Skip to main content
SpringerLink
Log in
Book cover

Hemorrhagic Fever Viruses pp 209–215Cite as

Hemorrhagic Fever Virus Budding Studies

  • Protocol
  • First Online:

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1604))

Abstract

Independent expression of the VP40 or Z matrix proteins of filoviruses (marburgviruses and ebolaviruses) and arenaviruses (Lassa fever and Junín), respectively, gives rise to the production and release of virus-like particles (VLPs) that are morphologically identical to infectious virions. We can detect and quantify VLP production and egress in mammalian cells by transient transfection, SDS-PAGE, Western blotting, and live cell imaging techniques such as total internal reflection fluorescence (TIRF) microscopy. Since the VLP budding assay accurately mimics budding of infectious virus, this BSL-2 assay is safe and useful for the interrogation of both viral and host determinants required for budding and can be used as an initial screen to identify and validate small molecule inhibitors of virus release and spread.

This is a preview of subscription content, 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   99.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD   129.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD   179.99
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

Learn about institutional subscriptions

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Ascenzi P, Bocedi A, Heptonstall J, Capobianchi MR, Di Caro A, Mastrangelo E, Bolognesi M, Ippolito G (2008) Ebolavirus and Marburgvirus: insight the Filoviridae family. Mol Aspects Med 29:151–185. doi:10.1016/j.mam.2007.09.005

    Article  CAS  PubMed  Google Scholar 

  2. Bray M, Murphy FA (2007) Filovirus research: knowledge expands to meet a growing threat. J Infect Dis 196(Suppl 2):S438–S443. doi:10.1086/520552

    Article  PubMed  Google Scholar 

  3. Casillas AM, Nyamathi AM, Sosa A, Wilder CL, Sands H (2003) A current review of Ebola virus: pathogenesis, clinical presentation, and diagnostic assessment. Biol Res Nurs 4:268–275

    Article  PubMed  Google Scholar 

  4. Feldmann H, Klenk HD, Sanchez A (1993) Molecular biology and evolution of filoviruses. Arch Virol Suppl 7:81–100

    Article  CAS  PubMed  Google Scholar 

  5. Peters CJ, Khan AS (1999) Filovirus diseases. Curr Top Microbiol Immunol 235:85–95

    CAS  PubMed  Google Scholar 

  6. Bausch DG, Sprecher AG, Jeffs B, Boumandouki P (2008) Treatment of Marburg and Ebola hemorrhagic fevers: a strategy for testing new drugs and vaccines under outbreak conditions. Antiviral Res 78:150–161. doi:10.1016/j.antiviral.2008.01.152

    Article  CAS  PubMed  Google Scholar 

  7. Bray M, Paragas J (2002) Experimental therapy of filovirus infections. Antiviral Res 54:1–17

    Article  CAS  PubMed  Google Scholar 

  8. Adalja AA, Henderson DA (2014) Optimization of interventions in Ebola: differential contagion. Biosecur Bioterror 12:299–300. doi:10.1089/bsp.2014.0925

    Article  PubMed  PubMed Central  Google Scholar 

  9. Marzi A, Feldmann H, Geisbert TW, Falzarano D (2011) Vesicular stomatitis virus-based vaccines for prophylaxis and treatment of filovirus infections. J Bioterror Biodef S1(4):2157–2526-S1-004. doi:10.4172/2157-2526.S1-004

  10. Kuhn JH, Dodd LE, Wahl-Jensen V, Radoshitzky SR, Bavari S, Jahrling PB (2011) Evaluation of perceived threat differences posed by filovirus variants. Biosecur Bioterror 9:361–371. doi:10.1089/bsp.2011.0051

    Article  PubMed  PubMed Central  Google Scholar 

  11. Okumura A, Rasmussen AL, Halfmann P, Feldmann F, Yoshimura A, Feldmann H, Kawaoka Y, Harty RN, Katze MG (2015) Suppressor of cytokine signaling 3 is an inducible host factor that regulates virus egress during Ebola virus infection. J Virol 89:10399–10406. doi:10.1128/JVI.01736-15

