RESUMO
Certain groups of physically linked genes remain linked over long periods of evolutionary time. The general view is that such evolutionary conservation confers 'fitness' to the species. Why gene order confers 'fitness' to the species is incompletely understood. For example, linkage of IL26 and IFNG is preserved over evolutionary time yet Th17 lineages express IL26 and Th1 lineages express IFNG. We considered the hypothesis that distal enhancer elements may be shared between adjacent genes, which would require linkage be maintained in evolution. We test this hypothesis using a bacterial artificial chromosome transgenic model with deletions of specific conserved non-coding sequences. We identify one enhancer element uniquely required for IL26 expression but not for IFNG expression. We identify a second enhancer element positioned between IL26 and IFNG required for both IL26 and IFNG expression. One function of this enhancer is to facilitate recruitment of RNA polymerase II to promoters of both genes. Thus, sharing of distal enhancers between adjacent genes may contribute to evolutionary preservation of gene order.
Assuntos
Elementos Facilitadores Genéticos , Evolução Molecular , Interferon gama/genética , Interleucinas/genética , Animais , Linhagem da Célula/genética , Linhagem da Célula/imunologia , Cromossomos Artificiais Bacterianos/genética , Sequência Conservada , Ordem dos Genes , Histonas/metabolismo , Humanos , Camundongos , Camundongos Transgênicos , Modelos Genéticos , Regiões Promotoras Genéticas , RNA Polimerase II/metabolismo , RNA não Traduzido/genética , RNA não Traduzido/metabolismo , Deleção de Sequência , Células Th1/imunologia , Células Th17/imunologia , Interleucina 22RESUMO
Six amino-terminal deletion mutants of the NH2-terminally anchored (type II orientation) hemagglutinin-neuraminidase (HN) protein of parainfluenza virus type 3 were expressed in tissue culture by recombinant SV-40 viruses. The mutations consisted of progressive deletions of the cytoplasmic domain and, in some cases, of the hydrophobic signal/anchor. Three activities were dissociated for the signal/anchor: membrane insertion, translocation, and anchoring/transport. HN protein lacking the entire cytoplasmic tail was inserted efficiently into the membrane of the endoplasmic reticulum but was translocated inefficiently into the lumen. However, the small amounts that were successfully translocated appeared to be processed subsequently in a manner indistinguishable from that of parental HN. Thus, the cytoplasmic domain was not required for maturation of this type II glycoprotein. Progressive deletions into the membrane anchor restored efficient translocation, indicating that the NH2-terminal 44 amino acids were fully dispensable for membrane insertion and translocation and that a 10-amino acid hydrophobic signal sequence was sufficient for both activities. These latter HN molecules appeared to be folded authentically as assayed by hemagglutination activity, reactivity with a conformation-specific antiserum, correct formation of intramolecular disulfide bonds, and homooligomerization. However, most (85-90%) of these molecules accumulated in the ER. This showed that folding and oligomerization into a biologically active form, which presumably represents a virion spike, occurs essentially to completion within that compartment but is not sufficient for efficient transport through the exocytotic pathway. Protein transport also appeared to depend on the structure of the membrane anchor. These latter mutants were not stably integrated in the membrane, and the small proportion (10-15%) that was processed through the exocytotic pathway was secreted. The maturation steps and some of the effects of mutations described here for a type II glycoprotein resemble previous observations for prototypic type I glycoproteins and are indicative of close similarities in these processes for proteins of both membrane orientations.
Assuntos
Proteína HN/genética , Vírus da Parainfluenza 3 Humana/genética , Processamento de Proteína Pós-Traducional , Respirovirus/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Códon/genética , Dissulfetos/metabolismo , Proteína HN/biossíntese , Proteína HN/metabolismo , Substâncias Macromoleculares , Dados de Sequência Molecular , Peso Molecular , Mutação , Sondas de Oligonucleotídeos , Biossíntese de Proteínas , Mapeamento por Restrição , Vírus 40 dos Símios/genéticaRESUMO
Fetal membranes are postulated to play a role in paracrine signaling during the initiation of labor in women. We developed a dual chamber-fetal membrane-uterine muscle model to study the effect of human fetal membranes on spontaneous uterine contractions. In this model, full-thickness fetal membranes (amnion, chorion, and maternal decidua) are sealed into a Plexiglass chamber. The membranes partition the chamber into a maternal and fetal compartment. Chorion and decidua face the maternal side, and amnion faces the fetal side. An estrogenized rat uterine muscle strip is anchored into the maternal side as a bioassay to measure effects of fetal membranes on uterine contractions. Fetal membranes cause a 40% decrease in uterine contractions compared to basal condition (no membranes). Inhibition is reversible after removal of the membranes. The inhibition is specific to the chorion/decidual side because reversal of membranes with amnion toward the muscle did not show inhibition. Uterine contractions did not change over time in control chambers in which Parafilm substituted for membranes. A model for studying paracrine regulation of uterine contractions by human fetal membranes has been developed. The model provides evidence that fetal membranes inhibit uterine contractions. This inhibitory effect may contribute to uterine quiescence during pregnancy.
Assuntos
Membranas Extraembrionárias/fisiologia , Contração Uterina , Animais , Feminino , Humanos , Técnicas In Vitro , Gravidez , Ratos , Ratos WistarRESUMO
Prostaglandin (PG) production by fetal membranes has been implicated in the initiation of human parturition, but its regulation is not well understood. We used an in vitro system to study paracrine control of term, fetal membrane PG production. Using a modified Ussing chamber, full thickness fetal membranes with attached decidua were sealed into a chamber so that each hemichamber was a compartment for either the fetal (amnion) or maternal (chorion/decidua) side. Released PGs from maternal and fetal sides were then measured after exposure of the amnion to either buffer or amniotic fluid. We found that basal release of PGs from both the fetal and maternal sides was 2- to 3-fold higher in membranes obtained after labor compared to those obtained before labor. When amnion obtained after labor was exposed to amniotic fluid, we found a 3- to 5-fold increase in the net release of PGE2 from the amnion; however, the maternal side showed an unexpected relative decrease in PGE2 and PGF2 alpha release. This was a paracrine effect, since direct exposure of chorion/decidua to amniotic fluid caused increased release of the PG precursor, arachidonic acid. Direct transfer of radiolabeled PG from fetal to maternal side was minimal.
Assuntos
Âmnio/metabolismo , Líquido Amniótico/fisiologia , Córion/metabolismo , Decídua/metabolismo , Prostaglandinas/metabolismo , Ácido Araquidônico/metabolismo , Feminino , Humanos , Troca Materno-Fetal , GravidezRESUMO
A total of 13 respiratory syncytial (RS) virus specific polypeptides were identified by pulse-chase metabolic labeling of infected HEp-2 cells. Ten of the 13 proteins were shown to be unique. They were the L, G, F (F1, F2), N, P, M, 24K, 14K, 11K and 9.5K proteins. These conclusions were based on peptide mapping and on previous work showing that each of 10 polypeptides are coded for by a unique mRNA. The seven largest proteins, L, G, F (F1, F2), N, P, M and 24K were identified clearly as virion structural proteins. The 24K protein was characterized by detergent and salt dissociation studies as an envelope-associated protein, bringing to four (G, F (F1, F2), M and 24K) the number of membrane associated proteins for RS virus. A fourth membrane-associated protein has not been described previously for any other paramyxovirus. Of the three smallest proteins, the 14K and 11K were characterized as non-structural proteins. The 9.5K protein was detected in low amounts in highly purified preparations of virions.
Assuntos
Vírus Sinciciais Respiratórios/análise , Proteínas do Envelope Viral/análise , Proteínas Virais/análise , Carcinoma de Células Escamosas , Linhagem Celular , Eletroforese em Gel de Poliacrilamida , Humanos , Neoplasias Pulmonares , Peso Molecular , Peptídeos/análise , Proteínas Estruturais Virais , Vírion/análiseRESUMO
The replication of RSV in unimmunized cotton rats was evaluated by quantitating the amount of infectious virus in the lung and the number of RSV infected cells in a histopathological section of lung by in situ hybridization. RSV infected cells were detected only in alveoli and bronchioles and constituted only a small minority of the cell population. The temporal patterns of rise to the peak number of infected cells (day 4) and the peak titer of infectious virus (day 3) were similar. The clearance of both infected cells and infectious virus was nearly complete by day 7. In animals previously immunized with purified RSV glycoproteins or formalin-inactivated RSV there also was a good correlation between the number of infected cells detected by in situ hybridization and the amount of infectious virus recovered. It was previously demonstrated that cotton rats immunized with formalin-inactivated vaccine developed enhanced pulmonary histopathology following challenge with RSV. In such animals, there was approximately a 90% reduction in the number of infected cells compared to control unimmunized, RSV-challenged animals. Formalin-inactivated RSV vaccine-enhanced lung histopathology developed despite the effective elimination of virus and virus-infected cells suggesting that the enhanced pathology is the result of an exaggeration of normal immune mechanisms involved in clearance of virus infection, an aberrant immune response during infection, or both.
