Hepatitis B virus (HBV), a member of a small family of hepatotropic viruses, afflicts over 200 million chronic HBV carriers worldwide with a wide spectrum of liver disease, including cancer. A variety of HBV viral particles have been observed, from enveloped 42 nm virions, to uncoated nucleocapsids and empty viral envelopes. Very little is known about the assembly of these structures. This proposal asks two related questions: 1. What molecular interactions drive capsid assembly? 2. How are the higher viral structures such as the replicative nucleocapsid and the virion assembled? These questions will mainly be addressed using Xenopus oocytes in which we have recently reconstituted and characterized the assembly of simple HBV capsids. To answer the first question we will: perform a detailed mutational analysis of the single HBV capsid protein (p21.5) to elucidate the protein-protein interactions involved in forming p21.5 dimers and capsids, and the protein-nucleic acid interactions mediating the binding of RNA and DNA; we will also create (in E. coli) the large amounts of capsids for biochemical and X-ray structural studies. To answer the second question, we will determine whether injection of a single pregenomic HBV RNA into oocytes can direct production of a replicative nucleocapsid, and we will study interactions between p21.5 and a genetically marked pol protein; to recreate HBV virions we will coinject oocytes with mixtures of synthetic mRNAs encoding the known virion components. These studies will supply a wealth of information about the p21.5 capsid protein and its functional, topographical and epitopic properties, and will reveal details of how p21.5 interacts with the viral envelope and pol proteins. It is hoped that this will lead to the design of strategies and reagents for therapeutic intervention in the HBV lifecycle.
