Skip to main content
SpringerLink
Log in

Intracytoplasmic Transport of Hepatitis B Virus Capsids

  • Protocol
  • First Online:
Hepatitis B Virus

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1540))

Abstract

The early steps of HBV entry remain largely unknown despite the recent discovery of an HBV-specific entry receptor. Following entry HBV capsids have to be transported through the cytoplasm to the nuclear periphery, followed by nuclear entry. These steps have to take place in a coordinated manner to allow delivery of the genome into the nucleus. Due to the viscosity of the cytoplasm, the intracytoplasmic translocation has to be active and directed.

Here, we describe protocols that can be applied to investigations of the HBV capsid with the cytoplasmic transport systems. We have chosen to present two independent experimental approaches, which allow avoiding artifacts. Aside of the specific capsid detection system, the protocols can be applied to any other viral structure.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 109.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 139.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 159.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Köck J, Schlicht HJ, Kock J (1993) Analysis of the earliest steps of hepadnavirus replication: genome repair after infectious entry into hepatocytes does not depend on viral polymerase activity. J Virol 67:4867–4874

    PubMed  PubMed Central  Google Scholar 

  2. Wodrich H, Cassany A, D’Angelo MA et al (2006) Adenovirus core protein pVII is translocated into the nucleus by multiple import receptor pathways. J Virol 80:9608–9618

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Shahin V, Hafezi W, Oberleithner H et al (2006) The genome of HSV-1 translocates through the nuclear pore as a condensed rod-like structure. J Cell Sci 119:23–30

    Article  CAS  PubMed  Google Scholar 

  4. Arhel NJ, Souquere-Besse S, Munier S et al (2007) HIV-1 DNA Flap formation promotes uncoating of the pre-integration complex at the nuclear pore. EMBO J 26:3025–3037

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Yan H, Zhong G, Xu G et al (2012) Sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis B and D virus. eLife 1:1–28

    Article  Google Scholar 

  6. Cooper A, Shaul Y (2006) Clathrin-mediated endocytosis and lysosomal cleavage of hepatitis B virus capsid-like core particles. J Biol Chem 281(24):16563–16569

    Article  CAS  PubMed  Google Scholar 

  7. Macovei A, Radulescu C, Lazar C et al (2010) Hepatitis B virus requires intact caveolin-1 function for productive infection in HepaRG cells. J Virol 84:243–253

    Article  CAS  PubMed  Google Scholar 

  8. Panté N, Kann M (2002) Nuclear pore complex is able to transport macromolecules with diameters of about 39 nm. Mol Biol Cell 13:425–434

    Article  PubMed  PubMed Central  Google Scholar 

  9. Rabe B, Vlachou A, Panté N et al (2003) Nuclear import of hepatitis B virus capsids and release of the viral genome. Proc Natl Acad Sci U S A 100:9849–9854

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Schmitz A, Schwarz A, Foss M et al (2010) Nucleoporin 153 arrests the nuclear import of hepatitis B virus capsids in the nuclear basket. PLoS Pathog 6:e1000741

    Article  PubMed  PubMed Central  Google Scholar 

  11. Lepock JR, Cheng KH, Campbell SD, Kruvv A (1983) Rotational diffusion of TEMPONE in the cytoplasm of Chinese hamster lung cells. Biophys J 44:405–412

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Mastro A, Babich M, Taylor W, Keith A (1984) Diffusion of a small molecule in the cytoplasm of mammalian cells. Proc Natl Acad Sci U S A 81:3414–3418

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Dix JA, Verkman AS (2008) Crowding effects on diffusion in solutions and cells. Annu Rev Biophys 37:247–263

    Article  CAS  PubMed  Google Scholar 

  14. Luby-Phelps K, Taylor DL, Lanni F (1986) Probing the structure of cytoplasm. J Cell Biol 102:2015–2022

    Article  CAS  PubMed  Google Scholar 

  15. van Loo ND, Fortunati E, Ehlert E et al (2001) Baculovirus infection of nondividing mammalian cells: mechanisms of entry and nuclear transport of capsids. J Virol 75:961–970

    Article  PubMed  PubMed Central  Google Scholar 

  16. Ohkawa T, Volkman LE, Welch MD (2010) Actin-based motility drives baculovirus transit to the nucleus and cell surface. J Cell Biol 190:187–195

