The Effects Of Hepatitis B X Protein (HBx) On Serum Ceruloplasmin(Cp) Through Protein-protein Interaction

Hepatitis B virus (HBV) has been showed a worldwide distribution and a seriouscontagious disease. HBV infection results in acute hepatitis, asymptomatic carrier state,chronic hepatitis, even develop liver cirrhosis and hepatocellular carcinoma (HCC)eventually. However, the pathogenic mechanism of HBV yet has not been fully clarified.HBV genome is a3.2kb, circular, partially double-stranded DNA molecule with fouropen reading frames (ORFs) named P, C, S, and X, coding for the viral polymerase,coreprotein, surface antigen, and X protein respectively. HBV X protein (HBx) is amultifunctional protein with a molecular weight of16.5kD, and plays an essential role in viralpathogenesis. We had screened for hepatocellular proteins that interacts with HBx byCytoTrap two-hybrid system and obtained some putatively positive clones. One of them wasthen identified as serum ceruloplasmin (Cp), and the interaction between Cp and HBx wasfirst confirmed in yeast. Then, we further confirmed their interaction in vitro and in vivo andelucidated the effects of HBx on Cp，and revealed a further mechanism for HBV-associatedpathogenesis.In the first part, the library prey plasmid was extracted from one of the putativelypositive clones, after analyzed by digestion and sequencing, the clone was identified as Cp.The bait and library prey plasmids were then re-transformed into cdc25H, and the interactionbetween Cp and HBx in yeast was confirmed by CytoTrap two-hybrid system.In the second part, the ineraction between Cp and HBx in vitro and in vivo was furtherconfirmed by GST pull-down and co-immunoprecipitation (Co-IP) respectively. And theresults of CytoTrap two-hybrid assay showed that HBx-Cp interaction was mediated by aminoacids51-80of HBx.The third part of this study was to explore both the interaction of HBx and Cp and its related functions. HBx and Cp were co-transfected into HepG2cells,and, the reactiveoxygen species,such as oxygen free radicals were detected, and then we found that, HBxcan inhibit the antioxidant effect of Cp through protein-protein interaction, and indirectlyinvolved in the pathogenesis of HBV, suggesting a new target for anti-HBV therapy.