Abstract
The cellular receptor for hepatitis B virus (HBV) has not yet been identified. A recent candidate is a homologue of squamous cell carcinoma antigen 1 (SCCA1), a serpin. This study confirms that transfection of SCCA1 into mammalian cells (both hepatocyte-derived and of non-hepatocyte origin) results in increased HBV binding. Furthermore, virus bound to transfected cells is protected significantly from degradation by trypsin (75% compared with 30% in untransfected cells). The possibility that HBV enters cells via the hepatic clearance system for serpin-enzyme complexes was investigated by analysis of the reactive site loop of SCCA1. Functional and deletion mutants of SCCA1 were constructed by site-directed mutagenesis and compared with the wild type construct. In no case was virus binding reduced by functional alterations or deletions within the reactive site loop. A possible role for the low density lipoprotein receptor-related protein (LRP) in binding virus was investigated. SCCA1 transfection of Huh7 cells was shown to result in up-regulation of LRP expression, reaching levels observed in total liver. However, the use of receptor-associated protein (RAP), a competitive ligand for LRP, suggests than LRP up-regulation is not responsible for enhanced virus binding to SCCA1-transfected cells.
Highlights
Hepatitis B virus (HBV)1 is a member of the hepadnavirus family and exhibits extreme host and tissue specificity, being able to infect only humans and higher primates and its replication being limited almost exclusively to hepatocytes
Cloning, Sequencing, and Expression of squamous cell carcinoma antigen 1 (SCCA1) cDNA—A full-length cDNA for SCCA1 was amplified from total RNA using a nested RT-PCR, and cloned as a fusion construct into the mammalian expression vector pCDNA 3.2 V5 His, to produce pCDNA SCCA1
Western blotting of lysates from cells transfected with pCDNA SCCA1 confirmed expression of a protein of ϳ44 kDa, which corresponds to the expected size of SCCA1 with the addition of a carboxylterminal V5 poly-His tag (Fig. 1)
Summary
Total RNA was purified from HepG2 cells using the SV Total RNA isolation system (Promega). Second round PCR utilized primers including restriction sites to facilitate cloning, SCCA1-XhoI (5Ј-gcgctcgagttcaccatgaattcactcagtgaagcc-3Ј) and SCCA2-His (5Ј-cgcgttcgaacggggatgagaatctgcca-3Ј). Sequencing was performed by Qiagen Sequencing Services, Germany The sequence of this clone has been submitted to the GenBankTM/ EMBL data base (accession number AJ515706). Sequencing (Qiagen) was performed to confirm that the sequence of the SCCA1 insert in pCDNA SCCA1 corresponded to the published SCCA1 sequence (accession number U19556). A single colony of E. coli BL21 cells was inoculated into L broth and incubated overnight at 37 °C, 225 rpm. This culture (500 l) was used to inoculate 100 ml of L broth, and incubated at 37 °C, 225 rpm until the culture reached an A600 of 0.6. Schechter and Berger numbering system [33], but for comparative purposes, numbering is relative to conserved P15Gly
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