, 100, and 200 ng) for either E1 F638A or E1 C640A, as indicated. (D) Abilities of the WT and mutant BPV1 E1 proteins to support DNA replication using the origin and/or E2 from HPV11. HPV11 or BPV1 DNA replication assays were performed with 5 and 10 ng of BPV1 E1 expression plasmid (wild-type or mutant proteins), together with 2.5 ng of origin plasmid (Ori) and 10 ng of E2 expression plasmid from either BPV1 or HPV11, as indicated at the top. DNA replication levels were determined 72 h posttransfection and are reported as percentages of the activity measured with wild-type E1, E2, and the origin from BPV1. Statistical significance was assessed by comparing the DNA replication activity of each BPV1 E1 mutant protein to that of wild-type E1, using one-way ANOVA followed by Dunnett's post hoc analysis. Significant differences are indicated (****, P ≤ 0.0001). Standard deviations are indicated by the error bars.