RESUMEN
BACKGROUND: Optineurin (OPTN) is associated with several human diseases, including amyotrophic lateral sclerosis (ALS), and is involved in various cellular processes, including autophagy. Optineurin regulates the expression of interferon beta (IFNß), which plays a central role in the innate immune response to viral infection. However, the role of optineurin in response to viral infection has not been fully clarified. It is known that optineurin-deficient cells produce more IFNß than wild-type cells following viral infection. In this study, we investigate the reasons for, and effects of, IFNß overproduction during optineurin deficiency both in vitro and in vivo. METHODS: To investigate the mechanism of IFNß overproduction, viral nucleic acids in infected cells were quantified by RT-qPCR and the autophagic activity of optineurin-deficient cells was determined to understand the basis for the intracellular accumulation of viral nucleic acids. Moreover, viral infection experiments using optineurin-disrupted (Optn-KO) animals were performed with several viruses. RESULTS: IFNß overproduction following viral infection was observed not only in several types of optineurin-deficient cell lines but also in Optn-KO mice and human ALS patient cells carrying mutations in OPTN. IFNß overproduction in Optn-KO cells was revealed to be caused by excessive accumulation of viral nucleic acids, which was a consequence of reduced autophagic activity caused by the loss of optineurin. Additionally, IFNß overproduction in Optn-KO mice suppressed viral proliferation, resulting in increased mouse survival following viral challenge. CONCLUSION: Our findings indicate that the combination of optineurin deficiency and viral infection leads to IFNß overproduction in vitro and in vivo. The effects of optineurin deficiency are elicited by viral infection, therefore, viral infection may be implicated in the development of optineurin-related diseases.
Asunto(s)
Esclerosis Amiotrófica Lateral , Proteínas de Ciclo Celular , Proteínas de Transporte de Membrana , Virosis , Animales , Humanos , Ratones , Esclerosis Amiotrófica Lateral/genética , Autofagia/genética , Proteínas de Ciclo Celular/genética , Inmunidad Innata , Interferón beta/genética , Factor de Transcripción TFIIIA/genética , Factor de Transcripción TFIIIA/metabolismo , Proteínas de Transporte de Membrana/genética , Ratones NoqueadosRESUMEN
Heat shock proteins (HSPs) are molecular chaperones that have recently been shown to function as host factors (HFs) for virus multiplication in fish as well as in mammals, plants, and insects. HSPs are classified into families, and each family has multiple isoforms. However, no comprehensive studies have been performed to clarify the biological importance of these multiple isoforms for fish virus multiplication. Betanodaviruses are the causative agents of viral nervous necrosis in cultured marine fish and cause very high mortality. Although the viral genome and encoded proteins have been characterized extensively, information on HFs for these viruses is limited. In this study, therefore, we focused on the HSP70 and HSP90 families to examine the importance of their isoforms for betanodavirus multiplication. We found that HSP inhibitors (17-AAG, radicicol, and quercetin) suppressed viral RNA replication and production of progeny virus in infected medaka (Oryzias latipes) cells. Thermal stress or virus infection resulted in increased expression of some isoform genes and facilitated virus multiplication. Furthermore, overexpression and knockdown of some isoform genes revealed that the isoforms HSP70-1, HSP70-2, HSP70-5, HSP90-α1, HSP90-α2, and HSP90-ß play positive roles in virus multiplication in medaka. Collectively, these results suggest that multiple isoforms of fish HPSs serve as HFs for betanodavirus multiplication.
Asunto(s)
Nodaviridae , Oryzias , Animales , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas HSP90 de Choque Térmico/genética , Proteínas HSP90 de Choque Térmico/metabolismo , Proteínas de Choque Térmico/metabolismo , Mamíferos , Chaperonas Moleculares , Nodaviridae/genética , Oryzias/metabolismo , Isoformas de Proteínas/genéticaRESUMEN
In spite of the growing attention given to medaka (Oryzias latipes) as an excellent vertebrate model, an effective gene knockdown system has not yet been established using cultured cells of this fish species. In this study, a gene knockdown system using short interfering RNA (siRNA) in medaka cell lines was established through the optimization of transfection conditions. By extensive screening of several medaka cell lines and transfection reagents, OLHNI-2 cells and X-tremeGENE siRNA Transfection Reagent were selected as the best combination to achieve high transfection efficiency of siRNA without cytotoxic effect. Knockdown conditions were then refined using the endogenous heat shock protein 90 (Hsp90) genes as the siRNA targets. Among the parameters tested, cell density, serum concentration in the culture medium, and duration of transfection improved knockdown efficiency, where the target mRNA in cells transfected with each of the siRNAs was reduced from 12.0% to 26.7% of the control level. Our results indicate that the established knockdown system using siRNA is a promising tool for functional analysis of medaka genes in vitro.
