HEPATITIS B VIRUS GENE EXPRESSION

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The absence of a tissue culture system and a convenient laboratory animal model system to propagate hepatitis B virus (HBV) has restricted the analysis of the events occurring during the HBV life cycle. The long-term objective, therefore, is to develop recombinant expression systems to analyze the viral and cellular factors which regulate HBV transcription, replication and assembly. In addition, since it is assumed that liver damage resulting from HBV Infection is mediated by a cellular immune response to hepatocytes expressing viral antigens, an amphotropoic retroviral expression system will be developed to generate autologous human cytolytic T-lymphocyte (CTL) target/stimulator cells expressing the various HBV antigens. The production of these cells will permit the analysis of this postulated mechanism of hepatocellular injury. Characterization of the four HBV open reading frames (ORFs), the surface, core, X and polymerase genes, using an amphotropic retroviral expression system, has been initiated. The analysis indicates that the pre-S(1) region of the surface antigen (HBsAg) has the capacity to sequester HBsAg in the pre-golgi or early golgi cellular compartment, the pre-core region has the properties of a signal peptide for protein secretion and the core polypeptide (HBcAg) contains signal peptide for protein secretion and the core polypeptide (HBcAg) contains signal sequences for the localization of HBcAg to the nucleus. Analysis of the cellular compartmentalization of HBV/marker protein fusions expressed using the retroviral expression vector should permit the identification of the various signal domains in these polypeptides. Using retroviral and SV40 expression vectors, the endogenous HBsAg and HBcAg promoters appear to be transcriptionally active. A detailed deletion analysis of these transcription units will permit the identification of regulatory sequences involved in the expression of these antigens. In addition, analysis of the endogenous HBV promoter activities in the cell lines expressing HBV antigens will determine if these polypeptides possess trans- acting regulatory activity. The introduction of mouse cell lines expressing HBV antigens into syngeneic mice will be used as a model system to determine which antigens can act as CTL targets. using the amphotropic expression vectors, the ability to transmit efficiently HBV antigen expression via retroviral infection to primary human cells will be developed so that the presence of HBV antigen specific CTL during viral infection might be investigated in man.