Investigations Into the Surface Antigens of Hepatitis B Virus

Clayton, Reginald Francis (2000) Investigations Into the Surface Antigens of Hepatitis B Virus. PhD thesis, University of Glasgow.

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The small (S), medium (M), and large (L) surface antigens of Hepatitis B virus (HBV) are found in the virion envelope. They are initially synthesised in infected cells as multispanning transmembrane proteins of the endoplasmic reticulum (ER). These proteins perform many roles in the life cycle of the virus. Specifically, they play a crucial role in initiating the infection through virus attachment and entry into the target cells, and subsequently in the assembly and morphogenesis of the HBV virion. The surface antigens mediate these processes through interaction with each other, with HBV core and viral nucleic acid, and with host cell proteins. The major aim of this study was to investigate the protein-protein interactions of the HBV structural proteins in order to enhance our understanding of the mechanisms involved in these processes. To carry out this work, an extensive array of specific reagents was initially generated in house and characterised. These include monoclonal and polyclonal antibodies (MAbs and PAbs, respectively) to the preS1 domain of the HBV L protein, and novel cell lines derived from primary human hepatocytes. Additional reagents were obtained from elsewhere. Furthermore, the genes encoding the HBV surface antigens and core were cloned and expressed using a recombinant vaccinia virus and bacterial systems. Using specific antibodies, the heterologous expression of the HBV proteins was confirmed in several assays. HBV displays a strong tropism for human hepatocytes and cannot be propagated efficiently in cell culture. However, under certain conditions, the virus can replicate in cell lines of hepatic origin. Most hepatocyte cell lines in existence are derived from tumours, thus are likely to display aberrant gene expression in comparison with healthy hepatocytes. The failure of the virus to replicate efficiently in cell culture may be due to the lack of certain hepatocyte-specific functions in the cells. In this study, two hepatocyte cell lines, H5 and HI6 were generated in house from healthy liver tissue and were examined for expression of certain proteins known to be associated with hepatocyte phenotype. The results suggested that cell lines H5 and H16 are likely to have retained at least some of the hepatocyte gene expression, and have not reverted to a fibroblast phenotype. Both cell lines H5 and HI6, as well as established cell lines (such as HepG2) were used for the study of the protein-protein interactions of the HBV structural antigens. A detailed antigenic characterisation of the anti-L MAbs showed that all recognised a linear epitope on the preS1 domain of the L protein. Interestingly, a previously uncharacterised anti-S MAb, 6B1, specific for a conformation-sensitive epitope in recognised S and M proteins, but not L in immunoprecipitation and immunofluorescence assays. These results provide the first biochemical evidence for the existence of important structural differences in the S moiety of L from that in M and S. Such differences have not been predicted in existing models of HBV surface antigen topology. The significance of a difference in the topology or conformation of the S domain of L in comparison with that of HBsAg is discussed. The intracellular distribution of the HBV proteins expressed in this study was in keeping with previously published data. Recent studies show that amino acid peptides corresponding to regions within L and S bind HBV core in in vitro assays. However, to-date these interactions have not been demonstrated in cell culture. In this study, the interaction of the surface antigens with the core protein was investigated. Confocal microscopy analysis showed that the core protein when expressed singly localised throughout the cytoplasm and the nucleus. However, in the presence of the L protein the intracellular distribution of core changed to a tightly defined perinuclear region where L is normally localised, indicating colocalisation of core with the L protein. Furthermore, the core protein also colocalised with a secreted form of L (Lx) protein. In confocal microscopy assays, the L (or Lx) protein co-localised with core was readily detected by three out of four of our anti-L MAbs. However, one MAb, RC28, failed to recognise L colocalised with core. This indicated that the amino acid residues in the epitope of preS1 recognised by MAb RC28 may play a critical role in L-core interaction. Alternatively, the failure of MAb RC28 to recognise L bound to core may be due to conformational changes in the preS1 domain of L induced by the interaction. In contrast with L (and Lx), the M and S proteins failed to affect the cellular distribution of core. The failure of core to co-localise with M or S in the confocal microscopy assay is not consistent with the published in vitro binding data. It is likely that L is fulfilling a scaffolding role in the virion morphogenesis process, where there is a small contribution to the L-core binding from the S domain of L. It is furthermore likely that S is unable to bind sufficiently strongly to the core to facilitate an intracellular redistribution, pointing to a minor role, if any, of S in the morphogenesis process. Taken together, this data adds considerably to the existing body of literature, and enhances our understanding of the virion morphogenesis process.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: Arvind Patel
Keywords: Virology
Date of Award: 2000
Depositing User: Enlighten Team
Unique ID: glathesis:2000-75816
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 19 Nov 2019 18:08
Last Modified: 19 Nov 2019 18:08

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