metallothionein and Hepatitis-B

To screen and identify the protein interacting with HBV antigen in hepatocytes. Then investigate the biological functions of hepatitis B virus antigen in the pathogenesis of hepatitis B and seek effective methods to prevent and treat it.. The yeast two-hybrid system-3 technique was used to construct HBV PreS2, HBeAg, HBcAg, HBxAg bait plasmids. The bait plasmids transformed the yeast AH109 and expressed themselves in it. After being identified by SDS-PAGE and Western blot, the AH109 yeast was mated with yeast Y187 containing liver cDNA library plasmid in 2 x YPDA medium to form diploid yeast and was then plated on synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) and synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) containing x-alpha-gal for screening. Plasmids of blue colonies were extracted and transformed into Escherichia coli, then analyzed by DNA sequencing and bioinformatics. To further prove the interaction between HBV antigen and metallothionein, translation was performed by using reticulocyte lysate and coimmunoprecipitation was displayed in vitro.. Genes coding for HBV antigen binding protein were successfully cloned and metallothionein was found in that protein. The interaction between HBeAg, HBcAg and HBxAg and metallothionein were further proved by coimmunoprecipitation in vitro.. The interaction between HBV antigen and metallothionein indicates that metallothionein may participate in the pathogenesis of hepatitis B

Topics: Hepatitis B; Hepatitis B Antigens; Hepatocytes; Humans; Metallothionein; Protein Interaction Mapping; Two-Hybrid System Techniques

The basal core promoter (BCP) of hepatitis B virus (HBV) controls the transcription of both the precore RNA and the core RNA. The precore RNA codes for the secreted e antigen, while the core RNA codes for the major core protein and the DNA polymerase and also is the pregenomic RNA. The double mutation of nucleotides 1762 and 1764 in the BCP from A and G to T and A, respectively, is frequently observed in HBV sequences isolated from chronic patients. Several papers have reported conflicting results regarding whether this double mutation is important for e antigen expression. In order to address this issue, we have introduced this double mutation into the HBV genome and studied its effects on HBV gene expression and replication. Our results indicate that the mutated BCP can no longer bind a liver-enriched transcription factor(s) and that the transcription of only precore RNA and, consequently, the expression of e antigen were reduced. The reduction of precore gene expression was accompanied by an increase in progeny virus production. This increase was found to occur at or immediately prior to the encapsidation of the pregenomic RNA. Thus, the results of our in vitro study resolve the discrepancy of previous clinical observations and indicate that this double mutation suppresses but does not abolish the e antigen phenotype. The implications of these findings in the pathogenesis of HBV are discussed.

Topics: Animals; Base Sequence; Binding Sites; DNA-Directed DNA Polymerase; Enhancer Elements, Genetic; Genes, Immunoglobulin; Growth Hormone; HeLa Cells; Hepatitis B; Hepatitis B Core Antigens; Hepatitis B e Antigens; Hepatitis B virus; Humans; Immunoglobulin kappa-Chains; Metallothionein; Methylation; Mice; Molecular Sequence Data; NF-kappa B; Oligodeoxyribonucleotides; Point Mutation; Promoter Regions, Genetic; Recombinant Proteins; RNA, Viral; Transcription, Genetic; Transfection; Virus Replication

Transgenic mice were obtained that contained a gene of human hepatitis B surface antigen (HBsAg). The integrated HBsAg DNA sequences were inherited in the normal Mendelian fashion. 6 of 25 investigated transgenic mice expressed the HBsAg. The expression was detected in the serum and within the cytoplasm of hepatocytes. Specific tissue pathology was shown in these animals, including systemic hyperplasia of lymphoid tissue and degenerative changes in the liver and kidney parenchymatous cell elements.

Topics: Animals; DNA Restriction Enzymes; Gene Expression Regulation, Viral; Genes; Genes, Viral; Hepatitis B; Hepatitis B Surface Antigens; Hepatitis B virus; Humans; Metallothionein; Mice; Mice, Inbred C57BL; Mice, Inbred CBA; Mice, Transgenic; Plasmids; Promoter Regions, Genetic

Difficulties were encountered with the orcein method currently being used to demonstrate hepatitis B surface antigen and copper-associated protein in the liver when a new batch of dye was introduced. A survey of published material produced a plethora of methods with many contradictory recommendations. A number of methods and a variety of orceins were compared to determine which methods and orcein solutions would give the most consistent results. Two methods gave equally satisfactory results and can be recommended for routine use in screening paraffin sections of liver for hepatitis B surface antigen and copper-associated protein.

Topics: Hepatitis B; Hepatitis B Surface Antigens; Histocytochemistry; Humans; Liver; Liver Cirrhosis; Metallothionein; Oxazines; Staining and Labeling