RESUMEN
We have investigated the entry pathway of Borna disease virus (BDV). Virus entry was assessed by detecting early viral replication and transcription. Lysosomotropic agents (ammonium chloride, chloroquine, and amantadine), as well as energy depletion, prevented BDV infection, indicating that BDV enters host cells by endocytosis and requires an acidic intracellular compartment to allow membrane fusion and initiate infection. Consistent with this hypothesis, we observed that BDV-infected cells form extensive syncytia upon low-pH treatment. Entry of enveloped viruses into animal cells usually requires the membrane-fusing activity of viral surface glycoproteins (GPs). BDV GP is expressed as two products of 84 and 43 kDa (GP-84 and GP-43, respectively). We show here that only GP-43 is present at the surface of BDV-infected cells and therefore is likely the viral polypeptide responsible for triggering fusion events. We also present evidence that GP-43, which corresponds to the C terminus of GP-84, is generated by cleavage of GP-84 by the cellular protease furin. Hence, we propose that BDV GP-84 is involved in attachment to the cell surface receptor whereas its furin-cleaved product, GP-43, is involved in pH-dependent fusion after internalization of the virion by endocytosis.
Asunto(s)
Virus de la Enfermedad de Borna/patogenicidad , Amantadina/farmacología , Cloruro de Amonio/farmacología , Animales , Virus de la Enfermedad de Borna/genética , Virus de la Enfermedad de Borna/fisiología , Línea Celular , Cloroquina/farmacología , Efecto Citopatogénico Viral , Endocitosis , Células Gigantes , Concentración de Iones de Hidrógeno , Lisosomas/efectos de los fármacos , Fusión de Membrana , Ratas , Transcripción Genética , Proteínas Virales/fisiología , Replicación Viral/efectos de los fármacosRESUMEN
Borna disease virus (BDV) is a nonsegmented negative-stranded (NNS) RNA virus, prototype of a new taxon in the Mononegavirales order. BDV causes neurologic disease manifested by behavioral abnormalities in several animal species, and evidence suggests that it may be a human pathogen. To improve our knowledge about the biology of this novel virus, we have identified and characterized the product of BDV open reading frame IV (BVp56). Based on sequence features, BVp56 encodes a virus surface glycoprotein. Glycoproteins play essential roles in the biology of NNS RNA viruses. Expression of BVp56 resulted in the generation of two polypeptides with molecular masses of about 84 and 43 kDa (GP-84 and GP-43). GP-84 and GP-43 likely correspond to the full-length BVp56 gene and to its C terminus, respectively. Endoglycosidase studies demonstrated that both products were glycosylated and that this process was required for the stabilization of newly synthesized products. Moreover, our results suggested that GP-43 is generated by cleavage of GP-84 by a cellular protease. Subcellular localization studies demonstrated that GP-84 accumulates in the ER, whereas GP-43 reaches the cell surface. Both BVp56 products were found to be associated with infectious virions, and antibodies to BVp56 had neutralizing activity. Our findings suggest that BVp56 exhibits a novel form of processing for an animal NNS RNA virus surface glycoprotein, which might influence the assembly and budding of BDV.
Asunto(s)
Virus de la Enfermedad de Borna/fisiología , Glicoproteínas/fisiología , Proteínas Virales/fisiología , Animales , Virus de la Enfermedad de Borna/genética , Cricetinae , Expresión Génica , Glicoproteínas/genética , Glicosilación , Humanos , Péptidos/metabolismo , Ratas , Células Tumorales Cultivadas , Proteínas Virales/genética , Virión/metabolismoRESUMEN
We have investigated the antiviral activity of amantadine (AD) against Borna disease virus (BDV) in several culture cell systems. We present evidence that AD, in the range 5 to 10 microM, does not have antiviral activity against BDV. Treatment of BDV infected cells with AD for six days caused neither a reduction in the number of infected cells, nor a decrease in steady state levels of BDV RNA or proteins. Moreover, treatment of cells with AD prior infection did not affect BDV multiplication, whereas influenza A virus yield was less than 1% with respect to that obtained in untreated control cells.
Asunto(s)
Amantadina/farmacología , Antivirales/farmacología , Virus de la Enfermedad de Borna/efectos de los fármacos , Amantadina/análogos & derivados , Animales , Antígenos Virales/metabolismo , Virus de la Enfermedad de Borna/genética , Virus de la Enfermedad de Borna/metabolismo , Línea Celular , Perros , ARN Viral/metabolismo , Conejos , Ratas , Células Tumorales Cultivadas , Proteínas Virales/metabolismoRESUMEN
In several vertebrate species, Borna disease virus (BDV), the prototype of a new group of animal viruses, causes central nervous system disease accompanied by diverse behavioral abnormalities. Seroepidemiological data indicate that BDV may contribute to the pathophysiology of certain human mental disorders. This hypothesis is further supported by the detection of both BDV antigens and BDV RNA in peripheral blood mononuclear cells (PBMCs) of patients with psychiatric disorders and the isolation of BDV from such PBMCs. Here we describe serological and molecular epidemiological studies on psychiatric patients and healthy individuals from the area of Homburg, Germany. Using a novel Western blot (immunoblot) assay, we found a BDV seroprevalence of 9.6% among 416 neuropsychiatric patients, which is significantly higher than the 1.4% found among 203 healthy control individuals. Human sera displayed a prominent immunoreactivity against the virus nucleoprotein, the p40 antigen. Reverse transcriptase-mediated PCR analysis of RNA extracted from PBMCs of a subset of 26 of the neuropsychiatric patients revealed that 50% were BDV RNA positive. Three of the 13 BDV RNA-positive patients also had BDV-positive serology, whereas one patient with serum antibodies to BDV p40 antigen did not harbor detectable BDV RNA in PBMCs. BDV p40 and p24 sequences derived from human PBMCs exhibited both a high degree of inter- and intrapatient conservation and a close genetic relationship to animal-derived BDV sequences.
Asunto(s)
Anticuerpos Antivirales/sangre , Antígenos Virales/genética , Enfermedad de Borna/virología , Virus de la Enfermedad de Borna/aislamiento & purificación , Secuencia Conservada , Trastornos Mentales/virología , ARN Viral/análisis , Proteínas Virales/genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Animales , Anticuerpos Antivirales/inmunología , Antígenos Virales/inmunología , Secuencia de Bases , Western Blotting , Enfermedad de Borna/sangre , Enfermedad de Borna/inmunología , Virus de la Enfermedad de Borna/genética , Virus de la Enfermedad de Borna/inmunología , Línea Celular , ADN Viral/análisis , Femenino , Humanos , Leucocitos Mononucleares/virología , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Conejos , Análisis de Secuencia de ADN , Spodoptera/citología , Proteínas Virales/inmunologíaRESUMEN
Borna disease virus (BDV) causes a central nervous system disease in several vertebrate animal species, which is manifest by behavioral abnormalities. Seroepidemiologic data suggest that BDV might infect humans, possibly being associated with certain mental disorders. This is further supported by the detection of both BDV-specific antigens and RNA sequences in peripheral blood mononuclear cells (PBMCs) of psychiatric patients. For the first time the sequence characterization of human BDV is documented here. BDV was recovered by co-cultivation techniques from the PBMCs of three hospitalized psychiatric patients. BDV was unequivocally identified based on sequence identification of BDV open reading frames (ORFs) p24, p16 and p56, as well as of the predicted catalytic domain of the BDV L polymerase. Each human BDV isolate had an unique sequence, but they displayed a high degree of sequence conservation with respect of BDV isolates from naturally infected animals of different species.
Asunto(s)
Virus de la Enfermedad de Borna/genética , Adulto , Secuencia de Bases , Enfermedad de Borna/virología , Virus de la Enfermedad de Borna/aislamiento & purificación , ADN Viral , Femenino , Expresión Génica , Humanos , Leucocitos Mononucleares/virología , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , ARN Viral , Homología de Secuencia de Ácido Nucleico , Proteínas Virales/genéticaRESUMEN
Borna disease virus (BDV) causes a central nervous system disease in several vertebrate species which is characterized by behavioral disturbances. Seroepidemiological data indicate an association of BDV infection with certain human mental disorders. Sclerosis of the hippocampus and astrocytosis constitute histopathological hallmarks of BDV infection in animals. Therefore, we searched for human brain autopsy cases with such histopathological features. Five of 600 cases examined were identified as having hippocampus sclerosis and astrocytosis. Using immunocytochemistry, RT-PCR, and in situ hybridization, we detected both BDV antigen and RNA in autopsy brain samples from 4 of these 5 patients, who presented with a clinical history of mental disorders involving memory loss and depression. This is the first demonstration that BDV can infect human brain tissue, possibly contributing to the pathophysiology of specific human neuropsychiatric disorders.
Asunto(s)
Virus de la Enfermedad de Borna/aislamiento & purificación , Encéfalo/virología , Trastornos Neurocognitivos/virología , Animales , Antígenos Virales/análisis , Astrocitos/patología , Autopsia , Western Blotting , Virus de la Enfermedad de Borna/genética , Virus de la Enfermedad de Borna/inmunología , Encéfalo/patología , Hipocampo/patología , Hipocampo/virología , Humanos , Inmunohistoquímica , Hibridación in Situ , Reacción en Cadena de la Polimerasa , ARN Viral/aislamiento & purificación , Esclerosis/patología , Proteínas Virales/inmunologíaRESUMEN
We recently demonstrated that Borna disease virus (BDV) has a negative non-segmented single stranded (NNS) RNA genome, whose organization is similar to that of other members of the Mononegavirales order. However, we have also documented that in contrast to the rest of the NNS-RNA animal viruses, BDV replication and transcription occur in the nucleus of infected cells. Here, we provide evidence that BDV uses the host nuclear splicing machinery to generate some of the viral mRNAs, representing the first documentation of RNA splicing in NNS-RNA animal viruses. Possible implications of RNA splicing for the regulation of BDV gene expression are discussed.
Asunto(s)
Virus de la Enfermedad de Borna/genética , Regulación Viral de la Expresión Génica , Empalme del ARN , ARN Mensajero/biosíntesis , ARN Viral/genética , Replicación Viral , Secuencia de Bases , Virus de la Enfermedad de Borna/fisiología , Núcleo Celular/virología , ADN Complementario/genética , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Reacción en Cadena de la Polimerasa , ARN Mensajero/genética , ARN Viral/biosíntesisRESUMEN
Borna disease virus (BDV) causes neurological disease in a wide range of animal species, providing an important model for studies of persistent viral infection of the central nervous system. In addition, the detection of antibodies that react with BDV antigen in serum from psychiatric patients suggests a role for BDV, or related viruses, in human mental disorders, providing further reason for study of this poorly characterized neurotropic virus. We present evidence that BDV has a nonsegmented negative single-strand RNA genome with the property that viral replication and transcription take place in the nuclei of infected cells where infectious BDV ribonucleoproteins are present. Our results support the view that BDV has unique biological features among animal viruses. Furthermore, the finding that BDV ribonucleoproteins are able to infect susceptible cells raises interesting questions regarding the mechanisms by which some neurotropic viruses may spread through the central nervous system of the infected host without requiring the production of mature infectious virus.
Asunto(s)
Virus de la Enfermedad de Borna/crecimiento & desarrollo , Encéfalo/microbiología , Núcleo Celular/microbiología , Ribonucleoproteínas/análisis , Animales , Antígenos Virales/análisis , Secuencia de Bases , Compartimento Celular , Núcleo Celular/química , Expresión Génica , Genoma Viral , Datos de Secuencia Molecular , Neuroglía/microbiología , ARN Viral/biosíntesis , Ratas , Fracciones Subcelulares , Transfección , Replicación ViralRESUMEN
We have previously demonstrated that Borna disease virus (BDV) has a negative nonsegmented single-stranded (NNS) RNA genome that replicates in the nucleus of infected cells. Here we report for the first time the cloning and complete sequence of the BDV genome. Our results revealed that BDV has a genomic organization similar to that of other members of the Mononegavirales order. We have identified five main open reading frames (ORFs). The largest ORF, V, is located closest to the 5' end in the BDV genome and, on the basis of strong homology with other NNS-RNA virus polymerases, is a member of the L-protein family. The intercistronic regions vary in length and nucleotide composition and contain putative transcriptional start and stop signals. BDV untranslated 3' and 5' RNA sequences resemble those of other NNS-RNA viruses. Using a set of overlapping probes across the BDV genome, we identified nine in vivo synthesized species of polyadenylated subgenomic RNAs complementary to the negative-strand RNA genome, including monocistronic transcripts corresponding to ORFs I, II, and IV, as well as six polycistronic polyadenylated BDV RNAs. Interestingly, although ORFs III and V were detected within polycistronic transcripts, their corresponding monocistronic transcripts were not detected. Our data indicate that BDV is a member of the Mononegavirales, specially related to the family Rhabdoviridae. However, in contrast to the rest of the NNS-RNA animal viruses, BDV replication and transcription occur in the nucleus of infected cells. These findings suggest a possible relationship between BDV and the plant rhabdoviruses, which also replicate and transcribe in the nucleus.
