The Pfaffl method was used for relative quantification. cell viability of HTLV-1 transformed cells without affecting primary control CD4+ cells and HTLV-1 negative cell lines, suggesting that the huge excess of AEU might provide news targets for treating ATLL. == Conclusions == Taken together, these data reveal that HTLV-1 significantly modifies the structure of cellular transcripts and unmask new putative leukemogenic pathways and possible therapeutic targets. == Electronic supplementary material == The online version of this article (doi:10.1186/s12977-014-0119-3) contains supplementary material, which is available to authorized users. Keywords:HTLV-1, Adult T-cell leukemia, Alternative splicing == Background == HTLV-1 causes chronic infection that relies on the persistent clonal expansion of infected CD4+and CD8+T-cells. Reprogramming of gene transcription sustains HTLV-1 viral persistence and ultimately leads to the development of adult T-cell leukemia/lymphoma (ATLL) from a CD4+-infected clone in a minority of carriers after a prolonged latency [1]. Transcriptome analyses using microarray technology has provided key insights into the biological processes involved in the pre-malignant and malignant expansion of infected cells [2-14]. HTLV-1 has been found to deregulate the expression of numerous genes involved in key cellular pathways such as the cell cycle, apoptosis, telomeres/telomerase, DNA repair, and immune and inflammatory responses through expression of theTaxoncoprotein [2-6]. Recent works have highlighted that in addition to their quantitative effects on gene expression, numerous pathogenic processes, such as persistent viral infections [15] or tumor development [16,17], rely on acquired alternate exon usage (AEU) events. As for other retroviruses, alternative splicing plays a GS-626510 pivotal role in HTLV-1 expression. From its 5LTR, HTLV-1 transcribes a single polycistronic pre-mRNA that codes for structural and enzymatic proteins required for viral particle production. This pre-mRNA also undergoes multiple alternative splicing events that generate mono-spliced transcripts coding for the regulatory proteins p21Rex, p12 and p13, and double-spliced transcripts coding for Tax, p27Rex and p30 [18,19]. Similarly, minus-strand transcription initiated from the 3LTR generates spliced and unspliced RNA isoforms of HBZ that synthesize HBZ proteins with distinct properties on cell proliferation [20-22]. How HTLV-1 intervenes in alternative splicing processes is still incompletely understood. It has been reported that the RNA-binding protein Rex regulates viral splicing through interacting with host splicing machinery to inhibit viral RNA splicing and export unspliced and single-spliced transcripts [23-25]. Gene-by-gene analyses have further shown that HTLV-1 may affect alternative splicing of cellular genes including CD44 [26] and GS-626510 IL-6- and IL-2-receptors [27,28], proposing that HTLV-1-induced GS-626510 AEUs might contribute to molecular mechanisms that underlie the clonal expansion and the malignant transformation of infected cells. However, no systematic study has been hitherto conducted to ascertain the extent of alternative splicing modifications upon HTLV-1 infection. Here, by using integrative analysis of exon expression profiles and gene ontology of CD4+T-cells derived from infected individuals with and without malignancy, we show that HTLV-1 induces multiple AEU alterations that unmask new putative leukemogenic pathways and possible therapeutic targets. == Results and discussion == Comparative microarray analysis of exon expression profiles was performed with three ATLL samples and 12 untransformed CD4+T-cell clones (six infected) derived from HTLV-1-infected individuals with no clinical signs of malignancy. ATLL samples were obtained from patients with an acute form of ATLL (>95% circulating malignant cells). CD4+clones were obtained through cloning by limiting dilution of peripheral blood mononuclear cells (PBMCs) derived from three HTLV-1-infected individuals with tropical spastic paraparesis/HTLV-1-associated myelopathy with a disease duration of 6, 11, and >26 years. This study was conducted according to the principles outlined in the Declaration of Helsinki, and approved by the Institutional Review Board of the Hospices Civils de Lyon (France). As previously described [29,30], CD4+clones were submitted toex vivoculture for Kcnj12 one month prior to HTLV-1 screening and RNA extraction. At this time, infected and uninfected cells remained non-immortalized and required IL-2 for continued growth. Given thatin vitroT-cell activation is known to modify GS-626510 AEU [31,32], exon array analysis of untransformed CD4+clones was carried out with RNAs extracted from unstimulated and phytohemagglutinin (PHA)-stimulated CD4+T-cells (Additional file1: Table S1). By this approach that take into account the in vitro cell culture, we assumed that significant changes in exon expression between infected and uninfected clones mainly resulted from the global impact of HTLV-1 infection,.