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Han Z, Madara JJ, Liu Y, Liu W, Ruthel G, Freedman BD, Harty RN (2015) ALIX rescues budding of a double PTAP/PPEY L-domain deletion mutant of Ebola VP40: a role for ALIX in Ebola virus egress. J Infect Dis 212(Suppl 2):S138–S145. doi:10.1093/infdis/jiu838

    Article  PubMed  PubMed Central  Google Scholar 

  13. Han Z, Madara JJ, Herbert A, Prugar LI, Ruthel G, Lu J, Liu Y, Liu W, Liu X, Wrobel JE, Reitz AB, Dye JM, Harty RN, Freedman BD (2015) Calcium regulation of hemorrhagic fever virus budding: mechanistic implications for host-oriented therapeutic intervention. PLoS Pathog 11:e1005220. doi:10.1371/journal.ppat.1005220

    Article  PubMed  PubMed Central  Google Scholar 

  14. Stahelin RV (2014) Membrane binding and bending in Ebola VP40 assembly and egress. Front Microbiol 5:300. doi:10.3389/fmicb.2014.00300

    PubMed  PubMed Central  Google Scholar 

  15. Lu J, Han Z, Liu Y, Liu W, Lee MS, Olson MA, Ruthel G, Freedman BD, Harty RN (2014) A host-oriented inhibitor of Junin Argentine hemorrhagic fever virus egress. J Virol 88:4736–4743. doi:10.1128/JVI.03757-13

    Article  PubMed  PubMed Central  Google Scholar 

  16. Han Z, Lu J, Liu Y, Davis B, Lee MS, Olson MA, Ruthel G, Freedman BD, Schnell MJ, Wrobel JE, Reitz AB, Harty RN (2014) Small-molecule probes targeting the viral PPxY-Host Nedd4 interface block egress of a broad range of RNA viruses. J Virol 88:7294–7306. doi:10.1128/JVI.00591-14

    Article  PubMed  PubMed Central  Google Scholar 

  17. Lu J, Qu Y, Liu Y, Jambusaria R, Han Z, Ruthel G, Freedman BD, Harty RN (2013) Host IQGAP1 and Ebola virus VP40 interactions facilitate virus-like particle egress. J Virol 87:7777–7780. doi:10.1128/JVI.00470-13

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Warfield KL, Aman MJ (2011) Advances in virus-like particle vaccines for filoviruses. J Infect Dis 204(Suppl 3):S1053–S1059. doi:10.1093/infdis/jir346

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Makino A, Yamayoshi S, Shinya K, Noda T, Kawaoka Y (2011) Identification of amino acids in Marburg virus VP40 that are important for virus-like particle budding. J Infect Dis 204(Suppl 3):S871–S877. doi:10.1093/infdis/jir309

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Liu Y, Lee MS (2011) Olson MA and Harty RN (2011) bimolecular complementation to visualize filovirus VP40-host complexes in live mammalian cells: toward the identification of budding inhibitors. Adv Virol 2011:341816. doi:10.1155/2011/341816

    Article  PubMed  PubMed Central  Google Scholar 

  21. Liu Y, Cocka L, Okumura A, Zhang YA, Sunyer JO, Harty RN (2010) Conserved motifs within Ebola and Marburg virus VP40 proteins are important for stability, localization, and subsequent budding of virus-like particles. J Virol 84:2294–2303. doi:10.1128/JVI.02034-09

    Article  CAS  PubMed  Google Scholar 

  22. Groseth A, Wolff S, Strecker T, Hoenen T, Becker S (2010) Efficient budding of the tacaribe virus matrix protein z requires the nucleoprotein. J Virol 84:3603–3611. doi:10.1128/JVI.02429-09

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Dolnik O, Kolesnikova L, Stevermann L, Becker S (2010) Tsg101 is recruited by a late domain of the nucleocapsid protein to support budding of Marburg virus-like particles. J Virol 84:7847–7856. doi:10.1128/JVI.00476-10

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Okumura A, Pitha PM, Harty RN (2008) ISG15 inhibits Ebola VP40 VLP budding in an L-domain-dependent manner by blocking Nedd4 ligase activity. Proc Natl Acad Sci U S A 105:3974–3979. doi:10.1073/pnas.0710629105

