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Title: An investigation of the complexes formed between the hepatitis C virus E1 and E2 glycoproteins
Author: Patel, Janisha
ISNI:       0000 0001 3475 6349
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 1999
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Hepatitis C virus (HCV) encodes two glycoproteins, termed El and E2, which are presumed to constitute the proteinaceous components of the virion envelope. From a number of studies, El and E2 associate to form a complex however, the nature of the types of interaction between the proteins is the subject of controversy. Two types of complex, termed the native and aggregated forms, have been identified. In native complexes, El and E2 associate by non-covalent interactions while disulphide bonds form intermolecular links between the glycoproteins in aggregated complexes. The aim of my project was to characterise the complex formed by El and E2 from a local genotype 1a strain, called strain Glasgow. Studies were later expanded to include analysis of the glycoproteins from strain H77, another genotype 1a strain that is infectious in chimpanzees. Due to the lack of efficient virus replication in tissue culture, the Semliki Forest virus vector was used for expression of the glycoproteins. This involved the introduction of in vitro transcribed RNA molecules into mammalian cells by electroporation and conditions to optimise the system were ascertained. In the absence of sufficient immunological reagents during the early phases of the project, a histidine tag was inserted at the N-terminus of El from strain Glasgow (El his) to provide a means for purification by metal chelate chromatography. This method co-eluted E2 with El his which was later verified by the ability of polyclonal sera against E2 to co-precipitate El with E2. Thus, a heteromeric complex formed between El and E2 from strain Glasgow, a finding which is in agreement with studies on the glycoproteins from other strains. Using the available reagents for identifying El and E2 and by analysis under reducing and non-reducing electrophoretic conditions, most of the complex was in the form of disulphide-linked aggregates with little detectable native complex. The requirement for disulphide bonds to enable El and E2 association was examined by treatment of cells synthesising the proteins with the reducing agent dithiothreitol (DTT). Addition of DTT to cells did not prevent cleavage and processing of the glycoproteins. Significantly, the intermolecular disulphide links in aggregates were disrupted by DTT but El and E2 continued to associate, indicating that both covalent and non-covalent intermolecular interactions occur in the aggregated complex. El and E2, synthesised in the presence of DTT, also formed a complex and therefore disulphide bond formation is not a prerequisite for interactions between the proteins. Comparisons between strain Glasgow and strain H77 showed that native E1E2 complexes were more readily detected from strain H77. It is proposed that this may be a consequence of alterations in the number of glycosylation sites in E2 between the strains as well as changes in amino acid sequences in El and E2. Moreover, El from strain Glasgow had reduced mobility as compared to strain H77 El protein which could be not be accounted by changes in the relative sizes of the nascent proteins or glycosylation patterns. Two deletion mutants in El from both strains also displayed higher apparent molecular weights than predicted. The behaviour of these mutant proteins may result from the deletion of hydrophobic segments in El. However, the apparent molecular weight difference between El from strain Glasgow and strain H77 could not be explained. In an attempt to identify regions of El and E2 in strain Glasgow that may account for the high level of aggregation by these proteins, deletion mutants were created in El and E2; for comparative purposes, an identical set of deletion mutants were created also for El from strain H77. Immunoprecipitation studies revealed that multiple regions were involved in interactions between El and E2. No single deletion was able to completely abolish E1E2 interactions although native complex formation in strain H77 was decreased on removal of sequences from El. Further analysis of complexes and the role of disulphide bond formation was undertaken by constructing a series of mutants in which the cysteine residues in El were replaced with either serine or alanine. With each of these mutants, El and E2 continued to associate but the amounts of native, non-aggregated E2 were reduced to undetectable levels. Hence, native complexes of El and E2 were highly sensitive to changes in either protein such that alteration even at a single cysteine residue was sufficient to reduce the abundance of native complex. Examination of El expressed in the presence and absence of E2 provided a set of criteria for assessing the folding status of the protein. In the absence of E2, El remained in the reduced state and formed homo-oligomeric molecules that could be identified under reducing electrophoretic conditions. The formation and stability of these homo-oligomers were not entirely dependent on disulphide bond formation. Moreover, there was an increase in the quantities of glycosylated forms of E1 in the absence of E2.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Virion envelope; Protein interaction; Aggregation