Application of fluorescent-based technology detecting protein-protein interactions to monitor the binding of hepatitis B virus X protein to DNA-damage-binding protein 1.

The hepatitis B virus X protein (HBx) and the V protein of paramyxovirus simian virus 5 (SV5-V) interact with DNA damage-binding protein 1 (DDB1), a cellular enzyme involved in DNA repair and cell cycle regulation, to stimulate viral activity. DDB1 has several cellular substrates, and the amino acid sequences of the binding sites in the viral proteins and their substrates are notably dissimilar. To determine whether HBx binds preferentially to DDB1, despite differences in the amino acid sequences, we developed a system to monitor DDB1 binding in living cells through a protein-protein visuali-zation system, designated fluorescent-based technology detecting protein-protein interactions (Fluoppi). HBx in association with DDB1 formed clear fluorescent puncta. The number of these fluorescent puncta increased with an increase in the amount of HBx. The binding of HBx to DDB1 inhibited the cellular substrate DDB1-CUL4A-associated factor 9 (DCAF9) from binding to DDB1. The inhibitor nitazoxanide prevented the viral proteins HBx and SV5-V from binding to DDB1 but did not inhibit the binding of DCAF9 or HBx(ΔNC), which constitutes the binding site of HBx. Our results demonstrate that the Fluoppi system is useful for monitoring the binding of HBx to DDB1 as well as for examining the effect of drugs on DDB1-Hbx binding.