RESUMEN
BACKGROUND: Betanodaviruses, members of the family Nodaviridae, have bipartite, positive-sense RNA genomes and are the causal agents of viral nervous necrosis in many marine fish species. Recently, the viruses were shown to infect a few freshwater fish species including a model fish medaka (Oryzias latipes). Although virological study using cultured medaka cells would provide a lot of insight into virus-fish interactions in molecular aspects, no such cells have yet been tested for virus susceptibility. RESULTS: We tested ten medaka cell lines for susceptibilities to redspotted grouper nervous necrosis virus (RGNNV). Although the viral coat protein was detected in all the cell lines inoculated, the levels of cytopathic effect development and viral propagation were quite different among the cell lines. Those levels were especially high in OLHNI-1 and OLHNI-2 cells, but were extremely low in OLME-104 cells. Some cell lines entered into antiviral state after RGNNV infections probably because of inducing an antiviral system. This is the first report to examine the susceptibilities of cultured medaka cells against a virus. CONCLUSION: OLHNI-1 and OLHNI-2 cells are candidates of new standard cells for betanodavirus study because of their high susceptibilities to the virus and their several advantages as model fish cells.
Asunto(s)
Susceptibilidad a Enfermedades , Enfermedades de los Peces/virología , Nodaviridae/fisiología , Infecciones por Virus ARN/veterinaria , Animales , Línea Celular , Modelos Biológicos , Oryzias , Infecciones por Virus ARN/virologíaRESUMEN
Betanodaviruses, the causative agents of viral nervous necrosis in marine fish, have bipartite positive-sense RNA genomes. The larger genomic segment, RNA1 (~3.1 kb), encodes an RNA-dependent RNA polymerase (protein A), and the smaller genomic segment RNA2 (~1.4 kb) codes for the coat protein. These viruses can be classified into four genotypes, designated striped jack nervous necrosis virus (SJNNV), redspotted grouper nervous necrosis virus (RGNNV), tiger puffer nervous necrosis virus (TPNNV), and barfin flounder nervous necrosis virus (BFNNV), based on similarities in their partial RNA2 sequences. The optimal temperatures for the growth of these viruses are 20-25°C (SJNNV), 25-30°C (RGNNV), 20°C (TPNNV), and 15-20°C (BFNNV). However, little is known about the mechanisms underlying the temperature sensitivity of these viruses. We first constructed two reassortants between SJNNV and RGNNV to test their temperature sensitivity. The levels of viral growth and RNA replication of these reassortants and parental viruses in cultured fish cells were similar at 25°C. However, the levels of all of the viruses but RGNNV were markedly reduced at 30°C. These results indicate that both RNA1 and RNA2 control the temperature sensitivity of betanodaviruses by modulating RNA replication or earlier viral growth processes. We then constructed ten mutated RGNNVs, the RNA1 segments of which were chimeric between SJNNV and RGNNV, and showed that only chimeric viruses bearing the RGNNV RNA1 region, encoding amino acid residues 1-445, grew similarly to the parental RGNNV at 30°C. This portion of protein A is known to serve as a mitochondrial-targeting signal rather than functioning as an enzymatic domain.
