Skip to main content
SpringerLink
Log in

TILDA: Tat/Rev Induced Limiting Dilution Assay

  • Protocol
  • First Online:
HIV Reservoirs

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2407))

Abstract

Recently the Tat/rev Induced Limiting Dilution Assay, or TILDA, has been proposed as a possible alternative method to quantify the HIV-1 reservoir. TILDA estimates the frequency of latently infected cells by probing, in a limiting dilution format, the presence or inducibility of tat and rev multiply spliced HIV-1 RNA. In doing so, TILDA reduces overestimation of reservoir size compared to HIV-1 DNA measurements because multiply spliced HIV-1 RNA is less likely to be transcribed from dysfunctional genomes with replication defects. TILDA is easy to perform, requires a very low input number of cells and has a fast turnaround time, making it ideal for use in clinical settings. Here we describe the execution of TILDA with particular emphasis on cell preparation and the limiting dilution scheme.

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 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 199.99
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. Falcinelli SD, Ceriani C, Margolis DM, Archin NM (2019) New frontiers in measuring and characterizing the HIV reservoir. Front Microbiol 10:2878. https://doi.org/10.3389/fmicb.2019.02878

    Article  PubMed  PubMed Central  Google Scholar 

  2. Laird GM, Rosenbloom DI, Lai J, Siliciano RF, Siliciano JD (2016) Measuring the frequency of latent HIV-1 in resting CD4(+) T cells using a limiting dilution coculture assay. Methods Mol Biol 1354:239–253. https://doi.org/10.1007/978-1-4939-3046-3_16

    Article  CAS  PubMed  Google Scholar 

  3. Siliciano JD, Siliciano RF (2005) Enhanced culture assay for detection and quantitation of latently infected, resting CD4+ T-cells carrying replication-competent virus in HIV-1-infected individuals. Methods Mol Biol 304:3–15. https://doi.org/10.1385/1-59259-907-9:003

    Article  PubMed  Google Scholar 

  4. Eriksson S, Graf EH, Dahl V, Strain MC, Yukl SA, Lysenko ES, Bosch RJ, Lai J, Chioma S, Emad F, Abdel-Mohsen M, Hoh R, Hecht F, Hunt P, Somsouk M, Wong J, Johnston R, Siliciano RF, Richman DD, O’Doherty U, Palmer S, Deeks SG, Siliciano JD (2013) Comparative analysis of measures of viral reservoirs in HIV-1 eradication studies. PLoS Pathog 9(2):e1003174. https://doi.org/10.1371/journal.ppat.1003174

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Ho YC, Shan L, Hosmane NN, Wang J, Laskey SB, Rosenbloom DI, Lai J, Blankson JN, Siliciano JD, Siliciano RF (2013) Replication-competent noninduced proviruses in the latent reservoir increase barrier to HIV-1 cure. Cell 155(3):540–551. https://doi.org/10.1016/j.cell.2013.09.020

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Rosenbloom DIS, Bacchetti P, Stone M, Deng X, Bosch RJ, Richman DD, Siliciano JD, Mellors JW, Deeks SG, Ptak RG, Hoh R, Keating SM, Dimapasoc M, Massanella M, Lai J, Sobolewski MD, Kulpa DA, Busch MP, Reservoir Assay V, Evaluation Network Study G (2019) Assessing intra-lab precision and inter-lab repeatability of outgrowth assays of HIV-1 latent reservoir size. PLoS Comput Biol 15(4):e1006849

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Alidjinou EK, Bocket L, Hober D (2015) Quantification of viral DNA during HIV-1 infection: a review of relevant clinical uses and laboratory methods. Pathol Biol (Paris) 63(1):53–59. https://doi.org/10.1016/j.patbio.2014.07.007

    Article  CAS  Google Scholar 

  8. Avettand-Fenoel V, Hocqueloux L, Ghosn J, Cheret A, Frange P, Melard A, Viard JP, Rouzioux C (2016) Total HIV-1 DNA, a marker of viral Reservoir dynamics with clinical implications. Clin Microbiol Rev 29(4):859–880. https://doi.org/10.1128/CMR.00015-16

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Anderson EM, Maldarelli F (2018) Quantification of HIV DNA using droplet digital PCR techniques. Curr Protoc Microbiol 51(1):e62. https://doi.org/10.1002/cpmc.62

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Vandergeeten C, Fromentin R, Merlini E, Lawani MB, DaFonseca S, Bakeman W, McNulty A, Ramgopal M, Michael N, Kim JH, Ananworanich J, Chomont N (2014) Cross-clade ultrasensitive PCR-based assays to measure HIV persistence in large-cohort studies. J Virol 88(21):12385–12396. https://doi.org/10.1128/jvi.00609-14

    Article  PubMed  PubMed Central  Google Scholar 

  11. Pasternak AO, Lukashov VV, Berkhout B (2013) Cell-associated HIV RNA: a dynamic biomarker of viral persistence. Retrovirology 10:41. https://doi.org/10.1186/1742-4690-10-41

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Pace MJ, Graf EH, Agosto LM, Mexas AM, Male F, Brady T, Bushman FD, O’Doherty U (2012) Directly infected resting CD4+T cells can produce HIV gag without spreading infection in a model of HIV latency. PLoS Pathog 8(7):e1002818. https://doi.org/10.1371/journal.ppat.1002818

