Chronic hepatitis B virus (HBV) infection is associated with a 25% lifetime risk of primary hepatocellular carcinoma (PHC). Though the mechanism of carcinogenesis is not well understood, it is generally believed that chronic liver damage mediated by immune effector cells is a major component of the process. This has led, in turn, to the idea that attenuating or eliminating the hepatitis that accompanies infection would reduce the chances of developing PHC. At present, them are no effective forms of treatment for chronic HBV-associated hepatitis. Agents which prevent infection of regenerating hepatocytes, in combination with drugs that inhibit HBV replication, may allow clearance of HBV-infected hepatocytes from the liver and avert the onset of PHC. The immediate objective of this study is to define the molecular interactions that occur between hepadnaviruses and hepatocytes to initiate infection. The long term goal is to incorporate this information into new antihepadnaviral therapies. Our analysis of the infection process will be carried out with the duck hepatitis B virus (DHBV) since both virus and tissues can be readily obtained, and an in vitro infection system is available using primary cultures of duck hepatocytes.
Our specific aims are to: 1) Identify and characterize the hepatocyte receptor for DHBV; 2) Develop stable cell lines that are susceptible to hepadnavirus infection; 3) Define the process of uptake of DHBV particles during infection, and 4) Identify liver specific cell surface proteins. Identification of the receptor for DHBV will allow us, first, to assess its role in determining virus tissue tropism and, second, may reveal the normal physiologic function of this protein, as well as the identity of the homologous human molecule. Moreover, production of a stable cell line expressing receptor protein might provide an efficient, permissive cell culture system for studies on hepadnavirus uptake. We will also attempt to achieve this goal by methods that do not require prior identification of the hepadnavirus receptor. Investigation of the pathway of virus uptake following adsorption may provide clues to the mechanism by which virus envelope fusion occurs to release cores into the cytosol, and hence, assist in the design of strategies to interfere with virus infection in vivo. Towards these goals we have developed an in vitro infection system for DHBV, used it to demonstrate that infection is receptor mediated, generated a monoclonal antibody to a duck hepatocyte plasma membrane protein that blocks infection of DHBV in vitro, produced a cDNA library and isolated a clone from this library using the blocking monoclonal. We also have preliminary data to suggest that DHBV infection in vitro is via a low-pH independent pathway.
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