Skip to main content
SpringerLink
Log in

Protocol for Detection of HIV-Tat Protein in Cerebrospinal Fluid by a Sandwich Enzyme-Linked Immunosorbent Assay

  • Protocol
HIV Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1354))

Abstract

The human immunodeficiency virus (HIV) transactivator of transcription (Tat) is a virally produced protein that is required for efficient viral replication. Once formed inside an infected cell, Tat is secreted into the extracellular space where it has pathophysiological consequences on cells it interacts with. Tat has been demonstrated to be neurotoxic and is produced even under the pressures of anti-retroviral therapy; therefore Tat is suspected to contribute to the development of HIV-associated neurocognitive disorders. In this chapter, we describe a sandwich enzyme-linked immunosorbent assay protocol for the detection of Tat from cerebrospinal fluid samples.

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 84.99
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 109.99
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. Fields BN, Knipe DM, Howley PM, Griffin DE (2001) Fields’ virology, 4th edn. Lippincott Williams & Wilkins, Philadelphia, PA

    Google Scholar 

  2. Rayne F, Debaisieux S, Yezid H, Lin YL, Mettling C, Konate K, Chazal N, Arold ST, Pugniere M, Sanchez F, Bonhoure A, Briant L, Loret E, Roy C, Beaumelle B (2010) Phosphatidylinositol-(4,5)-bisphosphate enables efficient secretion of HIV-1 Tat by infected T-cells. EMBO J 29:1348–1362

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Li W, Huang Y, Reid R, Steiner J, Malpica-Llanos T, Darden TA, Shankar SK, Mahadevan A, Satishchandra P, Nath A (2008) NMDA receptor activation by HIV-Tat protein is clade dependent. J Neurosci 28:12190–12198

    Article  CAS  PubMed  Google Scholar 

  4. Vendeville A, Rayne F, Bonhoure A, Bettache N, Montcourrier P, Beaumelle B (2004) HIV-1 Tat enters T cells using coated pits before translocating from acidified endosomes and eliciting biological responses. Mol Biol Cell 15:2347–2360

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Ensoli B, Barillari G, Salahuddin SZ, Gallo RC, Wong-Staal F (1990) Tat protein of HIV-1 stimulates growth of cells derived from Kaposi’s sarcoma lesions of AIDS patients. Nature 345:84–86

    Article  CAS  PubMed  Google Scholar 

  6. Rayne F, Debaisieux S, Bonhoure A, Beaumelle B (2010) HIV-1 Tat is unconventionally secreted through the plasma membrane. Cell Biol Int 34:409–413

    Article  CAS  PubMed  Google Scholar 

  7. Johnson TP, Patel K, Johnson KR, Maric D, Calabresi PA, Hasbun R, Nath A (2013) Induction of IL-17 and nonclassical T-cell activation by HIV-Tat protein. Proc Natl Acad Sci U S A 110:13588–13593

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Xiao H, Neuveut C, Tiffany HL, Benkirane M, Rich EA, Murphy PM, Jeang KT (2000) Selective CXCR4 antagonism by Tat: implications for in vivo expansion of coreceptor use by HIV-1. Proc Natl Acad Sci U S A 97:11466–11471

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Frankel AD, Pabo CO (1988) Cellular uptake of the tat protein from human immunodeficiency virus. Cell 55:1189–1193

    Article  CAS  PubMed  Google Scholar 

  10. Ferrari A, Pellegrini V, Arcangeli C, Fittipaldi A, Giacca M, Beltram F (2003) Caveolae-mediated internalization of extracellular HIV-1 tat fusion proteins visualized in real time. Mol Ther 8:284–294

    Article  CAS  PubMed  Google Scholar 

  11. Fittipaldi A, Ferrari A, Zoppe M, Arcangeli C, Pellegrini V, Beltram F, Giacca M (2003) Cell membrane lipid rafts mediate caveolar endocytosis of HIV-1 Tat fusion proteins. J Biol Chem 278:34141–34149

    Article  CAS  PubMed  Google Scholar 

  12. Liu Y, Jones M, Hingtgen CM, Bu G, Laribee N, Tanzi RE, Moir RD, Nath A, He JJ (2000) Uptake of HIV-1 tat protein mediated by low-density lipoprotein receptor-related protein disrupts the neuronal metabolic balance of the receptor ligands. Nat Med 6:1380–1387

    Article  CAS  PubMed  Google Scholar 

  13. Tyagi M, Rusnati M, Presta M, Giacca M (2001) Internalization of HIV-1 tat requires cell surface heparan sulfate proteoglycans. J Biol Chem 276:3254–3261

    Article  CAS  PubMed  Google Scholar 

  14. Eguchi A, Akuta T, Okuyama H, Senda T, Yokoi H, Inokuchi H, Fujita S, Hayakawa T, Takeda K, Hasegawa M, Nakanishi M (2001) Protein transduction domain of HIV-1 Tat protein promotes efficient delivery of DNA into mammalian cells. J Biol Chem 276:26204–26210

    Article  CAS  PubMed  Google Scholar 

  15. Wadia JS, Dowdy SF (2002) Protein transduction technology. Curr Opin Biotechnol 13:52–56

    Article  CAS  PubMed  Google Scholar 

  16. Lewin M, Carlesso N, Tung CH, Tang XW, Cory D, Scadden DT, Weissleder R (2000) Tat peptide-derivatized magnetic nanoparticles allow in vivo tracking and recovery of progenitor cells. Nat Biotechnol 18:410–414

    Article  CAS  PubMed  Google Scholar 

  17. Abbas W, Herbein G (2013) T-Cell Signaling in HIV-1 Infection. Open Virol J 7:57–71

    Article  PubMed  PubMed Central  Google Scholar 

  18. Nicoli F, Finessi V, Sicurella M, Rizzotto L, Gallerani E, Destro F, Cafaro A, Marconi P, Caputo A, Ensoli B, Gavioli R (2013) The HIV-1 Tat protein induces the activation of CD8(+) T cells and affects in vivo the magnitude and kinetics of antiviral responses. PLoS One 8:e77746

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Secchiero P, Zella D, Curreli S, Mirandola P, Capitani S, Gallo RC, Zauli G (2000) Pivotal role of cyclic nucleoside phosphodiesterase 4 in Tat-mediated CD4+ T cell hyperactivation and HIV type 1 replication. Proc Natl Acad Sci U S A 97:14620–14625

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Eugenin EA, King JE, Nath A, Calderon TM, Zukin RS, Bennett MV, Berman JW (2007) HIV-tat induces formation of an LRP-PSD-95-NMDAR-nNOS complex that promotes apoptosis in neurons and astrocytes. Proc Natl Acad Sci U S A 104:3438–3443. doi:10.1073/pnas.0611699104

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Hargus NJ, Thayer SA (2013) Human immunodeficiency virus-1 Tat protein increases the number of inhibitory synapses between hippocampal neurons in culture. J Neurosci 33:17908–17920

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Magnuson DS, Knudsen BE, Geiger JD, Brownstone RM, Nath A (1995) Human immunodeficiency virus type 1 tat activates non-N-methyl-D-aspartate excitatory amino acid receptors and causes neurotoxicity. Ann Neurol 37:373–380

    Article  CAS  PubMed  Google Scholar 

  23. Hahn YK, Podhaizer EM, Farris SP, Miles MF, Hauser KF, Knapp PE (2015) Effects of chronic HIV-1 Tat exposure in the CNS: heightened vulnerability of males versus females to changes in cell numbers, synaptic integrity, and behavior. Brain Struct Funct 220:605

    Article  PubMed  PubMed Central  Google Scholar 

  24. Paris JJ, Singh HD, Ganno ML, Jackson P, McLaughlin JP (2014) Anxiety-like behavior of mice produced by conditional central expression of the HIV-1 regulatory protein, Tat. Psychopharmacology 231:2349–2360

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Hudson L, Liu J, Nath A, Jones M, Raghavan R, Narayan O, Male D, Everall I (2000) Detection of the human immunodeficiency virus regulatory protein tat in CNS tissues. J Neurovirol 6:145–155

    Article  CAS  PubMed  Google Scholar 

  26. Bachani M, Sacktor N, McArthur JC, Nath A, Rumbaugh J (2013) Detection of anti-tat antibodies in CSF of individuals with HIV-associated neurocognitive disorders. J Neurovirol 19:82–88

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Mediouni S, Darque A, Baillat G, Ravaux I, Dhiver C, Tissot-Dupont H, Mokhtari M, Moreau H, Tamalet C, Brunet C, Paul P, Dignat-George F, Stein A, Brouqui P, Spector SA, Campbell GR, Loret EP (2012) Antiretroviral therapy does not block the secretion of the human immunodeficiency virus tat protein. Infect Disord Drug Targets 12:81–86

    Article  CAS  PubMed  Google Scholar 

  28. Westendorp MO, Frank R, Ochsenbauer C, Stricker K, Dhein J, Walczak H, Debatin KM, Krammer PH (1995) Sensitization of T cells to CD95-mediated apoptosis by HIV-1 Tat and gp120. Nature 375:497–500

    Article  CAS  PubMed  Google Scholar 

  29. Valvatne H, Szilvay AM, Helland DE (1996) A monoclonal antibody defines a novel HIV type 1 Tat domain involved in trans-cellular trans-activation. AIDS Res Hum Retroviruses 12:611–619

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Section of Infections of the Nervous System, National Institute of Neurological Diseases and Stroke, National Institutes of Health, Room 7C-103, Bldg 10; 10 Center Drive, Bethesda, MD, 20892, USA

    Tory P. Johnson Ph.D. & Avindra Nath

Authors
  1. Tory P. Johnson Ph.D.
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Avindra Nath
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Tory P. Johnson Ph.D. .

Editor information

Editors and Affiliations

  1. Department of Microbiology & Immunology, Albert Einstein College of Medicine, BRONX, New York, USA

    Vinayaka R. Prasad

  2. Department of Genetics, Albert Einstein College of Medicine, BRONX, New York, USA

    Ganjam V. Kalpana

Rights and permissions

Reprints and permissions

Copyright information

© 2016 Springer Science+Business Media New York

About this protocol

Cite this protocol

Johnson, T.P., Nath, A. (2016). Protocol for Detection of HIV-Tat Protein in Cerebrospinal Fluid by a Sandwich Enzyme-Linked Immunosorbent Assay. In: Prasad, V., Kalpana, G. (eds) HIV Protocols. Methods in Molecular Biology, vol 1354. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3046-3_23

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-3046-3_23

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-3045-6

  • Online ISBN: 978-1-4939-3046-3

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation