RESUMEN
Rift Valley fever (RVF) is an emerging viral zoonosis that primarily affects ruminants and humans. We have previously shown that wild-derived MBT/Pas mice are highly susceptible to RVF virus and that part of this phenotype is controlled by a locus located on distal Chromosome 11. Using congenic strains, we narrowed down the critical interval to a 530 kb region containing five protein-coding genes among which Rnf213 emerged as a potential candidate. We generated Rnf213-deficient mice by CRISPR/CAS9 on the C57BL/6 J background and showed that they were significantly more susceptible to RVF than control mice, with an average survival time post-infection reduced from 7 to 4 days. The human RNF213 gene had been associated with the cerebrovascular Moyamoya disease (MMD or MYMY) but the inactivation of this gene in the mouse resulted only in mild anomalies of the neovascularization. This study provides the first evidence that the Rnf213 gene may also impact the resistance to infectious diseases such as RVF.
Asunto(s)
Adenosina Trifosfatasas/genética , Resistencia a la Enfermedad/genética , Interacciones Huésped-Patógeno/genética , Fiebre del Valle del Rift/genética , Fiebre del Valle del Rift/virología , Virus de la Fiebre del Valle del Rift/fisiología , Ubiquitina-Proteína Ligasas/genética , Animales , Sistemas CRISPR-Cas , Mapeo Cromosómico , Modelos Animales de Enfermedad , Ratones , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
Infection of mice with Rift Valley fever virus (RVFV) reproduces major pathological features of severe human disease, notably the early-onset hepatitis and delayed-onset encephalitis. We previously reported that the Rvfs2 locus from the susceptible MBT/Pas strain reduces survival time after RVFV infection. Here, we used BALB/cByJ (BALB) mice congenic for Rvfs2 (C.MBT-Rvfs2) to investigate the pathophysiological mechanisms impacted by Rvfs2. Clinical, biochemical and histopathological features indicated similar liver damage in BALB and C.MBT-Rvfs2 mice until day 5 after infection. However, while C.MBT-Rvfs2 mice succumbed from acute liver injury, most BALB mice recovered and died later of encephalitis. Hepatocytes of BALB infected liver proliferated actively on day 6, promoting organ regeneration and recovery from liver damage. By comparison with C.MBT-Rvfs2, BALB mice had up to 100-fold lower production of infectious virions in the peripheral blood and liver, strongly decreased RVFV protein in liver and reduced viral replication in primary cultured hepatocytes, suggesting that the BALB Rvfs2 haplotype limits RVFV pathogenicity through decreased virus replication. Moreover, bone marrow chimera experiments showed that both hematopoietic and non-hematopoietic cells are required for the protective effect of the BALB Rvfs2 haplotype. Altogether, these results indicate that Rvfs2 controls critical events which allow survival to RVFV-induced hepatitis.
Asunto(s)
Cromosomas Humanos Par 11/genética , Sitios Genéticos , Hepatitis/mortalidad , Encefalitis Infecciosa/mortalidad , Fiebre del Valle del Rift/genética , Virus de la Fiebre del Valle del Rift/patogenicidad , Animales , Proliferación Celular , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades , Hepatitis/virología , Humanos , Encefalitis Infecciosa/virología , Hígado/citología , Hígado/virología , Masculino , Ratones , Ratones Congénicos , Ratones Endogámicos BALB C , Fiebre del Valle del Rift/complicaciones , Fiebre del Valle del Rift/mortalidadRESUMEN
Rift Valley fever virus (RVFV) leads to varied clinical manifestations in animals and in humans that range from moderate fever to fatal illness, suggesting that host immune responses are important determinants of the disease severity. We investigated the immune basis for the extreme susceptibility of MBT/Pas mice that die with mild to acute hepatitis by day 3 post-infection compared to more resistant BALB/cByJ mice that survive up to a week longer. Lower levels of neutrophils observed in the bone marrow and blood of infected MBT/Pas mice are unlikely to be causative of increased RVFV susceptibility as constitutive neutropenia in specific mutant mice did not change survival outcome. However, whereas MBT/Pas mice mounted an earlier inflammatory response accompanied by higher amounts of interferon (IFN)-α in the serum compared to BALB/cByJ mice, they failed to prevent high viral antigen load. Several immunological alterations were uncovered in infected MBT/Pas mice compared to BALB/cByJ mice, including low levels of leukocytes that expressed type I IFN receptor subunit 1 (IFNAR1) in the blood, spleen and liver, delayed leukocyte activation and decreased percentage of IFN-γ-producing leukocytes in the blood. These observations are consistent with the complex mode of inheritance of RVFV susceptibility in genetic studies.
Asunto(s)
Inmunidad Innata , Fiebre del Valle del Rift/inmunología , Fiebre del Valle del Rift/virología , Virus de la Fiebre del Valle del Rift/inmunología , Animales , Antígenos Virales/inmunología , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades , Hepatitis Viral Animal/genética , Hepatitis Viral Animal/inmunología , Hepatitis Viral Animal/virología , Recuento de Leucocitos , Hígado/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , Neutrófilos/inmunología , Neutrófilos/metabolismo , Fiebre del Valle del Rift/genética , Fiebre del Valle del Rift/patología , Bazo/inmunologíaRESUMEN
Targeted induction of double-strand breaks (DSBs) at natural endogenous loci was shown to increase the rate of gene replacement by homologous recombination in mouse embryonic stem cells. The gene encoding dopachrome tautomerase (Dct) is specifically expressed in melanocytes and their precursors. To construct a genetic tool allowing the replacement of Dct gene by any gene of interest, we generated an embryonic stem cell line carrying the recognition site for the yeast I-SceI meganuclease embedded in the Dct genomic segment. The embryonic stem cell line was electroporated with an I-SceI expression plasmid, and a template for the DSB-repair process that carried sequence homologies to the Dct target. The I-SceI meganuclease was indeed able to introduce a DSB at the Dct locus in live embryonic stem cells. However, the level of gene targeting was not improved by the DSB induction, indicating a limited capacity of I-SceI to mediate homologous recombination at the Dct locus. These data suggest that homologous recombination by meganuclease-induced DSB may be locus dependent in mammalian cells.
Asunto(s)
Daño del ADN , Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Células Madre Embrionarias/metabolismo , Recombinación Homóloga , Oxidorreductasas Intramoleculares/genética , Alelos , Animales , Secuencia de Bases , Cartilla de ADN , Marcación de Gen , Ratones , Reacción en Cadena de la PolimerasaRESUMEN
Susceptibility of inbred strains to infection with West Nile virus (WNV) has been genetically associated with an arginine-to-a nonsense codon substitution at position 253 (R253X) in the predicted sequence of the murine 2',5'-oligoadenylate synthetase 1B (OAS1B) protein. We introduced by transgenesis the Oas1b cDNA from MBT/Pas mice carrying the R253 codon (Oas1b(MBT)) into BALB/c mice homozygous for the X253 allele (Oas1b(BALB/c)). Overexpression of Oas1b(MBT) mRNA in the brain of transgenic mice prior and in the time course of infection provided protection against the neuroinvasive WNV strain IS-98-ST1. A 200-fold induction of Oas1b(MBT) mRNA in the brain of congenic BALB/c mice homozygous for a MBT/Pas segment encompassing the Oas1b gene was also efficient in reducing both viral growth and mortality, whereas a 200-fold induction of Oas1b(BALB/c) mRNA was unable to prevent virally-induced encephalitis, confirming the critical role of the R253X mutation on Oas1b activity in live mice.
Asunto(s)
2',5'-Oligoadenilato Sintetasa/genética , 2',5'-Oligoadenilato Sintetasa/metabolismo , Predisposición Genética a la Enfermedad , Fiebre del Nilo Occidental/genética , Virus del Nilo Occidental/fisiología , Animales , Regulación Enzimológica de la Expresión Génica , Ingeniería Genética , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Fiebre del Nilo Occidental/inmunología , Fiebre del Nilo Occidental/virologíaRESUMEN
The recessive mutation oligotriche (olt) affects the coat and male fertility in the mouse. In homozygous (olt/olt) mutants, the coat is sparse, most notably in the inguinal and medial femoral region. In these regions, almost all hair shafts are bent and distorted in their course through the dermis and rarely penetrate the epidermis because the hair cortex is not fully keratinized. During hair follicle morphogenesis, mutant hair follicles exit from anagen one day before those of normal littermates and show a prolongation of the catagen stage. The oligotriche (olt) locus was mapped to distal chromosome 9 within a 5-Mbp interval distal to D9Mit279. Analysis of candidate gene expression revealed that olt/olt mutant mice do not express functional phospholipase C delta 1 (Plcd1) mRNA. This deficiency is the consequence of a 234-kbp deletion involving not only the Plcd1 locus but also the chromosomal segment harboring the genes Vill (villin-like), Dlec1 (deleted in lung and esophageal cancer 1), Acaa1b (acetyl-Coenzyme A acyltransferase 1B, synonym thiolase B), and parts of the genes Ctdspl (carboxy-terminal domain RNA polymerase II polypeptide A small phosphatase-like) and Slc22a14 (solute carrier family 22 member 14). Offspring of olt/olt females, mated with Plcd1 ( -/- ) knockout males, exhibit coat defects similar to those observed in homozygous olt/olt mutant mice but the spermiogenesis in male offspring is normal. We conclude that the 234-kbp deletion from chromosome 9 harbors a gene involved in spermiogenesis and we propose that the oligotriche mutant be used as a model for the study of the putative tumor suppressor genes Dlec1, Ctdspl, and Vill. We also suggest that the oligotriche locus be named Del(9Ctdspl-Slc22a14)1Pas.
