Influences Of Hepatitis B X Protein (HBx) On Hepatocyte Proteins Through Protein-protein Interactions

Hepatitis B virus (HBV) infection results in acute, chronic hepatitis, cirrhosis of the liver and closely associated with hepatocellular carcinoma (HCC),yet the exact molecular mechanism is not very clear. HBV has a genome of approximately 3.2 kb in length consisting of four open reading frames (ORFs) which encodes polymerase, core, surface and X protein respectively. HBx protein, with the molecular weigh of 154-amino-acid, is a multi-functional transcriptional transactivator of many cellular and viral genes, and plays pivotal role in viral pathogenesis. In this study, CytoTrap yeast two-hybrid system was employed to screen binding partners for HBx protein.After confirming of the interactions between HBx and evolutionarily conserved signaling intermediate in toll pathways (ECSIT) or valosin-containing protein (VCP), we investigate the influences of HBx on ECSIT or VCP.The first part of this study is to screen the cellular proteins which could interact with HBx by CytoTrap yeast two-hybrid system. After confirming the expression of Sos-HBx fusion in yeasts and exclusion of auto-activation of the fusion protein, a two-hybrid library screening was performed. 30 candidates were screened as followed: MYPK, TCTP, EKI1, ECSIT, MRLC2, VCP, USP15, PSMC1, PSMD4, YWHAH, FN1, NACA, MPP6, GSTO1, TOMM70A, ORM2, ITIH3, SF3A3, PREPL, PTPRA, SMARCC1, BRWD2, CP, ETNK1, PPM1A, LSM8, AMACR, AFM, IQWD1, RBX1.In the second part of this study, the direct interaction between HBx and ECSIT or VCP were confirmed in vitro and in vivo by the GST pull-down assay and the Co-immunoprecipitation(Co-IP) assay respectively. In addition, it was also showed by CytoTrap yeast two-hybrid assay that ECSIT-HBx interaction was mediated by amino acids 51-80 of HBx, and VCP-HBx interaction was mediated by amino acids 51-120 of HBx.The third part of this study is to investigate the influences of HBx on ECSIT and VCP by a dual luciferase reporter assay. The results demonstrated that HBx can enhance VCP or ECSIT-mediated NF-κB activation, suggesting that HBx may participate in the pathogenesis of HBV through the interaction with VCP or ECSIT.