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

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.

Impact of Intra- and Interspecies Variation of Occludin on Its Function as Coreceptor for Authentic Hepatitis C Virus Particles

“…To this end, cell lines deficient for individual key factors (8) or expressing functionally relevant variants may be very useful, especially when combined with innovative approaches that make the HCV cell entry process directly observable (10). A better understanding of the entry process may inform both the development of antiviral approaches and the development of better smallanimal models.…”

Entry and replication of recombinant hepatitis C viruses in cell culture

“…The most intuitive but work-intensive approach is to directly look at virus entry with live-cell microscopy. To this end, Coller et al labelled HCVcc with the DiD or DiI lipophilic dyes and monitored entry of single particles into target cells [154]. The main difficulties to overcome seem to be the low HCV titers and specific infectivity, making the distinction between infectious and non-infectious particles difficult.…”

“…Clathrin-dependent endocytosis -Internalization assay [146] -Inhibition by drugs and dominant-negative mutants -Live cell microscopy with labelled HCV [154] Low pH-mediated fusion in early endosomes -Inhibitors of endosomal acidi cation -Di erent fusion assays (see main text) Attachment and receptor interaction -Cell binding assays (CHO-SR-BI, CHO-CD81, etc) [118] -Pull-down assays [152] or ELISAs [132,153] with soluble CD81 or with heparin -Cell lines for receptor complementation (see Table 1) -Lentiviral expression vectors for the 4 speci c receptors and their homologs [104,116] H + Concanamycin A, Bafilomycin A1, Chloroquine, NH 4 Cl [69,146,147] Rab5 dominant negative mutant [145,146] Eps15 dominant negative mutant, Clathrin heavy chain siRNAs, Chlorpromazine [78,[145][146][147] Abs, Ezetimibe [32] NPC1L1 EGFR…”

Strategies to Preclude Hepatitis C Virus Entry

“…The HCV particle finally enters the cell via clathrin-mediated endocytosis [61]. The HCV-receptor complexes then migrate to endosomal compartments [62,63] where acidification occurs to induce membrane fusion, which allows the release of viral RNA into the host cytosol.…”