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Yamayoshi S, Kawaoka Y (2007) Mapping of a region of Ebola virus VP40 that is important in the production of virus-like particles. J Infect Dis 196(Suppl 2):S291–S295. doi:10.1086/520595

    Article  CAS  PubMed  Google Scholar 

  26. McCarthy SE, Johnson RF, Zhang YA, Sunyer JO, Harty RN (2007) Role for amino acids 212KLR214 of Ebola virus VP40 in assembly and budding. J Virol 81:11452–11460. doi:10.1128/JVI.00853-07

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Han Z, Harty RN (2007) Influence of calcium/calmodulin on budding of Ebola VLPs: implications for the involvement of the Ras/Raf/MEK/ERK pathway. Virus Genes 35:511–520. doi:10.1007/s11262-007-0125-9

    Article  CAS  PubMed  Google Scholar 

  28. Urata S, Noda T, Kawaoka Y, Yokosawa H, Yasuda J (2006) Cellular factors required for Lassa virus budding. J Virol 80:4191–4195. doi:10.1128/JVI.80.8.4191-4195.2006

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Johnson RF, Bell P, Harty RN (2006) Effect of Ebola virus proteins GP, NP and VP35 on VP40 VLP morphology. Virol J 3:31. doi:10.1186/1743-422X-3-31

    Article  PubMed  PubMed Central  Google Scholar 

  30. Kallstrom G, Warfield KL, Swenson DL, Mort S, Panchal RG, Ruthel G, Bavari S, Aman MJ (2005) Analysis of Ebola virus and VLP release using an immunocapture assay. J Virol Methods 127:1–9. doi:10.1016/j.jviromet.2005.02.015

    Article  CAS  PubMed  Google Scholar 

  31. Han Z, Harty RN (2005) Packaging of actin into Ebola virus VLPs. Virol J 2:92. doi:10.1186/1743-422X-2-92

    Article  PubMed  PubMed Central  Google Scholar 

  32. Swenson DL, Warfield KL, Kuehl K, Larsen T, Hevey MC, Schmaljohn A, Bavari S, Aman MJ (2004) Generation of Marburg virus-like particles by co-expression of glycoprotein and matrix protein. FEMS Immunol Med Microbiol 40:27–31

    Article  CAS  PubMed  Google Scholar 

  33. Yasuda J, Nakao M, Kawaoka Y, Shida H (2003) Nedd4 regulates egress of Ebola virus-like particles from host cells. J Virol 77:9987–9992

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Licata JM, Simpson-Holley M, Wright NT, Han Z, Paragas J, Harty RN (2003) Overlapping motifs (PTAP and PPEY) within the Ebola virus VP40 protein function independently as late budding domains: involvement of host proteins TSG101 and VPS-4. J Virol 77:1812–1819

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Harty RN, Brown ME, Wang G, Huibregtse J, Hayes FP (2000) A PPxY motif within the VP40 protein of Ebola virus interacts physically and functionally with a ubiquitin ligase: implications for filovirus budding. Proc Natl Acad Sci U S A 97:13871–13876. doi:10.1073/pnas.250277297

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Johnson KA, Taghon GJ, Scott JL, Stahelin RV (2016) The Ebola Virus matrix protein, VP40, requires phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) for extensive oligomerization at the plasma membrane and viral egress. Sci Rep 6:19125. doi:10.1038/srep19125

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgments

I would like to thank past and present members of the lab who contributed to this work. This work was supported in part by NIH grants to R.N.H.

Author information

Authors and Affiliations

  1. Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, 3800 Spruce St., Philadelphia, PA, 19104, USA

    Ronald N. Harty

Authors
  1. Ronald N. Harty
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Ronald N. Harty .

Editor information

Editors and Affiliations

  1. School of Medicine, University of Maryland, Baltimore, Maryland, USA

    Maria S. Salvato

Rights and permissions

Reprints and permissions

Copyright information

© 2018 Springer Science+Business Media LLC

About this protocol

Cite this protocol

Harty, R.N. (2018). Hemorrhagic Fever Virus Budding Studies. In: Salvato, M. (eds) Hemorrhagic Fever Viruses. Methods in Molecular Biology, vol 1604. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6981-4_15

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-6981-4_15

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6980-7

  • Online ISBN: 978-1-4939-6981-4

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation