RESUMO
It is known that the use of anti-TNF-α drugs is related to an increased incidence of infective diseases. This therapy can not be administered to patients having active infections and it has to be considered with caution in case of acquired or congenital immunodeficiency diseases. We report the case of a 28-years-old man affected by psoriatic arthritis; he developed some infections during treatment with TNF-α blockers. The infections were caused by a selective IgA deficiency, that was not evident before the anti-TNF-α blockers administration and disappeared after withdrawing the biological therapy. This case-report draws our attention to the possibility of cases of subclinical immunodeficiency, unknown by the patients, but important in the prognosis and in the therapeutic approach to these diseases. Therefore, it is important to evaluate carefully the immunologic status of patients during the pre-therapeutic screening for TNF-α blocking therapy.
Assuntos
Anticorpos Monoclonais Humanizados/efeitos adversos , Antirreumáticos/efeitos adversos , Artrite Psoriásica/tratamento farmacológico , Foliculite/etiologia , Herpes Labial/etiologia , Deficiência de IgA/diagnóstico , Imunossupressores/efeitos adversos , Micoses/etiologia , Estomatite/etiologia , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Adalimumab , Adulto , Anticorpos Monoclonais Humanizados/uso terapêutico , Artrite Psoriásica/complicações , Ciclosporina/uso terapêutico , Suscetibilidade a Doenças , Humanos , Deficiência de IgA/complicações , Hospedeiro Imunocomprometido , Imunossupressores/uso terapêutico , Síndrome do Intestino Irritável/complicações , Masculino , Metilprednisolona/uso terapêutico , Hipersensibilidade a Leite/complicaçõesRESUMO
OBJECTIVE: Analysis of the association between psoriatic arthritis (PsA) clinical forms and MICA gene transmembrane polymorphisms. METHODS: Patients were classified as having peripheral asymmetric oligoarthritis (AO), peripheral symmetric poly-arthritis (PA) and spondylitis (SP), or disease combinations (PA/SP, OA/SP). Two hundred and twenty-six patients with PsA were typed for MICA exon 5 microsatellite (TM) by heteroduplex analysis and compared with 225 normal controls. RESULTS: MICA-TM microsatellite typing revealed that, among the different clinical forms of PsA, only the combined PA/SP subset shows a significant positive association with MICA-A9 and a lower frequency of MICA-A4, A5 genotype in PsA patients with a decrease, only in the PA/SP cohort, of all MICA-A5 combinations except MICA-A5, -A9. CONCLUSION: These results suggest a role for genes within the HLA region in the pathogenesis of PsA, and reinforce the idea that the different forms of PsA may have heterogeneous genetic basis.
Assuntos
Artrite Psoriásica/genética , Antígenos de Histocompatibilidade Classe I/genética , Repetições de Microssatélites/genética , Polimorfismo de Nucleotídeo Único/genética , Artrite Psoriásica/classificação , Estudos de Casos e Controles , Estudos de Coortes , Frequência do Gene , Predisposição Genética para Doença , Haplótipos , Humanos , ItáliaRESUMO
OBJECTIVE: Two common factors, cigarette smoking and appendectomy, have been found to play a role in ulcerative colitis (UC). Data on their role in the development of extraintestinal manifestations (EIM) are scarce. METHODS: The relationship between cigarette smoking, appendectomy, and EIM was examined in a prospective study involving 535 (M/F = 319/216) consecutive UC patients followed up for 18 yr. We considered the major EIM: seronegative spondyloarthropathy, pyoderma gangrenosum/erythema nodosum, acute anterior uveitis, and primary sclerosing cholangitis. We excluded patients with a history of EIM or those colectomized before study entry, ex-smokers, and those who started to smoke during the course of UC. RESULTS: In UC patients, seronegative spondyloarthropathy and dermatologic complications were found increased in smokers (p < 0.0001; p = 0.001) or in subjects with appendectomy (p = 0.0003; p = 0.02), while acute anterior uveitis and primary sclerosing cholangitis did not differ. The Kaplan-Meier analysis showed 18-yr rates for EIM of 71% in smokers and 45% in nonsmokers (log-rank test, p = 0.0001), and of 85% in patients with appendectomy and 48% in those without (p = 0.0001). Cox proportional-hazard model showed that cigarette smoking and appendectomy are independent factors promoting EIM. In smokers with appendectomy the adjusted hazard ratio (3.197, 95% CI 1.529-6.684) was higher than in patients with appendectomy alone (2.617, 95% CI 1.542-4.442) or smoking alone (1.947, 95% CI 1.317-2.879). CONCLUSIONS: In UC patients, appendectomy and cigarette smoking are prognostic factors for the development of EIM. The unfavorable effect of cigarette smoking on EIM is additive to that of appendectomy.
Assuntos
Apendicectomia/efeitos adversos , Colite Ulcerativa/complicações , Fumar/efeitos adversos , Adolescente , Adulto , Criança , Colangite Esclerosante/etiologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico , Estudos Prospectivos , Fatores de Risco , Dermatopatias/etiologia , Osteofitose Vertebral/etiologia , Uveíte Anterior/etiologiaRESUMO
A bispecific Ab (BsAb) that binds the TCR on T cells and the G protein of the vesicular stomatitis virus (VSV) can redirect staphylococcal enterotoxin B (SEB)-activated T cells to kill VSV-infected cells and to inhibit VSV replication in vitro. Inhibition of virus replication in our system is dependent upon the specificity of the Ab for the viral protein. IFN-gamma does not play a very important role in this phenomenon, which is mainly mediated by the release of Pfp from CD8+ T cells. We have used a Stat1 knockout mouse model in which VSV infection is lethal. Infusion of staphylococcal enterotoxin-activated B T cells and bispecific Ab significantly slowed virus progression and prolonged the survival of VSV-infected Stat1 knockout mice in vivo.
Assuntos
Anticorpos Biespecíficos/farmacologia , Infecções por Rhabdoviridae/imunologia , Superantígenos/imunologia , Linfócitos T/imunologia , Vírus da Estomatite Vesicular Indiana/imunologia , Replicação Viral/imunologia , Animais , Anticorpos Biespecíficos/administração & dosagem , Anticorpos Biespecíficos/biossíntese , Antivirais/metabolismo , Antivirais/fisiologia , Células Cultivadas , Cricetinae , Testes Imunológicos de Citotoxicidade , Enterotoxinas/administração & dosagem , Enterotoxinas/genética , Enterotoxinas/imunologia , Injeções Intraperitoneais , Interferon gama/metabolismo , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infecções por Rhabdoviridae/terapia , Infecções por Rhabdoviridae/virologia , Especificidade da Espécie , Staphylococcus aureus/imunologia , Estomatite/imunologia , Estomatite/terapia , Estomatite/virologia , Superantígenos/administração & dosagem , Superantígenos/genética , Linfócitos T/metabolismo , Linfócitos T/virologia , Linfócitos T Citotóxicos/imunologia , Vírus da Estomatite Vesicular Indiana/classificação , Vírus da Estomatite Vesicular Indiana/fisiologiaRESUMO
Previous work (C.F. Spiropoulou and S.T. Nichol, 1993, J. Virol. 67, 3103-3110) has demonstrated the existence in cells infected with the New Jersey serotype of vesicular stomatitis virus (VSV) of two small carboxy-coterminal proteins encoded by the P mRNA. These proteins have been named C' and C. We are interested in studying the function of these proteins in the virus life cycle. Toward this end, we have cloned the ORF encoding the potential C' protein of the Indiana serotype as a histidine-tagged fusion protein, purified the expressed protein from Escherichia coli, and used the fusion protein as an immunogen to raise antiserum in a rabbit. We have used this anti-C' protein serum to demonstrate that both of the predicted C' and C proteins are synthesized in cells infected with the Indiana serotype of VSV. In addition we have localized a portion of these proteins to nucleocapsids isolated from infected cells, suggesting that they may play a role in RNA synthesis. Reconstitution of the viral polymerase activity by expressing the L and P protein subunits with or without the C proteins failed to demonstrate any effect of the presence of these latter proteins on reconstituted transcription using purified nucleocapsids as templates. However, we have been able to show a dramatic stimulation of the polymerase activity in purified virions by the addition of purified C' protein to in vitro transcription reactions. Both the level and the fidelity of mRNA synthesis are stimulated by this protein. Evidence for the specificity of this effect comes from the fact that stimulation appears to be serotype specific; C' protein of the Indiana serotype stimulates transcription by purified Indiana serotype virions but has a minimal effect on transcription by purified virions of the New Jersey serotype. We are continuing our studies to determine the mechanism of this stimulation.
Assuntos
Transcrição Gênica/fisiologia , Vírus da Estomatite Vesicular Indiana/metabolismo , Vesiculovirus , Proteínas não Estruturais Virais/fisiologia , Animais , Anticorpos Antivirais , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/genética , Genes Virais/fisiologia , Histidina/genética , Fases de Leitura Aberta/genética , Peptídeos/genética , RNA Mensageiro/biossíntese , RNA Viral/biossíntese , Coelhos , Proteínas Recombinantes de Fusão , Espermidina/farmacologia , Transcrição Gênica/efeitos dos fármacos , Vírus da Estomatite Vesicular Indiana/imunologia , Proteínas não Estruturais Virais/análise , Proteínas não Estruturais Virais/biossíntese , Proteínas não Estruturais Virais/farmacologia , Vírion/química , Vírion/metabolismoRESUMO
In order for the fusion protein (F) of the human parainfluenza virus type 3 (HPF3) to promote membrane fusion and viral entry, the haemagglutinin-neuraminidase (HN) glycoprotein must interact with its receptor. Sialoglycoconjugates are known to be the receptors for the HPF3 HN, however specific attachment factors or receptors for HPF3 have not been identified. In this report we describe the analysis of variants of HPF3 with increased fusion-promoting phenotypes that were selected by treatment with viral neuraminidase. The results suggest that for HPF3, the virus is specific in its use of sialic acid receptors; the majority of sialic-acid containing molecules are not targets for HPF3.
Assuntos
Vírus da Parainfluenza 3 Humana/química , Receptores Virais/análise , Variação Genética , Neuraminidase/metabolismo , Proteínas Virais de Fusão/análiseRESUMO
The growth of vesicular stomatitis virus requires two distinct RNA synthetic events: transcription of messenger RNA molecules and replication of the viral genome RNA. We report the use of a panel of monoclonal antibodies directed against the viral phosphoprotein P in an attempt to assess the role of this protein in RNA synthesis. Using extracts derived from virus-infected cells, we show that several anti-P monoclonal antibodies can have an inhibitory effect on genome RNA replication by binding to a soluble form of the P protein. We also show that the P protein to which one of these antibodies (6D11) is directed is not complexed with the N protein and that the amount of soluble P protein that binds to the 6D11 antibody in immunoprecipitation reactions can be increased by treating extracts with alkaline phosphatase. In addition, phosphatase treatment of infected cell extracts results in an increased level of genome RNA replication. These results suggest that a soluble subspecies of the P protein that functions in genome RNA replication exists in infected cells and that this species of the P protein is not required for transcription.
Assuntos
Fosfoproteínas , RNA Viral/biossíntese , Transcrição Gênica , Vírus da Estomatite Vesicular Indiana/fisiologia , Proteínas Estruturais Virais/fisiologia , Fosfatase Alcalina/metabolismo , Animais , Anticorpos Monoclonais/imunologia , Capsídeo/metabolismo , Linhagem Celular , Cricetinae , Fosforilação , Testes de Precipitina , RNA Viral/efeitos dos fármacos , Solubilidade , Vírus da Estomatite Vesicular Indiana/genética , Proteínas do Core Viral/metabolismo , Proteínas Estruturais Virais/imunologiaRESUMO
The expression of two small basic proteins (C and C') encoded by a second open reading frame of the New Jersey serotype of vesicular stomatitis virus (VSV) P gene was reported previously (Spiropoulou and Nichol, J. Virol., 67, 3103-3110, 1993). Here we found that the Indiana serotype virus also expressed C and C' proteins from this reading frame. We eliminated C and C' expression by making a single base change that introduced a stop codon in the C and C' coding sequence, but left the P-protein sequence unchanged. This mutated P gene supported normal replication and packaging of VSV minigenomes encoding G and M proteins. The mutated P gene was also recombined into an infectious clone of VSV that was used to recover virus. The mutant virus no longer expressed the C and C' proteins but showed growth kinetics identical to wild-type virus. The amounts of viral mRNAs and proteins synthesized were indistinguishable in mutant and wild-type virus infected cells as were the yields and composition of mutant and wild-type virus particles. The kinetics of host protein-synthesis shut-off were also identical for both viruses. Although the C and C' proteins were dispensable for VSV growth in tissue culture, they are known to be conserved in all vesiculoviruses, and thus perhaps play a role in viral pathogenesis or transmission by insect vectors.
Assuntos
Vírus da Estomatite Vesicular Indiana/fisiologia , Vesiculovirus , Proteínas não Estruturais Virais , Proteínas Estruturais Virais/fisiologia , Replicação Viral , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Cricetinae , DNA Viral , Genoma Viral , Dados de Sequência Molecular , Mutação Puntual , Coelhos , Fatores de Tempo , Transcrição Gênica , Vírus da Estomatite Vesicular Indiana/genética , Proteínas Estruturais Virais/genética , Replicação Viral/genéticaRESUMO
The ability of enveloped viruses to cause disease depends on their ability to enter the host cell via membrane fusion events. An understanding of these early events in infection, crucial for the design of methods of blocking infection, is needed for viruses that mediate membrane fusion at neutral pH, such as paramyxoviruses and human immunodeficiency virus. Sialic acid is the receptor for the human parainfluenza virus type 3 (HPF3) hemagglutinin-neuraminidase (HN) glycoprotein, the molecule responsible for binding of the virus to cell surfaces. In order for the fusion protein (F) of HPF3 to promote membrane fusion, the HN must interact with its receptor. In the present report, two variants of HPF3 with increased fusion-promoting phenotypes were selected and used to study the function of the HN glycoprotein in membrane fusion. Increased fusogenicity correlated with single amino acid changes in the HN protein that resulted in increased binding of the variant viruses to the sialic acid receptor. These results suggest that the avidity of binding of the HN protein to its receptor regulates the level of F protein-mediated fusion and begin to define one role of the receptor-binding protein of a paramyxovirus in the membrane fusion process.
Assuntos
Fusão Celular , Proteína HN/metabolismo , Vírus da Parainfluenza 3 Humana/patogenicidade , Receptores Virais/metabolismo , Ácidos Siálicos/metabolismo , Proteínas Virais de Fusão/metabolismo , Animais , Linhagem Celular , Chlorocebus aethiops , Técnicas In VitroRESUMO
Cells can be persistently infected with human parainfluenza virus type 3 (HPF3) by using a high multiplicity of infection (MOI) (> or = 5 PFU per cell). The persistently infected cells exhibit no cytopathic effects and do not fuse with each other, yet they readily fuse with uninfected cells. We have previously shown that the failure of the persistently infected cells to fuse with each other is due to the lack of a receptor on these cells for the viral hemagglutinin-neuraminidase glycoprotein, and we have established that both fusion and hemagglutinin-neuraminidase proteins are needed for cell fusion mediated by HPF3. We then postulated that the generation of persistent infection and the failure of cells infected with HPF3 at high MOI to form syncytia are both due to the action of viral neuraminidase in the high-MOI inoculum. In this report, we describe experiments to test this hypothesis and further investigate the receptor requirements for HPF3 infection and cell fusion. A normally cytopathic low-MOI HPF3 infection can be converted into a noncytopathic infection by the addition of exogenous neuraminidase, either in the form of a purified enzyme or as UV-inactivated HPF3 virions. Evidence is presented that the receptor requirements for an HPF3 virus particle to infect a cell are different from those for fusion between cells. By treating infected cells in culture with various doses of neuraminidase, we demonstrate that virus spreads from cell to cell in the complete absence of cell-cell fusion. We compare the outcome of HPF3 infection in the presence of excess neuraminidase with that of another paramyxovirus (simian virus 5) and provide evidence that these two viruses differ in their receptor requirements for mediating fusion.
Assuntos
Fusão Celular , Fusão de Membrana , Vírus da Parainfluenza 3 Humana/crescimento & desenvolvimento , Receptores Virais/metabolismo , Células Cultivadas , Hemaglutininas Virais/metabolismo , Humanos , Modelos Biológicos , Ácido N-Acetilneuramínico , Neuraminidase/metabolismo , Vírus da Parainfluenza 3 Humana/patogenicidade , Retrovirus dos Símios/metabolismo , Retrovirus dos Símios/patogenicidade , Ácidos Siálicos/metabolismo , Proteínas Virais de Fusão/metabolismo , Replicação ViralRESUMO
A cell-free system supporting transcription, replication, and nucleocapsid assembly of the genome RNA of human parainfluenza virus type 3 (HPF3) is described. Cytoplasmic extracts from infected CV-1 or BHK cells catalyzed the transcription of the entire HPF3 genome, the replication of genome RNA, and the assembly of this RNA into nucleocapsidlike structures. Newly replicated RNA was resistant to micrococcal nuclease digestion and was stable in CsCl gradients, exhibiting the density of authentic HPF3 nucleocapsids. After fractionation of the extracts, the nucleocapsid-containing pellet fraction synthesized viral mRNAs. Reconstitution with the soluble protein fraction was necessary for genome RNA replication and nucleocapsid assembly.
Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , Vírus da Parainfluenza 3 Humana/enzimologia , RNA Viral/biossíntese , RNA Polimerase Dependente de RNA/metabolismo , Transcrição Gênica , Animais , Capsídeo/biossíntese , Linhagem Celular , Centrifugação com Gradiente de Concentração , Fracionamento Químico , Eletroforese em Gel de Ágar , Humanos , Nuclease do Micrococo/metabolismo , Vírus da Parainfluenza 3 Humana/genética , Proteínas do Core Viral/biossínteseRESUMO
Cells persistently infected with human parainfluenza virus type 3 (HPF3) exhibit a novel phenotype. They are completely resistant to fusion with each other but readily fuse with uninfected cells. We demonstrate that the inability of these cells to fuse with each other is due to a lack of cell surface neuraminic acid. Neuraminic acid is the receptor for the HPF3 hemagglutinin-neuraminidase (HN) glycoprotein, the molecule responsible for binding of the virus to cell surfaces. Uninfected CV-1 cells were treated with neuraminidase and then tested for their ability to fuse with the persistently infected (pi) cells. Neuraminidase treatment totally abolished cell fusion. To extend this result, we used a cell line deficient in sialic acid and demonstrated that these cells, like the neuraminidase-treated CV-1 cells, were unable to fuse with pi cells. We then tested whether mimicking the agglutinating function of the HN molecule with lectins would result in cell fusion. We added a panel of five lectins to the neuraminic acid-deficient cells and showed that binding of these cells to the pi cells did not result in fusion; the lectins could not substitute for interaction of neuraminic acid with the HN molecule in promoting membrane fusion. These results provide compelling evidence that the HN molecule of HPF3 and its interaction with neuraminic acid participate in membrane fusion and that cell fusion is mediated by an interaction more complex than mere juxtaposition of the cell membranes.
Assuntos
Proteína HN/farmacologia , Fusão de Membrana/efeitos dos fármacos , Vírus da Parainfluenza 3 Humana/fisiologia , Proteínas Virais de Fusão/farmacologia , Animais , Células Cultivadas/efeitos dos fármacos , Efeito Citopatogênico Viral , Humanos , Lectinas/farmacologia , Ácidos Neuramínicos/farmacologia , Infecções por Paramyxoviridae/patologiaRESUMO
We studied the effect pH had on the N-NS protein complex to determine its role in vesicular stomatitis virus (VSV) genome replication, as we had previously shown that VSV genome replication in vitro requires the interaction of the viral N and NS proteins into a 1:1 complex. A previous report showed that the growth of VSV in L cells was sensitive to the pH of the environment (M. Fiszman, J. B. Leaute, C. Chany, and M. Girard, J. Virol. 13:801-808, 1974). We hypothesized that low pH might disrupt the N-NS protein complex, and so we investigated the molecular events leading to inhibition of viral RNA replication in vitro from extracts that were prepared from VSV-infected cells incubated at pH 6.6. We found that viral genome RNA synthesis in vitro was reduced when infected cells were maintained at pH 6.6. Through immunoprecipitation analysis of the viral soluble protein pool, we found that a complex that usually exists between the N and NS proteins at pH 7.4 was altered in extracts from infected cells maintained at pH 6.6, and this was responsible for the observed effects on viral replication. The effect of low pH on the N-NS protein complex could not be abolished by increasing the concentration of the altered complex, indicating that the effects is more than simply a decrease in the level of the protein complex in the cell. Our data provide additional evidence that the 1:1 N-NS protein complex, and not the N protein alone, serves as the substrate for viral RNA replication in vivo.
Assuntos
Capsídeo/fisiologia , Vírus da Estomatite Vesicular Indiana/crescimento & desenvolvimento , Proteínas do Core Viral/fisiologia , Replicação Viral , Capsídeo/análise , Capsídeo/imunologia , Células Cultivadas , Concentração de Íons de Hidrogênio , Testes de Precipitina , RNA Viral/biossíntese , Proteínas do Core Viral/análise , Proteínas do Core Viral/imunologia , Proteínas não Estruturais ViraisRESUMO
The template for transcription and replication of negative-stranded RNA viruses is a ribonucleoprotein structure, the nucleocapsid. We have developed a system that supports assembly of the negative-stranded RNA genome of a defective interfering (DI) particle of vesicular stomatitis virus (VSV) into a nucleocapsid in vitro. This system uses extracts from wild-type VSV-infected cells as a source of proteins to encapsidate the RNA. In vitro assembled nucleocapsids were compared to in vivo-derived nucleocapsids by the following characteristics: nuclease resistance of the encapsidated RNA, CsCl density banding of labeled RNA in a position coincident with nucleocapsids, correct sedimentation rate in sucrose gradients, the presence of the nucleocapsid protein on the nucleocapsids, and the infectivity of the in vitro assembled nucleocapsids. We conclude that the system we present is capable of assembling the isolated genome of a rhabdovirus DI particle into nucleocapsids indistinguishable from those produced during the course of intracellular DI replication.
Assuntos
Capsídeo/biossíntese , Vírus Defeituosos/genética , RNA Viral/metabolismo , Vírus da Estomatite Vesicular Indiana/genética , Proteínas do Core Viral/biossíntese , Animais , Capsídeo/genética , Linhagem Celular , Centrifugação com Gradiente de Concentração , Eletroforese em Gel de Ágar , Nuclease do Micrococo/metabolismo , RNA Viral/genética , Moldes Genéticos , Vírus da Estomatite Vesicular Indiana/metabolismo , Proteínas do Core Viral/genéticaRESUMO
Multiple forms of the vesicular stomatitis virus nucleocapsid protein N have been detected in infected cells. One form is complexed with the viral NS protein in a 1:1 molar ratio, and the other forms are distinguished by their more rapid sedimentation rates on glycerol gradients. I performed a series of experiments designed to analyze the relationships between these forms of the N protein. Pulse-chase experiments demonstrate that the N protein is made first as the form which binds to the NS protein, forming a 1-to-1 molar complex, and that with increasing times of chase it is either assembled into nucleocapsids or converted to the two higher sedimenting forms. Using a newly developed quantitative immunoblotting procedure, I have quantitated the three differentially sedimenting species of the N protein and have shown that at later times postinfection (6 to 7 h), the faster-sedimenting forms of the N protein account for as much as 50% of the soluble N protein in the cell. The activity of these forms has been assessed, with only the 1-to-1 molar N-NS complex demonstrating the ability to support the replication and encapsidation of viral genomic RNA. A model for the conversion of the N protein from the active N-NS complex into the other forms of the protein is presented, and the possible function of the N-protein self-complexes is discussed.
Assuntos
Capsídeo/fisiologia , Vírus da Estomatite Vesicular Indiana/fisiologia , Proteínas do Core Viral/fisiologia , Cinética , Peso Molecular , Ligação Proteica , Precursores de Proteínas/metabolismo , RNA Viral/metabolismo , Solubilidade , Ultracentrifugação , Vírus da Estomatite Vesicular Indiana/ultraestrutura , Proteínas não Estruturais Virais , Replicação ViralRESUMO
The viral proteins required for VSV RNA replication have been partially purified. With the use of monoclonal antibodies specific for the VSV N protein we have identified a putative N/NS complex present in the soluble protein fraction of infected cells. The complex is stable upon partial purification, contains the N and NS proteins in a 1:1 molar ratio, and has an elongated shape based on its hydrodynamic properties. Depletion of the N/NS complex from the infected cell soluble protein fraction results in the loss of the ability of this fraction to support RNA replication suggesting that the complex is required for this reaction. The ability to support viral genome RNA replication indeed cochromatographs with the N/NS protein complex through several steps of purification. Only the N protein of the N/NS complex appears to be bound to RNA during encapsidation with the release of NS protein.
