Etude du transcriptome des leucémies induites par les delta-rétrovirus par séquençage à haut débit: de microARNs à macroARNs

Plus de 20 million de personnes à travers le monde sont infectées par le virus T-lymphotrope humain de type 1 (HTLV-1), causant des leucémies à cellules T dans ~5 % des individus infectés. Le virus de la leucémie bovine (BLV), structurellement et fonctionnellement proche de HTLV-1, induit des leucémies à cellules B dans des modèles animaux suite à une infection naturelle (bovin) ou expérimentale (mouton). Les mécanismes moléculaires responsables du potentiel oncogène de ces deux virus restent largement incompris. Dans les deux maladies, leucémies T chez l’homme, leucémies B chez l’animal, le site intégration du virus dans les cellules leucémiques est très variable. Il est donc généralement admis que le potentiel oncogène du provirus est principalement lié à l’activité de l’oncoprotéine virale Tax. De manière paradoxale cependant, ni HTLV-1 ni BLV n’expriment de protéines virales au stade tumoral. Dans ce travail, nous avons étudié le transcriptome non codant des leucémies induites par BLV et HTLV-1 par séquençage à haut débit. Dans la première partie, nous démontrons que le provirus BLV n’est en fait pas silencieux dans les cellules tumorales. BLV produit un ensemble de dix microARNs (miRNAs) très abondants et extrêmement conservés dans toutes les tumeurs. Cette observation constitue la première description de miRNAs encodés par un rétrovirus. Les microARNs encodés par BLV sont transcrits par la RNA Polymérase III, stratégie qui permet leur production de façon indépendante de celle des messagers viraux ainsi que leur expression abondante dans le contexte tumoral caractérisé par l’absence d’activité RNA Polymérase II. Nous avons ensuite montré que, comme HTLV-1, BLV produit des transcrits encodés par le brin négatif du provirus. L’analyse par séquençage ARN à haut débit (RNA-Seq) de tumeurs induites par BLV montre l’absence d’expression virale à partir du promoteur viral situé dans le LTR 5’. Cependant, elle révèle la présence de deux transcrits viraux anti-sens non codants (AS1 et AS2) produits par le LTR 3’. Nous avons identifié ces transcrits dans toutes les tumeurs BLV analysées. Enfin, l’analyse RNA-Seq de tumeurs induites par HTLV-1 et BLV a révélé la présence d’interactions transcriptionnelles virus-hôte. Les gènes hôtes affectés sont significativement enrichis en gènes liés au cancer. Ces résultats suggèrent que les transcrits HTLV hbz et BLV AS1 jouent un rôle essentiel dans la tumorigenèse en interagissant avec le génome de l’hôte. Nous avons également détecté ce type de perturbation à des temps précoces dans le modèle expérimental BLV chez le mouton. Ces observations suggèrent que ces interactions virus-hôte constituent des événements précoces qui procurent un avantage sélectif aux clones associés, mais que d’autres altérations -génétiques et/ou épigenetiques- sont nécessaires à l’établissement de la tumeur. En conclusion, nos travaux vont permettre de mieux comprendre le rôle des interactions virus-génome hôte dans l’oncogenèse ainsi que la fonction de transcrits non codants dans le développement des cancers qu’ils soient ou non d’étiologie virale.More than 20 million people are infected by Human T-cell Lymphotropic Virus type 1 (HTLV-1) worldwide and this will cause T-cell leukemia in 5% of them. Yet the molecular mechanisms that underlie the oncogenic potential of this virus remain largely unknown. Bovine Leukemia Virus (BLV) is closely related to HTLV1 and causes a very similar B-cell leukemia in cattle and sheep. As for HTLV1, the oncogenic mechanisms underlying BLV-induced leukemia remain poorly understood. In both diseases, leukemic cells harbor mainly one integrated provirus, yet the integration sites are very variable. As a consequence, it is generally assumed that the oncogenic effect of the provirus is largely mediated by the virally encoded Tax protein. Paradoxically, however, both HTLV1 and BLV proviruses are found to be epigenetically silenced in tumor cells. Thus Tax, as any other virally encoded protein, is not expressed in leukemic cells suggesting that other factors are involved in tumorigenesis. In this study we made three observations that might dramatically change the prevalent dogma of HTLV1 and BLV-induced leukemia. First, we demonstrated that the BLV provirus is not silent at all in tumor cells. A cluster of BLV-encoded microRNAs (miRNAs) is highly expressed, accounting for 40% of the miRNAs present in leukemic cells. This finding is the first description of retroviral-encoded miRNAs. BLV miRNAs are transcribed from five independent RNA Pol III units and are exceedingly conserved across BLV isolates (more than the protein coding genes), strongly supporting an essential yet still unknown function. Next we showed that – as HTLV1 – BLV strongly expresses antisense RNAs. High-throughput sequencing of RNA libraries (RNA-seq) from BLV associated tumors, as expected, showed no expression of viral mRNA from the 5’ LTR. However, it did reveal the presence of two novel non-coding antisense transcripts originating in the 3’ LTR of BLV. Finally, RNA-Seq analysis of HTLV-1 and BLV-induced tumors revealed that the viral 3’ LTR-driven antisense RNAs produced by both viruses interact with host genes localized in the vicinity of proviral integration. Enrichment analysis of affected host genes suggests a significant bias towards cancer-related genes. Host gene perturbations were also found at early stages post-infection in the BLV experimental model in sheep, suggesting that provirus-dependent cancer driver gene perturbations trigger initial amplification of the corresponding clones, requiring additional genetic and/or epigenetic changes to develop full blown leukemia. Overall, our findings reveal an unexpected role for BLV and HTLV antisense transcripts and contribute to the understanding of non-coding RNA-mediated mechanisms in leukemogenesis.Doctorat en Sciences biomédicales et pharmaceutiquesinfo:eu-repo/semantics/nonPublishe

Improving the methodology for high throughput mapping of proviral integration sites

Bovine Leukemia Virus (BLV) and Human T-cell leukemia virus-1 (HTLV-1) are closely related delta-retroviruses provoking a polyclonal expansion of infected B- and T-cells respectively, with monoclonal leukemia/lymphoma developing in about ~5% of infected individuals. To date, the molecular mechanisms leading to cellular transformation remain unclear. Both proviruses are largely transcriptionally silent in tumors and their integration sites in the host genome appear very variable. Mapping proviral insertion sites using high throughput sequencing techniques has provided insights into the evolution of BLV/HTLV-1 infections and the expansion of transformed clones in delta-retrovirus induced leukemia/lymphoma. The methods currently used have a number of limitations such as the utilisation of custom sequencing primers, relatively high sequencing costs, no examination of the 5’LTR host flanking region and a limited dynamic range for determining clone abundance. We have developed an alternative high-throughput sequencing protocol for tracking proviral integration sites and determining clonal abundance in BLV and HTLV-1 infected individuals. We implemented the use of off-the-shelf Illumina primers for the addition of adapters and indexes, which facilitates library multiplexing and avoids the need for custom sequencing primers. Our method reduces the amount of sequencing of PCR duplicates by reducing the number of PCR cycles via an enrichment of BLV- and HTLV-1-carrying DNA fragments. Moreover, in addition to the proviral 3’LTR, our approach also assays the 5’LTR, providing additional information on the frequency of 5’-end deletions in proviruses and increasing the dynamic range of the assay. We have tested the approach on over 100 BLV and HTLV-1 samples, representing both tumors and asymptomatic stages. Our approach allowed for a more accurate determination of clone abundance in tumors and by assaying the provirus 5’ end, identified clones overlooked with previously published methods. Finally, by facilitating greater multiplexing of libraries we have reduced the cost to a level where the technique may be attractive in a clinical setting

MONITORING METHOD FOR ADULT T-CELL LEUKEMIA/LYMPHOMA (ATL)

publication date: 2018-10-11; filing date: 2017-04-06The present invention refers to a method for preparing a linear PCR product from genomic DNA derived from cells of a host subject infected with an retrovirus or a subject suffering from a disease associated with said retrovirus, wherein the PCR product contains a target sequence comprising an integration site of the retrovirus in the host genomic DNA of the cells, said integration site comprising at least the terminal end of 3'-LTR or 5'-LTR sequence of the retrovirus and the adjacent host genomic DNA sequence, wherein the PCR product comprises a first terminus and a second terminus and sequences in the following order: sequences specific for the first terminus, a sequence comprising at least6 consecutive random nucleotides followed by a linker sequence, host genomic DNA sequence, at least the terminal endof 3'-LTR or 5' -LTR sequence of the retrovirus, sequences specific for the second terminus; wherein the PCR product is prepared by specific steps. The present invention also refers to a method for determining and longitudinally monitor the dominant leukemic T lymphocyte clone in subjects suffering from Adult T-cell leukemia/lymphoma (ATL), wherein a linear PCR product is prepared by the method according to the first aspect of the present invention, said PCR product is subjected to multiplex sequencing thereby determining all insertion sites and all shearing sites, the shearing sites are correlated to the respective insertion site, followed by counting the number of different shear sites for each insertion site representing a specific T lymphocyte clone, removing any PCR duplicate from consideration by eliminating reads that have the same insertion site and the same random tag, and determining the abundance of each specific T lymphocyte clone therefrom