The Effects Of Ubiquitin Specific Peptidase 15(USP15) Onhepatitis B X Protein(HBx)through Protein-protein Interaction

Worldwide, an estimated 350 million people are chronically infected with the Hepatitis B Virus(HBV); chronic infection with HBV is associated with the development of severe liver diseases including hepatitis and cirrhosis. Individuals who are chronically infected with HBV also have a significantly higher risk of developing hepatocellular carcinoma(HCC) than uninfected individuals. However, the pathogenic mechanism of HBV yet has not been fully clarified.HBV genome is a 3.2-kb, circular, partially double-stranded DNA molecule with four open reading frames(ORFs) named P, C, S, and X, coding for the viral polymerase, core protein, surface antigen, and X protein respectively. HBV X protein(HBx) is a protein with a molecular weight of 16.5kD, and plays an essential role in viral pathogenesis. In our previous study, by yeast CytoTrap two-hybrid screening, ubiquitin specific peptidase 15(USP15) had been identified as a candidate hepatocellular protein that could interact with HBx. In this research, we further confirmed their interaction in vivo and elucidated the effects of USP15 on HBx.The first part of this study, we constructed the expression vectors, pHBx-Flag and pUSP15 and confirmed the ineraction between USP15 and HBx in vivo by co-immunoprecipitation(Co-IP).The second part of this study was to investigate the effect of USP15 on the expression level, half-life and the ubiquitination of HBx. To address these issues, USP15 and HBx were over-expressed, and the HBx protein was detected by Western-Blot, The result show that over expression of USP15 lead to the elevated protein level, half-life, but decreased HBx ubiquitination levels.The third part of this study was to investigate the the effect of USP15 on transactivation capacity of HBx. To address this issue, pHBx-Flag and pUSP15 were co-expressed together with the luciferase reporting plasmids including pAP-1- Luc, pAP-2-Luc, pAP-3-Luc, pSP-1-Luc, pNF-?B-Luc, pCRE-Luc or pGRE-Luc, by detecting firefly luciferase luminescent substrate and calculated the relative luciferase units(RLU). The results showed that over-expression of USP15 could increase the RLU of all reporter plasmids, which indicated that USP15 could increase the trans-activation capability of HBx.In summary, our study demonstrated that USP15 could de-ubiquitinate the HBx through protein-protein interaction, hence reduced degradation of HBx and sequencially improved its trans-activation capacity.