Assuntos
Antígenos Virais/imunologia , Formaldeído/farmacologia , Proteína HN , Pulmão/microbiologia , Vírus Sinciciais Respiratórios/crescimento & desenvolvimento , Infecções por Respirovirus/imunologia , Proteínas Virais , Vacinas Virais/imunologia , Ativação Viral/efeitos dos fármacos , Animais , Relação Dose-Resposta Imunológica , Pulmão/patologia , Hibridização de Ácido Nucleico , RNA Mensageiro/análise , Ratos , Vírus Sinciciais Respiratórios/efeitos dos fármacos , Vírus Sinciciais Respiratórios/genética , Proteínas do Envelope Viral , Replicação ViralRESUMO
RSV and PIV3 are responsible for about 30% of severe viral respiratory tract disease leading to hospitalization of infants and children. For this reason, there is a need to develop vaccines effective against these viruses. Since these viruses cause severe disease in early infancy, vaccines must be effective in the presence of maternal antibody. Currently, several strategies for immunization against these viruses are being explored including peptide vaccines, subunit vaccines, vectored vaccines (e.g., vaccinia-RSV or adenovirus-RSV recombinants), and live attenuated virus vaccines. The current status of these approaches is reviewed. In addition, the immunologic basis for the disease potentiation seen in vaccinees immunized with formalin-inactivated RSV during subsequent RSV infection is reviewed. The efficacy of immunization in the presence of maternal antibody is discussed. Much progress for a RSV and PIV3 vaccine has been made and successful immunization against each of these pathogens should be achieved within this decade.
Assuntos
Vacinas contra Influenza , Vírus da Parainfluenza 3 Humana/imunologia , Vírus Sinciciais Respiratórios/imunologia , Vacinas Virais , Adulto , Animais , Anticorpos Anti-Idiotípicos/imunologia , Anticorpos Antivirais/biossíntese , Anticorpos Antivirais/imunologia , Ensaios Clínicos como Assunto , Humanos , ISCOMs , Imunidade Materno-Adquirida , Lactente , Recém-Nascido , Vacinas contra Influenza/imunologia , Vacinas contra Influenza/toxicidade , Influenza Humana/prevenção & controle , Camundongos , Pan troglodytes , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/imunologia , Infecções por Vírus Respiratório Sincicial/prevenção & controle , Sigmodontinae , Vacinação , Vacinas Atenuadas , Vacinas Sintéticas , Vacinas Virais/imunologia , Vacinas Virais/toxicidadeRESUMO
Vaccines against parainfluenza (PIV) and respiratory syncytial viruses (RSV) that are currently being developed include both live and subunit vaccines. Candidate live PIV vaccines that have been found to be attenuated and efficacious in rodents or primate models are (1) cold-adapted, temperature-sensitive mutants of PIV-type 3 that have been serially passaged at low temperature (20 degrees C) in simian kidney tissue culture; (2) protease-activation mutants (PIV-1-Sendai), which have mutations that decrease the cleavability of their F glycoprotein by host cell protease; (3) an animal virus, bovine PIV-3 virus, which is antigenically related to the human PIV-3 virus, and (4) vaccinia recombinant viruses bearing RSV or PIV-3 glycoproteins. Subunit RSV and PIV-3 viruses are being produced and evaluated as immunogens. A major concern with these vaccines is the possibility of disease potentiation following virus infection as occurred previously with formalin-inactivated measles and RSV vaccines. Studies indicate that PIV-3 and RSV glycoprotein vaccines are immunogenic and efficacious in animals but insufficient data exist to estimate their capacity to potentiate disease. However, since a cotton rat model is available to detect potentiated disease resulting from infection of cotton rats previously immunized with formalin-inactivated RSV vaccine, it is now possible to systematically evaluate new vaccines in experimental animals for disease potentiation before studies are initiated in humans. It is likely within the next several years that one or more of these PIV or RSV vaccines will be tested in humans for safety and immunogenicity.
