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We study the influence of spatial heterogeneity on the antiviral activity of mouse embryonic fibroblasts (MEF) infected with influenza A. MEF of type Ube1L-/- are composed of two distinct sub-populations, the strong type that sustains a strong viral infection and the weak type, sustaining a weak viral load. We present new data on the virus load infection of Ube1L-/-, which have been micro-printed in a checker board pattern of different sizes of the inner squares. Surprisingly, the total viral load at one day after inoculation significantly depends on the sizes of the inner squares. We explain this observation by using a reaction diffusion model and we show that mathematical homogenization can explain the observed inhomogeneities. If the individual patches are large, then the growth rate and the carrying capacity will be the arithmetic means of the patches. For finer and finer patches the average growth rate is still the arithmetic mean, however, the carrying capacity uses the harmonic mean. While fitting the PDE to the experimental data, we also predict that a discrepancy in virus load would be unobservable after only half a day. Furthermore, we predict the viral load in different inner squares that had not been measured in our experiment and the travelling distance the virions can reach after one day.
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Virus de la Influenza A , Gripe Humana , Animales , Antivirales/uso terapéutico , Fibroblastos , Humanos , Gripe Humana/tratamiento farmacológico , Ratones , Carga ViralRESUMEN
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Microfabrication methods have widely been used to control the local cellular environment on a micron scale. However, accurately mimicking the complexity of the in vivo tissue architecture while maintaining the freedom of form and design is still a challenge when co-culturing multiple types of cells on the same substrate. For the first time, we present a drop-on-demand inkjet printing method to directly pattern living cells into a cell-friendly liquid environment. High-resolution control of cell location is achieved by precisely optimizing printing parameters with high-speed imaging of cell jetting and impacting behaviors. We demonstrated the capabilities of the direct cell printing method by co-printing different cells into various designs, including complex gradient arrangements. Finally, we applied this technique to investigate the influence of the heterogeneity and geometry of the cell population on the infectivity of seasonal H1N1 influenza virus (PR8) by generating A549 and HeLa cells printed in checkboard patterns of different sizes in a medium-filled culture dish. Direct inkjet cell patterning can be a powerful and versatile tool for both fundamental biology and applied biotechnology.
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Bioimpresión/métodos , Técnicas de Cocultivo , Medios de Cultivo , Microtecnología , Células A549 , Proliferación Celular , Supervivencia Celular , Técnicas de Cocultivo/instrumentación , Diseño de Equipo , Células HeLa , Humanos , Subtipo H1N1 del Virus de la Influenza A , Gripe Humana/patología , Gripe Humana/fisiopatología , Microtecnología/instrumentación , Microtecnología/métodosRESUMEN
RIG-I is a cytosolic receptor recognizing virus-specific RNA structures and initiates an antiviral signaling that induces the production of interferons and proinflammatory cytokines. Because inappropriate RIG-I signaling affects either viral clearance or immune toxicity, multiple regulations of RIG-I have been investigated since its discovery as the viral RNA detector. In this review, we describe the recent progress in research on the regulation of RIG-I activity or abundance. Specifically, we focus on the mechanism that modulates RIG-I-dependent antiviral response through post-translational modifications of or protein-protein interactions with RIG-I.
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Proteína 58 DEAD Box/genética , Proteína 58 DEAD Box/inmunología , Transducción de Señal , Virosis/inmunología , Virus/inmunología , Animales , Regulación de la Expresión Génica , Interacciones Huésped-Patógeno , Humanos , Unión Proteica , Receptores Inmunológicos , Virosis/genética , Virosis/virología , Virus/genéticaRESUMEN
A population often contains distinct sub-populations, thereby increasing the complexity of the overall heterogeneity. However, the cellular origin and biological relevance of sub-populations in cell population have not been clearly identified. Here we demonstrated the novel roles of ISGylation, which is an IFN-induced post-translational modification, controlling heterogeneity at the population level in cultured adherent cells. Without UBE1L, an E1 enzyme of ISGylation, mouse embryonic fibroblasts (MEF) exhibited low viral resistance despite high STAT1 and ISG expression compared with the wild-type MEF. We observe that Ube1l-/- MEF populations consist of two behaviorally distinguishable sub-populations with distinct basal STAT1 activity, while wild-type MEF populations are unimodal. This population heterogeneity in Ube1l knock-out cells was perturbed by tyrosine kinase inhibitors, AG490 and PF431396. In contrast, the neutralization of type I IFN did not affect population heterogeneity. Based on these results, we concluded that UBE1L functions to adjust basal immunological states with the regulation of population heterogeneity.
