Fig 2.

The p80-binding domain of E1 is dispensable for the assembly of the E1-E2-ori complex. (A) Interaction of HPV31 E1 and E2 in vivo. 3F-tagged E1 WT, the ATPase mutant K463A (ATP), or p80-binding mutants (WF and C40), alone or in combination, were cotransfected in C33A with RFP-tagged E2. Cells were harvested 48 h posttransfection, and whole-cell extracts were submitted to immunoprecipitation using an anti-Flag antibody. The immunoprecipitates were analyzed by Western blotting using anti-RFP and anti-Flag antibodies. (B) Inhibition of HPV DNA replication by the ATPase-negative K463A E1 and derivatives containing the WF or C40 mutations. DNA replication was performed using 5 ng of WT E1 expression plasmid and the indicated amounts of E1-inhibitory plasmid. The error bars represent the standard deviations of duplicate values. The results are representative of at least three independent experiments. (C) Western blot showing the expression of mutant E1 proteins. β-Tubulin was used as a loading control.