Moore, Penelope L; (2003) SCCA1-mediated binding of hepatitis B virus to host cells. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The recent report by De Falco et al. that SCCA1 may play a role in the infection of host cells by HBV was the basis of the work described in this thesis. The purpose of this study was to confirm the findings described in that paper, and then to investigate the mechanism whereby SCCA1 mediates the binding of HBV to host cells. A cDNA for SCCA1 was cloned, its sequence analysed, and the effect of SCCA1 expression on virus-cell binding assayed in hepatocyte-derived cells. Virus internalisation and replication in SCCA1-transfected cells were also investigated. Recombinant SCCA1 was produced in a bacterial expression system and used in competitive binding experiments with HBV. The possibility that the protease inhibitory activity of SCCA1 was important in the mediation of enhanced virus-cell binding was of interest, especially considering previous reports that treatment of HBV with the V8 protease enhances infectivity. Protease inhibition is mediated by the reactive site loop (RSL) of a serpin. Site-directed mutagenesis was utilised to introduce mutations into the RSL thereby abrogating the ability of the serpin to inhibit proteases. Infectivity assays using the RSL mutants showed that the RSL is not required for enhanced virus-cell binding. The known hepatic clearance of serpin-enzyme complexes was intriguing, and led to investigations into the possibility that HBV subverts this clearance system in order to gain access to hepatocytes. Therefore, studies were performed also to investigate the effect of SCCA1 expression on the receptor proposed to mediate hepatic clearance of serpins, the LDL receptor related protein (LRP). Real-time PCR showed that SCCA1 expression in hepatocyte-derived cells (Huh7) resulted in up-regulation of LRP. However, competitive binding assays which were performed with RAP (receptor associated protein), a well-characterised ligand of the LRP, showed that SCCA1- mediated virus-cell binding does not result from enhanced expression of LRP in SCCA1-transfected cells.

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