Study Of HBx Interacting Proteins And Receptor For HBV

HBx, a transcriptional transactivating protein of hepatitis B virus (HBV), is required for viral infection and has been implicated in virus-mediated liver oncogenesis. However, the molecular mechanism for its influence on cell remains largely unknown. It was proved that HBx need the help of host cell proteins to exert its function by binding to them. During purifying of GSTX (fusion protein of GST and HBx) expressed in E. coli, we found that it can bind specifically with GrpE (HSP60) and DnaK (HSP70) of E. coli while GST cannot. Using GST pull-down, two-dimensional gel electrophoresis and mass spectrum, we found that GSTX can also bind to human mitochondrial HSP60 and HSP70, which are homologues of GrpE and DnaK. These interactions between HBx and mitochondrial HSP60 and HSP70 are supported by the result of co-immunoprecipitation experiment. It means thatHBxcan form complex with E. coli and human HSP60 and HSP70. The implication of HBx, HSP60 and HSP70 complex in molecular mechanism of virus infection is discussed. We also used the yeast two-hybrid system to identify that HBx interacts with MIF directly. Macrophage migration inhibitory factor (MIF) is implicated in the regulation of inflammation, cell growth and even tumor formation. The interaction between HBx and MIF was verified with co-immunoprecipitation, GST pull-down and cellular colocalization. The expression of MIF was up-regulated in HBV particle producing cell 2.2.15 compared with HepG2 cell. Both HBx and MIF cause HepG2 cells G₀/G₁ phase arrest, proliferation inhibition and apoptosis. However, MIF can counteract the apoptotic effect of HBx. These results may provide evidence to explain the link between HBV infection and hepatocellular carcinoma (HCC).Due to lack of reliable in vitro culture system for human hepatitis B virus (HBV), the mechanism of its infection remains large elusive. In order to facilitate the investigation of the early events of HBV infection, a pseudotype system based on human immunodeficiency virus (HIV) were developed in this study. To do this, expression plasmids for three membrane proteins, large S, middle S, small S and a mutant with PreS2 domain deletion were constructed. Pseudotype particles were produced by co-transfection...