PATHOGENESIS OF HEPATITIS B VIRUS IN TRANSGENIC MICE

Scripps Research Institute, La Jolla CA

Investigators

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Abstract

DESCRIPTION: (Adapted from the investigator's abstract) Hepatitis B virus (HBV) is a noncytopathic DNA virus that causes acute and chronic hepatitis and hepatocellular carcinoma. The long term objective of our research is to discover the mechanisms responsible for viral clearance and disease pathogensis with the hope that this information will be used to develop new methods to prevent and cure HBV infection. We recently proved that viral hepatitis has an immunologocal basis by showing that HBV-specific cytotoxic T lymphocytes (CTLs) cause acute hepatitis when injected into HBV transgenic mice. We also discovered that CTLs inhibit HBV replication and gene expression in the liver noncytopathically by secreting interferon gamma and tumor necrosis factor alpha when they recognize antigen. The cytokines inhibit HBV replication by deleting RNA-containing capsid particles from the hepatocyte; and they inhibit HBV gene expression by destabilizing the viral RNA. The central objectives of this application are to identify the cytokine-induced cellular genes and mechanisms that mediate these antiviral effects. Using cytokine-responsive, immortalized, transgenic hepatocyte lines that replicate HBV, we will determine if the cytokines inhibit HBV replication by preventing capsid assembly or by stimulating capsid degradation. We will use this information to design yeast two-hybrid, co-immunoprecipitation and GST pull-down experiments to identify cytokine-inducible cellular proteins that bind the core protein or capsid particles. Then we will overexpress and/or delete these proteins to prove that they inhibit HBV replication in vitro and in transgenic mice. In conceptually similar studies, we will define the cytokine-inducible cellular mechanisms that destroy HBV mRNA in hepatocytes. We will use mutagenesis to define the HBV mRNA sequence element(s) that mediate their responsiveness, and then use these sequences in uv-crosslinking and yeast three-hybrid assays to identify cytokine-regulated hepatocellular proteins that bind to the viral mRNA. When the HBV RNA-binding proteins are identified, we will determine if they destabilize the viral RNA in vitro and in transgenic mice. Finally, we will use genomic analysis to identify cytokine-inducible cellular proteins that inhibit HBV replication and gene expression, irrespective of their ability to bind directly to viral proteins, particles or RNA. If we succeed in these experiments, our understanding of the host-virus interactions that control HBV replication and gene expression will be greatly enhanced. In addition, we hope to identify hepatocellular proteins and viral target elements that can serve as a starting point to develop antiviral drugs to prevent and/or treat this disease that affects over 350 million people worldwide, and kills over 1 million people every year.