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Calor , Nodaviridae/crecimiento & desarrollo , Nodaviridae/genética , ARN Viral/genética , Replicación Viral/efectos de la radiación , Animales , Línea Celular , Peces , Viabilidad Microbiana/efectos de la radiación , Nodaviridae/efectos de la radiación , Virus Reordenados , Recombinación GenéticaRESUMEN
Interferons (IFNs) are cytokines that induce an antiviral state in vertebrate cells. The Atlantic salmon (Salmo salar) IFN gene (SasaIFN-alpha1) was introduced in potato and rice plants by Agrobacterium-mediated transformation to produce a biologically active fish IFN in these plants. The transgenes and their transcripts were detected by PCR and Northern blot analysis. Western blot analysis showed the existence of SasaIFN-alpha1in transgenic plants. The antiviral activity of the SasaIFN-alpha1 protein expressed in these plants was determined by the survival rates of pre-treated cultured fish cells against pancreatic necrosis virus infection. The survival rate of cells pre-treated with transgenic samples was up to 95% but was reduced to 30-47% when cells were pre-treated with non-transgenic samples. These results demonstrated an antiviral effect of the SasaIFN-alpha1 protein derived from transgenic plants. Plant-derived IFNs may be suitable as components of functional feeds because such IFNs are free of animal pathogens and can be produced at a lower cost compared with those from transgenic mammalian and bacterial cells. This is the first study describing the production of a biologically active fish IFN using transgenic plants.
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Interferón-alfa/biosíntesis , Oryza/fisiología , Proteínas de Plantas/fisiología , Plantas Modificadas Genéticamente/metabolismo , Ingeniería de Proteínas/métodos , Salmo salar/fisiología , Solanum tuberosum/metabolismo , Animales , Interferones/metabolismo , Proteínas Recombinantes/metabolismoRESUMEN
Diseased wild redspotted grouper Epinephelus akaara were collected from Seto Inland Sea, Ehime Prefecture, in August 2002. Fish showed erratic swimming behavior and inflation of the swim bladder. The fish brains were positive for nodavirus in both RT-PCR and nested PCR. The sequence of the nested PCR product (177 nt) was closely related to that of a known betanodavirus, redspotted grouper nervous necrosis virus. When juvenile sevenband grouper E. septemfasciatus were challenged intravitreously with virus, abnormal swimming behavior and high mortality were observed. This is the first report on viral nervous necrosis in a wild population of redspotted grouper with clinical signs.
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Lubina/virología , Proteínas de la Cápside/genética , Enfermedades de los Peces/virología , Nodaviridae/genética , Nodaviridae/patogenicidad , Infecciones por Virus ARN/veterinaria , Animales , Encéfalo/virología , Proteínas de la Cápside/clasificación , Genes Virales , Japón , Nodaviridae/clasificación , Nodaviridae/aislamiento & purificación , Filogenia , Infecciones por Virus ARN/virología , Virulencia/genéticaRESUMEN
Betanodaviruses, the causative agents of viral nervous necrosis in marine fish, have bipartite positive-sense RNA genomes and have been classified (based on analysis of RNA2 sequences) into 4 genotypes: tiger puffer nervous necrosis virus (TPNNV), barfin flounder nervous necrosis virus (BFNNV), striped jack nervous necrosis virus (SJNNV), and redspotted grouper nervous necrosis virus (RGNNV). Full-length genomes of TPNNV and BFNNV were sequenced for the first time in this study. Their sequence data and those of SJNNV and RGNNV retrieved from GenBank were compared in order to investigate the relationships among the 4 genotypes. Between TPNNV and BFNNV, sequence identities were relatively high in RNA1 and encoded Protein A, but were not significantly high in RNA2 or the coat protein (CP). Similarly, between BFNNV and RGNNV, the amino acid sequences of CP were highly similar, but identities of RNA1, RNA2, and Protein A sequences were not especially high. Furthermore, multiple alignment data of the 4 genotypes of RNA2 sequences revealed that the TPNNV and SJNNV sequences have the same sizes of gaps and extra sequences at the same positions. Collectively, these apparent contradictions in sequence identity suggest that betanodavirus genomes have been constructed via complex evolutionary processes.