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Pasternak AO, DeMaster LK, Kootstra NA, Reiss P, O’Doherty U, Berkhout B (2016) Minor contribution of chimeric host-HIV readthrough transcripts to the level of HIV cell-associated gag RNA. J Virol 90(2):1148–1151. https://doi.org/10.1128/jvi.02597-15

    Article  CAS  PubMed  Google Scholar 

  14. Imamichi H, Dewar RL, Adelsberger JW, Rehm CA, O’Doherty U, Paxinos EE, Fauci AS, Lane HC (2016) Defective HIV-1 proviruses produce novel protein-coding RNA species in HIV-infected patients on combination antiretroviral therapy. Proc Natl Acad Sci U S A 113(31):8783–8788. https://doi.org/10.1073/pnas.1609057113

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Procopio FA, Fromentin R, Kulpa DA, Brehm JH, Bebin AG, Strain MC, Richman DD, O’Doherty U, Palmer S, Hecht FM, Hoh R, Barnard RJ, Miller MD, Hazuda DJ, Deeks SG, Sekaly RP, Chomont N (2015) A novel assay to measure the magnitude of the inducible viral reservoir in HIV-infected individuals. EBioMedicine 2(8):874–883. https://doi.org/10.1016/j.ebiom.2015.06.019

    Article  PubMed  PubMed Central  Google Scholar 

  16. Fisher AG, Feinberg MB, Josephs SF, Harper ME, Marselle LM, Reyes G, Gonda MA, Aldovini A, Debouk C, Gallo RC et al (1986) The trans-activator gene of HTLV-III is essential for virus replication. Nature 320(6060):367–371. https://doi.org/10.1038/320367a0

    Article  CAS  PubMed  Google Scholar 

  17. Laspia MF, Rice AP, Mathews MB (1989) HIV-1 tat protein increases transcriptional initiation and stabilizes elongation. Cell 59(2):283–292. https://doi.org/10.1016/0092-8674(89)90290-0

    Article  CAS  PubMed  Google Scholar 

  18. Kao SY, Calman AF, Luciw PA, Peterlin BM (1987) Anti-termination of transcription within the long terminal repeat of HIV-1 by tat gene product. Nature 330(6147):489–493. https://doi.org/10.1038/330489a0

    Article  CAS  PubMed  Google Scholar 

  19. Blissenbach M, Grewe B, Hoffmann B, Brandt S, Uberla K (2010) Nuclear RNA export and packaging functions of HIV-1 rev revisited. J Virol 84(13):6598–6604. https://doi.org/10.1128/jvi.02264-09

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Razooky BS, Pai A, Aull K, Rouzine IM, Weinberger LS (2015) A hardwired HIV latency program. Cell 160(5):990–1001. https://doi.org/10.1016/j.cell.2015.02.009

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Hu Y, Smyth GK (2009) ELDA: extreme limiting dilution analysis for comparing depleted and enriched populations in stem cell and other assays. J Immunol Methods 347(1–2):70–78. https://doi.org/10.1016/j.jim.2009.06.008

    Article  CAS  PubMed  Google Scholar 

  22. Autran B, Descours B, Avettand-Fenoel V, Rouzioux C (2011) Elite controllers as a model of functional cure. Curr Opin HIV AIDS 6(3):181–187. https://doi.org/10.1097/COH.0b013e328345a328

    Article  PubMed  Google Scholar 

  23. Colby DJ, Trautmann L, Pinyakorn S, Leyre L, Pagliuzza A, Kroon E, Rolland M, Takata H, Buranapraditkun S, Intasan J, Chomchey N, Muir R, Haddad EK, Tovanabutra S, Ubolyam S, Bolton DL, Fullmer BA, Gorelick RJ, Fox L, Crowell TA, Trichavaroj R, O’Connell R, Chomont N, Kim JH, Michael NL, Robb ML, Phanuphak N, Ananworanich J (2018) Rapid HIV RNA rebound after antiretroviral treatment interruption in persons durably suppressed in Fiebig I acute HIV infection. Nat Med 24(7):923–926. https://doi.org/10.1038/s41591-018-0026-6

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Rebrikov DV, Trofimov DY (2006) Real-time PCR: a review of approaches to data analysis. Appl Biochem Microbiol 42(5):455–463. https://doi.org/10.1134/S0003683806050024

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Viroscience, Erasmus University Medical Center, Rotterdam, Netherlands

    Cynthia Lungu

  2. Department of Immunology and Allergy, Lausanne University Hospital, Lausanne, Switzerland

    Francesco A. Procopio

Authors
  1. Cynthia Lungu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Francesco A. Procopio
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Francesco A. Procopio .

Editor information

Editors and Affiliations

  1. School of Medicine and Human Immuno-Virology (H.I.V.) Group, San Raffaele University and Scientific Institute, Milano, Italy

    Guido Poli

  2. Viral Pathogenesis and Biosafety Group, San Raffaele Scientific Institute, Milano, Italy

    Elisa Vicenzi

  3. Department of Molecular and Comparative Pathobiology, Johns Hopkins University School of Medicine, Baltimore, MD, USA

    Fabio Romerio

Rights and permissions

Reprints and permissions

Copyright information

© 2022 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Lungu, C., Procopio, F.A. (2022). TILDA: Tat/Rev Induced Limiting Dilution Assay. In: Poli, G., Vicenzi, E., Romerio, F. (eds) HIV Reservoirs. Methods in Molecular Biology, vol 2407. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1871-4_24

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-1871-4_24

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1870-7

  • Online ISBN: 978-1-0716-1871-4

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation