RESUMO
HIV-1 is a complex retrovirus that uses host machinery to promote its replication. Understanding cellular proteins involved in the multistep process of HIV-1 infection may result in the discovery of more adapted and effective therapeutic targets. Kinases and phosphatases are a druggable class of proteins critically involved in regulation of signal pathways of eukaryotic cells. Here, we focused on the discovery of kinases and phosphatases that are essential for HIV-1 replication but dispensable for cell viability. We performed an iterative screen in Jurkat T-cells with a short-hairpin-RNA (shRNA) library highly enriched for human kinases and phosphatases. We identified 14 new proteins essential for HIV-1 replication that do not affect cell viability. These proteins are described to be involved in MAPK, JNK and ERK pathways, vesicular traffic and DNA repair. Moreover, we show that the proteins under study are important in an early step of HIV-1 infection before viral integration, whereas some of them affect viral transcription/translation. This study brings new insights for the complex interplay of HIV-1/host cell and opens new possibilities for antiviral strategies.
Assuntos
HIV-1/fisiologia , Monoéster Fosfórico Hidrolases/genética , Fosfotransferases/genética , RNA Interferente Pequeno/genética , Western Blotting , Linhagem Celular , Sobrevivência Celular , Biblioteca Gênica , HIV-1/genética , Células HeLa , Interações Hospedeiro-Patógeno , Humanos , Células Jurkat , Leucemia de Células T/genética , Leucemia de Células T/patologia , Leucemia de Células T/virologia , Monoéster Fosfórico Hidrolases/metabolismo , Fosfotransferases/metabolismo , Interferência de RNA , Replicação Viral/genética , Replicação Viral/fisiologia , Produtos do Gene vif do Vírus da Imunodeficiência Humana/genética , Produtos do Gene vif do Vírus da Imunodeficiência Humana/metabolismoRESUMO
Exosomes are secreted membrane vesicles that share structural and biochemical characteristics with intraluminal vesicles of multivesicular endosomes (MVEs). Exosomes could be involved in intercellular communication and in the pathogenesis of infectious and degenerative diseases. The molecular mechanisms of exosome biogenesis and secretion are, however, poorly understood. Using an RNA interference (RNAi) screen, we identified five Rab GTPases that promote exosome secretion in HeLa cells. Among these, Rab27a and Rab27b were found to function in MVE docking at the plasma membrane. The size of MVEs was strongly increased by Rab27a silencing, whereas MVEs were redistributed towards the perinuclear region upon Rab27b silencing. Thus, the two Rab27 isoforms have different roles in the exosomal pathway. In addition, silencing two known Rab27 effectors, Slp4 (also known as SYTL4, synaptotagmin-like 4) and Slac2b (also known as EXPH5, exophilin 5), inhibited exosome secretion and phenocopied silencing of Rab27a and Rab27b, respectively. Our results therefore strengthen the link between MVEs and exosomes, and introduce ways of manipulating exosome secretion in vivo.
Assuntos
Comunicação Celular , Endossomos/fisiologia , Exossomos/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Membrana Celular/metabolismo , Citometria de Fluxo , Imunofluorescência , Inativação Gênica , Células HeLa , Humanos , Immunoblotting , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Frações Subcelulares , Proteínas de Transporte Vesicular/antagonistas & inibidores , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo , Proteínas rab de Ligação ao GTP/antagonistas & inibidores , Proteínas rab de Ligação ao GTP/genética , Proteínas rab27 de Ligação ao GTPRESUMO
Innate immune cells detect pathogens via pattern recognition receptors (PRRs), which signal for initiation of immune responses to infection. Studies with Dectin-1, a PRR for fungi, have defined a novel innate signaling pathway involving Syk kinase and the adaptor CARD9, which is critical for inducing Th17 responses to fungal infection. We show that another C-type lectin, Dectin-2, also signals via Syk and CARD9, and contributes to dendritic cell (DC) activation by fungal particles. Unlike Dectin-1, Dectin-2 couples to Syk indirectly, through association with the FcRgamma chain. In a model of Candida albicans infection, blockade of Dectin-2 did not affect innate immune resistance but abrogated Candida-specific T cell production of IL-17 and, in combination with the absence of Dectin-1, decreased Th1 responses to the organism. Thus, Dectin-2 constitutes a major fungal PRR that can couple to the Syk-CARD9 innate signaling pathway to activate DCs and regulate adaptive immune responses to fungal infection.
Assuntos
Candidíase/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Lectinas Tipo C/imunologia , Proteínas Tirosina Quinases/imunologia , Receptores de Reconhecimento de Padrão/metabolismo , Transdução de Sinais/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/genética , Animais , Proteínas Adaptadoras de Sinalização CARD , Citometria de Fluxo , Immunoblotting , Interleucina-17/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lectinas Tipo C/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/genética , Proteínas Tirosina Quinases/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Quinase SykRESUMO
The transition from naïve to activated T cells is marked by alternative splicing of pre-mRNA encoding the transmembrane phosphatase CD45. Using a short hairpin RNA interference screen, we identified heterogeneous ribonucleoprotein L-like (hnRNPLL) as a critical inducible regulator of CD45 alternative splicing. HnRNPLL was up-regulated in stimulated T cells, bound CD45 transcripts, and was both necessary and sufficient for CD45 alternative splicing. Depletion or overexpression of hnRNPLL in B and T cell lines and primary T cells resulted in reciprocal alteration of CD45RA and RO expression. Exon array analysis suggested that hnRNPLL acts as a global regulator of alternative splicing in activated T cells. Induction of hnRNPLL during hematopoietic cell activation and differentiation may allow cells to rapidly shift their transcriptomes to favor proliferation and inhibit cell death.