Hepatitis C Virus Is Primed by CD81 Protein for Low pH-dependent Fusion

Previous studies have shown that hepatitis C virus (HCV) enters human hepatic cells through interaction with a series of cellular receptors, followed by clathrin-mediated, pH-dependent endocytosis. Here, we investigated the mechanisms of HCV entry into multiple HCV-permissive human hepatocyte-derived cells using trans-complemented HCV particles (HCVtcp). Knockdown of CD81 and claudin-1, or treatment with bafilomycin A1, reduced infection in Huh-7 and Huh7.5.1 cells, suggesting that HCV entered both cell types via receptor-mediated, pH-dependent endocytosis. Interestingly, knockdown of the clathrin heavy chain or dynamin-2 (Dyn2), as well as expression of the dominant-negative form of Dyn2, reduced infection of Huh-7 cells with HCVtcp, whereas infectious entry of HCVtcp into Huh7.5.1 cells was not impaired. Infection of Huh7.5.1 cells with culture-derived HCV (HCVcc) via a clathrin-independent pathway was also observed. Knockdown of caveolin-1, ADP-ribosylation factor 6 (Arf6), flotillin, p21-activated kinase 1 (PAK1) and the PAK1 effector C-terminal binding protein 1 of E1A had no inhibitory effects on HCVtcp infection into Huh7.5.1 cells, thus suggesting that the infectious entry pathway of HCV into Huh7.5.1 cells was not caveolae-mediated, or Arf6- and flotillin-mediated endocytosis and macropinocytosis, but rather may have occurred via an undefined endocytic pathway. Further analysis revealed that HCV entry was clathrin- and dynamin-dependent in ORL8c and HepCD81/miR122 cells, but productive entry of HCV was clathrin- and dynamin-independent in Hep3B/miR122 cells. Collectively, these data indicated that HCV entered different target cells through different entry routes.

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Hepatitis C virus glycoproteins mediate pH-dependent cell entry of pseudotyped retroviral particles

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.0832180100 ◽

2003 ◽

Vol 100 (12) ◽

pp. 7271-7276 ◽

Cited By ~ 604

Author(s):

M. Hsu ◽

J. Zhang ◽

M. Flint ◽

C. Logvinoff ◽

C. Cheng-Mayer ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Cell Entry ◽

Dependent Cell ◽

Ph Dependent

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Time- and Temperature-Dependent Activation of Hepatitis C Virus for Low-pH-Triggered Entry

Journal of Virology ◽

10.1128/jvi.80.4.1734-1741.2006 ◽

2006 ◽

Vol 80 (4) ◽

pp. 1734-1741 ◽

Cited By ~ 273

Author(s):

Donna M. Tscherne ◽

Christopher T. Jones ◽

Matthew J. Evans ◽

Brett D. Lindenbach ◽

Jane A. McKeating ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Viral Entry ◽

Ebola Virus ◽

Virus Production ◽

Low Ph ◽

Acidic Ph ◽

Prolonged Incubation ◽

Diarrhea Virus ◽

Concanamycin A

ABSTRACT Hepatitis C virus (HCV) is an important human pathogen associated with chronic liver disease. Recently, based on a genotype 2a isolate, tissue culture systems supporting complete replication and infectious virus production have been developed. In this study, we used cell culture-produced infectious HCV to analyze the viral entry pathway into Huh-7.5 cells. Bafilomycin A1 and concanamycin A, inhibitors of vacuolar ATPases, prevented HCV entry when they were present prior to infection and had minimal effect on downstream replication events. HCV entry therefore appears to be pH dependent, requiring an acidified intracellular compartment. For many other enveloped viruses, acidic pH triggers an irreversible conformational change, which promotes virion-endosomal membrane fusion. Such viruses are often inactivated by low pH. In the case of HCV, exposure of virions to acidic pH followed by return to neutral pH did not affect their infectivity. This parallels the observation made for the related pestivirus bovine viral diarrhea virus. Low pH could activate the entry of cell surface-bound HCV but only after prolonged incubation at 37°C. This suggests that there are rate-limiting, postbinding events that are needed to render HCV competent for low-pH-triggered entry. Such events may involve interaction with a cellular coreceptor or other factors but do not require cathepsins B and L, late endosomal proteases that activate Ebola virus and reovirus for entry.

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Hepatitis C Virus Entry Requires a Critical Postinternalization Step and Delivery to Early Endosomes via Clathrin-Coated Vesicles

Journal of Virology ◽

10.1128/jvi.01717-06 ◽

2006 ◽

Vol 80 (23) ◽

pp. 11571-11578 ◽

Cited By ~ 210

Author(s):

Laurent Meertens ◽

Claire Bertaux ◽

Tatjana Dragic

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Low Ph ◽

Dominant Negative ◽

Coated Vesicles ◽

Target Cells ◽

Bafilomycin A1 ◽

Late Endosomes ◽

Early Endosomes ◽

Life Threatening

ABSTRACT Hepatitis C virus (HCV) is a major human pathogen associated with life-threatening liver disease. Entry into hepatocytes requires CD81 and a putative second receptor. In this study, we elucidated the postreceptor attachment stages of HCV entry using HCV pseudoparticles (HCVpp) as a model system. By means of dominant-negative mutants and short interfering RNAs of various cellular proteins, we showed that HCVpp enter via clathrin-coated vesicles and require delivery to early but not to late endosomes. However, the kinetics of HCV envelope glycoprotein-mediated fusion are delayed compared to those of other viruses that enter in early endosomes. Entry of HCVpp can be efficiently blocked by bafilomycin A1, which neutralizes the pH in early endosomes and impairs progression of endocytosis beyond this stage. However, low-pH exposure of bafilomycin A1-treated target cells does not induce entry of HCVpp at the plasma membrane or in the early stages of endocytosis. These observations indicate that, subsequent to internalization, HCVpp entry necessitates additional, low-pH-dependent interactions, modifications, or trafficking, and that these events are irreversibly disrupted by bafilomycin A1 treatment.

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