Sec24C-Dependent Transport of Claudin-1 Regulates Hepatitis C Virus Entry

ABSTRACT Claudin-1 is a hepatitis C virus (HCV) coreceptor required for viral entry. Although extensive studies have focused on claudin-1 as an anti-HCV target, little is known about how the level of claudin-1 at the cell surface is regulated by host vesicular transport. Here, we identified an interaction between claudin-1 and Sec24C, a cargo-sorting component of the coat protein complex II (COPII) vesicular transport system. By interacting with Sec24C through its C-terminal YV, claudin-1 is transported from the endoplasmic reticulum (ER) and is eventually targeted to the cell surface. Blocking COPII transport inhibits HCV entry by reducing the level of claudin-1 at the cell surface. These findings provide mechanistic insight into the role of COPII vesicular transport in HCV entry. IMPORTANCE Tight junction protein claudin-1 is one of the cellular receptors for hepatitis C virus, which infects 185 million people globally. Its cellular distribution plays important role in HCV entry; however, it is unclear how the localization of claudin-1 to the cell surface is controlled by host transport pathways. In this paper, we not only identified Sec24C as a key host factor for HCV entry but also uncovered a novel mechanism by which the COPII machinery transports claudin-1 to the cell surface. This mechanism might be extended to other claudins that contain a C-terminal YV or V motif.

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Hepatitis C virus depends on E-cadherin as an entry factor and regulates its expression in epithelial-to-mesenchymal transition

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1602701113 ◽

2016 ◽

Vol 113 (27) ◽

pp. 7620-7625 ◽

Cited By ~ 35

Author(s):

Qisheng Li ◽

Catherine Sodroski ◽

Brianna Lowey ◽

Cameron J. Schweitzer ◽

Helen Cha ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Cell Surface ◽

Viral Entry ◽

Epithelial To Mesenchymal Transition ◽

Hcv Infection ◽

Hcv Rna ◽

Entry Site ◽

Mesenchymal Transition ◽

E Cadherin

Hepatitis C virus (HCV) enters the host cell through interactions with a cascade of cellular factors. Although significant progress has been made in understanding HCV entry, the precise mechanisms by which HCV exploits the receptor complex and host machinery to enter the cell remain unclear. This intricate process of viral entry likely depends on additional yet-to-be-defined cellular molecules. Recently, by applying integrative functional genomics approaches, we identified and interrogated distinct sets of host dependencies in the complete HCV life cycle. Viral entry assays using HCV pseudoparticles (HCVpps) of various genotypes uncovered multiple previously unappreciated host factors, including E-cadherin, that mediate HCV entry. E-cadherin silencing significantly inhibited HCV infection in Huh7.5.1 cells, HepG2/miR122/CD81 cells, and primary human hepatocytes at a postbinding entry step. Knockdown of E-cadherin, however, had no effect on HCV RNA replication or internal ribosomal entry site (IRES)-mediated translation. In addition, an E-cadherin monoclonal antibody effectively blocked HCV entry and infection in hepatocytes. Mechanistic studies demonstrated that E-cadherin is closely associated with claudin-1 (CLDN1) and occludin (OCLN) on the cell membrane. Depletion of E-cadherin drastically diminished the cell-surface distribution of these two tight junction proteins in various hepatic cell lines, indicating that E-cadherin plays an important regulatory role in CLDN1/OCLN localization on the cell surface. Furthermore, loss of E-cadherin expression in hepatocytes is associated with HCV-induced epithelial-to-mesenchymal transition (EMT), providing an important link between HCV infection and liver cancer. Our data indicate that a dynamic interplay among E-cadherin, tight junctions, and EMT exists and mediates an important function in HCV entry.

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Generation of fluorescent HCV pseudoparticles to study early viral entry events-involvement of Rab1a in HCV entry

10.1101/2021.03.11.434898 ◽

2021 ◽

Author(s):

Chayan Bhattacharjee ◽

Aparna Mukhopadhyay

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Confocal Microscopy ◽

Viral Entry ◽

Envelope Glycoprotein ◽

List Type ◽

Enveloped Viruses ◽

Early Entry ◽

Gfp Tag

AbstractUnderstanding the early events in viral biology holds the key to the development of preventives. In this study fluorescent Hepatitis C Virus pseudoparticles have been generated where the envelope glycoprotein has a GFP tag. Using these pseudoparticles entry assays were conducted where the entry of the pseudoparticles was tracked via confocal microscopy. Using this system, fusion of host and viral membranes is predicted to occur within 15 minutes of entry in HCV. Using cells with a knockdown for Rab1a, HCV trafficking was observed to be altered, indicating a role of Rab1a in HCV trafficking. In conclusion, this study reports the generation and use of fluorescent pseudoparticles which may be used to understand the early events of viral entry. This system may be adapted for the study of other enveloped viruses as well.HighlightsFluorescent HCV pseudoparticles have been created to study early entry events.HCV entry tracking via confocal microscopy reveals fusion within 15 minutes.Rab1a is important for HCV trafficking within the cell.

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Neglected but Important Role of Apolipoprotein E Exchange in Hepatitis C Virus Infection

Journal of Virology ◽

10.1128/jvi.01353-16 ◽

2016 ◽

Vol 90 (21) ◽

pp. 9632-9643 ◽

Cited By ~ 30

Author(s):

Zaili Yang ◽

Xiaoning Wang ◽

Xiumei Chi ◽

Fanfan Zhao ◽

Jinxu Guo ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Cell Surface ◽

Apolipoprotein E ◽

Virus Assembly ◽

Hcv Infection ◽

Apoe Level ◽

New Perspective ◽

Apoe Expression

ABSTRACT Hepatitis C virus (HCV) is a major cause of chronic liver disease, infecting approximately 170 million people worldwide. HCV assembly is tightly associated with the lipoprotein pathway. Exchangeable apolipoprotein E (apoE) is incorporated on infectious HCV virions and is important for infectious HCV virion morphogenesis and entry. Moreover, the virion apoE level is positively correlated with its ability to escape E2 antibody neutralization. However, the role of apoE exchange in the HCV life cycle is unclear. In this study, the relationship between apoE expression and cell permissiveness to HCV infection was assessed by infecting apoE knockdown and derived apoE rescue cell lines with HCV. Exchange of apoE between lipoproteins and HCV lipoviral particles (LVPs) was evaluated by immunoprecipitation, infectivity testing, and viral genome quantification. Cell and heparin column binding assays were applied to determine the attachment efficiency of LVPs with different levels of incorporated apoE. The results showed that cell permissiveness for HCV infection was determined by exogenous apoE-associated lipoproteins. Furthermore, apoE exchange did occur between HCV LVPs and lipoproteins, which was important to maintain a high apoE level on LVPs. Lipid-free apoE was capable of enhancing HCV infectivity for apoE knockdown cells but not apoE rescue cells. A higher apoE level on LVPs conferred more efficient LVP attachment to both the cell surface and heparin beads. This study revealed that exogenous apoE-incorporating lipoproteins from uninfected hepatocytes safeguarded the apoE level of LVPs for more efficient attachment during HCV infection. IMPORTANCE In this study, a neglected but important role of apoE exchange in HCV LVP infectivity after virus assembly and release was identified. The data indicated that apoE expression level in uninfected cells is important for high permissiveness to HCV infection. Secreted apoE-associated lipoprotein specifically enhances infection of HCV LVPs. apoE exchange between HCV LVP and lipoproteins is important to maintain an adequate apoE level on LVPs for their efficient attachment to cell surface. These data defined for the first time an extracellular role of exchangeable apoE in HCV infection and suggested that exchangeable apolipoproteins reach a natural equilibrium between HCV LVPs and lipoprotein particles, which provides a new perspective to the understanding of the heterogeneity of HCV LVPs in composition.

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Diminished viral replication and compartmentalization of hepatitis C virus in hepatocellular carcinoma tissue

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1516879113 ◽

2016 ◽

Vol 113 (5) ◽

pp. 1375-1380 ◽

Cited By ~ 32

Author(s):

Djamila Harouaka ◽

Ronald E. Engle ◽

Kurt Wollenberg ◽

Giacomo Diaz ◽

Ashley B. Tice ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Viral Population ◽

Hcv Rna ◽

Carcinoma Tissue ◽

Insight Into ◽

Hepatocellular Carcinoma Tissue ◽

Comprehensive Study

Analysis of hepatitis C virus (HCV) replication and quasispecies distribution within the tumor of patients with HCV-associated hepatocellular carcinoma (HCC) can provide insight into the role of HCV in hepatocarcinogenesis and, conversely, the effect of HCC on the HCV lifecycle. In a comprehensive study of serum and multiple liver specimens from patients with HCC who underwent liver transplantation, we found a sharp and significant decrease in HCV RNA in the tumor compared with surrounding nontumorous tissues, but found no differences in multiple areas of control non-HCC cirrhotic livers. Diminished HCV replication was not associated with changes in miR-122 expression. HCV genetic diversity was significantly higher in livers containing HCC compared with control non-HCC cirrhotic livers. Tracking of individual variants demonstrated changes in the viral population between tumorous and nontumorous areas, the extent of which correlated with the decline in HCV RNA, suggesting HCV compartmentalization within the tumor. In contrast, compartmentalization was not observed between nontumorous areas and serum, or in controls between different areas of the cirrhotic liver or between liver and serum. Our findings indicate that HCV replication within the tumor is restricted and compartmentalized, suggesting segregation of specific viral variants in malignant hepatocytes.

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Identification of Piperazinylbenzenesulfonamides as New Inhibitors of Claudin-1 Trafficking and Hepatitis C Virus Entry

Journal of Virology ◽

10.1128/jvi.01982-17 ◽

2018 ◽

Vol 92 (10) ◽

Cited By ~ 7

Author(s):

Laura Riva ◽

Ok-ryul Song ◽

Jannick Prentoe ◽

François Helle ◽

Laurent L'homme ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Cell Surface ◽

Serotonin Receptor ◽

Chemical Compounds ◽

Biologically Active ◽

Independent Manner ◽

Active Chemical ◽

Junction Protein ◽

Pka Pathway

ABSTRACT Hepatitis C virus (HCV) infection causes 500,000 deaths annually, in association with end-stage liver diseases. Investigations of the HCV life cycle have widened the knowledge of virology, and here we discovered that two piperazinylbenzenesulfonamides inhibit HCV entry into liver cells. The entry of HCV into host cells is a complex process that is not fully understood but is characterized by multiple spatially and temporally regulated steps involving several known host factors. Through a high-content virus infection screening analysis with a library of 1,120 biologically active chemical compounds, we identified SB258585, an antagonist of serotonin receptor 6 (5-HT6), as a new inhibitor of HCV entry in liver-derived cell lines as well as primary hepatocytes. A functional characterization suggested a role for this compound and the compound SB399885, which share similar structures, as inhibitors of a late HCV entry step, modulating the localization of the coreceptor tight junction protein claudin-1 (CLDN1) in a 5-HT6-independent manner. Both chemical compounds induced an intracellular accumulation of CLDN1, reflecting export impairment. This regulation correlated with the modulation of protein kinase A (PKA) activity. The PKA inhibitor H89 fully reproduced these phenotypes. Furthermore, PKA activation resulted in increased CLDN1 accumulation at the cell surface. Interestingly, an increase of CLDN1 recycling did not correlate with an increased interaction with CD81 or HCV entry. These findings reinforce the hypothesis of a common pathway, shared by several viruses, which involves G-protein-coupled receptor-dependent signaling in late steps of viral entry. IMPORTANCE The HCV entry process is highly complex, and important details of this structured event are poorly understood. By screening a library of biologically active chemical compounds, we identified two piperazinylbenzenesulfonamides as inhibitors of HCV entry. The mechanism of inhibition was not through the previously described activity of these inhibitors as antagonists of serotonin receptor 6 but instead through modulation of PKA activity in a 5-HT6-independent manner, as proven by the lack of 5-HT6 in the liver. We thus highlighted the involvement of the PKA pathway in modulating HCV entry at a postbinding step and in the recycling of the tight junction protein claudin-1 (CLDN1) toward the cell surface. Our work underscores once more the complexity of HCV entry steps and suggests a role for the PKA pathway as a regulator of CLDN1 recycling, with impacts on both cell biology and virology.

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The Tight Junction Proteins Claudin-1, -6, and -9 Are Entry Cofactors for Hepatitis C Virus

Journal of Virology ◽

10.1128/jvi.01977-07 ◽

2008 ◽

Vol 82 (7) ◽

pp. 3555-3560 ◽

Cited By ~ 140

Author(s):

Laurent Meertens ◽

Claire Bertaux ◽

Lisa Cukierman ◽

Emmanuel Cormier ◽

Dimitri Lavillette ◽

...

Keyword(s):

Endothelial Cells ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Tight Junction ◽

Tight Junction Proteins ◽

Junction Protein ◽

Cellular Factors ◽

Cd81 Tetraspanin ◽

Junction Proteins

ABSTRACT Hepatitis C virus (HCV) is a major cause of liver disease in humans. The CD81 tetraspanin is necessary but not sufficient for HCV penetration into hepatocytes, and it was recently reported that the tight junction protein claudin-1 is a critical HCV entry cofactor. Here, we confirm the role of claudin-1 in HCV entry. In addition, we show that claudin-6 and claudin-9 expressed in CD81+ cells also enable the entry of HCV pseudoparticles derived from six of the major genotypes. Whereas claudin-1, -6, and -9 function equally well as entry cofactors in endothelial cells, claudin-1 is more efficient in hepatoma cells. This suggests that additional cellular factors modulate the ability of claudins to function as HCV entry cofactors. Our work has generated novel and essential means to investigate the mechanism of HCV penetration into hepatocytes and the role of the claudin protein family in HCV dissemination, replication, and pathogenesis.

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