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. David R (2010) Cytoskeleton: baculoviruses “ride” actin. Nat Rev Mol Cell Biol 11:606

    Article  CAS  PubMed  Google Scholar 

  18. Tilney LG, DeRosier DJ, Tilney MS (1992) How Listeria exploits host cell actin to form its own cytoskeleton: I. Formation of a tail and how that tail might be involved in movement. J Cell Biol 118:71–81

    Article  CAS  PubMed  Google Scholar 

  19. Rabe B, Glebe D, Kann M (2006) Lipid-mediated introduction of hepatitis B virus capsids into nonsusceptible cells allows highly efficient replication and facilitates the study of early infection events. J Virol 80:5465–5473

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Nogales E, Wolf SG, Downing KH (1998) Structure of the alpha beta tubulin dimer by electron crystallography. Nature 391:199–203

    Article  CAS  PubMed  Google Scholar 

  21. Nogales E, Whittaker M, Milligan RA, Downing KH (1999) High-resolution model of the microtubule. Cell 96:79–88

    Article  CAS  PubMed  Google Scholar 

  22. Zhai Y, Kronebusch PJ, Simon PM, Borisy GG (1996) Microtubule dynamics at the G2/M transition: abrupt breakdown of cytoplasmic microtubules at nuclear envelope breakdown and implications for spindle morphogenesis. J Cell Biol 135:201–214

    Article  CAS  PubMed  Google Scholar 

  23. Giannakakou P, Sackett DL, Ward Y et al (2000) p53 is associated with cellular microtubules and is transported to the nucleus by dynein. Nat Cell Biol 2:709–717

    Article  CAS  PubMed  Google Scholar 

  24. Hirokawa N, Noda Y, Tanaka Y, Niwa S (2009) Kinesin superfamily motor proteins and intracellular transport. Nat Rev Mol Cell Biol 10:682–696

    Article  CAS  PubMed  Google Scholar 

  25. Hirokawa N, Takemura R (2005) Molecular motors and mechanisms of directional transport in neurons. Nat Rev Neurosci 6:201–214

    Article  CAS  PubMed  Google Scholar 

  26. Hirokawa N, Noda Y (2008) Intracellular transport and kinesin superfamily proteins, KIFs: structure, function, and dynamics. Physiol Rev 88:1089–1118

    Article  CAS  PubMed  Google Scholar 

  27. Mandelkow EM (1995) Microtubules and microtubule-associated proteins. Curr Opin Cell Biol 7:72–81

    Article  CAS  PubMed  Google Scholar 

  28. Holzbaur EL, Vallee RB (1994) DYNEINS: molecular structure and cellular function. Annu Rev Cell Biol 10:339–372

    Article  CAS  PubMed  Google Scholar 

  29. Vlach J, Lipov J, Rumlová M et al (2008) D-retrovirus morphogenetic switch driven by the targeting signal accessibility to Tctex-1 of dynein. Proc Natl Acad Sci U S A 105:10565–10570

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Merino-Gracia J, García-Mayoral MF, Rodríguez-Crespo I (2011) The association of viral proteins with host cell dynein components during virus infection. FEBS J 278:2997–3011

    Article  CAS  PubMed  Google Scholar 

  31. Rapali P, Szenes Á, Radnai L et al (2011) DYNLL/LC8: a light chain subunit of the dynein motor complex and beyond. FEBS J 278:2980–2996

    Article  CAS  PubMed  Google Scholar 

  32. Rapali P, Radnai L, Süveges D et al (2011) Directed evolution reveals the binding motif preference of the LC8/DYNLL hub protein and predicts large numbers of novel binders in the human proteome. PLoS One 6:e18818

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Wanschers B, van de Vorstenbosch R, Wijers M et al (2008) Rab6 family proteins interact with the dynein light chain protein DYNLRB1. Cell Motil Cytoskeleton 65:183–196

    Article  CAS  PubMed  Google Scholar 

  34. Tang Q (2002) A novel transforming growth factor-beta receptor-interacting protein that is also a light chain of the motor protein dynein. Mol Biol Cell 13:4484–4496

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Karki S, Holzbaur EL (1999) Cytoplasmic dynein and dynactin in cell division and intracellular transport. Curr Opin Cell Biol 11:45–53

    Article  CAS  PubMed  Google Scholar 

  36. Schroer TA (2004) Dynactin. Annu Rev Cell Dev Biol 20:759–779

    Article  CAS  PubMed  Google Scholar 

  37. Zlotnick A, Cheng N, Stahl SJ et al (1997) Localization of the C terminus of the assembly domain of hepatitis B virus capsid protein: implications for morphogenesis and organization of encapsidated RNA. Proc Natl Acad Sci U S A 94:9556–9561

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Kann M, Sodeik B, Vlachou A et al (1999) Phosphorylation-dependent binding of hepatitis B virus core particles to the nuclear pore complex. J Cell Biol 145:45–55

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Gerelsaikhan T, Tavis JE, Bruss V (1996) Hepatitis B virus nucleocapsid envelopment does not occur without genomic DNA synthesis. J Virol 70:4269–4274

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Kim J, Wu J (2014) A theoretical study of SRPK interaction with the flexible domains of hepatitis B capsids. Biophys J 107:1453–1461

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Miller RH, Tran CT, Robinson WS (1984) Hepatitis B virus particles of plasma and liver contain viral DNA-RNA hybrid molecules. Virology 139:53–63

    Article  CAS  PubMed  Google Scholar 

  42. Ning X, Nguyen D, Mentzer L et al (2011) Secretion of genome-free hepatitis B virus—single strand blocking model for virion morphogenesis of para-retrovirus. PLoS Pathog 7:e1002255

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Stray SJ, Bourne CR, Punna S et al (2005) A heteroaryldihydropyrimidine activates and can misdirect hepatitis B virus capsid assembly. Proc Natl Acad Sci U S A 102:8138–8143

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Wickstead B, Gull K (2012). Evolutionary Biology of Dyneins. Chapter 2, pp.88-121. In: Dyneins. Structure, Biology and Disease (1st edition). Stephen M. King (ed). Academic Press, London, Waltham, San Diego. doi:10.1016/B978-0-12-382004-4.10002-0

  45. Cohen S, Au S, Panté N (2009) Microinjection of Xenopus laevis oocytes. J Vis Exp (24):e1106

    Google Scholar 

  46. Rabe B, Delaleau M, Bischof A et al (2009) Nuclear entry of hepatitis B virus capsids involves disintegration to protein dimers followed by nuclear reassociation to capsids. PLoS Pathog 5:e1000563

    Article  PubMed  PubMed Central  Google Scholar 

  47. Ceres P, Zlotnick A (2002) Weak protein-protein interactions are sufficient to drive assembly of hepatitis B virus capsids. Biochemistry. 41(39):11525–11531

    Google Scholar 

Download references

Acknowledgments

This work was supported by a grant of the ANRS (French National Agency for Research on AIDS and Viral hepatitis) to Q.O., further support was obtained by a grant of “la region Aquitaine.”

Author information

Authors and Affiliations

  1. University of Bordeaux, Microbiologie Fondamentale et Pathogénicité, UMR 5234, Bordeaux, France

    Quentin Osseman & Michael Kann

  2. CNRS, Microbiologie Fondamentale et Pathogénicité, UMR 5234, Bordeaux, France

    Quentin Osseman & Michael Kann

  3. Centre Hospitalier Universitaire de Bordeaux, Service de Virologie, Bordeaux, France

    Michael Kann

Authors
  1. Quentin Osseman
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Michael Kann
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Michael Kann .

Editor information

Editors and Affiliations

  1. Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, Indiana, USA

    Haitao Guo

  2. Arbutus Biopharma, Inc., Doylestown, Pennsylvania, USA

    Andrea Cuconati

Rights and permissions

Reprints and permissions

Copyright information

© 2017 Springer Science+Business Media LLC

About this protocol

Cite this protocol

Osseman, Q., Kann, M. (2017). Intracytoplasmic Transport of Hepatitis B Virus Capsids. In: Guo, H., Cuconati, A. (eds) Hepatitis B Virus. Methods in Molecular Biology, vol 1540. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6700-1_4

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-6700-1_4

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6698-1

  • Online ISBN: 978-1-4939-6700-1

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation