Schwarz, Anne-Katrin (2009)
Ph.D. thesis, University of Birmingham.
Hepatitis C virus (HCV) is a major human pathogen and the leading cause of cirrhosis and liver cancer worldwide. HCV entry is clathrin- and pH-dependent, and requires CD81, Scavenger receptor BI (SR-BI), and the tight junction (TJ) proteins Claudin-1 and Occludin. Primary HCV strains cannot be efficiently cultured in vitro, making the evaluation of potential antiviral compounds in a biologically relevant context extremely difficult. Despite being suitable for high-throughput screening, most cell-based reporter assays rely on the secretion of serine alkaline phosphatase and thus do not allow the selection of HCV infected cells, or the screening of patient samples to identify cell culture infectious viral strains. We aimed to develop a cell-based reporter assay, which utilizes the viral NS3/4A protease to cleave and activate a fluorescent reporter protein constitutively expressed in Huh-7.5 hepatoma cells. HCV tropism is restricted to the liver, where hepatocytes are polarized and form TJ, which are indispensable for normal liver functionality. We demonstrate that in confluent cells, SR-BI and Claudin-1 expression is increased and that HCV entry is enhanced when cellular contact is established. Furthermore, cell junction formation and SR-BI overexpression, respectively, accelerated virus entry, suggesting a key role for SR-BI in HCV internalization. The mechanisms underlying HCV-associated hepatic injury are poorly understood, however, it is thought that HCV may disrupt TJ integrity, thus compromising hepatocyte polarity and function. We demonstrate that the HCV structural proteins modulate the expression and localization of TJ proteins, leading to their redistribution to cytoplasmic vesicles with possible consequences for TJ integrity in vivo.
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