Skip to main content
SpringerLink
Log in

Deciphering Virus Entry with Fluorescently Labeled Viral Particles

  • Protocol
  • First Online:
Influenza Virus

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1836))

Abstract

To infect host cells, viruses have to gain access to the intracellular compartment. The infection process starts with the attachment of viruses to the cell surface. Then a complex series of events, highly dynamic, tightly intricate, and often hard to investigate, follows. This includes virus displacement at the plasma membrane, binding to receptors, signaling, internalization, and release of the viral genome and material into the cytosol. In the past decades, the emergence of sensitive, accurate fluorescence-based technologies has opened new perspectives of investigations in the field. Visualization of single viral particles in fixed and living cells as well as quantification of each virus entry step has been made possible. Here we describe the procedure to fluorescently label viral particles. We also illustrate how to use this powerful tool to decipher the entry of viruses with the most recent fluorescence-based techniques such as high-speed confocal and total internal reflection microscopy, flow cytometry, and fluorimetry.

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 189.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 249.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 249.99
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. White JM, Whittaker GR (2016) Fusion of enveloped viruses in endosomes. Traffic 17(6):593–614. https://doi.org/10.1111/tra.12389

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  2. Day PM, Schelhaas M (2014) Concepts of papillomavirus entry into host cells. Curr Opin Virol 4:24–31. https://doi.org/10.1016/j.coviro.2013.11.002

    Article  PubMed  CAS  Google Scholar 

  3. Melikyan GB (2014) HIV entry: a game of hide-and-fuse? Curr Opin Virol 4:1–7. https://doi.org/10.1016/j.coviro.2013.09.004

    Article  PubMed  CAS  Google Scholar 

  4. Lozach PY, Huotari J, Helenius A (2011) Late-penetrating viruses. Curr Opin Virol 1(1):35–43. https://doi.org/10.1016/j.coviro.2011.05.004

    Article  PubMed  CAS  Google Scholar 

  5. Leger P, Lozach PY (2015) Bunyaviruses: from transmission by arthropods to virus entry into the mammalian host first-target cells. Future Virol 10(7):859–881. https://doi.org/10.2217/Fvl.15.52

    Article  CAS  Google Scholar 

  6. Oker-Blom N, Salminen A, Brummer-Korvenkontio M, Kaeaeriaeinen L, Weckstroem P (1964) Isolation of some viruses other than typical tick-borne encephalitis viruses from Ixodes Ricinus ticks in Finland. Ann Med Exp Biol Fenn 42:109–112

    PubMed  CAS  Google Scholar 

  7. Major L, Linn ML, Slade RW, Schroder WA, Hyatt AD, Gardner J, Cowley J, Suhrbier A (2009) Ticks associated with macquarie island penguins carry arboviruses from four genera. PLoS One 4(2):e4375. https://doi.org/10.1371/journal.pone.0004375

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  8. Xu B, Liu L, Huang X, Ma H, Zhang Y, Du Y, Wang P, Tang X, Wang H, Kang K, Zhang S, Zhao G, Wu W, Yang Y, Chen H, Mu F, Chen W (2011) Metagenomic analysis of fever, thrombocytopenia and leukopenia syndrome (FTLS) in Henan Province, China: discovery of a new bunyavirus. PLoS Pathog 7(11):e1002369. https://doi.org/10.1371/journal.ppat.1002369

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  9. Rezelj VV, Overby AK, Elliott RM (2015) Generation of mutant Uukuniemi viruses lacking the nonstructural protein NSs by reverse genetics indicates that NSs is a weak interferon antagonist. J Virol 89(9):4849–4856. https://doi.org/10.1128/JVI.03511-14

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  10. McMullan LK, Folk SM, Kelly AJ, MacNeil A, Goldsmith CS, Metcalfe MG, Batten BC, Albarino CG, Zaki SR, Rollin PE, Nicholson WL, Nichol ST (2012) A new phlebovirus associated with severe febrile illness in Missouri. N Engl J Med 367(9):834–841. https://doi.org/10.1056/NEJMoa1203378

    Article  PubMed  CAS  Google Scholar 

  11. Yu XJ, Liang MF, Zhang SY, Liu Y, Li JD, Sun YL, Zhang L, Zhang QF, Popov VL, Li C, Qu J, Li Q, Zhang YP, Hai R, Wu W, Wang Q, Zhan FX, Wang XJ, Kan B, Wang SW, Wan KL, Jing HQ, Lu JX, Yin WW, Zhou H, Guan XH, Liu JF, Bi ZQ, Liu GH, Ren J, Wang H, Zhao Z, Song JD, He JR, Wan T, Zhang JS, Fu XP, Sun LN, Dong XP, Feng ZJ, Yang WZ, Hong T, Zhang Y, Walker DH, Wang Y, Li DX (2011) Fever with thrombocytopenia associated with a novel bunyavirus in China. N Engl J Med 364(16):1523–1532. https://doi.org/10.1056/NEJMoa1010095

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  12. Albornoz A, Hoffmann AB, Lozach PY, Tischler ND (2016) Early bunyavirus-host cell interactions. Viruses 8(5). https://doi.org/10.3390/v8050143

  13. Spiegel M, Plegge T, Pohlmann S (2016) The role of phlebovirus glycoproteins in viral entry, assembly and release. Viruses 8(7). https://doi.org/10.3390/v8070202

  14. Leger P, Tetard M, Youness B, Cordes N, Rouxel RN, Flamand M, Lozach PY (2016) Differential use of the C-type lectins L-SIGN and DC-SIGN for phlebovirus endocytosis. Traffic 17(6):639–656. https://doi.org/10.1111/tra.12393

    Article  PubMed  CAS  Google Scholar 

  15. Lozach PY, Kuhbacher A, Meier R, Mancini R, Bitto D, Bouloy M, Helenius A (2011) DC-SIGN as a receptor for phleboviruses. Cell Host Microbe 10(1):75–88. https://doi.org/10.1016/j.chom.2011.06.007

    Article  PubMed  CAS  Google Scholar 

  16. Lozach PY, Mancini R, Bitto D, Meier R, Oestereich L, Overby AK, Pettersson RF, Helenius A (2010) Entry of bunyaviruses into mammalian cells. Cell Host Microbe 7(6):488–499. https://doi.org/10.1016/j.chom.2010.05.007

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  17. Ronka H, Hilden P, Von Bonsdorff CH, Kuismanen E (1995) Homodimeric association of the spike glycoproteins G1 and G2 of Uukuniemi virus. Virology 211(1):241–250. https://doi.org/10.1006/viro.1995.1397

    Article  PubMed  CAS  Google Scholar 

  18. Bitto D, Halldorsson S, Caputo A, Huiskonen JT (2016) Low pH and anionic lipid-dependent fusion of Uukuniemi phlebovirus to liposomes. J Biol Chem 291(12):6412–6422. https://doi.org/10.1074/jbc.M115.691113

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  19. Sun E, He J, Zhuang X (2013) Live cell imaging of viral entry. Curr Opin Virol 3(1):34–43. https://doi.org/10.1016/j.coviro.2013.01.005

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  20. Whitt MA, Mire CE (2011) Utilization of fluorescently-labeled tetracysteine-tagged proteins to study virus entry by live cell microscopy. Methods 55(2):127–136. https://doi.org/10.1016/j.ymeth.2011.09.002

    Article  PubMed  CAS  Google Scholar 

  21. Sakin V, Paci G, Lemke EA, Muller B (2016) Labeling of virus components for advanced, quantitative imaging analyses. FEBS Lett 590(13):1896–1914. https://doi.org/10.1002/1873-3468.12131

    Article  PubMed  CAS  Google Scholar 

  22. Stolp B, Melican K (2016) Microbial pathogenesis revealed by intravital microscopy: pros, cons and cautions. FEBS Lett 590(13):2014–2026. https://doi.org/10.1002/1873-3468.12122

    Article  PubMed  CAS  Google Scholar 

  23. Ewers H, Schelhaas M (2012) Analysis of virus entry and cellular membrane dynamics by single particle tracking. Methods Enzymol 506:63–80. https://doi.org/10.1016/B978-0-12-391856-7.00028-7

    Article  PubMed  CAS  Google Scholar 

  24. Ruthardt N, Lamb DC, Brauchle C (2011) Single-particle tracking as a quantitative microscopy-based approach to unravel cell entry mechanisms of viruses and pharmaceutical nanoparticles. Mol Ther 19(7):1199–1211. https://doi.org/10.1038/mt.2011.102

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  25. Sivaraman D, Biswas P, Cella LN, Yates MV, Chen W (2011) Detecting RNA viruses in living mammalian cells by fluorescence microscopy. Trends Biotechnol 29(7):307–313. https://doi.org/10.1016/j.tibtech.2011.02.006

    Article  PubMed  CAS  Google Scholar 

  26. Ward EN, Pal R (2017) Image scanning microscopy: an overview. J Microsc. https://doi.org/10.1111/jmi.12534

  27. Combs CA, Shroff H (2017) Fluorescence microscopy: a concise guide to current imaging methods. Curr Protoc Neurosci 79:2.1.1–2.1.25. https://doi.org/10.1002/cpns.29

  28. Meier R, Franceschini A, Horvath P, Tetard M, Mancini R, von Mering C, Helenius A, Lozach PY (2014) Genome-wide small interfering RNA screens reveal VAMP3 as a novel host factor required for Uukuniemi virus late penetration. J Virol 88(15):8565–8578. https://doi.org/10.1128/JVI.00388-14

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  29. Snijder B, Sacher R, Ramo P, Liberali P, Mench K, Wolfrum N, Burleigh L, Scott CC, Verheije MH, Mercer J, Moese S, Heger T, Theusner K, Jurgeit A, Lamparter D, Balistreri G, Schelhaas M, De Haan CA, Marjomaki V, Hyypia T, Rottier PJ, Sodeik B, Marsh M, Gruenberg J, Amara A, Greber U, Helenius A, Pelkmans L (2012) Single-cell analysis of population context advances RNAi screening at multiple levels. Mol Syst Biol 8:579. https://doi.org/10.1038/msb.2012.9

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  30. Mercer J, Schelhaas M, Helenius A (2010) Virus entry by endocytosis. Annu Rev Biochem 79:803–833. https://doi.org/10.1146/annurev-biochem-060208-104626

    Article  PubMed  CAS  Google Scholar 

  31. Engel S, Heger T, Mancini R, Herzog F, Kartenbeck J, Hayer A, Helenius A (2011) Role of endosomes in simian virus 40 entry and infection. J Virol 85(9):4198–4211. https://doi.org/10.1128/JVI.02179-10

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  32. Johannsdottir HK, Mancini R, Kartenbeck J, Amato L, Helenius A (2009) Host cell factors and functions involved in vesicular stomatitis virus entry. J Virol 83(1):440–453. https://doi.org/10.1128/JVI.01864-08

    Article  PubMed  CAS  Google Scholar 

  33. Stauffer S, Feng Y, Nebioglu F, Heilig R, Picotti P, Helenius A (2014) Stepwise priming by acidic pH and a high K+ concentration is required for efficient uncoating of influenza A virus cores after penetration. J Virol 88(22):13029–13046. https://doi.org/10.1128/JVI.01430-14

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  34. Banerjee I, Miyake Y, Nobs SP, Schneider C, Horvath P, Kopf M, Matthias P, Helenius A, Yamauchi Y (2014) Influenza A virus uses the aggresome processing machinery for host cell entry. Science 346(6208):473–477. https://doi.org/10.1126/science.1257037

    Article  PubMed  CAS  Google Scholar 

  35. Schelhaas M, Shah B, Holzer M, Blattmann P, Kuhling L, Day PM, Schiller JT, Helenius A (2012) Entry of human papillomavirus type 16 by actin-dependent, clathrin- and lipid raft-independent endocytosis. PLoS Pathog 8(4):e1002657. https://doi.org/10.1371/journal.ppat.1002657

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  36. Pettersson R, Kaariainen L (1973) The ribonucleic acids of Uukuniemi virus, a noncubical tick-borne arbovirus. Virology 56(2):608–619

    Article  CAS  PubMed  Google Scholar 

  37. Mazelier M, Rouxel RN, Zumstein M, Mancini R, Bell-Sakyi L, Lozach PY (2016) Uukuniemi Virus as a tick-borne virus model. J Virol 90(15):6784–6798. https://doi.org/10.1128/JVI.00095-16

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  38. Schneider CA, Rasband WS, Eliceiri KW (2012) NIH image to ImageJ: 25 years of image analysis. Nat Methods 9(7):671–675

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  39. Sbalzarini IF, Koumoutsakos P (2005) Feature point tracking and trajectory analysis for video imaging in cell biology. J Struct Biol 151(2):182–195. https://doi.org/10.1016/j.jsb.2005.06.002

    Article  PubMed  CAS  Google Scholar 

  40. Boulant S, Stanifer M, Lozach PY (2015) Dynamics of virus-receptor interactions in virus binding, signaling, and endocytosis. Viruses 7(6):2794–2815. https://doi.org/10.3390/v7062747

    Article  PubMed  PubMed Central  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported by grants from CellNetworks Research Group funds and from DFG LO-2338/1-1.

Author information

Authors and Affiliations

  1. From CellNetworks Cluster of Excellence and Department of Infectious Diseases, Virology, University Hospital Heidelberg, Heidelberg, Germany

    Anja B. Hoffmann, Magalie Mazelier, Psylvia Léger & Pierre-Yves Lozach

Authors
  1. Anja B. Hoffmann
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Magalie Mazelier
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Psylvia Léger
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Pierre-Yves Lozach
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Pierre-Yves Lozach .

Editor information

Editors and Affiliations

  1. School of Cellular and Molecular Medicine, University of Bristol, Bristol, UK

    Yohei Yamauchi

Rights and permissions

Reprints and permissions

Copyright information

© 2018 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Hoffmann, A.B., Mazelier, M., Léger, P., Lozach, PY. (2018). Deciphering Virus Entry with Fluorescently Labeled Viral Particles. In: Yamauchi, Y. (eds) Influenza Virus. Methods in Molecular Biology, vol 1836. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8678-1_8

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-8678-1_8

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-8677-4

  • Online ISBN: 978-1-4939-8678-1

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation