Characterisation of the Hepatitis C Virus NS3 Serine Protease

Thompson, Russell John (1998) Characterisation of the Hepatitis C Virus NS3 Serine Protease. PhD thesis, University of Glasgow.

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Hepatitis C Virus (HCV) is the major cause of post-transfusion non-A. non-B hepatitis. HCV is a member of the Flaviviridae, a family of viruses characterised by a single-stranded, positive sense RNA genome. The genome of HCV is approximately 9.6kb and contains a large open reading frame (ORF) that encodes the viral polyprotein. The viral polyprotein contains the structural proteins (Core, El and E2) at the N-terminus and the non-structural proteins (NS2-NS5B) within the remainder of the polyprotein. The HCV polyprotein is processed by cellular and viral-encoded proteases. A serine protease, contained within the NS3 protein, is responsible for the majority of cleavage events within the non-structural region of the polyprotein. A cell-based assay to study the cleavage of an HCV substrate in trans by the NS3 serine protease was developed, and using this assay characterisation of the substrate specificity of the NS3 serine protease was carried out. HCV proteins were expressed from plasmid DNAs in vTF7.3 infected mammalian cells and the cells lysed and immuneprecipitated before analysis by SDS-PAGE. A truncated NS5 substrate was constructed with an epitope tag at both termini to enable immuneprecipitation of the substrate and cleavage products. The consensus amino acid motif for cleavage by NS3 had been previously determined (D/E X X X X C/T S/A) and mutagenesis within an HCV NS5 substrate confirmed the importance of these residues (P6, PI, PI'). The importance of the P6 residue appeared to be more important for cleavage at the NS5A/NS5B cleavage site than at other cleavage sites within the HCV polyprotein. The amino acid sequence of the HCV G1a polyprotein was searched for the consensus NS3 cleavage site motif. In addition to the sites cleaved by NS3 an additional three potential sites were found. Analysis of one of these sites demonstrated that an acidic residue at P5 was important for efficient cleavage in trans by the NS3 serine protease. In addition to this the presence of an additional serine residue in close proximity to the PI and PP residues was observed to have a negative effect on the efficiency of processing. The protease requirement for processing of an HCV NS5 substrate was investigated. The NS5A/NS5B cleavage site was processed when the NS5 substrates were coexpressed with NS34A. Coexpression with NS3 and NS3P did not result in the generation of cleavage products. When specific amino acids were substituted with residues found within other HCV isolates the NS3 alone was able to process in trans. These investigations illustrated that additional residues to the catalytic triad within the NS3 protease are important for cleavage in trans, at least for the NS3 protein alone. An NS3 serine protease from a different HCV subtype was shown to be able to process the HCV G1a NS5 substrate.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: Richard Elliott
Keywords: Virology
Date of Award: 1998
Depositing User: Enlighten Team
Unique ID: glathesis:1998-75904
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 19 Nov 2019 17:38
Last Modified: 19 Nov 2019 17:38

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