RESUMEN
Viral tumor models have significantly contributed to our understanding of oncogenic mechanisms. How transforming delta-retroviruses induce malignancy, however, remains poorly understood, especially as viral mRNA/protein are tightly silenced in tumors. Here, using deep sequencing of broad windows of small RNA sizes in the bovine leukemia virus ovine model of leukemia/lymphoma, we provide in vivo evidence of the production of noncanonical RNA polymerase III (Pol III)-transcribed viral microRNAs in leukemic B cells in the complete absence of Pol II 5'-LTR-driven transcriptional activity. Processed from a cluster of five independent self-sufficient transcriptional units located in a proviral region dispensable for in vivo infectivity, bovine leukemia virus microRNAs represent â¼40% of all microRNAs in both experimental and natural malignancy. They are subject to strong purifying selection and associate with Argonautes, consistent with a critical function in silencing of important cellular and/or viral targets. Bovine leukemia virus microRNAs are strongly expressed in preleukemic and malignant cells in which structural and regulatory gene expression is repressed, suggesting a key role in tumor onset and progression. Understanding how Pol III-dependent microRNAs subvert cellular and viral pathways will contribute to deciphering the intricate perturbations that underlie malignant transformation.
Asunto(s)
Leucosis Bovina Enzoótica/genética , Leucosis Bovina Enzoótica/virología , Virus de la Leucemia Bovina/genética , Leucemia de Células B/genética , Leucemia de Células B/virología , Linfoma de Células B/genética , Linfoma de Células B/virología , MicroARNs/genética , ARN Viral/genética , Animales , Proteínas Argonautas/metabolismo , Secuencia de Bases , Bovinos , Línea Celular Tumoral , Modelos Animales de Enfermedad , Expresión Génica , Proteínas del Grupo de Alta Movilidad/genética , Virus Linfotrópico T Tipo 1 Humano/genética , Humanos , Leucemia de Células B/veterinaria , Leucemia-Linfoma de Células T del Adulto/genética , Leucemia-Linfoma de Células T del Adulto/virología , Linfoma de Células B/veterinaria , MicroARNs/química , MicroARNs/metabolismo , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , ARN Polimerasa III/metabolismo , ARN Neoplásico/genética , ARN Neoplásico/metabolismo , ARN Viral/química , ARN Viral/metabolismo , Análisis de Secuencia de ARN , Homología de Secuencia de Ácido Nucleico , Ovinos , Enfermedades de las Ovejas/genética , Enfermedades de las Ovejas/virología , Secuencias Repetidas TerminalesRESUMEN
BACKGROUND: Reprogramming cellular gene transcription sustains HTLV-1 viral persistence that ultimately leads to the development of adult T-cell leukemia/lymphoma (ATLL). We hypothesized that besides these quantitative transcriptional effects, HTLV-1 qualitatively modifies the pattern of cellular gene expression. RESULTS: Exon expression analysis shows that patients' untransformed and malignant HTLV-1(+) CD4(+) T-cells exhibit multiple alternate exon usage (AEU) events. These affect either transcriptionally modified or unmodified genes, culminate in ATLL, and unveil new functional pathways involved in cancer and cell cycle. Unsupervised hierarchical clustering of array data permitted to isolate exon expression patterns of 3977 exons that discriminate uninfected, infected, and transformed CD4(+) T-cells. Furthermore, untransformed infected CD4+ clones and ATLL samples shared 486 exon modifications distributed in 320 genes, thereby indicating a role of AEUs in HTLV-1 leukemogenesis. Exposing cells to splicing modulators revealed that Sudemycin E reduces 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.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/virología , Exones , Regulación de la Expresión Génica , Interacciones Huésped-Patógeno , Virus Linfotrópico T Tipo 1 Humano/fisiología , Leucemia-Linfoma de Células T del Adulto/patología , Virus Linfotrópico T Tipo 1 Humano/crecimiento & desarrollo , Humanos , Transcripción GenéticaRESUMEN
Viruses disrupt the host cell microRNA (miRNA) network to facilitate their replication. Human T-cell leukemia virus type I (HTLV-1) replication relies on the clonal expansion of its host CD4(+) and CD8(+) T cells, yet this virus causes adult T-cell leukemia/lymphoma (ATLL) that typically has a CD4(+) phenotype. The viral oncoprotein Tax, which is rarely expressed in ATLL cells, has long been recognized for its involvement in tumor initiation by promoting cell proliferation, genetic instability, and miRNA dysregulation. Meanwhile, HBZ is expressed in both untransformed infected cells and ATLL cells and is involved in sustaining cell proliferation and silencing virus expression. Here, we show that an HBZ-miRNA axis promotes cell proliferation and genetic instability, as indicated by comet assays that showed increased numbers of DNA-strand breaks. Expression profiling of miRNA revealed that infected CD4(+) cells, but not CD8(+) T cells, overexpressed oncogenic miRNAs, including miR17 and miR21. HBZ activated these miRNAs via a posttranscriptional mechanism. These effects were alleviated by knocking down miR21 or miR17 and by ectopic expression of OBFC2A, a DNA-damage factor that is downregulated by miR17 and miR21 in HTLV-1-infected CD4(+) T cells. These findings extend the oncogenic potential of HBZ and suggest that viral expression might be involved in the remarkable genetic instability of ATLL cells.