RESUMEN
Recent methods for transcriptome-wide N6-methyladenosine (m6A) profiling have facilitated investigations into the RNA methylome and established m6A as a dynamic modification that has critical regulatory roles in gene expression and may play a role in human disease. However, bioinformatics resources available for the analysis of m6A sequencing data are still limited. Here, we describe m6aViewer-a cross-platform application for analysis and visualization of m6A peaks from sequencing data. m6aViewer implements a novel m6A peak-calling algorithm that identifies high-confidence methylated residues with more precision than previously described approaches. The application enables data analysis through a graphical user interface, and thus, in contrast to other currently available tools, does not require the user to be skilled in computer programming. m6aViewer and test data can be downloaded here: http://dna2.leeds.ac.uk/m6a.
Asunto(s)
Adenosina/análogos & derivados , Biología Computacional/métodos , Análisis de Secuencia de ARN/métodos , Programas Informáticos , Adenosina/análisis , Interfaz Usuario-ComputadorRESUMEN
BACKGROUND: Lethal fetal akinesia deformation sequence (FADS) describes a clinically and genetically heterogeneous phenotype that includes fetal akinesia, intrauterine growth retardation, arthrogryposis and developmental anomalies. Affected babies die as a result of pulmonary hypoplasia. We aimed to identify the underlying genetic cause of this disorder in a family in which there were three affected individuals from two sibships. METHODS: Autosomal-recessive inheritance was suggested by a family history of consanguinity and by recurrence of the phenotype between the two sibships. We performed exome sequencing of the affected individuals and their unaffected mother, followed by autozygosity mapping and variant filtering to identify the causative gene. RESULTS: Five autozygous regions were identified, spanning 31.7 Mb of genomic sequence and including 211 genes. Using standard variant filtering criteria, we excluded all variants as being the likely pathogenic cause, apart from a single novel nonsense mutation, c.188C>A p.(Ser63*) (NM_002478.4), in MYOD1. This gene encodes an extensively studied transcription factor involved in muscle development, which has nonetheless not hitherto been associated with a hereditary human disease phenotype. CONCLUSIONS: We provide the first description of a human phenotype that appears to result from MYOD1 mutation. The presentation with FADS is consistent with a large body of data demonstrating that in the mouse, MyoD is a major controller of precursor cell commitment to the myogenic differentiation programme.
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Artrogriposis/genética , Retardo del Crecimiento Fetal/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Proteína MioD/genética , Feto Abortado , Animales , Artrogriposis/patología , Exoma/genética , Femenino , Retardo del Crecimiento Fetal/patología , Humanos , Pulmón/patología , Ratones , Mutación , Linaje , Fenotipo , EmbarazoRESUMEN
MOTIVATION: Exome sequencing has become a de facto standard method for Mendelian disease gene discovery in recent years, yet identifying disease-causing mutations among thousands of candidate variants remains a non-trivial task. RESULTS: Here we describe a new variant prioritization tool, OVA (ontology variant analysis), in which user-provided phenotypic information is exploited to infer deeper biological context. OVA combines a knowledge-based approach with a variant-filtering framework. It reduces the number of candidate variants by considering genotype and predicted effect on protein sequence, and scores the remainder on biological relevance to the query phenotype.We take advantage of several ontologies in order to bridge knowledge across multiple biomedical domains and facilitate computational analysis of annotations pertaining to genes, diseases, phenotypes, tissues and pathways. In this way, OVA combines information regarding molecular and physical phenotypes and integrates both human and model organism data to effectively prioritize variants. By assessing performance on both known and novel disease mutations, we show that OVA performs biologically meaningful candidate variant prioritization and can be more accurate than another recently published candidate variant prioritization tool. AVAILABILITY AND IMPLEMENTATION: OVA is freely accessible at http://dna2.leeds.ac.uk:8080/OVA/index.jsp. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online. CONTACT: umaan@leeds.ac.uk.
Asunto(s)
Algoritmos , Ontologías Biológicas , Biología Computacional/métodos , Enfermedad/genética , Fenotipo , Polimorfismo de Nucleótido Simple/genética , Exoma/genética , Genotipo , Humanos , TranscriptomaRESUMEN
BACKGROUND: The widespread adoption of high-throughput sequencing technologies by genetic diagnostic laboratories has enabled significant expansion of their testing portfolios. Rare autosomal recessive conditions have been a particular focus of many new services. Here we report a cohort of 26 patients referred for genetic analysis of Joubert (JBTS) and Meckel-Gruber (MKS) syndromes, two clinically and genetically heterogeneous neurodevelopmental conditions that define a phenotypic spectrum, with MKS at the severe end. METHODS: Exome sequencing was performed for all cases, using Agilent SureSelect v5 reagents and Illumina paired-end sequencing. For two cases medium-coverage (9×) whole genome sequencing was subsequently undertaken. RESULTS: Using a standard analysis pipeline for the detection of single nucleotide and small insertion or deletion variants, molecular diagnoses were confirmed in 12 cases (4%). Seeking to determine whether our cohort harboured pathogenic copy number variants (CNV), in JBTS- or MKS-associated genes, targeted comparative read-depth analysis was performed using FishingCNV. These analyses identified a putative intragenic AHI1 deletion that included three exons spanning at least 3.4 kb and an intergenic MPP4 to TMEM237 deletion that included exons spanning at least 21.5 kb. Whole genome sequencing enabled confirmation of the deletion-containing alleles and precise characterisation of the mutation breakpoints at nucleotide resolution. These data were validated following development of PCR-based assays that could be subsequently used for "cascade" screening and/or prenatal diagnosis. CONCLUSIONS: Our investigations expand the AHI1 and TMEM237 mutation spectrum and highlight the importance of performing CNV screening of disease-associated genes. We demonstrate a robust increasingly cost-effective CNV detection workflow that is applicable to all MKS/JBTS referrals.
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Cerebelo/anomalías , Mapeo Cromosómico , Trastornos de la Motilidad Ciliar/diagnóstico , Trastornos de la Motilidad Ciliar/genética , Encefalocele/diagnóstico , Encefalocele/genética , Exoma , Enfermedades Renales Poliquísticas/diagnóstico , Enfermedades Renales Poliquísticas/genética , Retina/anomalías , Anomalías Múltiples/diagnóstico , Anomalías Múltiples/genética , Alelos , Estudios de Cohortes , Variaciones en el Número de Copia de ADN , Exones , Anomalías del Ojo/diagnóstico , Anomalías del Ojo/genética , Pruebas Genéticas , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Enfermedades Renales Quísticas/diagnóstico , Enfermedades Renales Quísticas/genética , Diagnóstico Prenatal , Retinitis Pigmentosa , Análisis de Secuencia de ADN , Eliminación de SecuenciaRESUMEN
Autozygosity mapping is a powerful technique for the identification of rare, autosomal recessive, disease-causing genes. The ease with which this category of disease gene can be identified has greatly increased through the availability of genome-wide SNP genotyping microarrays and subsequently of exome sequencing. Although these methods have simplified the generation of experimental data, its analysis, particularly when disparate data types must be integrated, remains time consuming. Moreover, the huge volume of sequence variant data generated from next generation sequencing experiments opens up the possibility of using these data instead of microarray genotype data to identify disease loci. To allow these two types of data to be used in an integrated fashion, we have developed AgileVCFMapper, a program that performs both the mapping of disease loci by SNP genotyping and the analysis of potentially deleterious variants using exome sequence variant data, in a single step. This method does not require microarray SNP genotype data, although analysis with a combination of microarray and exome genotype data enables more precise delineation of disease loci, due to superior marker density and distribution.
Asunto(s)
Variación Genética , Genotipo , Secuenciación de Nucleótidos de Alto Rendimiento , Polimorfismo de Nucleótido Simple , Programas Informáticos , Mapeo Cromosómico/métodos , Biología Computacional/métodos , Consanguinidad , Exoma , Estudios de Asociación Genética , Humanos , Patrón de Herencia , LinajeRESUMEN
Targeted hybridization enrichment prior to next-generation sequencing is a widespread method for characterizing sequence variation in a research setting, and is being adopted by diagnostic laboratories. However, the number of variants identified can overwhelm clinical laboratories with strict time constraints, the final interpretation of likely pathogenicity being a particular bottleneck. To address this, we have developed an approach in which, after automatic variant calling on a standard unix pipeline, subsequent variant filtering is performed interactively, using AgileExomeFilter and AgilePindelFilter (http://dna.leeds.ac.uk/agile), tools designed for clinical scientists with standard desktop computers. To demonstrate the method's diagnostic efficacy, we tested 128 patients using (1) a targeted capture of 36 cancer-predisposing genes or (2) whole-exome capture for diagnosis of the genetically heterogeneous disorder primary ciliary dyskinesia (PCD). In the cancer cohort, complete concordance with previous diagnostic data was achieved across 793 variant genotypes. A high yield (42%) was also achieved for exome-based PCD diagnosis, underscoring the scalability of our method. Simple adjustments to the variant filtering parameters further allowed the identification of a homozygous truncating mutation in a presumptive new PCD gene, DNAH8. These tools should allow diagnostic laboratories to expand their testing portfolios flexibly, using a standard set of reagents and techniques.
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Dineínas Axonemales/genética , Dineínas/genética , Pruebas Genéticas/métodos , Síndrome de Kartagener/diagnóstico , Neoplasias/diagnóstico , Codón sin Sentido , Genes Relacionados con las Neoplasias , Predisposición Genética a la Enfermedad , Variación Genética , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Síndrome de Kartagener/genética , Neoplasias/genética , Polimorfismo de Nucleótido Simple , Reproducibilidad de los Resultados , Programas Informáticos , Interfaz Usuario-ComputadorRESUMEN
Whole genome sequencing (WGS) has the potential to report on all types of genetic abnormality, thus converging diagnostic testing on a single methodology. Although WGS at sufficient depth for robust detection of point mutations is still some way from being affordable for diagnostic purposes, low-coverage WGS is already an excellent method for detecting copy number variants ("CNVseq"). We report on a family in which individuals presented with a presumed autosomal recessive syndrome of severe intellectual disability and epilepsy. Array comparative genomic hybridization (CGH) analysis had revealed a homozygous deletion apparently lying within intron 3 of CNTNAP2. Since this was too small for confirmation by FISH, CNVseq was used, refining the extent of this mutation to approximately 76.8 kb, encompassing CNTNAP2 exon 3 (an out-of-frame deletion). To characterize the precise breakpoints and provide a rapid molecular diagnostic test, we resequenced the CNVseq library at medium coverage and performed split read mapping. This yielded information for a multiplex polymerase chain reaction (PCR) assay, used for cascade screening and/or prenatal diagnosis in this family. This example demonstrates a rapid, low-cost approach to converting molecular cytogenetic findings into robust PCR-based tests.
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Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/genética , Proteínas del Tejido Nervioso/deficiencia , Proteínas del Tejido Nervioso/genética , Nucleótidos/genética , Eliminación de Secuencia/genética , Adolescente , Variaciones en el Número de Copia de ADN/genética , Exones/genética , Femenino , Humanos , Masculino , Técnicas de Diagnóstico Molecular/métodos , Mutación/genética , Linaje , Análisis de Secuencia de ADN/métodosRESUMEN
Massively parallel ("next generation") DNA sequencing (NGS) has quickly become the method of choice for seeking pathogenic mutations in rare uncharacterized monogenic diseases. Typically, before DNA sequencing, protein-coding regions are enriched from patient genomic DNA, representing either the entire genome ("exome sequencing") or selected mapped candidate loci. Sequence variants, identified as differences between the patient's and the human genome reference sequences, are then filtered according to various quality parameters. Changes are screened against datasets of known polymorphisms, such as dbSNP and the 1000 Genomes Project, in the effort to narrow the list of candidate causative variants. An increasing number of commercial services now offer to both generate and align NGS data to a reference genome. This potentially allows small groups with limited computing infrastructure and informatics skills to utilize this technology. However, the capability to effectively filter and assess sequence variants is still an important bottleneck in the identification of deleterious sequence variants in both research and diagnostic settings. We have developed an approach to this problem comprising a user-friendly suite of programs that can interactively analyze, filter and screen data from enrichment-capture NGS data. These programs ("Agile Suite") are particularly suitable for small-scale gene discovery or for diagnostic analysis.
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Exoma/genética , Predisposición Genética a la Enfermedad , Variación Genética , Análisis de Secuencia de ADN/métodos , Programas Informáticos , Biología Computacional/métodos , Genoma Humano/genética , Humanos , Polimorfismo de Nucleótido Simple/genéticaRESUMEN
The Kaposi's sarcoma-associated herpesvirus (KSHV) replication and transcription activator (RTA) protein regulates the latent-lytic switch by transactivating a variety of KSHV lytic and cellular promoters. RTA is a novel E3 ubiquitin ligase that targets a number of transcriptional repressor proteins for degradation by the ubiquitin proteasome pathway. Herein, we show that RTA interacts with the cellular transcriptional repressor protein Hey1. We demonstrate that Hey1 is a target for RTA-mediated ubiquitination and is subsequently degraded by the proteasome. Moreover, a Cys-plus-His-rich region within RTA is important for RTA-mediated degradation of Hey1. We confirm that Hey1 represses the RTA promoter and, furthermore, show that Hey1 binds to the RTA promoter. An interaction was observed between Hey1 and the corepressor mSin3A, and this interaction was abolished in the presence of RTA. Additionally, mSin3A associated with the RTA promoter in nonreactivated, but not reactivated, BCBL1 cells. Small interfering RNA knockdown of Hey1 in HEK 293T cells latently infected with the recombinant virus rKSHV.219 led to increased levels of RTA expression upon reactivation but was insufficient to induce complete lytic reactivation. These results suggest that other additional transcriptional repressors are also important in maintenance of KSHV latency. Taken together, our results suggest that Hey1 has a contributory role in the maintenance of KSHV latency and that disruption of the Hey1 repressosome by RTA-targeted degradation may be one step in the mechanism to regulate lytic reactivation.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Herpesvirus Humano 8/metabolismo , Proteínas Inmediatas-Precoces/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas Represoras/metabolismo , Transactivadores/metabolismo , Línea Celular , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Infecciones por Herpesviridae/metabolismo , Herpesvirus Humano 8/fisiología , Humanos , Regiones Promotoras Genéticas , Complejo Correpresor Histona Desacetilasa y Sin3 , Ubiquitinación , Latencia del VirusRESUMEN
Kaposi's sarcoma-associated herpesvirus 'replication transcriptional activator' (Rta) plays a critical role in the switch from latency to lytic replication. Rta upregulates several lytic KSHV genes, including its own, through multiple mechanisms. We demonstrate that cellular HMGB1 binds and synergistically upregulates the ORF50 promoter in conjunction with Rta. No direct interaction between Rta and HMGB1 was observed, however a ternary complex is formed in the presence of Oct1. Furthermore, deletion of an Oct-1 binding site within the ORF50 promoter ablates the HMGB1-mediated synergistic response. These results suggest Rta autostimulation may be mediated by a transient complex involving Oct1 and HMGB1.
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Regulación Viral de la Expresión Génica , Proteína HMGB1/metabolismo , Herpesvirus Humano 8/genética , Proteínas Inmediatas-Precoces/metabolismo , Transactivadores/metabolismo , Activación Transcripcional , Sitios de Unión , Línea Celular , Humanos , Sistemas de Lectura Abierta , Transportador 1 de Catión Orgánico/metabolismo , Regiones Promotoras Genéticas , Eliminación de SecuenciaRESUMEN
The positive strand RNA coronavirus, infectious bronchitis virus (IBV), induces a G2/M phase arrest and reduction in the G1 and G1/S phase transition regulator cyclin D1. Quantitative real-time RT-PCR and Western blot analysis demonstrated that cyclin D1 was reduced post-transcriptionally within infected cells independently of the cell-cycle stage at the time of infection. Confocal microscopy revealed that cyclin D1 decreased in IBV-infected cells as infection progressed and inhibition studies indicated that a population of cyclin D1 could be targeted for degradation by a virus mediated pathway. In contrast to the SARS-coronavirus, IBV nucleocapsid protein did not interact with cyclin D1.
