Abstract
HTLV-1 and HTLV-2 viruses express Tax transactivator proteins required for viral genome transcription and capable of transforming cells in vivo and in vitro. Although Tax oncogenic potential needs to be further elucidated, it is well established that Tax proteins activate, among others, transcription factors of the NF-ĸB family, which are involved in immune and inflammatory responses, cell growth, apoptosis, stress responses and oncogenesis. Here, we describe a reporter gene assay applied for quantitative analysis of Tax-dependent NF-ĸB activation. The procedure is based on co-transfection of two individual vectors containing the cDNA for firefly and Renilla luciferase enzymes and vectors expressing Tax proteins. The luciferase expression is driven by cis-NF-ĸB promoter regulatory elements responsive to Tax transactivating factor. This assay is particularly useful to investigate Tax influence on NF-ĸB activation mediated by viral or host factors.
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References
Ciminale V, Rende F, Bertazzoni U, Romanelli MG (2014) HTLV-1 and HTLV-2: highly similar viruses with distinct oncogenic properties. Front Microbiol 29(5):398. doi:10.3389/fmicb.2014.00398
Romanelli MG, Diani E, Bergamo E, Casoli C, Ciminale V, Bex F, Bertazzoni U (2013) Highlights on distinctive structural and functional properties of HTLV Tax proteins. Front Microbiol 9(4):271. doi:10.3389/fmicb.2013.00271
Rende F, Cavallari I, Romanelli MG, Diani E, Bertazzoni U, Ciminale V (2012) Comparison of the genetic organization, expression strategies and oncogenic potential of HTLV-1 and HTLV-2. Leuk Res Treatment 2012:876153. doi:10.1155/2012/876153.2012
Bertazzoni U, Turci M, Avesani F, Di Gennaro G, Bidoia C, Romanelli MG (2011) Intracellular localization and cellular factors interaction of HTLV-1 and HTLV-2 Tax proteins: similarities and functional differences. Viruses. 3:541–560. doi:10.3390/v3050541
Pilotti E, Bianchi MV, De Maria A, Bozzano F, Romanelli MG, Bertazzoni U, Casoli C (2013) HTLV-1/−2 and HIV-1 co-infections: retroviral interference on host immune status. Front Microbiol 23(4):372. doi:10.3389/fmicb.2013.00372
Matsuoka M, Yasunaga J (2013) Human T-cell leukemia virus type 1: replication, proliferation and propagation by Tax and HTLV-1 bZIP factor. Curr Opin Virol 3:684–691. doi:10.1016/j.coviro.2013.08.010
Yasunaga J, Matsuoka M (2011) Molecular mechanisms of HTLV-1 infection and pathogenesis. Int J Hematol 94:435–442. doi:10.1007/s12185-011-0937-1
Qu Z, Xiao G (2011) Human T-cell lymphotropic virus: a model of NF-ĸB-associated tumorigenesis. Viruses 3:714–749. doi:10.3390/v3060714
Harhaj EW, Sun SC (1999) IKKgamma serves as a docking subunit of the IkappaB kinase (IKK) and mediates interaction of IKK with the human T-cell leukemia virus Tax protein. J Biol Chem 274:22911–22914
Avesani F, Romanelli MG, Turci M, Di Gennaro G, Sampaio C, Bidoia C, Bertazzoni U, Bex F (2010) Association of HTLV Tax proteins with TAK1-binding protein 2 and RelA in calreticulin-containing cytoplasmic structures participates in Tax-mediated NF-ĸB activation. Virology 408:39–48. doi:10.1016/j
Diani E, Avesani F, Bergamo E, Cremonese G, Bertazzoni U, Romanelli MG (2015) HTLV-1 Tax protein recruitment into IKKε and TBK1 kinase complexes enhances IFN-I expression. Virology 476:92–99. doi:10.1016/j
Bex F, McDowall A, Burny A, Gaynor R (1997) The human T-cell leukemia virus type 1 transactivator protein tax colocalizes in unique nuclear structures with NF-kB proteins. J Virol 71:3484–3497
Shibata Y, Tanaka Y, Gohda J, Inoue J (2011) Activation of the IĸB kinase complex by HTLV-1 tax requires cytosolic factors involved in tax-induced polyubiquitination. J Biochem 150:679–686
Xiao G (2012) NF-kappaB activation: Tax sumoylation is out, but what about Tax ubiquitination? Retrovirology 25(9):78. doi:10.1186/1742-4690-9-78
Turci M, Lodewick J, Di Gennaro G, Rinaldi AS, Marin O, Diani E, Sampaio C, Bex F, Bertazzoni U, Romanelli MG (2012) Ubiquitination and sumoylation of the HTLV-2 Tax-2B protein regulate its NF-ĸB activity: a comparative study with the HTLV-1 Tax-1 protein. Retrovirology 7(9):102. doi:10.1186/1742-4690-9-102.2012
Journo C, Bonnet A, Favre-Bonvin A, Turpin J, Vinera J, Côté E, Chevalier SA, Kfoury Y, Bazarbachi A, Pique C, Mahieux R (2013) Human T cell leukemia virus type 2 tax-mediated NF-ĸB activation involves a mechanism independent of Tax conjugation to ubiquitin and SUMO. J Virol 87:1123–1136. doi:10.1128/JVI.01792-12
Zhi H, Yang L, Kuo YL, Ho YK, Shih HM, Giam CZ (2011) NF-ĸB hyper-activation by HTLV-1 tax induces cellular senescence, but can be alleviated by the viral anti-sense protein HBZ. PLoS Pathog 7:e1002025. doi:10.1371/journal.ppat.1002025
Turci M, Romanelli MG, Lorenzi P, Righi P, Bertazzoni U (2006) Localization of human T-cell lymphotropic virus type II Tax protein is dependent upon a nuclear localization determinant in the N-terminal region. Gene 365:119–124
Turci M, Lodewick J, Righi P, Polania A, Romanelli MG, Bex F, Bertazzoni U (2009) HTLV-2B Tax oncoprotein is modified by ubiquitination and sumoylation and display intracellular localization similar to its homologue HTLV-1 Tax. Virology 30:6–11. doi:10.1016/j.virol.2009.01.003
Shifera AS, Hardin JA (2010) Factors modulating expression of Renilla luciferase from control plasmids used in luciferase reporter gene assays. Anal Biochem 396:167–172. doi:10.1016/j.ab
Lwa TR, Lee J, Ng CH, Lew QJ, Hia HC, Chao SH (2011) Human T-lymphotropic virus tax activates human cytomegalovirus major-immediate early promoter and improves production of recombinant proteins in HEK293 cells. Biotechnol Prog 27:751–756. doi:10.1002/btpr.571
Acknowledgment
This work was supported by the following grants to M.G.R.: AIRC-Cariverona-2008 “An integrated approach to chronic lymphoproliferative disorders: B-CLL and virus related neoplasia”, FUR 2014 University of Verona.
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Bergamo, E., Diani, E., Bertazzoni, U., Romanelli, M.G. (2017). A Luciferase Functional Quantitative Assay for Measuring NF-ĸB Promoter Transactivation Mediated by HTLV-1 and HTLV-2 Tax Proteins. 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_6
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DOI: https://doi.org/10.1007/978-1-4939-6872-5_6
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