Assuntos
Vírus Sinciciais Respiratórios/imunologia , Respirovirus/imunologia , Vacinas Virais/isolamento & purificação , Animais , Antígenos Virais , Humanos , Infecções por Paramyxoviridae/prevenção & controle , Infecções por Respirovirus/prevenção & controleRESUMO
BACKGROUND: Necrotizing fasciitis is an uncommon, rapidly progressive, life-threatening infection involving the subcutaneous tissue and fascia. Usually, it is a synergistic polymicrobic infection that occurs in patients with coexisting factors predisposing them to bacterial inoculation and the spread of infection. CASES: We report a monomicrobial variant of necrotizing fasciitis affecting three otherwise healthy pregnant or postpartum women. The necrotizing fasciitis involved either the lower extremity or the abdominal wall. The causative bacteria were Streptococcus pyogenes (two cases) and Staphylococcus aureus (one). All patients presented with an acute fulminant infection, including one woman who died from overwhelming sepsis. CONCLUSION: These cases raise a question about the possible role of increased bacterial virulence and the immunologic changes of pregnancy as potential predisposing factors in the development of necrotizing fasciitis.
Assuntos
Fasciite Necrosante/microbiologia , Complicações Infecciosas na Gravidez/microbiologia , Infecção Puerperal/microbiologia , Infecções Estafilocócicas/epidemiologia , Streptococcus pyogenes/patogenicidade , Adulto , Causalidade , Fasciite Necrosante/epidemiologia , Feminino , Humanos , Idade Materna , Gravidez , Complicações Infecciosas na Gravidez/epidemiologia , Gravidez de Alto Risco , Infecção Puerperal/epidemiologia , Staphylococcus aureus/patogenicidade , VirulênciaRESUMO
Prostaglandin (PG) production by human amnion has been postulated to have a role in the onset of labor. Previous work by ourselves and others has demonstrated that oxytocin, phorbol esters and epidermal growth factor (EGF) increase PGE2 production in human amnion cells by activation of the Phospholipase C/Protein Kinase C (PKC) cascade system. The present study was undertaken to determine the effect of prior activation of the Adenylate Cyclase cascade system upon subsequent stimulation of PGE2 production by oxytocin, phorbol 12-myristate-13-acetate (PMA) or EGF in amnion cells and membrane discs. Isoproterenol, forskolin and dibutyryl cyclic adenosine monophosphate (dbcAMP) were utilized to activate the Adenylate Cyclase system at the receptor, enzyme and second messenger level. In control amnion cells, oxytocin, PMA and EGF each provoked dose dependent increases in PGE2 production. In cells preincubated with dbcAMP, forskolin or isoproterenol, agonist stimulated PGE2 production was markedly (50-90%) inhibited (p < 0.01). Inhibition was dose dependent upon preincubator concentrations. Maximal inhibition by adenylate cyclase activators occurred with 2-4 h of preincubation. In membrane discs, forskolin preincubation also inhibited oxytocin, PMA and EGF stimulation of PGE2 production. Activation of the Adenylate Cyclase system in human amnion cells or membrane discs inhibits the subsequent action of potent stimulators of PGE2 production in human amnion.
