Expression of HTLV-1 Genes in T-Cells Using RNA Electroporation

  • Mariangela Manicone
  • Francesca Rende
  • Ilaria Cavallari
  • Andrea K. Thoma-Kress
  • Vincenzo CiminaleEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1582)


Human T-cell leukemia virus type 1 (HTLV-1) infects about 20 million people world-wide. Around 5% of the infected individuals develop adult T-cell leukemia (ATL) or a neurological disease termed tropical spastic paraparesis (TSP) after a clinical latency of years to decades. Through the use of two promoters and alternative splicing HTLV-1 expresses at least 12 different proteins. HTLV-1 establishes a life-long persistent infection by inducing the clonal expansion of infected cells, a property largely ascribed to the viral genes Tax and HBZ. However, the fact that ATL arises in a minority of infected individuals after a long clinical latency suggests the existence of factors counterbalancing the oncogenic potential of HTLV-1 in the context of natural infection.

To study the role of the different HTLV-1 gene products in the HTLV-1 life cycle, we optimized a transfection protocol for primary T-cells using an approach based on the electroporation of in vitro-transcribed RNA. Results showed that the RNA transfection technique combines a high transfection efficiency with low toxicity, not only in Jurkat T-cells but also in primary T-cells. These findings suggest that RNA electroporation is preferable for experiments aimed at investigating the role of HTLV-1 gene products in the context of primary T-cells, which represent the main target of HTLV-1 in vivo.

Key words

HTLV-1 ATL In vitro transcription RNA electroporation Peripheral blood mononuclear cells (PBMCs) 



The authors are grateful to Prof. Ugur Sahin (Research Center for Immunotherapy (FZI), Mainz, Germany; TRON—Translational Oncology at the University Medical Center of Johannes Gutenberg University, Mainz, Germany; Biopharmaceutical New Technologies (BioNTech) Corporation, Mainz, Germany) for providing the plasmid pST1-eGFPmut-2hBgUTR-A120. We are grateful to Armin Ensser and Benjamin Vogel (Institute of Clinical and Molecular Virology, Erlangen, Germany) for helpful discussions.


  1. 1.
    Lairmore M, Franchini G (2007) Human T-cell leukemia virus types 1 and 2. In: Knipe DM, Howley PM (eds) Fields virology, 5th edn. Williams and Wilkins, Lippincott, Philadelphia, PA, pp 2071–2106Google Scholar
  2. 2.
    Ciminale V et al (1992) Complex splicing in the human T-cell leukemia virus (HTLV) family of retroviruses: novel mRNAs and proteins produced by HTLV type I. J Virol 66(3):1737–1745PubMedPubMedCentralGoogle Scholar
  3. 3.
    Koralnik IJ et al (1992) Protein isoforms encoded by the pX region of human T-cell leukemia/lymphotropic virus type I. Proc Natl Acad Sci U S A 89(18):8813–8817CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Larocca D et al (1989) Human T-cell leukemia virus minus strand transcription in infected T-cells. Biochem Biophys Res Commun 163(2):1006–1013CrossRefPubMedGoogle Scholar
  5. 5.
    Murata K et al (2006) A novel alternative splicing isoform of human T-cell leukemia virus type 1 bZIP factor (HBZ-SI) targets distinct subnuclear localization. J Virol 80(5):2495–2505CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Cavanagh MH et al (2006) HTLV-I antisense transcripts initiating in the 3’LTR are alternatively spliced and polyadenylated. Retrovirology 3:15CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Holtkamp S et al (2006) Modification of antigen-encoding RNA increases stability, translational efficacy, and T-cell stimulatory capacity of dendritic cells. Blood 108(13):4009–4017CrossRefPubMedGoogle Scholar
  8. 8.
    Van Tendeloo VF et al (2001) Highly efficient gene delivery by mRNA electroporation in human hematopoietic cells: superiority to lipofection and passive pulsing of mRNA and to electroporation of plasmid cDNA for tumor antigen loading of dendritic cells. Blood 98(1):49–56CrossRefPubMedGoogle Scholar
  9. 9.
    Zhao Y et al (2006) High-efficiency transfection of primary human and mouse T lymphocytes using RNA electroporation. Mol Ther 13(1):151–159CrossRefPubMedGoogle Scholar
  10. 10.
    Rowley J et al (2009) Expression of IL-15RA or an IL-15/IL-15RA fusion on CD8+ T cells modifies adoptively transferred T-cell function in cis. Eur J Immunol 39(2):491–506CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Silic-Benussi M et al (2010) Redox regulation of T-cell turnover by the p13 protein of human T-cell leukemia virus type 1: distinct effects in primary versus transformed cells. Blood 116(1):54–62CrossRefPubMedGoogle Scholar
  12. 12.
    Krieg PA (1990) Improved synthesis of full-length RNA probe at reduced incubation temperatures. Nucleic Acids Res 18(21):6463CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Ben Aziz R, Soreq H Improving poor in vitro transcription from G,C-rich genes. Nucleic Acids Res 18(11):3418Google Scholar
  14. 14.
    Kim JA et al (2008) A novel electroporation method using a capillary and wire-type electrode. Biosens Bioelectron 23(9):1353–1360CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Mariangela Manicone
    • 1
  • Francesca Rende
    • 1
  • Ilaria Cavallari
    • 3
  • Andrea K. Thoma-Kress
    • 2
  • Vincenzo Ciminale
    • 1
    • 3
    Email author
  1. 1.Department of Surgery, Oncology and GastroenterologyUniversity of PadovaPadovaItaly
  2. 2.Institute of Clinical and Molecular Virology, Friedrich-Alexander-Universität Erlangen-NürnbergErlangenGermany
  3. 3.Istituto Oncologico Veneto, IRCCSPadovaItaly

Personalised recommendations