Asunto(s)
Genoma Viral/genética , Nodaviridae/genética , Secuencia de Bases , Genotipo , Datos de Secuencia Molecular , ARN Viral/genética , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Proteínas Virales/química , Proteínas Virales/genéticaRESUMEN
Betanodaviruses, the causative agents of viral nervous necrosis in marine fish, have bipartite positive-sense RNA genomes. The viruses have been classified into 4 distinct types based on nucleotide sequence similarities in the variable region (the so-called T4 region) of the smaller genomic segment RNA2 (1.4 kb). Betanodaviruses have marked host specificity, although the primary structures of the viral RNAs and encoded proteins are similar among the viruses. We have previously demonstrated, using reassortants between striped jack nervous necrosis virus (SJNNV) and redspotted grouper nervous necrosis virus (RGNNV), that RNA2, which encodes the coat protein, strictly controls host specificity. However, because RNA2 is large, we were unable to propose a mechanism underlying this RNA2-based host specificity. To identify the RNA2 region that controls host specificity, we constructed RNA2 chimeric viruses from SJNNV and RGNNV and tested their infectivity in the original host fish, striped jack Pseudocaranx dentex and sevenband grouper Epinephelus septemfasciatus. Among these chimeric viruses, SJNNV mutants containing the variable region of RGNNV RNA2 infected sevenband grouper larvae in a manner similar to RGNNV, while RGNNV mutants containing the variable region of SJNNV RNA2 infected striped jack larvae in a manner similar to SJNNV. Immunofluorescence microscopic studies using anti-SJNNV polyclonal antibodies revealed that these chimeric viruses multiplied in the brains, spinal cords and retinas of the infected fish, as in infections by the parental viruses. These results indicate that the variable region of RNA2 is sufficient to control host specificity in SJNNV and RGNNV.
Asunto(s)
Enfermedades de los Peces/virología , Nodaviridae/genética , Nodaviridae/patogenicidad , Perciformes/virología , ARN Viral/genética , Animales , Proteínas de la Cápside/genética , Especificidad de la Especie , Proteínas Virales/genética , VirulenciaRESUMEN
Betanodaviruses, the causative agents of viral nervous necrosis in marine fish, have bipartite positive-sense RNA genomes. Because the genomes are the smallest and simplest among viruses, betanodaviruses have been well studied using a reversed genetics system as model viruses. However, studies of virus-host interactions have progressed slowly because permissive hosts for betanodaviruses (basically larvae and juveniles of marine fish) are only available for limited periods of the year and are not suitable for the construction of a genetic engineering system. To obtain a model fish species that are not subject to these problems, 21 freshwater fish species were injected intramuscularly with a betanodavirus (redspotted grouper nervous necrosis virus) and tested for their susceptibility to the virus. Based on their responses, the tested fish were classified into 3 groups: 4 susceptible fish, 10 less susceptible fish, and 7 resistant fish. The susceptible fish, celebes rainbowfish Telmatherina ladigesi, threadfin rainbowfish Iriatherina werneri, dwarf rainbowfish Melanotaenia praecox, and medaka Oryzias latipes, exhibited erratic swimming and eventually died within 10 d post-inoculation. The virus was specifically localized in the brains, spinal cords, and retinas of the infected fish, similar to the pattern of infection in naturally infected marine fish. We believe that these susceptible freshwater fish species could act as good host models for betanodavirus-fish interaction studies.
Asunto(s)
Susceptibilidad a Enfermedades/veterinaria , Enfermedades de los Peces/virología , Nodaviridae/patogenicidad , Infecciones por Virus ARN/veterinaria , Smegmamorpha , Animales , Encéfalo/patología , Encéfalo/virología , Susceptibilidad a Enfermedades/virología , Enfermedades de los Peces/patología , Agua Dulce , Interacciones Huésped-Patógeno , Nodaviridae/aislamiento & purificación , Oryzias/virología , Infecciones por Virus ARN/patología , Infecciones por Virus ARN/virología , Retina/patología , Retina/virología , Smegmamorpha/clasificación , Smegmamorpha/virología , Especificidad de la Especie , Médula Espinal/patología , Médula Espinal/virología , Replicación ViralRESUMEN
Edwardsiella tarda is a pathogen with a broad host range that infects both animals and humans. Resistance to phagocytic killing may be involved in the pathogenicity of this bacterium. Here we show that intracellular replication of E. tarda in murine macrophages is dependent on the type III secretion system and induces an anti-apoptotic effect by up-regulating anti-apoptotic NF-kappaB target genes. The wild-type strain replicates within the phagosomal membrane of macrophages; whereas the type III mutant does not. Microarray analysis shows the mRNA expression level of NF-kappaB target genes (e.g. pro-inflammatory cytokines and anti-apoptotic genes) in macrophages infected with the wild-type strain were up-regulated compared to macrophages infected with the type III mutant. Up-regulation of Bcl2a1a, Bcl2a1b, cIAP-2, and TRAF1 genes induced expression of anti-apoptotic proteins to protect macrophages from apoptosis induced by staurosporine. Further, this protection was inhibited by adding kamebakaurin, an inhibitor of NF-kappaB activation and was confirmed using an NF-kappaB reporter gene assay. Up-regulation of anti-apoptotic NF-kappaB target genes is responsible for the anti-apoptotic activity of E. tarda and is required for intracellular replication in murine macrophages.
