Membrane Fusion Assays for Studying Entry Hepatitis C Virus into Cells

  • Solène Denolly
  • François-Loïc CossetEmail author
  • Natalia Freitas
Part of the Methods in Molecular Biology book series (MIMB, volume 1911)


The membrane fusion properties of HCV envelope glycoproteins can be evaluated using several assays. Fusion assays generally require contacts between glycoproteins expressed on a donor membrane, such as those from a cell or a viral particle, and an acceptor membrane that may or may not express cognate viral receptor, such as those from an indicator cell or a liposome. In this chapter, we describe three well-established methods in the field that use either cell surface expression of glycoproteins, HCV pseudoparticles (HCVpp), or cell culture-grown HCV (HCVcc) particles for donor membrane and cells or liposomes as acceptor membrane in which specific components can be included to monitor and quantify fusion. We provide details of cell-cell fusion assay, virus-liposome fusion assay, and finally virus-plasma membrane fusion assay. We also describe inhibitors that can block HCV envelope membrane fusion.

Key words

Fusion Hepatitis C Liposomes Inhibitors Plasma membrane Cell-cell fusion 



Our work is supported by the French “Agence Nationale de la Recherche sur le SIDA et les hépatites virales” (ANRS), the European Research Council (ERC-2008-AdG-233130-HEPCENT), and the LabEx ECOFECT (ANR-11-LABX-0048).


  1. 1.
    Douam F, Lavillette D, Cosset FL (2015) The mechanism of HCV entry into host cells. Prog Mol Biol Transl Sci 129:63–107CrossRefGoogle Scholar
  2. 2.
    Perez-Berna AJ, Moreno MR, Guillen J, Bernabeu A, Villalain J (2006) The membrane-active regions of the hepatitis C virus E1 and E2 envelope glycoproteins. Biochemistry 45:3755–3768CrossRefGoogle Scholar
  3. 3.
    Takikawa S, Ishii K, Aizaki H, Suzuki T, Asakura H, Matsuura Y et al (2000) Cell fusion activity of hepatitis C virus envelope proteins. J Virol 74:5066–5074CrossRefGoogle Scholar
  4. 4.
    Bartosch B, Dubuisson J, Cosset FL (2003) Infectious hepatitis C virus pseudo-particles containing functional E1-E2 envelope protein complexes. J Exp Med 197:633–642CrossRefGoogle Scholar
  5. 5.
    Lavillette D, Bartosch B, Nourrisson D, Verney G, Cosset FL, Penin F et al (2006) Hepatitis C virus glycoproteins mediate low pH-dependent membrane fusion with liposomes. J Biol Chem 281:3909–3917CrossRefGoogle Scholar
  6. 6.
    Lavillette D, Pecheur EI, Donot P, Fresquet J, Molle J, Corbau R et al (2007) 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. J Virol 81:8752–8765CrossRefGoogle Scholar
  7. 7.
    Tscherne DM, Jones CT, Evans MJ, Lindenbach BD, McKeating JA, Rice CM (2006) Time- and temperature-dependent activation of hepatitis C virus for low-pH-triggered entry. J Virol 80:1734–1741CrossRefGoogle Scholar
  8. 8.
    Haid S, Pietschmann T, Pecheur EI (2009) Low pH-dependent hepatitis C virus membrane fusion depends on E2 integrity, target lipid composition, and density of virus particles. J Biol Chem 284:17657–17667CrossRefGoogle Scholar
  9. 9.
    Pecheur EI, Lavillette D, Alcaras F, Molle J, Boriskin YS, Roberts M et al (2007) Biochemical mechanism of hepatitis C virus inhibition by the broad-spectrum antiviral arbidol. Biochemistry 46:6050–6059CrossRefGoogle Scholar
  10. 10.
    Owsianka AM, Tarr AW, Keck ZY, Li TK, Witteveldt J, Adair R et al (2008) Broadly neutralizing human monoclonal antibodies to the hepatitis C virus E2 glycoprotein. J Gen Virol 89:653–659CrossRefGoogle Scholar
  11. 11.
    Matsumura T, Hu Z, Kato T, Dreux M, Zhang YY, Imamura M et al (2009) Amphipathic DNA polymers inhibit hepatitis C virus infection by blocking viral entry. Gastroenterology 137:673–681CrossRefGoogle Scholar
  12. 12.
    Chi X, Niu Y, Cheng M, Liu X, Feng Y, Zheng F et al (2016) Identification of a Potent and Broad-Spectrum Hepatitis C Virus Fusion Inhibitory Peptide from the E2 Stem Domain. Sci Rep 6:25224CrossRefGoogle Scholar
  13. 13.
    Perin PM, Haid S, Brown RJ, Doerrbecker J, Schulze K, Zeilinger C et al (2016) Flunarizine prevents hepatitis C virus membrane fusion in a genotype-dependent manner by targeting the potential fusion peptide within E1. Hepatology 63:49–62CrossRefGoogle Scholar
  14. 14.
    Ashfaq UA, Javed T, Rehman S, Nawaz Z, Riazuddin S (2011) Lysosomotropic agents as HCV entry inhibitors. Virol J 8:163CrossRefGoogle Scholar
  15. 15.
    Cavrois M, De Noronha C, Greene WC (2002) A sensitive and specific enzyme-based assay detecting HIV-1 virion fusion in primary T lymphocytes. Nat Biotechnol 20:1151–1154CrossRefGoogle Scholar
  16. 16.
    Yonezawa A, Cavrois M, Greene WC (2005) Studies of ebola virus glycoprotein-mediated entry and fusion by using pseudotyped human immunodeficiency virus type 1 virions: involvement of cytoskeletal proteins and enhancement by tumor necrosis factor alpha. J Virol 79:918–926CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Solène Denolly
    • 1
  • François-Loïc Cosset
    • 1
    Email author
  • Natalia Freitas
    • 1
  1. 1.CIRI–International Center for Infectiology Research, Team EVIR, Inserm, U1111, Université Claude Bernard Lyon 1, CNRS, UMR5308, Ecole Normale Supérieure de Lyon, Univ LyonLyonFrance

Personalised recommendations