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Citocinas/genética , Procesamiento Proteico-Postraduccional , Proteínas Tirosina Quinasas/genética , Factor de Transcripción STAT1/genética , Enzimas Activadoras de Ubiquitina/genética , Animales , Linaje de la Célula/efectos de los fármacos , Linaje de la Célula/genética , Citocinas/metabolismo , Embrión de Mamíferos , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Citometría de Flujo , Humanos , Inmunofenotipificación , Ratones , Ratones Noqueados , Cultivo Primario de Células , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/metabolismo , Pirimidinas/farmacología , Factor de Transcripción STAT1/metabolismo , Transgenes , Tirfostinos/farmacología , Enzimas Activadoras de Ubiquitina/metabolismo , Ubiquitinas/genética , Ubiquitinas/metabolismoRESUMEN
RIG-I is a key cytosolic RNA sensor that mediates innate immune defense against RNA virus. Aberrant RIG-I activity leads to severe pathological states such as autosomal dominant multi-system disorder, inflammatory myophathies and dermatomyositis. Therefore, identification of regulators that ensure efficient defense without harmful immune-pathology is particularly critical to deal with RIG-I-associated diseases. Here, we presented the inflammatory inducible FAT10 as a novel negative regulator of RIG-I-mediated inflammatory response. In various cell lines, FAT10 protein is undetectable unless it is induced by pro-inflammatory cytokines. FAT10 non-covalently associated with the 2CARD domain of RIG-I, and inhibited viral RNA-induced IRF3 and NF-kB activation through modulating the RIG-I protein solubility. We further demonstrated that FAT10 was recruited to RIG-I-TRIM25 to form an inhibitory complex where FAT10 was stabilized by E3 ligase TRIM25. As the result, FAT10 inhibited the antiviral stress granules formation contains RIG-I and sequestered the active RIG-I away from the mitochondria. Our study presented a novel mechanism to dampen RIG-I activity. Highly accumulated FAT10 is observed in various cancers with pro-inflammatory environment, therefore, our finding which uncovered the suppressive effect of the accumulated FAT10 during virus-mediated inflammatory response may also provide molecular clue to understand the carcinogenesis related with infection and inflammation.
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Proteína 58 DEAD Box/metabolismo , Inflamación/metabolismo , Inflamación/virología , Transducción de Señal , Ubiquitinas/metabolismo , Células HEK293 , Humanos , Factor 3 Regulador del Interferón/metabolismo , FN-kappa B/metabolismo , Receptores Inmunológicos , Factores de Transcripción/metabolismo , Proteínas de Motivos Tripartitos/metabolismo , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Singleton-Merten syndrome (SMS) is an autosomal-dominant multi-system disorder characterized by dental dysplasia, aortic calcification, skeletal abnormalities, glaucoma, psoriasis, and other conditions. Despite an apparent autosomal-dominant pattern of inheritance, the genetic background of SMS and information about its phenotypic heterogeneity remain unknown. Recently, we found a family affected by glaucoma, aortic calcification, and skeletal abnormalities. Unlike subjects with classic SMS, affected individuals showed normal dentition, suggesting atypical SMS. To identify genetic causes of the disease, we performed exome sequencing in this family and identified a variant (c.1118A>C [p.Glu373Ala]) of DDX58, whose protein product is also known as RIG-I. Further analysis of DDX58 in 100 individuals with congenital glaucoma identified another variant (c.803G>T [p.Cys268Phe]) in a family who harbored neither dental anomalies nor aortic calcification but who suffered from glaucoma and skeletal abnormalities. Cys268 and Glu373 residues of DDX58 belong to ATP-binding motifs I and II, respectively, and these residues are predicted to be located closer to the ADP and RNA molecules than other nonpathogenic missense variants by protein structure analysis. Functional assays revealed that DDX58 alterations confer constitutive activation and thus lead to increased interferon (IFN) activity and IFN-stimulated gene expression. In addition, when we transduced primary human trabecular meshwork cells with c.803G>T (p.Cys268Phe) and c.1118A>C (p.Glu373Ala) mutants, cytopathic effects and a significant decrease in cell number were observed. Taken together, our results demonstrate that DDX58 mutations cause atypical SMS manifesting with variable expression of glaucoma, aortic calcification, and skeletal abnormalities without dental anomalies.
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Enfermedades de la Aorta/genética , ARN Helicasas DEAD-box/genética , Hipoplasia del Esmalte Dental/genética , Glaucoma/genética , Metacarpo/anomalías , Modelos Moleculares , Enfermedades Musculares/genética , Odontodisplasia/genética , Osteoporosis/genética , Calcificación Vascular/genética , Adulto , Enfermedades de la Aorta/patología , Secuencia de Bases , Células Cultivadas , Preescolar , Proteína 58 DEAD Box , ARN Helicasas DEAD-box/química , Hipoplasia del Esmalte Dental/patología , Exoma/genética , Femenino , Genes Dominantes/genética , Humanos , Masculino , Metacarpo/patología , Datos de Secuencia Molecular , Enfermedades Musculares/patología , Anomalías Musculoesqueléticas/diagnóstico por imagen , Anomalías Musculoesqueléticas/genética , Mutación Missense/genética , Odontodisplasia/diagnóstico por imagen , Odontodisplasia/patología , Osteoporosis/patología , Linaje , Polimorfismo de Nucleótido Simple/genética , Radiografía , Receptores Inmunológicos , Análisis de Secuencia de ADN , Calcificación Vascular/patologíaRESUMEN
The cellular RIG-I-like receptor (RLR) senses pathogenic RNA molecular patterns and transmits signals for type I interferon (IFN) production. It acts as a center for antiviral responses, and large numbers of RIG-I (retinoic acid inducible gene-I) interacting proteins are identified as signaling regulators. In the present study, we report PRKRIR, a negative regulator of PKR inhibitor, as a novel RIG-I interacting protein. In HEK293FT cells, PRKRIR synergistically enhances type I IFN production mediated by a signal activated- or constitutively active form of RIG-I. The C-terminal domain of the PRKRIR was required for physical interaction and the signal augmentation. The PRKRIR blocks poly-ubiquitination and protein degradation of RIG-I, thereby increasing cellular levels of RIG-I proteins. Furthermore, overexpression of PRKRIR, along with a signal activated- or constitutively active form of RIG-I, efficiently inhibits virus replication in the infected host. In conclusion, PRKRIR provides a novel positive regulator controlling the RIG-I-IFN production system through protein stability control.