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Coronavirus , Ciclina D1/análisis , Ciclina D1/metabolismo , Virus de la Bronquitis Infecciosa , Animales , Western Blotting , Chlorocebus aethiops , Ciclina D1/genética , Regulación hacia Abajo , ARN Mensajero/análisis , ARN Mensajero/metabolismo , ARN Viral/análisis , ARN Viral/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células VeroRESUMEN
Three isolates APMV/gull/Kazakhstan/5976/2014, APMV/gull/Kazakhstan/ 5977/2014 and APMV/gull/Kazakhstan/5979/2014, were obtained from independent samples during annual surveillance for avian influenza and paramyxoviruses in wild birds from the Caspian Sea coast in Western Kazakhstan, and were initially identified as putative paramyxoviruses on the basis of electron microscopy. Hemagglutination Inhibition Assays with antisera to nine known APMV serotypes (APMV1-9) indicated no relation to any of them. Next generation sequencing of whole genome sequences indicated the three isolates were genetically identical, and had a nucleotide structure typical for all APMVs, consisting of six genes 3'-NP-P-M-F-HN-L-5'. Phylogenetic analyses, and assessment of amino acid identities, suggested the most closely related lineages to be APMV-2, 8, 10 and 15, but the novel isolate had less than 64% identity to them and all other known avian paramyxoviruses. This value was above levels considered to generally define other APMV serotypes. Estimates of the evolutionary divergence of the nucleotide sequences of the genomes of APMVs have shown that novel Kazakhstan APMV strain was closest to APMV-2, APMV-8, APMV-10 and APMV-15, with calculated distance values of 2.057, 2.058, 2.026 and 2.286 respectively, which is above values considered to differentiate other serotypes (observed minimum was 1.108 between APMV-1 and recently isolated APMV/UPO216/Korea). Together, the data suggest that isolate APMV/gull/Kazakhstan/5976/2014 and other two should be considered as the first representative of a novel APMV-20 group, and is the first time that avian paramyxoviruses have been found infecting members of the gull family, extending the known taxonomic host range.
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Avulavirus/aislamiento & purificación , Charadriiformes/virología , Animales , Avulavirus/genética , Genoma Viral , KazajstánRESUMEN
Next generation sequencing methodologies are facilitating the rapid characterisation of novel structural variants at nucleotide resolution. These approaches are particularly applicable to variants initially identified using alternative molecular methods. We report a child born with bilateral postaxial syndactyly of the feet and bilateral fifth finger clinodactyly. This was presumed to be an autosomal recessive syndrome, due to the family history of consanguinity. Karyotype analysis revealed a homozygous pericentric inversion of chromosome 7 (46,XX,inv(7)(p15q21)x2) which was confirmed to be heterozygous in both unaffected parents. Since the resolution of the karyotype was insufficient to identify any putatively causative gene, we undertook medium-coverage whole genome sequencing using paired-end reads, in order to elucidate the molecular breakpoints. In a two-step analysis, we first narrowed down the region by identifying discordant read-pairs, and then determined the precise molecular breakpoint by analysing the mapping locations of "soft-clipped" breakpoint-spanning reads. PCR and Sanger sequencing confirmed the identified breakpoints, both of which were located in intergenic regions. Significantly, the 7p15 breakpoint was located 523 kb upstream of HOXA13, the locus for hand-foot-genital syndrome. By inference from studies of HOXA locus control in the mouse, we suggest that the inversion has delocalised a HOXA13 enhancer to produce the phenotype observed in our patient. This study demonstrates how modern genetic diagnostic approach can characterise structural variants at nucleotide resolution and provide potential insights into functional regulation.
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Anomalías Múltiples/genética , Inversión Cromosómica , Cromosomas Humanos Par 7/genética , Deformidades Congénitas del Pie/genética , Deformidades Congénitas de la Mano/genética , Proteínas de Homeodominio/genética , Anomalías Urogenitales/genética , Puntos de Rotura del Cromosoma , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Recién Nacido , Análisis de Secuencia de ADNRESUMEN
Herpesvirus saimiri (HVS) establishes a persistent infection in which the viral genome persists as a circular non-integrated episome. ORF73 tethers HVS episomes to host mitotic chromosomes, allowing episomal persistence via an interaction with the chromosome-associated protein, MeCP2. Here we demonstrate that ORF73 also interacts with the linker histone H1 via its C terminus, suggesting it associates with multiple chromosome-associated proteins. In addition, we show that the C terminus is also required for the ability of ORF73 to bind the terminal repeat region of the HVS genome. These results suggest that the ORF73 C terminus contains all the necessary elements required for HVS episomal persistence. Using a range of ORF73 C terminus deletions to rescue the episomal maintenance properties of a HVSDelta73 recombinant virus, we show that a C terminus region comprising residues 285-407 is sufficient to maintain the HVS episome in a dividing cell population.
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Antígenos Virales/genética , Proteínas Portadoras/genética , Proteínas Nucleares/genética , Proteínas Virales/genética , Animales , Antígenos Virales/química , Proteínas Portadoras/química , División Celular , Línea Celular , Genes Reporteros , Herpesvirus Saimiriino 2 , Humanos , Proteínas Nucleares/química , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Saimiri/virología , Transfección , Proteínas Virales/química , Replicación ViralRESUMEN
The nucleolus is a dynamic sub-nuclear structure which is involved in ribosome subunit biogenesis, modulation of cell growth and response to cell stress. The nucleolar proteome varies particularly with regard to the cell cycle. Viral proteins can localise to the nucleolus and using the coronavirus nucleocapsid (N) protein as a model, the cell cycle dependent trafficking of viral proteins to the nucleolus was investigated. Cell synchronisation studies coupled to live cell confocal microscopy indicated that nucleolar localisation of N protein was greater in the G2/M phase of the cell cycle than at other stages. This result was reinforced when FRAP and FLIP analysis indicated that N protein was more mobile within the nucleoplasm and nucleolus in the G2/M phase of the cell cycle. The data suggested that viral nucleolar proteins can also localise to the nucleolus in a cell cycle dependent manner and this may be related to dynamic trafficking.
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Ciclo Celular/genética , Nucléolo Celular/metabolismo , Coronavirus/metabolismo , Proteínas de la Nucleocápside/metabolismo , Transporte Activo de Núcleo Celular/fisiología , Animales , Compartimento Celular/fisiología , Nucléolo Celular/genética , Chlorocebus aethiops , Coronavirus/genética , Infecciones por Coronavirus/genética , Infecciones por Coronavirus/metabolismo , Proteínas de la Nucleocápside de Coronavirus , Recuperación de Fluorescencia tras Fotoblanqueo , Fase G2/fisiología , Microscopía Confocal , Proteínas de la Nucleocápside/genética , Transporte de Proteínas/fisiología , Células VeroRESUMEN
The avian coronavirus infectious bronchitis virus (IBV) is a major economic pathogen of domestic poultry that, despite vaccination, causes mortality and significant losses in production. During replication of the RNA genome there is a high frequency of mutation and recombination, which has given rise to many strains of IBV and results in the potential for new and emerging strains. Currently the live-attenuated vaccine gives poor cross-strain immunity. Effective antiviral agents may therefore be advantageous in the treatment of IBV. Lithium chloride (LiCl) is a potent inhibitor of the DNA virus herpes simplex virus but not RNA viruses. The effect of LiCl on the replication of IBV was examined in cell culture using two model cell types; Vero cells, an African Green monkey kidney-derived epithelial cell line; and DF-1 cells, an immortalized chicken embryo fibroblast cell line. When treated with a range of LiCl concentrations, IBV RNA and protein levels and viral progeny production were reduced in a dose-dependent manner in both cell types, and the data indicated that inhibition was a cellular rather than a virucidal effect. Host cell protein synthesis still took place in LiCl-treated cells and the level of a standard cellular housekeeping protein remained unchanged, indicating that the effect of LiCl was specifically against IBV.
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Virus de la Bronquitis Infecciosa/efectos de los fármacos , Cloruro de Litio/farmacología , Animales , Embrión de Pollo , Chlorocebus aethiops , Relación Dosis-Respuesta a Droga , Regulación Viral de la Expresión Génica/efectos de los fármacos , ARN Viral/metabolismo , Células Vero , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
The nucleocapsid (N) protein of infectious bronchitis virus (IBV) localizes to the cytoplasm and nucleolus and contains an eight-amino-acid nucleolar retention motif. In this study, a leucine-rich nuclear export signal (NES) (291-LQLDGLHL-298) present in the C-terminal region of the IBV N protein was analyzed by using alanine substitution and deletion mutagenesis to investigate the relative contributions that leucine residues make to nuclear export and where these residues are located on the structure of the IBV N protein. The analysis indicated that Leu296 and Leu298 are required for efficient nuclear export of the protein. Structural information indicated that both of these amino acids are available for interaction with protein complexes involved in this process. However, export of N protein from the nucleus/nucleolus was not inhibited by leptomycin B treatment, indicating that N protein nuclear export is independent of the CRM1-mediated export pathway.