Asunto(s)
Apoptosis/fisiología , Edwardsiella tarda/crecimiento & desarrollo , Infecciones por Enterobacteriaceae/microbiología , Macrófagos/microbiología , FN-kappa B/genética , Estaurosporina/farmacología , Animales , Apoptosis/efectos de los fármacos , Apoptosis/genética , Western Blotting , Línea Celular , ADN Bacteriano/genética , Diterpenos/farmacología , Edwardsiella tarda/genética , Edwardsiella tarda/metabolismo , Edwardsiella tarda/patogenicidad , Regulación Bacteriana de la Expresión Génica , Macrófagos/citología , Macrófagos/efectos de los fármacos , Macrófagos/fisiología , Ratones , Microscopía Electrónica de Transmisión , Antígenos de Histocompatibilidad Menor , Mutagénesis Insercional , FN-kappa B/antagonistas & inhibidores , FN-kappa B/biosíntesis , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Proteínas Proto-Oncogénicas c-bcl-2/genética , Regulación hacia ArribaRESUMEN
Betanodaviruses, the causal agents of viral nervous necrosis in marine fish, have bipartite, positive-sense RNA genomes. As their genomes are the smallest and simplest among viruses, betanodaviruses have been studied in detail as model viruses by using a genetic-engineering system, as has occurred with the insect alphanodaviruses, the other members of the family Nodaviridae. However, studies of virus-host interactions have been limited, as betanodaviruses basically infect marine fish at early developmental stages (larval and juvenile). These fish are only available for a few months of the year and are not suitable for the construction of a reverse-genetics system. To overcome these problems, several freshwater fish species were tested for their susceptibility to betanodaviruses. It was found that adult medaka (Oryzias latipes), a well-known model fish, was susceptible to both Striped jack nervous necrosis virus (the type species of the genus Betanodavirus) and Redspotted grouper nervous necrosis virus (RGNNV), which have different host specificities in marine fish species. Infected medaka exhibited erratic swimming and the viruses were localized specifically in the brain, spinal cord and retina of the infected fish, similar to the pattern of infection in naturally infected marine fish. Moreover, medaka were susceptible to RGNNV at the larval stage. This is the first report of a model virus-model host infection system in fish. This system should facilitate elucidation of the mechanisms underlying RNA virus infections in fish.
Asunto(s)
Modelos Animales de Enfermedad , Enfermedades de los Peces/virología , Nodaviridae , Oryzias/virología , Infecciones por Virus ARN , Animales , Encéfalo/virología , Enfermedades de los Peces/patología , Enfermedades de los Peces/fisiopatología , Nodaviridae/aislamiento & purificación , Infecciones por Virus ARN/patología , Infecciones por Virus ARN/fisiopatología , Retina/virología , Médula Espinal/virologíaRESUMEN
Striped jack nervous necrosis virus (SJNNV), which infects fish, is the type species of the genus Betanodavirus. This virus has a bipartite genome of positive-strand RNAs, designated RNAs 1 and 2. A small RNA (ca. 0.4 kb) has been detected from SJNNV-infected cells, which was newly synthesized and corresponded to the 3'-terminal region of RNA1. Rapid amplification of cDNA ends analysis showed that the 5' end of this small RNA (designated RNA3) initiated at nt 2730 of the corresponding RNA1 sequence and contained a 5' cap structure. Substitution of the first nucleotide of the subgenomic RNA sequence within RNA1 selectively inhibited production of the positive-strand RNA3 but not of the negative-strand RNA3, which suggests that RNA3 may be synthesized via a premature termination model. The single RNA3-encoded protein (designated protein B2) was expressed in Escherichia coli, purified and used to immunize a rabbit to obtain an anti-protein B2 polyclonal antibody. An immunological test showed that the antigen was specifically detected in the central nervous system and retina of infected striped jack larvae (Pseudocaranx dentex), and in the cytoplasm of infected cultured E-11 cells. These results indicate that SJNNV produces subgenomic RNA3 from RNA1 and synthesizes protein B2 during virus multiplication, as reported for alphanodaviruses. In addition, an Agrobacterium co-infiltration assay established in transgenic plants that express green fluorescent protein showed that SJNNV protein B2 has a potent RNA silencing-suppression activity, as discovered for the protein B2 of insect-infecting alphanodaviruses.
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Genoma Viral , Nodaviridae/metabolismo , ARN Viral/genética , Transcripción Genética , Proteínas Virales/metabolismo , Animales , Enfermedades de los Peces/virología , Peces/virología , Nodaviridae/genética , Nodaviridae/patogenicidad , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Viral/metabolismo , ARN Polimerasa Dependiente del ARN/metabolismo , Proteínas Virales/genéticaRESUMEN
A green fluorescent protein-based in vivo expression technology leaf array was used to identify genes in Erwinia chrysanthemi 3937 that were specifically upregulated in plants compared with growth in a laboratory culture medium. Of 10,000 E. chrysanthemi 3937 clones, 61 were confirmed as plant upregulated. On the basis of sequence similarity, these were recognized with probable functions in metabolism (20%), information transfer (15%), regulation (11%), transport (11%), cell processes (11%), and transposases (2%); the function for the remainder (30%) is unknown. Upregulated genes included transcriptional regulators, iron uptake systems, chemotaxis components, transporters, stress response genes, and several already known or new putative virulence factors. Ten independent mutants were constructed by insertions in these plant-upregulated genes and flanking genes. Two different virulence assays, local leaf maceration and systemic invasion in African violet, were used to evaluate these mutants. Among these, mutants of a purM homolog from Escherichia coli (purM::Tn5), and hrpB, hrcJ, and a hrpD homologs from the Erwinia carotovorum hrpA operon (hrpB::Tn5, hrcJ::Tn5, and hrpD::Tn5) exhibited reduced abilities to produce local and systemic maceration of the plant host. Mutants of rhiT from E. chrysanthemi (rhiT::Tn5), and an eutR homolog from Salmonella typhimurium (eutR::TnS) showed decreased ability to cause systemic inva sion on African violet. However, compared with the wild-type E. chrysanthemi 3937, these mutants exhibited no significant differences in local leaf maceration. The pheno type of hrpB::Tn5, hrcC::Tn5, and hrpD::Tn5 mutants further confirmed our previous findings that hrp genes are crucial virulence determinants in E. chrysanthemi 3937.
Asunto(s)
Dickeya chrysanthemi/genética , Regulación de la Expresión Génica de las Plantas , Secuencia de Bases , Cartilla de ADN , Genes Reporteros , Genoma de Planta , Hojas de la Planta/genética , Hojas de la Planta/microbiología , Regiones Promotoras Genéticas/genética , Transcripción GenéticaRESUMEN
The resistance of Japanese flounder (Paralichthys olivaceus Temminck et Schlegel) against a viral haemorrhagic septicaemia virus (VHSV) challenge induced by a preceding non-lethal aquabirnavirus (ABV) challenge was investigated through experimental dual-infections with different intervals between the two challenges. The non-specific protection conferred by the primary ABV infection against the secondary VHSV infection commenced at Day 3 and persisted up to Day 14 but vanished at Day 21 post-ABV challenge. The in vitro assay using HINAE (hirame natural embryo) cells demonstrated anti-VHSV activity in the serum of ABV-challenged flounder from Day 1 to Day 14 but not at Day 21 post-ABV challenge. A high expression of a Mx gene, a molecular marker of type I interferon(s) (IFN) occurred in the head kidneys of ABV-challenged flounder from Day 1 to Day 7. These results suggest that the non-specific protection against the secondary VHSV infection in flounder was due to IFN(s) induced by the primary ABV infection.
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Aquabirnavirus/inmunología , Expresión Génica , Septicemia Hemorrágica Viral/inmunología , Novirhabdovirus/inmunología , Animales , Ensayo de Inmunoadsorción Enzimática , Lenguado , Proteínas de Unión al GTP/genética , Proteínas de Unión al GTP/metabolismo , Inmunidad Innata , Interferones/sangre , Riñón/metabolismo , Riñón/virología , Cinética , Proteínas de Resistencia a Mixovirus , Factores de TiempoRESUMEN
Betanodaviruses, the causal agents of viral nervous necrosis in marine fish, have bipartite positive-sense RNAs as genomes. The larger genomic segment, RNA1 (3.1 kb), encodes an RNA-dependent RNA polymerase, and the smaller genomic segment, RNA2 (1.4 kb), codes for the coat protein. Betanodaviruses have marked host specificity, although the primary structures of the viral RNAs and encoded proteins are similar among betanodaviruses. However, no mechanism underlying the host specificity has yet been reported. To evaluate viral factors that control host specificity, we first constructed a cDNA-mediated infectious RNA transcription system for sevenband grouper nervous necrosis virus (SGNNV) in addition to that for striped jack nervous necrosis virus (SJNNV), which was previously established by us. We then tested two reassortants between SJNNV and SGNNV for infectivity in the host fish from which they originated. When striped jack and sevenband grouper larvae were bath challenged with the reassortant virus comprising SJNNV RNA1 and SGNNV RNA2, sevenband groupers were killed exclusively, similar to inoculation with SGNNV. Conversely, inoculations with the reassortant virus comprising SGNNV RNA1 and SJNNV RNA2 killed striped jacks but did not affect sevenband groupers. Immunofluorescence microscopic studies using anti-SJNNV polyclonal antibodies revealed that both of the reassortants multiplied in the brains, spinal cords, and retinas of infected fish, similar to infections with parental virus inoculations. These results indicate that viral RNA2 and/or encoded coat protein controls host specificity in SJNNV and SGNNV.
Asunto(s)
Peces/virología , Nodaviridae/patogenicidad , Perciformes/virología , Infecciones por Virus ARN/veterinaria , Virus Reordenados/patogenicidad , Animales , Células Cultivadas , Enfermedades de los Peces/mortalidad , Enfermedades de los Peces/virología , Larva/virología , Datos de Secuencia Molecular , Nodaviridae/genética , Infecciones por Virus ARN/mortalidad , Infecciones por Virus ARN/virología , ARN Viral/química , ARN Viral/genética , ARN Viral/metabolismo , Virus Reordenados/genética , Análisis de Secuencia de ADN , Especificidad de la Especie , VirulenciaRESUMEN
Brome mosaic virus (BMV) requires the coat protein (CP) not only for encapsidation but also for viral cell-to-cell and long-distance movement in barley plants. This suggests that BMV infection is controlled by interactions of CP with putative host factors as well as with viral components. To identify the host factors that interact with BMV CP, we screened a barley cDNA library containing 2.4 x 10(6) independent clones, using a yeast two-hybrid system. Using full-length and truncated BMV CPs as baits, four candidate cDNA clones were isolated. One of the candidate cDNAs encodes a unique oxidoreductase enzyme, designated HCP1. HCP1 was found predominantly in the soluble fractions after differential centrifugation of BMV-infected and mock-inoculated barley tissues. A two-hybrid binding assay using a series of truncated BMV CPs demonstrated that a C-terminal portion of CP is essential for its interaction with HCP1. Interestingly, experiments with CP mutants bearing single amino acid substitutions at the C-terminus revealed that the capacity for mutant CP-HCP1 binding correlates well with the infectivity of the corresponding mutant viruses in barley. These results indicate that CP-HCP1 binding controls BMV infection of barley, interacting directly with CP, probably in the cell cytoplasm.
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Bromovirus/fisiología , Proteínas de la Cápside/fisiología , Hordeum/virología , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Receptores Virales/genética , Receptores Virales/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Secuencia de Bases , Sitios de Unión , Clonación Molecular , Hordeum/genética , Datos de Secuencia Molecular , Mutagénesis , Proteínas de Plantas/química , Receptores Virales/química , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMEN
Blue flowers generally contain 3',5'-hydroxylated anthocyanins (delphinidin derivatives) as pigments, which are formed only in the presence of flavonoid 3',5'-hydroxylases (F3'5'H). Heterologous expression of a F3'5'H gene therefore provides an opportunity to produce novel blue flowers for a number of ornamental plants missing blue flowering varieties. However, our previous study indicated difficulties in obtaining good accumulation of delphinidin derivatives in plants expressing F3'5'H. Here we report the isolation of a putative F3'5'H cDNA (Ka1) from canterbury bells (Campanula medium) and its expression in tobacco. Surprisingly, compared with other F3'5'H cDNAs, Ka1 encoded a protein with a unique primary structure that conferred high competence in the accumulation of delphinidin derivatives (up to 99% of total anthocyanins) and produced novel purple flowers. These results suggest that, among F3'5' H cDNAs, Ka1 is the best genetic resource for the creation of fine blue flowers by genetic engineering.
Asunto(s)
Antocianinas/metabolismo , Campanulaceae/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , ADN Complementario/metabolismo , Oxigenasas de Función Mixta/metabolismo , Nicotiana/metabolismo , Antocianinas/química , Flores/química , Datos de Secuencia MolecularRESUMEN
The syringolide receptor P34 mediates avrD-Rpg4 gene-for-gene complementarity in soybean. However, the mechanism underlying P34 signal transmission after syringolide binding is unknown. In an effort to identify a second messenger for P34, soybean leaf proteins were run though a P34-affinity column. A 42-kDa protein which specifically bound to the column was identified as a putative plant NADH-dependent hydroxypyruvate reductase (HPR) by N-terminal peptide sequencing. HPR is an important enzyme involved in the plant photorespiration system. Screening of a soybean cDNA library yielded two distinct HPR clones that encoded proteins with 97% identity (P42-1 and P42-2). Surprisingly, only P42-2 displayed good binding with P34 in a yeast two-hybrid assay, indicating that P42-2, but not P42-1, is a potential second messenger for P34. Glycerate and its analogs, which are utilized in the photorespiration system, were tested for their inhibitory effect on syringolide-induced hypersensitive response (HR) to evaluate the biological significance of P42-2. Interestingly, the downstream products of HPR (glycerate and 3-phosphoglycerate) inhibited HR but the upstream compounds (hydroxypyruvate or serine) did not have a significant effect on HR. These results suggest that P42-2 is a primary target for a P34/syringolide complex and that P42-2 binding with the complex probably induces HR by inhibiting one or more HPR functions in soybean.
Asunto(s)
Deshidrogenasas de Carbohidratos/metabolismo , Glycine max/genética , Proteínas de Plantas/metabolismo , Alérgenos , Secuencia de Aminoácidos , Antígenos de Plantas , Sitios de Unión/genética , Deshidrogenasas de Carbohidratos/genética , Clonación Molecular , ADN Complementario/química , ADN Complementario/genética , Luz , Datos de Secuencia Molecular , Consumo de Oxígeno/fisiología , Consumo de Oxígeno/efectos de la radiación , Proteínas de Plantas/genética , Unión Proteica , Saccharomyces cerevisiae/genética , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Proteínas de Soja , Glycine max/enzimología , Técnicas del Sistema de Dos HíbridosRESUMEN
Microarray technology was used to identify genes in Erwinia chrysanthemi 3937 that are specifically up- or down-regulated in a plant host compared with growth in laboratory culture medium. Several genes were plant down-regulated, and almost all of them were homologues of well-known housekeeping genes, such as those encoding metabolic functions, oxidative phosphorylation components, and transcription or translation processes. On the other hand, almost all of the plant up-regulated genes were involved with specialized functions, including already known or new putative virulence factors, anaerobiosis, iron uptake, transporters or permeases, xenobiotic resistance, chemotaxis, and stress responses to reactive oxygen species and heat. A substantial number of the plant up-regulated genes do not appear to be directly involved in damaging the host, but are probably important in adapting the pathogen to the host environment. We constructed insertion mutations in several of the plant up-regulated E. chrysanthemi 3937 genes. Among these, mutations of Bacillus subtilis pps1, Escherichia coli purU, and Pseudomonas aeruginosa pheC homologues reduced virulence on African violet leaves. Thus, new insights were obtained into genes important in bacterial virulence.