RESUMEN
Host encounters with viruses lead to an innate immune response that must be rapid and broadly targeted but also tightly regulated to avoid the detrimental effects of unregulated interferon expression. Viral stimulation of host negative regulatory mechanisms is an alternate method of suppressing the host innate immune response. We examined three key mediators of the innate immune response: NF-KB, STAT1 and STAT2 during HCV infection in order to investigate the paradoxical induction of an innate immune response by HCV despite a multitude of mechanisms combating the host response. During infection, we find that all three are repressed only in HCV infected cells but not in uninfected bystander cells, both in vivo in chimeric mouse livers and in cultured Huh7.5 cells after IFNα treatment. We show here that HCV and Flaviviruses suppress the innate immune response by upregulation of PDLIM2, independent of the host interferon response. We show PDLIM2 is an E3 ubiquitin ligase that also acts to stimulate nuclear degradation of STAT2. Interferon dependent relocalization of STAT1/2 to the nucleus leads to PDLIM2 ubiquitination of STAT2 but not STAT1 and the proteasome-dependent degradation of STAT2, predominantly within the nucleus. CRISPR/Cas9 knockout of PDLIM2 results in increased levels of STAT2 following IFNα treatment, retention of STAT2 within the nucleus of HCV infected cells after IFNα stimulation, increased interferon response, and increased resistance to infection by several flaviviruses, indicating that PDLIM2 is a global regulator of the interferon response.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/fisiología , Infecciones por Flavivirus/inmunología , Flavivirus/inmunología , Hepacivirus/inmunología , Hepatitis C/inmunología , Inmunidad Innata/inmunología , Proteínas con Dominio LIM/fisiología , Factor de Transcripción STAT2/metabolismo , Animales , Antivirales/farmacología , Flavivirus/efectos de los fármacos , Infecciones por Flavivirus/tratamiento farmacológico , Infecciones por Flavivirus/virología , Hepacivirus/efectos de los fármacos , Hepatitis C/tratamiento farmacológico , Hepatitis C/virología , Humanos , Inmunidad Innata/efectos de los fármacos , Interferón-alfa/farmacología , Proteínas con Dominio LIM/genética , Proteínas con Dominio LIM/metabolismo , Ratones , Ratones Noqueados , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , FN-kappa B , Factor de Transcripción STAT2/genética , Transducción de SeñalRESUMEN
Mutations in H6-homeobox (HMX) genes are linked to neural mispatterning and neural tube closure defects in humans. We demonstrate that zebrafish Hmx4 regulates the signaling of two morphogens critical for neural development, retinoic acid (RA) and Sonic hedgehog (Shh). Hmx4-depleted embryos have a strongly narrowed eye field and reduced forebrain Shh target gene expression. hmx4 morphants fail to properly transcribe the Shh signal transducer gli3, and have reduced ventral forebrain specification. Hmx4-depleted embryos also have neural tube patterning defects that phenocopy RA-deficiency. We show that Hmx4 is required for the initiation and maintenance of aldh1a2, the principal RA-synthesizing gene. Loss of RA is the primary defect in Hmx4-depleted embryos, as RA treatment rescues a number of the neural patterning defects. Surprisingly, RA treatment also rescues forebrain morphology, gli3 transcription, and Shh signaling. We propose that Hmx4 is a critical regulator of retinoic acid synthesis in a developing embryo, and that this regulation is essential for controlling Shh signaling and forebrain development.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Proteínas Hedgehog/genética , Proteínas de Homeodominio/metabolismo , Prosencéfalo/embriología , Tretinoina/metabolismo , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo , Pez Cebra/embriología , Aldehído Deshidrogenasa/genética , Animales , Tipificación del Cuerpo/genética , Proteínas de Homeodominio/genética , Prosencéfalo/metabolismo , Transducción de Señal , Factores de Transcripción/genética , Pez Cebra/genética , Proteína Gli3 con Dedos de ZincRESUMEN
Ocular mal-development results in heterogeneous and frequently visually disabling phenotypes that include coloboma and microphthalmia. Due to the contribution of bone morphogenetic proteins to such processes, the function of the paralogue Growth Differentiation Factor 3 was investigated. Multiple mis-sense variants were identified in patients with ocular and/or skeletal (Klippel-Feil) anomalies including one individual with heterozygous alterations in GDF3 and GDF6. These variants were characterized, individually and in combination, through integrated biochemical and zebrafish model organism analyses, demonstrating appreciable effects with western blot analyses, luciferase based reporter assays and antisense morpholino inhibition. Notably, inhibition of the zebrafish co-orthologue of GDF3 accurately recapitulates patient phenotypes. By demonstrating the pleiotropic effects of GDF3 mutation, these results extend the contribution of perturbed BMP signaling to human disease and potentially implicate multi-allelic inheritance of BMP variants in developmental disorders.