Asunto(s)
Virus de la Enfermedad de Borna/genética , Genoma Viral , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Virus de la Enfermedad de Borna/clasificación , Células Cultivadas , Clonación Molecular , Datos de Secuencia Molecular , Mononegavirales/clasificación , Mononegavirales/genética , Sistemas de Lectura Abierta/genética , Poli A/genética , ARN Mensajero/genética , ARN Viral/genética , Ratas , Secuencias Reguladoras de Ácidos Nucleicos/genética , Análisis de Secuencia de ADN , Homología de Secuencia de AminoácidoRESUMEN
Cytotoxic T lymphocytes (CTL) recognize virus peptide fragments complexed with class I major histocompatibility complex (MHC) molecules on the surface of virus-infected cells. Recognition is mediated by a membrane-bound T-cell receptor (TCR) composed of alpha and beta chains. Studies of the CTL response to lymphocytic choriomeningitis virus (LCMV) in H-2b mice have revealed that three distinct viral epitopes are recognized by CTL of the H-2b haplotype and that all of the three epitopes are restricted by the Db MHC molecule. The immunodominant Db-restricted CTL epitope, located at LCMV glycoprotein amino acids 278 to 286, was earlier noted to be recognized by TCRs that consistently contained V alpha 4 segments but had heterogeneous V beta segments. Here we show that CTL clones recognizing the other two H-2Db-restricted epitopes, LCMV glycoprotein amino acids 34 to 40 and nucleoprotein amino acids 397 to 407 (defined in this study), utilize TCR alpha chains which do not belong to the V alpha 4 subfamily. Hence, usage of V alpha and V beta in the TCRs recognizing peptide fragments from one virus restricted by a single MHC molecule is not sufficiently homogeneous to allow manipulation of the anti-viral CTL response at the level of TCRs. The diversity of anti-viral CTL likely provides the host with a wider option for attacking virus-infected cells and prevents the emergence of virus escape mutants that might arise if TCRs specific for the virus were homogeneous.
Asunto(s)
Antígenos Virales/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Virus de la Coriomeningitis Linfocítica/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T Citotóxicos/inmunología , Secuencia de Aminoácidos , Animales , Northern Blotting , Células Cultivadas , Epítopos/inmunología , Complejo Mayor de Histocompatibilidad , Ratones , Datos de Secuencia MolecularRESUMEN
Measles virus (MV) inhibits lymphocyte function in patients, as well as in cells infected in vitro. The proliferation of phytohemagglutinin-stimulated T lymphocytes is suppressed by in vitro MV infection, as shown by the diminished incorporation of [3H]thymidine into DNA and the reduced frequency of cells in the S phase of the cell cycle, as compared with mock-infected cells. MV infection itself, however, does not completely block DNA synthesis in infected cells, because infected T cells expressing MV antigens on the cell surface, isolated by fluorescence-activated cell sorter, could still proliferate. Northern blot analysis indicated that the expression of genes induced during T cell activation, such as those encoding interleukin 2 (IL-2), c-myc, IL-2 receptor, IL-6, c-myb, and cdc-2, was not significantly suppressed in MV-infected cells, suggesting that MV does not interfere with the T cell activation process. When anti-MV serum or carbobenzoxy-D-Phe-L-Phe-Gly, a synthetic oligopeptide known to inhibit MV-induced fusion, was added 24 hr after infection, the inhibition of T cell proliferation was reversed in a dose-dependent manner. From these results we propose a model for the inhibition of T cell proliferation by MV; MV glycoproteins expressed on the cell surface of infected cells interact with the MV receptor or other molecules on the cell membrane of adjacent T cells, which in turn affects the proliferation of those T cells.