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.
Highlights
Hepatitis C virus (HCV) contains a small positive stranded RNA (,9600 bp) genome encased by a capsid and surrounded by a lipid bilayer envelope containing the glycoproteins, E1 and E2 [1]
We first identified siRNAs that inhibited infectious HCV production and re-tested these siRNAs using HCV pseudoparticles (HCVpp) that contain a luciferase reporter gene. The rationale for this approach was to do a primary evaluation of host cofactors using the most authentic system (HCVcc) and distinguish host genes involved in HCV endocytosis from those acting at later stages of the viral life cycle by using HCVpp
We combined an unbiased RNA interference (RNAi) analysis that identified 16 cellular proteins involved in HCV endocytosis with the first description of single particle tracking of HCV infection
Summary
Hepatitis C virus (HCV) contains a small positive stranded RNA (,9600 bp) genome encased by a capsid and surrounded by a lipid bilayer envelope containing the glycoproteins, E1 and E2 [1]. Subsequent internalization of HCV requires a complex set of receptor molecules including, the tetraspannin molecule CD81, scavenger receptor B-1 (SR-BI), and the tight junction proteins claudin(s) (CLDN)-1, -6, -9, and occludin (OCDN) to bind and enter into hepatocytes [2,3,4,5,6,7,8,9,10,11,12,13,14,22,23,24,25,26]. HCV internalization has never been visualized, but is postulated to occur at the tight junction where the entry receptors claudin-1 and occludin localize. Engagement of CD81 activates the Rho GTPase, CDC42, which may result in actin cytoskeletal rearrangements that would allow for trafficking of HCV to the tight junction [27]
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