Asunto(s)
Alopecia/genética , Deleción Cromosómica , Cromosomas Humanos Par 9/genética , Infertilidad Masculina/genética , Mutación , Animales , Modelos Animales de Enfermedad , Femenino , Folículo Piloso/metabolismo , Humanos , Masculino , Ratones , Ratones Pelados , Ratones Endogámicos C3H , Ratones Transgénicos , Fosfolipasa C delta/genética , EspermatogénesisRESUMEN
Positional cloning of two recessive mutations of the mouse that cause polysyndactyly (dan and mdig-Chr 2) confirmed that the gene encoding MEGF7/LRP4, a member of the low-density lipoprotein receptor family, plays an essential role in the process of digit differentiation. Pathologies observed in the mutant mice provide insight into understanding the function(s) of LRP4 as a negative regulator of the Wnt-beta-catenin signaling pathway and may help identify the genetic basis for common human disorders with similar phenotypes.
Asunto(s)
Miembro Anterior/anomalías , Receptores de LDL/genética , Animales , Animales Modificados Genéticamente , Secuencia de Bases , Análisis Mutacional de ADN , Femenino , Miembro Anterior/diagnóstico por imagen , Proteínas Relacionadas con Receptor de LDL , Masculino , Ratones , Datos de Secuencia Molecular , Mutagénesis Insercional , Mutación , Sistemas de Lectura Abierta , Radiografía , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Análisis de Secuencia de ADN , Sindactilia/diagnóstico , Sindactilia/genéticaRESUMEN
2',5'-Oligoadenylate synthetases (2',5'-OASs) are interferon-inducible enzymes. Some of these proteins play an important role in cellular physiology, in particular, in the innate defense mechanisms against RNA virus infections. In the present publication we report the complete genomic structure of the cluster of genes encoding mouse 2',5'-OAS, with all its transcription units, their predicted functions, and their evolutionary relationships. We found that mouse Oas2/Oas3 genes have a genomic structure similar to that of human OAS2/OAS3, while the mouse equivalent of human OAS1 is composed of eight (Oas1a to Oas1h) tandemly arranged transcription units. For all these eight genes a specific inducible promoter controls transcription. The possible functions of this family of proteins are discussed.
Asunto(s)
2',5'-Oligoadenilato Sintetasa/química , 2',5'-Oligoadenilato Sintetasa/genética , Evolución Molecular , Familia de Multigenes , Secuencia de Aminoácidos , Animales , Expresión Génica , Ligamiento Genético , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos , Datos de Secuencia Molecular , Filogenia , Mapeo Físico de Cromosoma , Isoformas de Proteínas , Alineación de Secuencia , Transcripción GenéticaRESUMEN
SCO-spondin is specifically expressed in the subcommissural organ (SCO), a secretory ependymal differentiation lining the roof of the third ventricular cavity of the brain. When released into the cerebro-spinal fluid (CSF), SCO-spondin aggregates and forms Reissner's fiber (RF), a structure present in the central canal of the spinal cord. SCO-spondin belongs to the superfamily of proteins exhibiting conserved motifs called TSRs for 'thrombospondin type 1 repeats' and involved in axonal pathfinding during development. The mouse SCO-spondin coding sequence was searched by alignement of the coding bovine SCO-spondin sequence with the mouse whole genome shotgun (WGS) supercontig (NW 000250). Compared to the bovine, mouse SCO-spondin shows 66.8% identity of amino acids. This extracellular matrix glycoprotein has a modular arrangement of several conserved domains including 25 TSRs, 10 low-density lipoprotein receptor (LDLr) type A repeats and cystein-rich regions in the -NH2 and -COOH ends. The spatio-temporal expression of SCO-spondin was analyzed using specific antisera and an homospecific SCO-spondin riboprobe. In the adult, the patterns obtained by in situ hybridization (ISH) and immunohistochemistry correlated well in the SCO, while Reissner's fiber and the ampulla caudalis were immunoreactive only. In the fetus, both the immuno and ISH reactions appeared between 14 and 15 days post coïtum (dpc) in the SCO anlage. In addition, the mouse SCO-spondin gene was located at chromosome 6, between marker D6Mit352 and D6Mit119, in a conserved syntenic region.
