RESUMO
Cell-autonomous induction of type I interferon must be stringently regulated. Rapid induction is key to control virus infection, whereas proper limitation of signaling is essential to prevent immunopathology and autoimmune disease. Using unbiased kinome-wide RNAi screening followed by thorough validation, we identified 22 factors that regulate RIG-I/IRF3 signaling activity. We describe a negative-feedback mechanism targeting RIG-I activity, which is mediated by death associated protein kinase 1 (DAPK1). RIG-I signaling triggers DAPK1 kinase activation, and active DAPK1 potently inhibits RIG-I stimulated IRF3 activity and interferon-beta production. DAPK1 phosphorylates RIG-I in vitro at previously reported as well as other sites that limit 5'ppp-dsRNA sensing and virtually abrogate RIG-I activation.
Assuntos
Proteínas Quinases Associadas com Morte Celular/metabolismo , RNA Interferente Pequeno/genética , Receptores do Ácido Retinoico/metabolismo , Células A549 , Animais , Células Cultivadas , Retroalimentação Fisiológica , Células HEK293 , Humanos , Camundongos , Fosforilação , Proteínas Quinases/metabolismo , Transdução de SinaisRESUMO
Specific types of human papillomaviruses (HPVs) cause cervical cancer. Cervical cancers exhibit aberrant cellular microRNA (miRNA) expression patterns. By genome-wide analyses, we investigate whether the intracellular and exosomal miRNA compositions of HPV-positive cancer cells are dependent on endogenous E6/E7 oncogene expression. Deep sequencing studies combined with qRT-PCR analyses show that E6/E7 silencing significantly affects ten of the 52 most abundant intracellular miRNAs in HPV18-positive HeLa cells, downregulating miR-17-5p, miR-186-5p, miR-378a-3p, miR-378f, miR-629-5p and miR-7-5p, and upregulating miR-143-3p, miR-23a-3p, miR-23b-3p and miR-27b-3p. The effects of E6/E7 silencing on miRNA levels are mainly not dependent on p53 and similarly observed in HPV16-positive SiHa cells. The E6/E7-regulated miRNAs are enriched for species involved in the control of cell proliferation, senescence and apoptosis, suggesting that they contribute to the growth of HPV-positive cancer cells. Consistently, we show that sustained E6/E7 expression is required to maintain the intracellular levels of members of the miR-17~92 cluster, which reduce expression of the anti-proliferative p21 gene in HPV-positive cancer cells. In exosomes secreted by HeLa cells, a distinct seven-miRNA-signature was identified among the most abundant miRNAs, with significant downregulation of let-7d-5p, miR-20a-5p, miR-378a-3p, miR-423-3p, miR-7-5p, miR-92a-3p and upregulation of miR-21-5p, upon E6/E7 silencing. Several of the E6/E7-dependent exosomal miRNAs have also been linked to the control of cell proliferation and apoptosis. This study represents the first global analysis of intracellular and exosomal miRNAs and shows that viral oncogene expression affects the abundance of multiple miRNAs likely contributing to the E6/E7-dependent growth of HPV-positive cancer cells.
Assuntos
Proteínas de Ligação a DNA/metabolismo , MicroRNAs/biossíntese , Proteínas Oncogênicas Virais/metabolismo , Proteínas Repressoras/metabolismo , Neoplasias do Colo do Útero/virologia , Apoptose/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Senescência Celular/genética , Exossomos/genética , Exossomos/metabolismo , Feminino , Estudo de Associação Genômica Ampla , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Immunoblotting , Microscopia Eletrônica de Transmissão , Infecções por Papillomavirus/complicações , Infecções por Papillomavirus/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção , Neoplasias do Colo do Útero/genéticaRESUMO
A polymerase chain reaction (PCR)-based diagnostic assay was developed that rapidly and reliably differentiates the sibling species of the Anopheles claviger complex, An. claviger s.s. and An. petragnani. The assay makes use of nucleotide differences in the internal transcribed spacer 2 ribosomal DNA sequences to generate PCR products of specific length for each of the two species. In evaluating the test, 580 of 592 field-collected An. claviger s.l. specimens were unambiguously identified as one of the two sibling species. Due to poor DNA quality, the remaining 12 specimens yielded no PCR product. Of the 592 mosquitoes, 407 larval specimens had been identified morphologically prior to species-specific DNA amplification, and in all instances PCR identification corroborated with morphologic identification. Mosquitoes identified as An. claviger s.s. came from various localities all over Europe and from Israel. Those identified as An. petragnani were collected in southern France and Spain. The species-diagnostic PCR assay would facilitate data collection on the temporal and spatial distribution of the two An. claviger sibling species because they represent possible vectors of disease in Europe, the Near and Middle East, and north Africa.
