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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
First Independent Research Support & Transition (FIRST) Awards (R29)
Project #
5R29AI025183-03
Application #
3454374
Study Section
Experimental Virology Study Section (EVR)
Project Start
1987-07-01
Project End
1992-06-30
Budget Start
1989-07-01
Budget End
1990-06-30
Support Year
3
Fiscal Year
1989
Total Cost
Indirect Cost
Name
Scripps Research Institute
Department
Type
DUNS #
City
San Diego
State
CA
Country
United States
Zip Code
92037
Zhang, P; Raney, A K; McLachlan, A (1993) Characterization of functional Sp1 transcription factor binding sites in the hepatitis B virus nucleocapsid promoter. J Virol 67:1472-81
Raney, A K; Le, H B; McLachlan, A (1992) Regulation of transcription from the hepatitis B virus major surface antigen promoter by the Sp1 transcription factor. J Virol 66:6912-21
Zhang, P; Raney, A K; McLachlan, A (1992) Characterization of the hepatitis B virus X- and nucleocapsid gene transcriptional regulatory elements. Virology 191:31-41
Raney, A K; Milich, D R; McLachlan, A (1991) Complex regulation of transcription from the hepatitis B virus major surface antigen promoter in human hepatoma cell lines. J Virol 65:4805-11
Raney, A K; Easton, A J; Milich, D R et al. (1991) Promoter-specific transactivation of hepatitis B virus transcription by a glutamine- and proline-rich domain of hepatocyte nuclear factor 1. J Virol 65:5774-81
Eckhardt, S G; Milich, D R; McLachlan, A (1991) Hepatitis B virus core antigen has two nuclear localization sequences in the arginine-rich carboxyl terminus. J Virol 65:575-82
Milich, D R; Jones, J E; McLachlan, A et al. (1990) Importance of subtype in the immune response to the pre-S(2) region of the hepatitis B surface antigen. II. Synthetic Pre-S(2) immunogen. J Immunol 144:3544-51
Raney, A K; Milich, D R; Easton, A J et al. (1990) Differentiation-specific transcriptional regulation of the hepatitis B virus large surface antigen gene in human hepatoma cell lines. J Virol 64:2360-8
Milich, D R; Hughes, J L; McLachlan, A et al. (1990) Importance of subtype in the immune response to the pre-S(2) region of the hepatitis B surface antigen. I. T cell fine specificity. J Immunol 144:3535-43
Raney, A K; Milich, D R; McLachlan, A (1989) Characterization of hepatitis B virus major surface antigen gene transcriptional regulatory elements in differentiated hepatoma cell lines. J Virol 63:3919-25

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