Identification, Isolation And Functional Analysis Of Cellular Proteins Associated With X Protein Of Hepatitis B Virus

Hepatitis B virus (HBV) is an enveloped hepatotropic DNA virus causing various liver diseases: acute to chronic hepatitis, cirrhosis and hepatocellular carcinoma (HCC). Thought the etiology of HCC was very perplexing, epidemiologically 80% of all HCC cases have been associated with long-standing HBV infection. The HBV X protein (HBx) is a multifunctional protein required for viral replication, displays a pleiotropic effect on variety of cellular functions, and plays critical roles in the development of HCC. Although the biological importance of HBx has been well established, the cellular and molecular bases of its function remain largely undefined. HBx acts as an indirect transcriptional transactivator to regulate the expression of many viral and cellular genes, suggesting multiple targets of HBx are involved in the virus-host cell interaction. However, the molecular mechanisms by which HBx effects pathogenesis and hepatocyte transformation largely remain elucidative. Identification and characterization of cellular targets of HBx remain an essential goal in the elucidation of molecular mechanisms of HBV infection and HCC development.In this study we utilized a yeast two-hybrid screen as a primary experimental approach to identify new HBx-associated cellular proteins. At first HBV DNA was isolated from a patient diagnosed hepatocellular carcinoma (HCC) with chronic viral infection. The HBV genome was cloned into the plasmid pGEM-T and sequenced. The phylogenetic analysis of HBV genome and its four ORF were performed with DNAstar. The results showed that the HBV isolate name WhuTj-37 belong to HBV B genotype according to the determined 8 genotypes, which is the predominate genotype mainly found in Asia. These results suggested this newly identified HBV isolate is a representative strain of genotype B, and therefore it is suitable for the study of molecular mechanisms of HBV infection among Asian population. A DNA fragment containing the HBx coding sequence was amplified and cloned into the Bait vector pGBKT7. Yeast cell AH 109 harboring the Bait vector expressing HBx protein that was fused to BD peptide was identified based on phenotype. Results indicated that the HBx protein was not toxic to yeast cell and not autoactivation.In order to screen cellular targets that are associated with the viral protein HBx, yeast cells AH109 with the vector expressing HBx protein fused in BD peptide were mated with y187 with the cDNA library fused AD peptides. We would be able to identify more than 80 potentialpositive candidates from two independent primary experiments. After verification by segregation analysis, 9 and 17 positive candidates, respectively, were farther conformed as positive candidates. Therefore, a total of 21 positive candidates were identified from the two screens. In vitro immunoprecipitation experiments were further used to eliminate the possibility that interaction between HBx and candidate proteins may occur through a yeast-derived bridged protein. Results indicated that several peptides, including Hepsin, aldolase B, hypothetical protein MGC13047 and complement component 8 were indeed interacted with HBx protein, respectively. In order to confirm the interaction of HBx protein and HBx-associated cellular proteins, the fragments inserted AD vector were obtained and cloned into the expressing vector. And the identified recombinant plasmids were co-transfected into cells. The interaction of them were determined by co-immunopreipriation and immunofluencsence co-focal staining.Entire ORF of Hepsin was isolated from adjacent live tissue of HCC with RT-PCR. Hepsin gene was inserted into expressing vector pCMV-flag-2B. The identified recombinant plasmid with hepsin gene was named pCMV-tag-HS. The recombinant vector pCMV-tag-HS was co-transfected into several liver tumor cell lines and normal liver cell line with plasmid encoding HBx protein. The functional changes of cells transfected with the appropriate plasmids were determined with MTT assay and FAC screening. The difference of HBV replication and expression of viral protein of HBV were determined using semi-quantitative PCR for HBV DNA and ELISA assay for HBsAg or HBeAg respectively. The results indicated that co-expression of HBx and Hepsin increased the expressing level of viral proteins and the copy number of HBV, which implicated that the association of HBx and Hepsin promoted the replication of HBV. The biological roles of interaction between HBx and hepsin were determined as promoting cell proliferation and blocking apoptosis in human liver tumor cell line.