RESUMEN
Effective drugs against SARS-CoV-2 are urgently needed to treat severe cases of infection and for prophylactic use. The main viral protease (nsp5 or 3CLpro) represents an attractive and possibly broad-spectrum target for drug development as it is essential to the virus life cycle and highly conserved among betacoronaviruses. Sensitive and efficient high-throughput screening methods are key for drug discovery. Here we report the development of a gain-of-signal, highly sensitive cell-based luciferase assay to monitor SARS-CoV-2 nsp5 activity and show that it is suitable for the screening of compounds in a 384-well format. A benefit of miniaturisation and automation is that screening can be performed in parallel on a wild-type and a catalytically inactive nsp5, which improves the selectivity of the assay. We performed molecular docking-based screening on a set of 14,468 compounds from an in-house chemical database, selected 359 candidate nsp5 inhibitors and tested them experimentally. We identified two molecules which show anti-nsp5 activity, both in our cell-based assay and in vitro on purified nsp5 protein, and inhibit SARS-CoV-2 replication in A549-ACE2 cells with EC50 values in the 4-8 µM range. The here described high-throughput-compatible assay will allow the screening of large-scale compound libraries for SARS-CoV-2 nsp5 inhibitors. Moreover, we provide evidence that this assay can be adapted to other coronaviruses and viruses which rely on a viral protease.
Asunto(s)
Tratamiento Farmacológico de COVID-19 , SARS-CoV-2 , Antivirales/química , Antivirales/farmacología , Humanos , Luciferasas/genética , Simulación del Acoplamiento Molecular , Péptido Hidrolasas , Inhibidores de Proteasas/farmacología , Proteasas ViralesRESUMEN
Effective drugs against SARS-CoV-2 are urgently needed to treat severe cases of infection and for prophylactic use. The main viral protease (nsp5 or 3CLpro) represents an attractive and possibly broad-spectrum target for drug development as it is essential to the virus life cycle and highly conserved among betacoronaviruses. Sensitive and efficient high-throughput screening methods are key for drug discovery. Here we report the development of a gain-of-signal, highly sensitive cell-based luciferase assay to monitor SARS-CoV-2 nsp5 activity and show that it is suitable for high-throughput screening of compounds in a 384-well format. A benefit of miniaturisation and automation is that screening can be performed in parallel on a wild-type and a catalytically inactive nsp5, which improves the selectivity of the assay. We performed molecular docking-based screening on a set of 14,468 compounds from an in-house chemical database, selected 359 candidate nsp5 inhibitors and tested them experimentally. We identified four molecules, including the broad-spectrum antiviral merimepodib/VX-497, which show anti-nsp5 activity and inhibit SARS-CoV-2 replication in A549-ACE2 cells with IC 50 values in the 4-21 µM range. The here described assay will allow the screening of large-scale compound libraries for SARS-CoV-2 nsp5 inhibitors. Moreover, we provide evidence that this assay can be adapted to other coronaviruses and viruses which rely on a viral protease.
RESUMEN
The major goals of Kawasaki disease (KD) therapy are to reduce inflammation and prevent thrombosis in the coronary arteries (CA), but some children do not respond to currently available non-specific therapies. New treatments have been difficult to develop because the molecular pathogenesis is unknown. In order to identify dysregulated gene expression in KD CA, we performed high-throughput RNA sequencing on KD and control CA, validated potentially dysregulated genes by real-time reverse transcription-polymerase chain reaction (RT-PCR) and localized protein expression by immunohistochemistry. Signalling lymphocyte activation molecule CD84 was up-regulated 16-fold (P < 0·01) in acute KD CA (within 2 months of onset) and 32-fold (P < 0·01) in chronic CA (5 months to years after onset). CD84 was localized to inflammatory cells in KD tissues. Genes associated with cellular proliferation, motility and survival were also up-regulated in KD CA, and immune activation molecules MX2 and SP140 were up-regulated in chronic KD. CD84, which facilitates immune responses and stabilizes platelet aggregates, is markedly up-regulated in KD CA in patients with acute and chronic arterial disease. We provide the first molecular evidence of dysregulated inflammatory responses persisting for months to years in CA significantly damaged by KD.
Asunto(s)
Antígenos CD/metabolismo , Antígenos Nucleares/metabolismo , Plaquetas/inmunología , Síndrome Mucocutáneo Linfonodular/inmunología , Proteínas de Resistencia a Mixovirus/metabolismo , Factores de Transcripción/metabolismo , Calcificación Vascular/inmunología , Enfermedad Aguda , Antígenos CD/genética , Antígenos Nucleares/genética , Procesos de Crecimiento Celular/genética , Movimiento Celular/genética , Supervivencia Celular/genética , Enfermedad Crónica , Vasos Coronarios/patología , Femenino , Ensayos Analíticos de Alto Rendimiento , Humanos , Lactante , Masculino , Síndrome Mucocutáneo Linfonodular/sangre , Síndrome Mucocutáneo Linfonodular/genética , Proteínas de Resistencia a Mixovirus/genética , Agregación Plaquetaria/genética , ARN Mensajero/análisis , Familia de Moléculas Señalizadoras de la Activación Linfocitaria , Factores de Transcripción/genética , Regulación hacia Arriba , Calcificación Vascular/sangre , Calcificación Vascular/genéticaRESUMEN
The PathoSystems Resource Integration Center (PATRIC) is one of eight Bioinformatics Resource Centers (BRCs) funded by the National Institute of Allergy and Infection Diseases (NIAID) to create a data and analysis resource for selected NIAID priority pathogens, specifically proteobacteria of the genera Brucella, Rickettsia and Coxiella, and corona-, calici- and lyssaviruses and viruses associated with hepatitis A and E. The goal of the project is to provide a comprehensive bioinformatics resource for these pathogens, including consistently annotated genome, proteome and metabolic pathway data to facilitate research into counter-measures, including drugs, vaccines and diagnostics. The project's curation strategy has three prongs: 'breadth first' beginning with whole-genome and proteome curation using standardized protocols, a 'targeted' approach addressing the specific needs of researchers and an integrative strategy to leverage high-throughput experimental data (e.g. microarrays, proteomics) and literature. The PATRIC infrastructure consists of a relational database, analytical pipelines and a website which supports browsing, querying, data visualization and the ability to download raw and curated data in standard formats. At present, the site warehouses complete sequences for 17 bacterial and 332 viral genomes. The PATRIC website (https://patric.vbi.vt.edu) will continually grow with the addition of data, analysis and functionality over the course of the project.
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Bioterrorismo , Bases de Datos Genéticas , Proteobacteria/genética , Virus ARN/genética , Genómica , Internet , Proteobacteria/metabolismo , Proteobacteria/patogenicidad , Proteómica , Virus ARN/metabolismo , Virus ARN/patogenicidad , Integración de Sistemas , Interfaz Usuario-ComputadorAsunto(s)
Coronavirus/metabolismo , Síndrome Mucocutáneo Linfonodular/virología , Enfermedad Aguda , Niño , Preescolar , Infecciones por Coronavirus/virología , Cartilla de ADN/química , Genoma Viral , Humanos , Lactante , Modelos Genéticos , Síndrome Mucocutáneo Linfonodular/etiología , ARN Viral/metabolismo , Infecciones del Sistema Respiratorio/virología , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
We describe a mathematical model of signal from single-channel direct hybridization microarray platforms. The model establishes a linear relationship between microarray signals and their standard deviations from a minimum set of assumptions. We use the model to precisely define important microarray quality characteristics: resolved fold change and dynamic range. The definitions lead to closed form expressions relating these characteristics to physical parameters of the microarray experiment in the case when both specific and nonspecific binding of target to probe are governed by the Langmuir hybridization isotherm. The predictions of the model are in close agreement to data obtained from spike-in experiments. Given the generality of the model, the introduced definitions of dynamic range and resolved concentration fold-change can be used to conduct cross-platform comparisons and to guide improvement of the microarray platform.
Asunto(s)
Biología Computacional , Modelos Genéticos , Análisis de Secuencia por Matrices de Oligonucleótidos/estadística & datos numéricos , Interpretación Estadística de DatosRESUMEN
The HUELLENLOS (HLL) gene participates in patterning and growth of the Arabidopsis ovule. We have isolated the HLL gene and shown that it encodes a protein homologous to the L14 proteins of eubacterial ribosomes. The Arabidopsis genome also includes a highly similar gene, HUELLENLOS PARALOG (HLP), and genes for both cytosolic (L23) and chloroplast ribosome L14 proteins. Phylogenetic analysis shows that HLL and HLP differ significantly from these other two classes of such proteins. HLL and HLP fusions to green fluorescent protein were localized to mitochondria. Ectopic expression of HLP complemented the hll mutant, indicating that HLP and HLL share redundant functions. We conclude that HLL and HLP encode L14 subunits of mitochondrial ribosomes. HLL mRNA was at significantly higher levels than HLP mRNA in pistils, with the opposite pattern in leaves. This differential expression can explain the confinement of effects of hll mutations to gynoecia and ovules. Our elucidation of the nature of HLL shows that metabolic defects can have specific effects on developmental patterning.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Mitocondrias/metabolismo , Proteínas Mitocondriales , Proteínas Recombinantes , Proteínas Ribosómicas/genética , Secuencia de Aminoácidos , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/clasificación , Proteínas de Arabidopsis/metabolismo , Mapeo Cromosómico , Regulación del Desarrollo de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Datos de Secuencia Molecular , Mutagénesis , Fenotipo , Filogenia , Tallos de la Planta/genética , Tallos de la Planta/crecimiento & desarrollo , Estructuras de las Plantas/genética , Estructuras de las Plantas/crecimiento & desarrollo , Proteínas Ribosómicas/clasificación , Proteínas Ribosómicas/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
Kawasaki Disease (KD) is a potentially fatal acute vasculitis of childhood. Although KD is the leading cause of acquired heart disease in children in developed nations, its pathogenesis remains unknown. We previously reported the novel observation that IgA plasma cells infiltrate the vascular wall in acute KD. We have now examined the clonality of this IgA response in vascular tissue from three fatal cases of KD to determine whether it is oligoclonal, suggesting an Ag-driven process, or polyclonal, suggesting nonspecific B cell activation or a response to a superantigen. We first sequenced VDJ junctions of 44 alpha genes isolated from a primary, unamplified KD vascular cDNA library. Five sets of clonally related alpha sequences were identified, comprising 34% (15 of 44) of the isolated alpha sequences. Furthermore, point mutations consistent with somatic mutation were detected in the related sequences. Next, using formalin-fixed coronary arteries from two additional fatal KD cases, we sequenced VDJ junctions of alpha genes isolated by RT-PCR, and a restricted pattern of CDR3 usage was observed in both. We conclude that the vascular IgA response in acute KD is oligoclonal. The identification of an oligoclonal IgA response in KD strongly suggests that the immune response to this important childhood illness is Ag-driven.
Asunto(s)
Vasos Coronarios/inmunología , Vasos Coronarios/metabolismo , Inmunoglobulina A/genética , Síndrome Mucocutáneo Linfonodular/genética , Síndrome Mucocutáneo Linfonodular/inmunología , Enfermedad Aguda , Secuencia de Aminoácidos , Secuencia de Bases , Niño , Células Clonales , Clonación Molecular , Femenino , Amplificación de Genes , Biblioteca de Genes , Reordenamiento Génico de Cadena Pesada de Linfocito B , Genes de Inmunoglobulinas , Humanos , Inmunoglobulina A/biosíntesis , Cadenas Pesadas de Inmunoglobulina/biosíntesis , Cadenas Pesadas de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/biosíntesis , Región Variable de Inmunoglobulina/genética , Cadenas alfa de Inmunoglobulina/biosíntesis , Cadenas alfa de Inmunoglobulina/genética , Lactante , Masculino , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa de Transcriptasa InversaAsunto(s)
Virus de la Hepatitis Murina/enzimología , Papaína/metabolismo , Precursores de Proteínas/metabolismo , ARN Polimerasa Dependiente del ARN/metabolismo , Proteínas Virales/metabolismo , Animales , Células HeLa , Humanos , Ratones , Virus de la Hepatitis Murina/genética , Plásmidos , TransfecciónAsunto(s)
Anticuerpos Antivirales/sangre , Inmunización/métodos , Virus de la Hepatitis Murina/inmunología , ARN Polimerasa Dependiente del ARN/inmunología , Vacunas de ADN/inmunología , Proteínas Virales/inmunología , Animales , Antígenos Virales/inmunología , Línea Celular , Células HeLa , Humanos , Virus de la Hepatitis Murina/enzimología , Virus de la Hepatitis Murina/genética , Plásmidos/genética , ARN Polimerasa Dependiente del ARN/genética , Conejos , Vacunas de ADN/administración & dosificación , Proteínas Virales/genéticaRESUMEN
The etiology and pathogenesis of Kawasaki disease (KD) remain unknown. As previously reported, in US patients with acute KD, IgA plasma cells (PCs) infiltrate the vascular wall. To determine whether IgA PCs are increased at mucosal sites in KD and to determine whether other nonvascular KD tissues are infiltrated by IgA PCs, the cells were immunolocalized and quantitated in tissue sections taken from 18 US and Japanese patients who died of acute KD and from 10 age-matched controls. IgA PCs were significantly increased in the trachea of patients who died of acute KD, compared with controls (P<.01), a finding that was similar to findings in children with fatal respiratory viral infection. IgA PCs also infiltrated coronary artery, pancreas, and kidney in all KD patients. These findings strongly support entry of the KD etiologic agent through the upper respiratory tract, resulting in an IgA immune response, with systemic spread to vascular tissue, pancreas, and kidney.
Asunto(s)
Inmunoglobulina A/análisis , Síndrome Mucocutáneo Linfonodular/inmunología , Síndrome Mucocutáneo Linfonodular/patología , Células Plasmáticas/patología , Enfermedad Aguda , Causas de Muerte , Niño , Preescolar , Vasos Coronarios/inmunología , Vasos Coronarios/patología , Etnicidad , Femenino , Humanos , Lactante , Japón , Riñón/inmunología , Riñón/patología , Masculino , Páncreas/inmunología , Páncreas/patología , Células Plasmáticas/inmunología , Sistema Respiratorio/inmunología , Sistema Respiratorio/patología , Estados UnidosRESUMEN
Mouse hepatitis virus (MHV) is a 31-kb positive-strand RNA virus that is replicated in the cytoplasm of infected cells by a viral RNA-dependent RNA polymerase, termed the replicase. The replicase is encoded in the 5'-most 22 kb of the genomic RNA, which is translated to produce a polyprotein of >800 kDa. The replicase polyprotein is extensively processed by viral and perhaps cellular proteinases to give rise to a functional replicase complex. To date, two of the MHV replicase-encoded proteinases, papain-like proteinase 1 (PLP1) and the poliovirus 3C-like proteinase (3CLpro), have been shown to process the replicase polyprotein. In this report, we describe the cloning, expression, and activity of the third MHV proteinase domain, PLP2. We show that PLP2 cleaves a substrate encoding the first predicted membrane-spanning domain (MP1) of the replicase polyprotein. Cleavage of MP1 and release of a 150-kDa intermediate, p150, are likely to be important for embedding the replicase complex in cellular membranes. Using an antiserum (anti-D11) directed against the C terminus of the MP1 domain, we verified that p150 encompasses the MP1 domain and identified a 44-kDa protein (p44) as a processed product of p150. Pulse-chase experiments showed that p150 is rapidly generated in MHV-infected cells and that p44 is processed from the p150 precursor. Protease inhibitor studies revealed that unlike 3CLpro activity, PLP2 activity is not sensitive to cysteine protease inhibitor E64d. Furthermore, coexpression studies using the PLP2 domain and a substrate encoding the MP1 cleavage site showed that PLP2 acts efficiently in trans. Site-directed mutagenesis studies confirmed the identification of cysteine 1715 as a catalytic residue of PLP2. This study is the first to report enzymatic activity of the PLP2 domain and to demonstrate that three distinct viral proteinase activities process the MHV replicase polyprotein.
Asunto(s)
Cisteína Endopeptidasas/aislamiento & purificación , Virus de la Hepatitis Murina/enzimología , ARN Polimerasa Dependiente del ARN/metabolismo , Proteínas no Estructurales Virales/aislamiento & purificación , Animales , Línea Celular , Clonación Molecular , Cisteína Endopeptidasas/genética , Cisteína Endopeptidasas/metabolismo , Inhibidores de Cisteína Proteinasa/farmacología , Células HeLa , Humanos , Leucina/análogos & derivados , Leucina/farmacología , Ratones , Virus de la Hepatitis Murina/genética , Virus de la Hepatitis Murina/metabolismo , Mutación , Procesamiento Proteico-Postraduccional , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismo , Ensayo de Placa ViralRESUMEN
The short integuments 2 (sin2) mutation arrests cell division during integument development of the Arabidopsis ovule and also has subtle pleiotropic effects on both sepal and pistil morphology. Genetic interactions between sin2 and other ovule mutations show that cell division, directionality of growth, and cell expansion represent at least partially independent processes during integument development. Double-mutant analyses also reveal that SIN2 shares functional redundancy with HUELLENLOS in ovule primordium outgrowth and proximal-distal patterning and with TSO1 in promotion of normal morphological development of the four whorls of primary floral organs. All of these observations are consistent with SIN2 being a promoter of growth and cell division during reproductive development, with a primary role in these processes during integument development. On the basis of the floral pleiotropic effects observed in a majority of ovule mutants, including sin2, we postulate a relationship between ovule genes and the evolutionary origin of some processes regulating flower morphology.
Asunto(s)
Arabidopsis/fisiología , Genes de Plantas , Mutación , Arabidopsis/genética , Arabidopsis/ultraestructura , Microscopía Electrónica de Rastreo , FenotipoRESUMEN
BACKGROUND: Adenoid cystic carcinoma (ACC) occurs not only as a neoplasm of salivary glands but also in the skin. Metastasis is rare, and metastasis to lymph nodes has not been reported in the English literature. Case Report A patient with a history of excisions of "cylindroma" of the scalp over the past 20 years was initially seen with 2 recurrent scalp nodules and a firm left neck mass. Both scalp lesions and multiple neck nodes were found to be ACC at resection. The patient underwent postoperative radiation therapy and is clinically free of disease at 4 years. CONCLUSIONS: We believe this represents the first reported case of nodal metastases from primary cutaneous ACC in the English literature.
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Carcinoma Adenoide Quístico/secundario , Neoplasias de Cabeza y Cuello/secundario , Ganglios Linfáticos/patología , Neoplasias Cutáneas/patología , Adulto , Biopsia con Aguja , Carcinoma Adenoide Quístico/radioterapia , Carcinoma Adenoide Quístico/cirugía , Terapia Combinada , Estudios de Seguimiento , Neoplasias de Cabeza y Cuello/radioterapia , Neoplasias de Cabeza y Cuello/cirugía , Humanos , Ganglios Linfáticos/cirugía , Metástasis Linfática , Masculino , Cuello , Neoplasias Cutáneas/radioterapia , Neoplasias Cutáneas/cirugía , Resultado del TratamientoRESUMEN
Murine hepatitis virus (MHV) gene 1, the 22-kb polymerase (pol) gene, is first translated into a polyprotein and subsequently processed into multiple proteins by viral autoproteases. Genetic complementation analyses suggest that the majority of the gene 1 products are required for viral RNA synthesis. However, there is no physical evidence supporting the association of any of these products with viral RNA synthesis. We have now performed immunofluorescent-staining studies with four polyclonal antisera to localize various MHV-A59 gene 1 products in virus-infected cells. Immunoprecipitation experiments showed that these antisera detected proteins representing the two papain-like proteases and the 3C-like protease encoded by open reading frame (ORF) 1a, the putative polymerase (p100) and a p35 encoded by ORF 1b, and their precursors. De novo-synthesized viral RNA was labeled with bromouridine triphosphate in lysolecithin-permeabilized MHV-infected cells. Confocal microscopy revealed that all of the viral proteins detected by these antisera colocalized with newly synthesized viral RNA in the cytoplasm, particularly in the perinuclear region of infected cells. Several cysteine and serine protease inhibitors, i.e., E64d, leupeptin, and zinc chloride, inhibited viral RNA synthesis without affecting the localization of viral proteins, suggesting that the processing of the MHV gene 1 polyprotein is tightly associated with viral RNA synthesis. Dual labeling with antibodies specific for cytoplasmic membrane structures showed that MHV gene 1 products and RNA colocalized with the Golgi apparatus in HeLa cells. However, in murine 17CL-1 cells, the viral proteins and viral RNA did not colocalize with the Golgi apparatus but, instead, partially colocalized with the endoplasmic reticulum. Our results provide clear physical evidence that several MHV gene 1 products, including the proteases and the polymerase, are associated with the viral RNA replication-transcription machinery, which may localize to different membrane structures in different cell lines.
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ARN Polimerasas Dirigidas por ADN/metabolismo , Membranas Intracelulares/metabolismo , Virus de la Hepatitis Murina/enzimología , ARN Viral/biosíntesis , Proteínas Virales/metabolismo , Animales , Línea Celular , Permeabilidad de la Membrana Celular , Cloruros/farmacología , Inhibidores de Cisteína Proteinasa/farmacología , ARN Polimerasas Dirigidas por ADN/genética , Células HeLa , Humanos , Leucina/análogos & derivados , Leucina/farmacología , Leupeptinas/farmacología , Ratones , Conejos , Coloración y Etiquetado , Factores de Tiempo , Proteínas Virales/genética , Compuestos de Zinc/farmacologíaRESUMEN
Paracoccus denitrificans and its near relative Paracoccus versutus (formerly known as Thiobacilllus versutus) have been attracting increasing attention because the aerobic respiratory system of P. denitrificans has long been regarded as a model for that of the mitochondrion, with which there are many components (e.g., cytochrome aa3 oxidase) in common. Members of the genus exhibit a great range of metabolic flexibility, particularly with respect to processes involving respiration. Prominent examples of flexibility are the use in denitrification of nitrate, nitrite, nitrous oxide, and nitric oxide as alternative electron acceptors to oxygen and the ability to use C1 compounds (e.g., methanol and methylamine) as electron donors to the respiratory chains. The proteins required for these respiratory processes are not constitutive, and the underlying complex regulatory systems that regulate their expression are beginning to be unraveled. There has been uncertainty about whether transcription in a member of the alpha-3 Proteobacteria such as P. denitrificans involves a conventional sigma70-type RNA polymerase, especially since canonical -35 and -10 DNA binding sites have not been readily identified. In this review, we argue that many genes, in particular those encoding constitutive proteins, may be under the control of a sigma70 RNA polymerase very closely related to that of Rhodobacter capsulatus. While the main focus is on the structure and regulation of genes coding for products involved in respiratory processes in Paracoccus, the current state of knowledge of the components of such respiratory pathways, and their biogenesis, is also reviewed.
Asunto(s)
Metabolismo Energético , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos , Consumo de Oxígeno/genética , Paracoccus/genética , Paracoccus/metabolismo , Secuencia de Bases , Transporte de Electrón , Datos de Secuencia Molecular , Oxidación-Reducción , Paracoccus/enzimologíaRESUMEN
The RNA polymerase gene of the murine coronavirus mouse hepatitis virus (MHV) encodes a polyprotein of greater than 750 kDa. The amino-terminal cleavage product of the MHV polymerase polyprotein, p28, has been shown to be cleaved from the polyprotein by the virus-encoded protease PCP-1. We aim to identify the MHV-JHM proteolytic products downstream of p28 and to determine which viral proteinase domains are responsible for generating each of them. To this end, we have generated antisera directed at specific MHV-JHM ORF1a regions and have used these antisera to identify six viral proteins, representing a large portion of ORF1a, from MHV-JHM-infected cells. These proteins include p28, p72, p65, p250, p210, and p27.
Asunto(s)
ARN Polimerasas Dirigidas por ADN/metabolismo , Virus de la Hepatitis Murina/enzimología , Procesamiento Proteico-Postraduccional , Proteínas/metabolismo , Proteínas Virales/metabolismo , Animales , Línea Celular , Endopeptidasas/metabolismo , Ratones , Precursores de Proteínas/metabolismo , ConejosRESUMEN
Recent studies suggest that infectious viruses and particularly persisting viral RNAs often exist as diverse populations or "quasispecies". We have developed an approach to characterize populations of the murine coronavirus mouse hepatitis virus (MHV) generated during persistent infection which has allowed us to begin to address the role of the viral quasispecies in MHV pathogenesis. We analyzed the population of persisting viral RNAs using reverse-transcription polymerase chain reaction amplification (RT-PCR) of the S1 "hypervariable" region of the spike gene followed by differential colony hybridization to identify spike deletion variants (SDVs) from acute and persistently infected mice. Sequence analysis revealed that mice with the most severe chronic paralysis harbored the most complex quasispecies. Mapping of the SDVs to the predicted RNA secondary structure of the spike RNA revealed that an isolated stem loop structure is frequently deleted. Overall, these results are consistent with high frequency recombination at sites of RNA secondary structure contributing to expansion of the viral quasispecies and persisting viral pathogenesis.
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Glicoproteínas de Membrana/genética , Virus de la Hepatitis Murina/genética , ARN Viral , Proteínas del Envoltorio Viral/genética , Latencia del Virus , Animales , Ratones , Ratones Endogámicos C57BL , Virus de la Hepatitis Murina/inmunología , Virus de la Hepatitis Murina/fisiología , Recombinación Genética , Glicoproteína de la Espiga del CoronavirusRESUMEN
Our understanding of the molecular mechanisms that regulate and integrate the temporal and spatial control of cell proliferation during organ ontogenesis, particularly of floral organs, continues to be primitive. The ovule, the progenitor of the seed, of Arabidopsis thaliana has been used to develop an effective model system for the analysis of plant organogenesis. A typical feature of a generalized ovule is the linear arrangement of at least three distinct elements, the funiculus, chalaza and nucellus, along a proximal-distal axis. This pattern is supposed to be established during the early proliferative phase of ovule development. We provide genetic evidence that the young ovule primordium indeed is a composite structure. Two genes, HUELLENLOS and AINTEGUMENTA have overlapping functions in the ovule and differentially control the formation of the central and proximal elements of the primordium. The results indicate that proximal-distal pattern formation in the Arabidopsis ovule takes place in a sequential fashion, starting from the distal end. Furthermore, we show that HUELLENLOS also regulates the initiation and/or maintenance of integument and embryo sac ontogenesis and interestingly prevents inappropriate cell death in the young ovule.