The nucleocapsid of Hepatitis B Virus is an icosahedral structure composed of 180 subunits of the viral Core Antigen (HBcAg). This protein is 183 amino acids in size and has a highly arginine-rich carboxy-terminal region. It can be expressed at high levels in <i>E.coli</i>, in which it forms nucleocapsid-like core particles which are morphologically indistinguishable from nucleocapsids isolated from infected individuals. The latter property was utilised in this work, the aim of which was to investigate the role of particular amino acids of HBcAg in the determination of the structure of the protein monomer and that of the core particle. Previous work (Stahl <i>et al</i>, 1982) had suggested the importance of the amino-terminal region of the protein in the determination of its structure. This was investigated further in this work by the use of site-directed mutagenesis to create a series of deletion and substitution mutations within this region. These were expressed in <i>E.coli</i> and the importance of amino acid 3 was demonstrated by immunological assay. HBcAg contains four cysteines, all of which are completely conserved among mammalian hepadnaviruses. The role of these cysteines in HBcAg and core particle structure determination and stability was investigated by the use of site-directed mutagenesis to create a series of mutants in which cysteine codons were replaced by serine codons in several combinations, and in the context of both the full-length protein and of a truncated protein lacking the arginine-rich carboxy-terminal region. These proteins were produced in <i>E.coli</i> and formed particles indistinguishable from wild-type particles in structure, even when no cysteines were present. Their behaviour during non-reducing SDS-polyacrylamide gel electrophoresis indicated the presence of different disulphide bond complements in different mutants and allowed a model to be deduced for the arrangement of disulphide bonds within the core particle.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:662454 |
| Date | January 1993 |
| Creators | Stewart, Fiona Jane |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/11433 |
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