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Journal of General Virology header logo Journal of General Virology

Volume 106, Issue 11

The architecture of membrane structures involved in hepatitis C virus genome replication revealed under close-to-native conditions by cryo-electron tomography Open Access

Abstract

Hepatitis C virus (HCV) infection induces extensive rearrangements of host cytoplasmic membranes, leading to the formation of multiple membranous structures that facilitate RNA replication. Current knowledge of these membranous structures has largely relied on correlative light and electron microscopy techniques using chemical fixation and resin embedding. To overcome these limitations, cryo-preserved cells were prepared using cryo-focused ion beam (cryo-FIB) milling and cryo-ultramicrotomy. For the first time, the contents within the membranous structures have been observed using cryo-electron tomography (cryo-ET) performed on lamellae (prepared via cryo-FIB) and on ultrathin sections (prepared via cryo-ultramicrotomy) from HCV subgenomic replicon-harbouring cells. Observations from 112 cryo-electron tomograms of cryo-FIB-derived samples revealed the presence of densities within the inner vesicles of a subset of single- and double-membrane vesicles, as well as within multi-vesicular bodies, which are consistent with the presence of the viral genome replication machinery. Notably, this study also presents the first direct visualization of densities within a multi-membrane vesicle observed by cryo-electron microscopy of vitreous sections. The cryo-ET methodologies established here lay the groundwork for future investigations into the architecture of the HCV replication complex, leveraging advanced computational tools for deeper structural and functional analyses.

  • Received:
  • Accepted:
  • Published Online:
Keyword(s): cryo-electron tomography (cryo-ET) , cryo-focused ion beam (cryo-FIB) milling , genome replication , hepatitis C virus (HCV) and membrane vesicles
Funding
This study was supported by the:
  • Medical Research Council (Award MR/S001026/1)
    • Principal Award Recipient: MarkHarris
  • Wellcome Trust (Award 222370/Z/21/Z)
    • Principal Award Recipient: UpasanaM Sykora
  • Wellcome Trust (Award 096670/Z/11/Z)
    • Principal Award Recipient: MarkHarris
© 2025 The Authors
  • This is an open-access article distributed under the terms of the Creative Commons Attribution License. This article was made open access via a Publish and Read agreement between the Microbiology Society and the corresponding author’s institution.
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2025-11-06
2025-12-15

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References

  1. Sykora UM, O’Sullivan TJ, Halfon Y, Fontana J, Harris M. The architecture of membrane structures involved in hepatitis c virus genome replication revealed in close-to-native conditions by cryo-electron tomography. Figshare 2025 [View Article]
     [Google Scholar]
  2. Martinello M, Solomon SS, Terrault NA, Dore GJ. Hepatitis C. Lancet 2023; 402:1085–1096 [View Article] [PubMed]
     [Google Scholar]
  3. Wolff G, Melia CE, Snijder EJ, Bárcena M. Double-membrane vesicles as platforms for viral replication. Trends Microbiol 2020; 28:1022–1033 [View Article] [PubMed]
     [Google Scholar]
  4. Ferraris P, Blanchard E, Roingeard P. Ultrastructural and biochemical analyses of hepatitis C virus-associated host cell membranes. J Gen Virol 2010; 91:2230–2237 [View Article] [PubMed]
     [Google Scholar]
  5. Lee J-Y, Cortese M, Haselmann U, Tabata K, Romero-Brey I et al. Spatiotemporal Coupling of the Hepatitis C Virus Replication Cycle by Creating a Lipid Droplet- Proximal Membranous Replication Compartment. Cell Rep 2019; 27:3602–3617 [View Article] [PubMed]
     [Google Scholar]
  6. Paul D, Madan V, Bartenschlager R. Hepatitis C virus RNA replication and assembly: living on the fat of the land. Cell Host & Microbe 2014; 16:569–579 [View Article]
     [Google Scholar]
  7. Romero-Brey I, Merz A, Chiramel A, Lee JY, Chlanda P et al. Three-dimensional architecture and biogenesis of membrane structures associated with hepatitis C virus replication. PLoS Pathog 2012; 8:e1003056 [View Article]
     [Google Scholar]
  8. Mohl B-P, Bartlett C, Mankouri J, Harris M. Early events in the generation of autophagosomes are required for the formation of membrane structures involved in hepatitis C virus genome replication. J Gen Virol 2016; 97:680–693 [View Article] [PubMed]
     [Google Scholar]
  9. Pérez-Berná AJ, Rodríguez MJ, Chichón FJ, Friesland MF, Sorrentino A et al. Structural changes in cells imaged by soft X-ray cryo-tomography during hepatitis C virus infection. ACS Nano 2016; 10:6597–6611 [View Article]
     [Google Scholar]
  10. Gosert R, Egger D, Lohmann V, Bartenschlager R, Blum HE et al. Identification of the hepatitis C virus RNA replication complex in Huh-7 cells harboring subgenomic replicons. J Virol 2003; 77:5487–5492 [View Article] [PubMed]
     [Google Scholar]
  11. Moradpour D, Gosert R, Egger D, Penin F, Blum HE et al. Membrane association of hepatitis C virus nonstructural proteins and identification of the membrane alteration that harbors the viral replication complex. Antiviral Res 2003; 60:103–109 [View Article] [PubMed]
     [Google Scholar]
  12. Lohmann V, Körner F, Koch J-O, Herian U, Theilmann L et al. Replication of subgenomic hepatitis C virus RNAs in a hepatoma cell line. Science 1999; 285:110–113 [View Article] [PubMed]
     [Google Scholar]
  13. Bartenschlager R, Lohmann V, Wilkinson T, Koch JO. Complex formation between the NS3 serine-type proteinase of the hepatitis C virus and NS4A and its importance for polyprotein maturation. J Virol 1995; 69:7519–7528 [View Article] [PubMed]
     [Google Scholar]
  14. Hughes M, Griffin S, Harris M. Domain III of NS5A contributes to both RNA replication and assembly of hepatitis C virus particles. J Gen Virol 2009; 90:1329–1334 [View Article] [PubMed]
     [Google Scholar]
  15. Shirota Y, Luo H, Qin W, Kaneko S, Yamashita T et al. Hepatitis C virus (HCV) NS5A binds RNA-dependent RNA polymerase (RdRP) NS5B and modulates RNA-dependent RNA polymerase activity. J Biol Chem 2002; 277:11149–11155 [View Article] [PubMed]
     [Google Scholar]
  16. Grünvogel O, Colasanti O, Lee J-Y, Klöss V, Belouzard S et al. Secretion of hepatitis C virus replication intermediates reduces activation of toll-like receptor 3 in hepatocytes. Gastroenterology 2018; 154:2237–2251 [View Article] [PubMed]
     [Google Scholar]
  17. Blanchard E, Roingeard P. The hepatitis C virus-induced membranous web in liver tissue. Cells 2018; 7:11 [View Article] [PubMed]
     [Google Scholar]
  18. Wakita T, Pietschmann T, Kato T, Date T, Miyamoto M et al. Production of infectious hepatitis C virus in tissue culture from a cloned viral genome. Nat Med 2005; 11:791–796 [View Article] [PubMed]
     [Google Scholar]
  19. Ross-Thriepland D, Mankouri J, Harris M. Serine phosphorylation of the hepatitis C virus NS5A protein controls the establishment of replication complexes. J Virol 2015; 89:3123–3135 [View Article] [PubMed]
     [Google Scholar]
  20. Wyles DL, Kaihara KA, Korba BE, Schooley RT, Beadle JR et al. The octadecyloxyethyl ester of (S)-9-[3-hydroxy-2-(phosphonomethoxy) propyl]adenine is a potent and selective inhibitor of hepatitis C virus replication in genotype 1A, 1B, and 2A replicons. Antimicrob Agents Chemother 2009; 53:2660–2662 [View Article] [PubMed]
     [Google Scholar]
  21. Moradpour D, Evans MJ, Gosert R, Yuan Z, Blum HE et al. Insertion of green fluorescent protein into nonstructural protein 5A allows direct visualization of functional hepatitis C virus replication complexes. J Virol 2004; 78:7400–7409 [View Article] [PubMed]
     [Google Scholar]
  22. Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M et al. Fiji: an open-source platform for biological-image analysis. Nat Methods 2012; 9:676–682 [View Article] [PubMed]
     [Google Scholar]
  23. Luft JH. Improvements in epoxy resin embedding methods. J Biophys Biochem Cytol 1961; 9:409–414 [View Article] [PubMed]
     [Google Scholar]
  24. Studer D, Klein A, Iacovache I, Gnaegi H, Zuber B. A new tool based on two micromanipulators facilitates the handling of ultrathin cryosection ribbons. J Struct Biol 2014; 185:125–128 [View Article] [PubMed]
     [Google Scholar]
  25. Schorb M, Briggs JAG. Correlated cryo-fluorescence and cryo-electron microscopy with high spatial precision and improved sensitivity. Ultramicroscopy 2014; 143:24–32 [View Article] [PubMed]
     [Google Scholar]
  26. Hagen WJH, Wan W, Briggs JAG. Implementation of a cryo-electron tomography tilt-scheme optimized for high resolution subtomogram averaging. J Struct Biol 2017; 197:191–198 [View Article] [PubMed]
     [Google Scholar]
  27. Zheng SQ, Palovcak E, Armache J-P, Verba KA, Cheng Y et al. MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat Methods 2017; 14:331–332 [View Article] [PubMed]
     [Google Scholar]
  28. Kremer JR, Mastronarde DN, McIntosh JR. Computer visualization of three-dimensional image data using IMOD. J Struct Biol 1996; 116:71–76 [View Article] [PubMed]
     [Google Scholar]
  29. Zheng S, Wolff G, Greenan G, Chen Z, Faas FGA et al. AreTomo: an integrated software package for automated marker-free, motion-corrected cryo-electron tomographic alignment and reconstruction. Journal of Structural Biology: X 2022; 6:100068 [View Article]
     [Google Scholar]
  30. Lamm L, Zufferey S, Zhang H, Righetto RD, Waltz F et al. MemBrain v2: an end-to-end tool for the analysis of membranes in cryo-electron tomography. Bioinformatics 2024 [View Article]
     [Google Scholar]
  31. Ferraris P, Beaumont E, Uzbekov R, Brand D, Gaillard J et al. Sequential biogenesis of host cell membrane rearrangements induced by hepatitis C virus infection. Cell Mol Life Sci 2013; 70:1297–1306 [View Article]
     [Google Scholar]
  32. Eyre NS, Fiches GN, Aloia AL, Helbig KJ, McCartney EM et al. Dynamic imaging of the hepatitis C virus NS5A protein during a productive infection. J Virol 2014; 88:3636–3652 [View Article] [PubMed]
     [Google Scholar]
  33. Romero-Brey I, Bartenschlager R. Viral infection at high magnification: 3D electron microscopy methods to analyze the architecture of infected cells. Viruses 2015; 7:6316–6345 [View Article] [PubMed]
     [Google Scholar]
  34. Berger C, Romero-Brey I, Radujkovic D, Terreux R, Zayas M et al. Daclatasvir-like inhibitors of NS5A block early biogenesis of hepatitis C virus-induced membranous replication factories, independent of RNA replication. Gastroenterology 2014; 147:1094–105 [View Article] [PubMed]
     [Google Scholar]
  35. Lam V, Villa E. Practical Approaches for Cryo-FIB Milling and Applications for Cellular Cryo-Electron Tomography BT - cryoEM: Methods and Protocols. In Gonen T, Nannenga BL. eds CryoEM Methods and Protocols New York, NY: Humana; 2021 pp 49–82 [View Article]
     [Google Scholar]
  36. Chlanda P, Sachse M. Cryo-electron microscopy of vitreous sections. Methods Mol Biol 2014; 1117:193–214 [View Article] [PubMed]
     [Google Scholar]
  37. Wagner FR, Watanabe R, Schampers R, Singh D, Persoon H et al. Preparing samples from whole cells using focused-ion-beam milling for cryo-electron tomography. Nat Protoc 2020; 15:2041–2070 [View Article] [PubMed]
     [Google Scholar]
  38. Bukong TN, Momen-Heravi F, Kodys K, Bala S, Szabo G. Exosomes from hepatitis C infected patients transmit HCV infection and contain replication competent viral RNA in complex with Ago2-miR122-HSP90. PLoS Pathog 2014; 10:e1004424 [View Article] [PubMed]
     [Google Scholar]
  39. Ramakrishnaiah V, Thumann C, Fofana I, Habersetzer F, Pan Q et al. Exosome-mediated transmission of hepatitis C virus between human hepatoma Huh7.5 cells. Proc Natl Acad Sci U S A 2013; 110:13109–13113 [View Article] [PubMed]
     [Google Scholar]
  40. Yin Y, Zhao Y, Chen Q, Chen Y, Mao L. Dual roles and potential applications of exosomes in HCV infections. Front Microbiol 2022; 13:1044832 [View Article] [PubMed]
     [Google Scholar]
  41. Matsui C, Yuliandari P, Deng L, Abe T, Shoji I. The role of chaperone-mediated autophagy in hepatitis C virus-induced pathogenesis. Front Cell Infect Microbiol 2021; 11:796664 [View Article] [PubMed]
     [Google Scholar]
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The architecture of membrane structures involved in hepatitis C virus genome replication revealed under close-to-native conditions by cryo-electron tomography
J. Gen. Virol. 106, 002168 (2025); https://doi.org/10.1099/jgv.0.002168
/content/journal/jgv/10.1099/jgv.0.002168
/content/journal/jgv/10.1099/jgv.0.002168
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