Characterization of Fusion Determinants Points to the Involvement of Three Discrete Regions of Both E1 and E2 Glycoproteins in the Membrane Fusion Process of Hepatitis C Virus

ABSTRACT Infection of eukaryotic cells by enveloped viruses requires the merging of viral and cellular membranes. Highly specific viral surface glycoproteins, named fusion proteins, catalyze this reaction by overcoming inherent energy barriers. Hepatitis C virus (HCV) is an enveloped virus that belongs to the genus Hepacivirus of the family Flaviviridae. Little is known about the molecular events that mediate cell entry and membrane fusion for HCV, although significant progress has been made due to recent developments in infection assays. Here, using infectious HCV pseudoparticles (HCVpp), we investigated the molecular basis of HCV membrane fusion. By searching for classical features of fusion peptides through the alignment of sequences from various HCV genotypes, we identified six regions of HCV E1 and E2 glycoproteins that present such characteristics. We introduced conserved and nonconserved amino acid substitutions in these regions and analyzed the phenotype of HCVpp generated with mutant E1E2 glycoproteins. This was achieved by (i) quantifying the infectivity of the pseudoparticles, (ii) studying the incorporation of E1E2 and their capacity to mediate receptor binding, and (iii) determining their fusion capacity in cell-cell and liposome/HCVpp fusion assays. We propose that at least three of these regions (i.e., at positions 270 to 284, 416 to 430, and 600 to 620) play a role in the membrane fusion process. These regions may contribute to the merging of viral and cellular membranes either by interacting directly with lipid membranes or by assisting the fusion process through their involvement in the conformational changes of the E1E2 complex at low pH.

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Aloperine inhibits hepatitis C virus entry into cells by disturbing internalisation from endocytosis to the membrane fusion process

European Journal of Pharmacology ◽

10.1016/j.ejphar.2020.173323 ◽

2020 ◽

Vol 883 ◽

pp. 173323 ◽

Cited By ~ 1

Author(s):

Xiao-Qin Lv ◽

Li-Li Zou ◽

Jia-Li Tan ◽

Hu Li ◽

Jian-Rui Li ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Membrane Fusion ◽

Virus Entry ◽

Fusion Process

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Neutralizing Host Responses in Hepatitis C Virus Infection Target Viral Entry at Post-Binding Steps and Membrane Fusion

Zeitschrift für Gastroenterologie ◽

10.1055/s-2008-1037626 ◽

2008 ◽

Vol 46 (01) ◽

Author(s):

A Haberstroh ◽

EK Schnober ◽

P Carolla ◽

B Gißler ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Virus Infection ◽

Membrane Fusion ◽

Viral Entry ◽

Host Responses ◽

Hepatitis C Virus Infection

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Characterization of Functional Hepatitis C Virus Envelope Glycoproteins

Journal of Virology ◽

10.1128/jvi.78.6.2994-3002.2004 ◽

2004 ◽

Vol 78 (6) ◽

pp. 2994-3002 ◽

Cited By ~ 157

Author(s):

Anne Op De Beeck ◽

Cécile Voisset ◽

Birke Bartosch ◽

Yann Ciczora ◽

Laurence Cocquerel ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Conformational Changes ◽

Structural Changes ◽

Envelope Proteins ◽

Viral Envelope ◽

Envelope Glycoproteins ◽

Functional Envelope

ABSTRACT Hepatitis C virus (HCV) encodes two envelope glycoproteins, E1 and E2, that assemble as a noncovalent heterodimer which is mainly retained in the endoplasmic reticulum. Because assembly into particles and secretion from the cell lead to structural changes in viral envelope proteins, characterization of the proteins associated with the virion is necessary in order to better understand how they mature to be functional in virus entry. There is currently no efficient and reliable cell culture system to amplify HCV, and the envelope glycoproteins associated with the virion have therefore not been characterized yet. Recently, infectious pseudotype particles that are assembled by displaying unmodified HCV envelope glycoproteins on retroviral core particles have been successfully generated. Because HCV pseudotype particles contain fully functional envelope glycoproteins, these envelope proteins, or at least a fraction of them, should be in a mature conformation similar to that on the native HCV particles. In this study, we used conformation-dependent monoclonal antibodies to characterize the envelope glycoproteins associated with HCV pseudotype particles. We showed that the functional unit is a noncovalent E1E2 heterodimer containing complex or hybrid type glycans. We did not observe any evidence of maturation by a cellular endoprotease during the transport of these envelope glycoproteins through the secretory pathway. These envelope glycoproteins were recognized by a panel of conformation-dependent monoclonal antibodies as well as by CD81, a molecule involved in HCV entry. The functional envelope glycoproteins associated with HCV pseudotype particles were also shown to be sensitive to low-pH treatment. Such conformational changes are likely necessary to initiate fusion.

