RNA Interference and Single Particle Tracking Analysis of Hepatitis C Virus Endocytosis

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Abstract
Hepatitis C virus (HCV) enters hepatocytes following a complex set of receptor interactions, culminating in internalization via clathrin-mediated endocytosis. However, aside from receptors, little is known about the cellular molecular requirements for infectious HCV entry. Therefore, we analyzed a siRNA library that targets 140 cellular membrane trafficking genes to identify host genes required for infectious HCV production and HCV pseudoparticle entry. This approach identified 16 host cofactors of HCV entry that function primarily in clathrin-mediated endocytosis, including components of the clathrin endocytosis machinery, actin polymerization, receptor internalization and sorting, and endosomal acidification. We next developed single particle tracking analysis of highly infectious fluorescent HCV particles to examine the co-trafficking of HCV virions with cellular cofactors of endocytosis. We observe multiple, sequential interactions of HCV virions with the actin cytoskeleton, including retraction along filopodia, actin nucleation during internalization, and migration of internalized particles along actin stress fibers. HCV co-localizes with clathrin and the ubiquitin ligase c-Cbl prior to internalization. Entering HCV particles are associated with the receptor molecules CD81 and the tight junction protein, claudin-1; however, HCV-claudin-1 interactions were not restricted to Huh-7.5 cell-cell junctions. Surprisingly, HCV internalization generally occurred outside of Huh-7.5 cell-cell junctions, which may reflect the poorly polarized nature of current HCV cell culture models. Following internalization, HCV particles transport with GFP-Rab5a positive endosomes, which is consistent with trafficking to the early endosome. This study presents technical advances for imaging HCV entry, in addition to identifying new host cofactors of HCV infection, some of which may be antiviral targets. Hepatitis C virus (HCV) chronically infects 130 million people and is a major cause of cirrhosis and liver cancer. The current antiviral therapy of pegylated interferon-2 alfa + ribavirin is successful in only half of treated patients. This has led to an intensive effort to design improved therapeutic strategies. The identification of cellular cofactors of HCV infection greatly expands the pool of potential targets for drug design. In this paper, we combine RNA interference analysis of HCV endocytosis with the development of live cell imaging of highly infectious HCV particles. We identify 16 host cofactors of HCV entry, most of which function in sequential stages of clathrin-mediated endocytosis. We observe the trafficking of fluorescent HCV particles with these cellular cofactors and their related pathways, including the actin cytoskeleton, known receptors CD81 and the tight junction protein claudin-1, clathrin, an E3 ubiquitin ligase, and early endosomes. Surprisingly, given the role of tight junction proteins as HCV entry factors, virion entry generally occurred outside of cell-cell junctions. This paper identifies novel host targets for therapeutic development, describes techniques to image HCV entry, and provides insights into HCV-cell interactions in the entry process.