Assuntos
Capsídeo/fisiologia , Vírus Defeituosos/genética , Vírus da Estomatite Vesicular Indiana/genética , Proteínas do Core Viral/fisiologia , Replicação Viral , Anticorpos Monoclonais , Capsídeo/imunologia , Cromatografia em Gel , Cromatografia por Troca Iônica , Peso Molecular , RNA Viral/genética , Proteínas do Core Viral/imunologiaRESUMO
Infection of several types of cultured cells with the paramyxoviruses simian virus 5 and Sendai virus stimulates synthesis of four polypeptides (I-IV) with molecular weights of approximately 99,000, 97,000, 86,000, and 78,000, respectively. That these are host polypeptides encoded in cellular mRNAs has been shown by the inhibition of their synthesis by actinomycin D and by the similarity of the peptide maps of them and of polypeptides with the same electrophoretic mobility from uninfected cells. Peptide mapping as well as identical migration in polyacrylamide gels has also indicated that polypeptides I, II, and IV are the same as plasma membrane polypeptides whose synthesis is enhanced in cells transformed by Rous sarcoma virus and in normal cells by glucose deprivation or treatment with 2-deoxyglucose. Polypeptides I and II appear to be the same polypeptides, with the observed differences in migration reflecting the glycosylation of polypeptide I, a relationship previously shown to exist between polypeptides in glucose-deprived and glucose-fed cells. Infection with paramyxoviruses does not significantly increase the transport of glucose by cells, and the maintenance of a high concentration of glucose in the medium does not prevent the enhanced synthesis of these polypeptides. This is in contrast to the situation in transformed cells in which stimulation of synthesis of these polypeptides is secondary to depletion of glucose in the medium due to increased glucose uptake by the cells. Thus, although paramyxovirus infection and transformation by Rous sarcoma virus result in stimulation of the synthesis of the same membrane polypeptides, the mechanism of stimulation differs.
Assuntos
Transformação Celular Viral , Glucose/metabolismo , Glicoproteínas/biossíntese , Proteínas de Membrana/biossíntese , Paramyxoviridae/metabolismo , Vírus do Sarcoma Aviário/metabolismo , Transporte Biológico , Dactinomicina/farmacologia , Glucose/farmacologia , Peso Molecular , Vírus da Parainfluenza 1 Humana/metabolismo , Fragmentos de Peptídeos/análiseRESUMO
Polypeptide synthesis in three different cell types infected with simian virus 5 has been examined using high-resolution polyacrylamide slab gel electrophoresis, and all of the known viral polypeptides have been identified above the host cell background. The polypeptides were synthesized in infected cells in unequal proportions, which are approximately the same as they are found in virions, suggesting that their relative rates of synthesis are controlled. The nucleocapsid polypeptide (NP) was the first to be detected in infected cells, and by 12 to 14 h the other virion structural polypeptides were identified, except for the polypeptides comprising the smaller glycoprotein (F). However, a glycosylated precursor (F(0)) with a molecular weight of 66,000 was found in each cell type, and pulse-chase experiments suggested that this precursor was cleaved to yield polypeptides F(1) and F(2). No other proteolytic processing was found. In addition to the structural polypeptides, the synthesis of five other polypeptides, designated I through V, has been observed in simian virus 5-infected cells. One of these (V), with a molecular weight of 24,000, was found in all cells examined and may be a nonstructural viral polypeptide. In contrast, there are polypeptides present in uninfected cells that correspond in size to polypeptides I through IV, and similar polypeptides have also been detected in increased amounts in cells infected with Sendai virus. These findings, and the fact that the synthesis of all four of these polypeptides is not increased in every cell type, suggest that they represent host polypeptides whose synthesis may be enhanced upon infection. When a high salt concentration was used to decrease host cell protein synthesis in infected cells, polypeptides IV and (to a lesser extent) I were synthesized in relatively greater amounts than other cellular polypeptides, as were the viral polypeptides. The possibility that these polypeptides may play some role in virus replication is discussed.