The SR-BI Partner PDZK1 Facilitates Hepatitis C Virus Entry

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Abstract
Entry of hepatitis C virus (HCV) into hepatocytes is a multi-step process that involves a number of different host cell factors. Following initial engagement with glycosaminoglycans and the low-density lipoprotein receptor, it is thought that HCV entry proceeds via interactions with the tetraspanin CD81, scavenger receptor class B type I (SR-BI), and the tight-junction proteins claudin-1 (CLDN1) and occludin (OCLN), culminating in clathrin-dependent endocytosis of HCV particles and their pH-dependent fusion with endosomal membranes. Physiologically, SR-BI is the major receptor for high-density lipoproteins (HDL) in the liver, where its expression is primarily controlled at the post-transcriptional level by its interaction with the scaffold protein PDZK1. However, the importance of interaction with PDZK1 to the involvement of SR-BI in HCV entry is unclear. Here we demonstrate that stable shRNA-knockdown of PDZK1 expression in human hepatoma cells significantly reduces their susceptibility to HCV infection, and that this effect can be reversed by overexpression of full length PDZK1 but not the first PDZ domain of PDZK1 alone. Furthermore, we found that overexpression of a green fluorescent protein chimera of the cytoplasmic carboxy-terminus of SR-BI (amino acids 479–509) in Huh-7 cells resulted in its interaction with PDZK1 and a reduced susceptibility to HCV infection. In contrast a similar chimera lacking the final amino acid of SR-BI (amino acids 479–508) failed to interact with PDZK1 and did not inhibit HCV infection. Taken together these results indicate an indirect involvement of PDZK1 in HCV entry via its ability to interact with SR-BI and enhance its activity as an HCV entry factor. Hepatitis C virus (HCV) infection is a major cause of serious liver disease, with approximately 170 million people infected worldwide. Although significant advances have been made in the characterization and development of novel therapeutics to combat HCV infection, there is still a great need for an improved understanding of the HCV lifecycle and potential future targets of antiviral therapy. HCV entry into hepatocytes involves numerous plasma membrane proteins including CD81, scavenger receptor class B type I (SR-BI), claudin-1 and occludin. Although these proteins may comprise the complete set of essential HCV entry factors, the secondary factors that influence the co-ordinated involvement of these proteins in HCV entry remain to be determined. Here we identify the SR-BI partner protein PDZK1 as an indirect regulator of HCV entry. Our results indicate that binding of PDZK1 to the cytoplasmic carboxy-terminus of SR-BI influences the receptor's involvement in HCV entry such that disruption of this interaction may represent a future target of therapeutic intervention.

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