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Hepatitis C virus entry into the hepatocyte

Open Life Sciences ◽

10.2478/s11535-011-0076-y ◽

2011 ◽

Vol 6 (6) ◽

pp. 933-945 ◽

Cited By ~ 3

Author(s):

Sandrine Belouzard ◽

Laurence Cocquerel ◽

Jean Dubuisson

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Current Knowledge ◽

Virus Entry ◽

Low Density Lipoprotein ◽

Scavenger Receptor ◽

Density Lipoprotein ◽

Surface Proteins ◽

Enveloped Virus ◽

Early Endosomes

AbstractHepatitis C virus (HCV) is a small enveloped virus with a positive stranded RNA genome belonging to the Flaviviridae family. The virion has the unique ability of forming a complex with lipoproteins, which is known as the lipoviroparticle. Lipoprotein components as well as the envelope proteins, E1 and E2, play a key role in virus entry into the hepatocyte. HCV entry is a complex multistep process involving sequential interactions with several cell surface proteins. The virus relies on glycosaminoglycans and possibly the low-density lipoprotein receptors to attach to cells. Furthermore, four specific entry factors are involved in the following steps which lead to virus internalization and fusion in early endosomes. These molecules are the scavenger receptor SRB1, tetraspanin CD81 and two tight junction proteins, Claudin-1 and Occludin. Although they are essential to HCV entry, the precise role of these molecules is not completely understood. Finally, hepatocytes are highly polarized cells and which likely affects the entry process. Our current knowledge on HCV entry is summarized in this review.

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Binding Dynamics of Hepatitis C Virus' NS5A Amphipathic Peptide to Cell and Model Membranes

Journal of Virology ◽

10.1128/jvi.02783-06 ◽

2007 ◽

Vol 81 (12) ◽

pp. 6682-6689 ◽

Cited By ~ 26

Author(s):

Nam-Joon Cho ◽

Kwang Ho Cheong ◽

ChoongHo Lee ◽

Curtis W. Frank ◽

Jeffrey S. Glenn

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Critical Role ◽

Membrane Binding ◽

Lipid Binding ◽

Model Membranes ◽

Cellular Membranes ◽

Kinetic Measurements ◽

Amphipathic Peptide ◽

Target Membrane

ABSTRACT Membrane association of the hepatitis C virus NS5A protein is required for viral replication. This association is dependent on an N-terminal amphipathic helix (AH) within NS5A and is restricted to a subset of host cell intracellular membranes. The mechanism underlying this specificity is not known, but it may suggest a novel strategy for developing specific antiviral therapy. Here we have probed the mechanistic details of NS5A AH-mediated binding to both cell-derived and model membranes by use of biochemical membrane flotation and quartz crystal microbalance (QCM) with dissipation. With both assays, we observed AH-mediated binding to model lipid bilayers. When cell-derived membranes were coated on the quartz nanosensor, however, significantly more binding was detected, and the QCM-derived kinetic measurements suggested the existence of an interacting receptor in the target membranes. Biochemical flotation assays performed with trypsin-treated cell-derived membranes exhibited reduced AH-mediated membrane binding, while membrane binding of control cytochrome b5 remained unaffected. Similarly, trypsin treatment of the nanosensor coated with cellular membranes abolished AH peptide binding to the cellular membranes but did not affect the binding of a control lipid-binding peptide. These results therefore suggest that a protein plays a critical role in mediating and stabilizing the binding of NS5A's AH to its target membrane. These results also demonstrate the successful development of a new nanosensor technology ideal both for studying the interaction between a protein and its target membrane and for developing inhibitors of that interaction.

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Hemagglutinin-Mediated Membrane Fusion: A Biophysical Perspective

Annual Review of Biophysics ◽

10.1146/annurev-biophys-070317-033018 ◽

2018 ◽

Vol 47 (1) ◽

pp. 153-173 ◽

Cited By ~ 14

Author(s):

Sander Boonstra ◽

Jelle S. Blijleven ◽

Wouter H. Roos ◽

Patrick R. Onck ◽

Erik van der Giessen ◽

...

Keyword(s):

Membrane Fusion ◽

Host Cell ◽

Conformational Changes ◽

Fusion Process ◽

Host Cell Membrane ◽

Activation Barriers ◽

Viral Membrane ◽

Influenza Hemagglutinin ◽

Working Together

Influenza hemagglutinin (HA) is a viral membrane protein responsible for the initial steps of the entry of influenza virus into the host cell. It mediates binding of the virus particle to the host-cell membrane and catalyzes fusion of the viral membrane with that of the host. HA is therefore a major target in the development of antiviral strategies. The fusion of two membranes involves high activation barriers and proceeds through several intermediate states. Here, we provide a biophysical description of the membrane fusion process, relating its kinetic and thermodynamic properties to the large conformational changes taking place in HA and placing these in the context of multiple HA proteins working together to mediate fusion. Furthermore, we highlight the role of novel single-particle experiments and computational approaches in understanding the fusion process and their complementarity with other biophysical approaches.

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