Asunto(s)
Encéfalo/embriología , Moléculas de Adhesión Celular Neuronal/genética , Secuencia de Aminoácidos , Animales , Northern Blotting , Encéfalo/crecimiento & desarrollo , Bovinos , Moléculas de Adhesión Celular Neuronal/metabolismo , ADN Complementario/química , ADN Complementario/genética , Regulación del Desarrollo de la Expresión Génica , Humanos , Inmunohistoquímica , Hibridación in Situ , Ratones , Datos de Secuencia Molecular , ARN Mensajero/genética , ARN Mensajero/metabolismo , Mapeo de Híbrido por Radiación , Secuencias Repetitivas de Ácidos Nucleicos/genética , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Trombospondina 1/genéticaRESUMEN
Over the past 7 years, West Nile zoonosis has been an emerging concern for public health in Europe, Middle East and more recently in North America. West Nile virus causes epidemic outbreaks in humans and infected patients may exhibit severe neurological symptoms. Because susceptibility and sensitivity to West Nile virus infections may depend on host genetic factors, a mouse model has been established to investigate the genetic determinism of host susceptibility to West Nile virus. A nonsense mutation in gene encoding the 1b isoform of the 2'-5'oligoadenylate synthetase (OAS1b) was constantly associated with the susceptibility of mouse strains to experimental West Nile virus infection. Oligoadenylate synthetase are interferon-inducible proteins playing a role in the endogeneous antiviral pathway. It was of interest to establish whether interferon-alpha and OAS 1B were sufficient to mediate resistance to West Nile virus infection. In the present study, we showed that interferon-alpha had the ability to modulate West Nile virus infection in mouse. In vitro, interferon-alpha protected mouse neuroblastoma cells against West Nile virus infection if cells have been pretreated with the cytokine for several hours. As a consequence of the presence of a stop codon, the Oas1b gene of the susceptible mice encodes a truncated and presumably inactive form, while resistant mice have a normal copy of the gene. Stable mouse neuroblastoma cell clones overexpressing mutant or wild-type OAS 1B were established. Replication of West Nile virus was less efficient in cells that produce the normal copy of OAS 1B as compared to those expressing the truncated form. Our data illustrate the notion that interferon-alpha and Oas genes may be critical for West Nile virus pathogenesis.
Asunto(s)
2',5'-Oligoadenilato Sintetasa/genética , Predisposición Genética a la Enfermedad , Neuronas/virología , Fiebre del Nilo Occidental/genética , Virus del Nilo Occidental/patogenicidad , 2',5'-Oligoadenilato Sintetasa/efectos de los fármacos , 2',5'-Oligoadenilato Sintetasa/inmunología , Animales , Células Cultivadas , Interferón-alfa/farmacología , Ratones , Mutación , Fiebre del Nilo Occidental/tratamiento farmacológico , Fiebre del Nilo Occidental/inmunología , Virus del Nilo Occidental/genética , Virus del Nilo Occidental/inmunologíaRESUMEN
A mouse model has been established to investigate the genetic determinism of host susceptibility to West Nile (WN) virus, a member of the genus flavivirus and family Flaviviridae. Whereas WN virus causes encephalitis and death in most laboratory inbred mouse strains after peripheral inoculation, most strains derived from recently trapped wild mice are completely resistant. The phenotype of resistance/susceptibility is determined by a major locus, Wnv, mapping to chromosome 5 within the 0.4-cM-wide interval defined by markers D5Mit408 and D5Mit242. We constructed a high resolution composite/consensus map of the interval by merging the data from the mouse T31 Radiation Hybrid map and those from the homologous region of human chromosome 12q, and found the cluster of genes encoding 2'-5'-oligoadenylate synthetases (2'-5'-OAS) to be the most prominent candidate. This cluster encodes a multimember family of IFN-inducible proteins that is known to play an important role in the established endogenous antiviral pathway. Comparing the cDNA sequences of 2'-5'-OAS L1, L2, and L3 isoforms, between susceptible and resistant strains, we identified a STOP codon in exon 4 of the gene encoding the L1 isoform in susceptible strains that can lead to a truncated form with amputation of one domain, whereas all resistant mice tested so far have a normal copy of this gene. The observation that WN virus sensitivity of susceptible mice was completely correlated with the occurrence of a point mutation in 2'-5'-OAS L1 suggests that this isoform may play a critical role in WN pathogenesis.