The overall objectives of this research proposal are to use the duck hepatitis B virus (DHBV) model of hepatitis B virus to investigate steps of hepadnaviral replication which might eventually be susceptible to inhibition by antivirals or provide clues to improved vaccines. The proposed experiments are derived from our previous work, including the development of a panel of murine monoclonal antibodies directed to DHBV surface antigens which neutralize virus infection in vitro the finding of ~ several missense mutations in the RNase H domain of the P gene which prevent encapsidation of viral RNA, and a long-term study of the disease associated with DHBV infection of Pekin ducks. Most of the experiments will use two cell culture systems, virion infection of primary duck hepatocytes, and infection of a chicken hepatoma cell line by transfection of cloned viral DNA.
The specific aims are: 1. To study the interaction between DHBV surface antigens and the host during early infection. We will continue to characterize the amino acid residues of DHBV surface antigens which are important in viral neutralization, first by delimiting the minimal number of residues that are important in binding the monoclonal antibodies in Western blots and then by site-specific mutagenesis of residues associated with the epitopes to determine which amino acids may be essential to the attachment or entry stages of viral infection. We will continue to study the mechanisms of neutralization of DHBV associated with the monoclonals directed to three epitopes on the pre-5 portion of the surface antigen proteins and with the monoclonal directed to the S portion to determine if any of the monoclonals prevent viral attachment to hepatocyte receptors. We will use several approaches to identify hepatocyte proteins or glycoproteins which interact with DHBV during virus binding and entry. These approaches will be a) development of monoclonal antibodies which are directed to hepatocyte membrane surfaces and which will block infection, to be used as above and b) development of antiidiotype monoclonal and polyclonal antibodies to existing monoclonal antibodies directed against DHBV surface antigen epitopes involved in viral neutralization and use of these anti- idiotype monoclonal antibodies to identify cellular proteins interacting with each viral epitope. If cellular proteins are identified as essential to DHBV-attachment or entry, they will be cloned and characterized. 2. Study the role of the P gene RNase H domain in packaging of the RNA pregenome. RNase H domain packaging mutants will be tested against wild type for functions of the P gene product that can be measured in assays in vitro. These include priming and extension of minus strand synthesis, binding of pregenomic RNA in Northwestern blots, and reactivity in RNase H activity gels. The goal is to understand the packaging activity of this domain by seeing if it is related to a function of the P gene product. 3. Continue long-term study of hepadnavirus-infected ducks to determine if persistent infection with DHBV is associated with development of HCC in ducks.
Showing the most recent 10 out of 15 publications
