Accurately mapping the location of the binding site for the interaction between hepatitis B virus X protein and cytochrome c oxidase III

The hepatitis B virus (HBV) X protein (HBx) plays an important pathogenetic role in hepatocarcinoma tumorigenesis. As HBx does not have the ability to bind to double-stranded DNA (dsDNA), protein-protein interaction is crucial for HBx functions. In a previous study, we screened a novel HBx-interacting protein, the cytochrome c oxidase subunit III (COXIII). In the present study, we aimed to accu- rately map the location of the binding site for the interaction of HBx with COXIII. Two fragments of HBx mutants (X1 aa1-72 and X2 aa1-117) were amplified by polymerase chain reac - tion (PCR) and separately inserted into the pAS2-1 plasmid. PCR and gene sequencing confirmed the correct insertion of the mutant fragments in the plasmid. The tanscription of the mutant fragments in yeast cells was demonstrated by RT-PCR and western blot analysis confirmed that they were accurately translated into fusion proteins. Hybridization on solid medium and the detection of β-galactosidase (β-gal) activity indicated that the binding site for the interaction between HBx and COXIII was located between aa72 and aa117. Specific interac - tions between the HBxX2 protein and COXIII were verified by co-immunoprecipitation. To the best of our knowledge, this is the first study showing to demonstrate that aa72-117 in HBx is the key region for binding with COXIII.