Figure 7. Interaction of HBx with cyclin A/E–cdk2 complex.

Huh7 cells were transfected with X0 or X9 and synchronized by serum starvation. Cell extracts were prepared for 6 h (for cyclin E), 8 h (for cdk2) or 15 h (for cyclin A) following serum stimulation. Cyclin–cdk complexes were immunoprecipitated (IP) either by anti-HBx antibody (A) or antisera against cyclin A and cyclin E (B), and analysed by Western blotting (WB) with indicated antibodies. To establish region D as site of interaction on HBx, cell extracts were immunoprecipitated with HBx polyclonal antisera followed by Western blotting for cyclin A, cyclin E and cdk2 (C). Domain structure of wild-type HBx (X0) and its deletion mutant X9 is shown schematically on top. (D) In vitro translated X0 and X9 proteins were incubated with control cell extracts, immunoprecipitated with anti-HBx antisera and Western blotted for cyclin E and cdk2. X0*, X0-transfected cell extract. (E) In vitro translated HBx (1 μg) was either directly incubated with recombinant cyclin A–cdk2 complex or after pre-incubation with anti-HBx mAb as described in the Experimental section. The samples were immunoprecipitated with anti-HBx mAb and Western blotted for cyclin A. PI, preimmune control; V, vector control; WCE, whole cell extract.