Assuntos
Adenilil Ciclases/metabolismo , Âmnio/metabolismo , Dinoprostona/biossíntese , Feto/metabolismo , Proteínas Quinases/metabolismo , Âmnio/citologia , Âmnio/efeitos dos fármacos , Bucladesina/farmacologia , Colforsina/farmacologia , Relação Dose-Resposta a Droga , Ativação Enzimática , Fator de Crescimento Epidérmico/farmacologia , Feminino , Feto/efeitos dos fármacos , Humanos , Técnicas In Vitro , Isoproterenol/farmacologia , Ocitocina/farmacologia , Gravidez , Terceiro Trimestre da Gravidez , Transdução de Sinais , Acetato de Tetradecanoilforbol/farmacologiaAssuntos
Vírus Sinciciais Respiratórios , Infecções Respiratórias/microbiologia , Infecções por Respirovirus , Animais , Criança , Humanos , Imunoterapia Ativa , Vírus Sinciciais Respiratórios/imunologia , Infecções Respiratórias/prevenção & controle , Infecções Respiratórias/terapia , Infecções por Respirovirus/imunologia , Infecções por Respirovirus/prevenção & controle , Infecções por Respirovirus/terapia , Vacinas ViraisRESUMO
cDNAs encoding the G glycoprotein of respiratory syncytial virus and the hemagglutinin-neuraminidase (HN) glycoprotein of parainfluenza virus type 3 were modified by site-specific mutagenesis and restriction fragment replacement to encode chimeric proteins consisting of the cytoplasmic and transmembrane domains of one protein fused to the ectodomain of the other. In the case of the HN ectodomain attached to the G transmembrane and cytoplasmic domains, cell surface expression of the chimera was reduced. Otherwise, the presence of the heterologous transmembrane and cytoplasmic domains had little effect on the processing of the HN or G ectodomain, as assayed by the acquisition of N-linked and O-linked carbohydrates, transport to the cell surface and, in the case of HN, folding, oligomerization, and hemadsorption activity. These results showed that the synthesis and processing of each ectodomain did not require the homologous transmembrane and cytoplasmic domains. In particular, O glycosylation of the G protein was specified fully by its ectodomain, even though this domain is highly divergent among the respiratory syncytial virus antigenic subgroups. In addition, whereas the cytoplasmic and transmembrane domains of the G protein were relatively highly conserved, they were nonetheless fully replaceable without significantly affecting processing.
Assuntos
Antígenos Virais/genética , Processamento de Proteína Pós-Traducional , Vírus Sinciciais Respiratórios/genética , Proteínas Virais , Sequência de Aminoácidos , Animais , Antígenos Virais/análise , Sequência de Bases , Linhagem Celular , Membrana Celular/metabolismo , Quimera , Códon/genética , Citoplasma/metabolismo , DNA Viral/genética , Imunofluorescência , Vetores Genéticos , Glicosilação , Proteína HN/análise , Proteína HN/genética , Dados de Sequência Molecular , Mutação , Vírus da Parainfluenza 3 Humana/genética , Mapeamento por Restrição , Vírus 40 dos Símios/genética , Proteínas do Envelope ViralRESUMO
The posttranslational maturation of the hemagglutinin-neuraminidase (HN) glycoprotein of human parainfluenza type 3 virus (PIV3) was investigated in pulse-chase experiments in which folding was monitored by immunoprecipitation with conformation-dependent antibodies and gel electrophoresis under nonreducing conditions and oligomerization was monitored by chemical cross-linking and sedimentation in sucrose gradients. The acquisition of mature immunoreactivity and the formation of correct intramolecular disulfide bonds were concurrent events, with half-times of approximately 10 to 15 min. The finding that newly synthesized HN had little reactivity with postinfection cotton rat serum or with most of the members of a panel of HN-specific monoclonal antibodies indicated that the major epitopes of the PIV3 HN protein are highly conformational in nature. Chemical cross-linking studies indicated that the mature HN protein is present in homoligomers, which are probably tetramers. These findings are consistent with recent observations for the HN protein of Sendai virus (S.D. Thompson, W.G. Laver, K.G. Murti, and A. Portner, J. Virol. 62:4653--4660, 1988; S. Vidal, G. Mottet, D. Kolakofsky, and L. Roux, J. Virol. 63:892--900, 1989). Surprisingly, analysis of pulse-labeled HN protein by sedimentation on sucrose gradients after labeling periods of as little as 2 min indicated that it was present intracellularly only in oligomeric form. The same results were obtained when the labeling period was preceded by a 1.5-h cycloheximide treatment to clear the endoplasmic reticulum of presynthesized HN protein, which indicated that the oligomerization did not involve the incorporation of newly synthesized monomers into partially assembled oligomers. Subsequent chase incubations did not significantly alter the sedimentation profile or stability of the oligomeric forms, suggesting that oligomers detected after short labeling periods were tetramers. Association with cellular proteins did not appear to be responsible for the sedimentation of newly synthesized HN protein as an oligomer. The absence of a detectable monomeric form of intracellular HN protein raised the possibility that oligomerization is cotranslational, and it is possible that the type II membrane orientation of the HN protein might be an important factor in its mode of oligomerization.
Assuntos
Dissulfetos/metabolismo , Proteína HN/metabolismo , Vírus da Parainfluenza 3 Humana/metabolismo , Processamento de Proteína Pós-Traducional , Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Centrifugação com Gradiente de Concentração , Reagentes de Ligações Cruzadas , Epitopos , Proteína HN/imunologia , Cinética , Vírus da Parainfluenza 3 Humana/imunologia , Polímeros , Testes de Precipitina , Conformação Proteica , Fatores de TempoRESUMO
Two major antigenic subgroups (A and B) have been described for human respiratory syncytial virus. The complete nucleotide sequence was determined for the fusion (F) mRNA of the subgroup B strain 18537 and the amino acid sequence of the F protein deduced, for comparison with the previously described sequences for the A2 strain of subgroup A. The F proteins (excluding the cleaved signal peptide) were 91% identical between the subgroups, consistent with the previously described high degree of antigenic relatedness. The greatest divergence occurred within the F2 subunit immediately preceding the cleavage activation site.
Assuntos
RNA Viral , Vírus Sinciciais Respiratórios/genética , Proteínas Virais de Fusão/genética , Sequência de Aminoácidos , Antígenos Virais/genética , Sequência de Bases , Humanos , Dados de Sequência Molecular , RNA Mensageiro , Vírus Sinciciais Respiratórios/imunologia , Especificidade da Espécie , Proteínas Virais de Fusão/imunologiaRESUMO
Intergenic and flanking gene regions for the 1C-1B, 1B-N, N-P, M-1A, G-F and F-22K gene junctions of respiratory syncytial virus strain 18537, representing antigenic subgroup B, were determined by dideoxynucleotide sequencing of polycistronic cDNAs and mRNAs. Comparison with their counterparts from the subgroup A strain A2 showed that the intergenic sequences were not conserved within or between the strains. Flanking non-coding gene sequences also were generally not conserved except for the highly conserved gene-start and gene-end transcription signals. The sequence of the overlap between the 22K and L genes was conserved almost exactly between the two subgroups.
Assuntos
Vírus Sinciciais Respiratórios/classificação , Sequência de Bases , Clonagem Molecular , Genes Virais , Dados de Sequência Molecular , RNA Mensageiro/genética , RNA Viral/genética , Vírus Sinciciais Respiratórios/genéticaRESUMO
The 1A mRNA is the smallest mRNA of human respiratory syncytial (RS) virus and encodes a single protein of approximate molecular weight 9500 as estimated previously by gel electrophoresis. The nucleotide sequence of the 1A mRNA, determined from several full-length cDNA clones, is reported. The 1A mRNA consists of 405 nucleotides, exclusive of poly(A), with relatively long nontranslated regions at the 5' and 3' ends (84 and 126 nucleotides, respectively). The sequences at the 5' and 3' termini of the 1A mRNA conform to the previously described conserved consensus sequences for RS virus mRNAs. The major open reading frame of the 1A mRNA codes for a hydrophobic polypeptide of 64 amino acids with a calculated molecular weight of 7536. The 5' terminus of the 1A mRNA was mapped and sequenced by primer extension under conditions for sequencing by partial chain termination. These experiments also identified a population of polycistronic RNA having the general structure: 5' M protein mRNA-1A mRNA 3'. This polytranscript was sequenced in order to determine the intergenic sequence. In the polytranscript, the nucleotide sequence of the M gene is followed by, in mRNA sense, six A residues and the intergenic sequence 5' ... UAUACACNN (N represents unidentified nucleotide).
Assuntos
RNA Mensageiro/genética , RNA Viral/genética , Vírus Sinciciais Respiratórios/genética , Sequência de Aminoácidos , Sequência de Bases , DNA , Genes , Genes Virais , Transcrição Gênica , Proteínas Virais/genéticaRESUMO
We recently determined that respiratory syncytial virus (strain A2) encodes a fourth unique envelope-associated virion protein that has molecular weight of approximately 24,000, as estimated by gel electrophoresis. The nucleotide sequence of the mRNA encoding this novel protein has now been determined from five cDNA clones, including three that contain the complete mRNA sequence. The complete mRNA sequence is 957 nucleotides, exclusive of polyadenylate, and contains two partially overlapping open reading frames. The 5'-proximal open reading frame is favored for utilization by the criteria of the location and sequence of its translational start site. Furthermore, the calculated molecular weight of the encoded protein, 22,153, is in agreement with the previous estimate of 24,000 for the authentic protein identified by hybrid selection and in vitro translation. The sequence of the predicted protein, now designated the 22K protein, contains 194 amino acids, is relatively hydrophilic, and appears to be the most basic of the respiratory syncytial virus proteins. The mRNA also contains a second, internal open reading frame which would encode a protein of 90 amino acids. However, no evidence for this translation product is known. The first nine nucleotides in the mRNA sequence, 5'-GGGGCAAAU, are identical to the conserved sequence identified previously at the 5' termini of seven other respiratory syncytial viral mRNAs; the sequence at the 3' end of the 22K mRNA, 5'. . . AGUUAUUU-polyadenylate, contains the elements of the previously identified 3'-terminal consensus sequence for respiratory syncytial virus mRNAs, AGUUAA(N)1-4-polyadenylate (P. L. Collins, Y. T. Huang, and G. W. Wertz, Proc. Natl. Acad. Sci. U.S.A. 81:7683-7687). In addition, we present and describe the intergenic sequence of a dicistronic RNA derived from readthrough of the F and 22K protein genes.
Assuntos
RNA Viral/genética , Vírus Sinciciais Respiratórios/genética , Proteínas do Envelope Viral/genética , Sequência de Aminoácidos , Sequência de Bases , DNA/genética , RNA Mensageiro/genética , SolubilidadeRESUMO
The complete nucleotide sequence of the P + C mRNA of human parainfluenza virus type 3 (PF3) was determined by sequencing cDNA, viral genomic RNA and mRNA. The P + C mRNA is 2009 nucleotides in length, exclusive of poly(A), and contains two overlapping open reading frames (ORFs). The P + C mRNA encodes two proteins, the 602 amino acid nucleocapsid phosphoprotein P and the 199 amino acid non-structural protein C. Peptide mapping confirmed that the two proteins are unrelated. Hybrid-arrest translation experiments assigned each of the two proteins to its respective ORF. These studies showed that the coding strategy of the PF3 P + C mRNA is similar to that of Sendai virus. Amino acid sequence alignment showed that the P and C proteins of PF3 and Sendai virus represent homologous pairs. However, these homologies are represented by high contents of accepted amino acid substitutions and by similarity in hydropathy profiles rather than by high contents of exact amino acid matches. Homology with the P and C proteins of measles, canine distemper and respiratory syncytial viruses was at the threshold of significance. The patterns of amino acid sequence homology among the paramyxovirus HN, F, NP, P and C proteins are compared.
Assuntos
Capsídeo/genética , Genes Virais , Vírus da Parainfluenza 3 Humana/genética , Paramyxoviridae/análise , RNA Viral/genética , Respirovirus/genética , Proteínas do Core Viral/genética , Proteínas Virais/genética , Sequência de Aminoácidos , Sequência de Bases , Capsídeo/análise , DNA , Vírus da Cinomose Canina/análise , Vírus da Cinomose Canina/genética , Humanos , Vírus do Sarampo/análise , Vírus do Sarampo/genética , Hibridização de Ácido Nucleico , Vírus da Parainfluenza 1 Humana/análise , Vírus da Parainfluenza 1 Humana/genética , Vírus da Parainfluenza 3 Humana/análise , Paramyxoviridae/genética , Mapeamento de Peptídeos , RNA Mensageiro/genética , Vírus Sinciciais Respiratórios/análise , Vírus Sinciciais Respiratórios/genética , Proteínas do Core Viral/análise , Proteínas Virais/análiseRESUMO
cDNA clones of mRNAs for the major nucleocapsid protein (NP), the nucleocapsid P protein plus the nonstructural C protein (P+C), and the matrix protein (M) of human parainfluenza virus type 3 (PF3) were identified by hybrid arrest and hybrid selection of in vitro translation. Previously, cDNA clones were identified and sequenced for the hemagglutinin-neuraminidase glycoprotein (HN) and the fusion glycoprotein (F) mRNAs (N. Elango, J. E. Coligan, R. C. Jambou, and S. Venkatesan, J. Virol. 57:481-489, 1986; M. K. Spriggs, R. A. Olmsted, S. Venkatesan, J. E. Coligan, and P. L. Collins, