Abstract
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.
*These authors contributed equally to this work.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
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 2071–2106
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):1737–1745
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):8813–8817
Larocca D et al (1989) Human T-cell leukemia virus minus strand transcription in infected T-cells. Biochem Biophys Res Commun 163(2):1006–1013
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):2495–2505
Cavanagh MH et al (2006) HTLV-I antisense transcripts initiating in the 3’LTR are alternatively spliced and polyadenylated. Retrovirology 3:15
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):4009–4017
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):49–56
Zhao Y et al (2006) High-efficiency transfection of primary human and mouse T lymphocytes using RNA electroporation. Mol Ther 13(1):151–159
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):491–506
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):54–62
Krieg PA (1990) Improved synthesis of full-length RNA probe at reduced incubation temperatures. Nucleic Acids Res 18(21):6463
Ben Aziz R, Soreq H Improving poor in vitro transcription from G,C-rich genes. Nucleic Acids Res 18(11):3418
Kim JA et al (2008) A novel electroporation method using a capillary and wire-type electrode. Biosens Bioelectron 23(9):1353–1360
Acknowledgment
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.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2017 Springer Science+Business Media LLC
About this protocol
Cite this protocol
Manicone, M., Rende, F., Cavallari, I., Thoma-Kress, A.K., Ciminale, V. (2017). Expression of HTLV-1 Genes in T-Cells Using RNA Electroporation. In: Casoli, C. (eds) Human T-Lymphotropic Viruses. Methods in Molecular Biology, vol 1582. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6872-5_12
Download citation
DOI: https://doi.org/10.1007/978-1-4939-6872-5_12
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-6870-1
Online ISBN: 978-1-4939-6872-5
eBook Packages: Springer Protocols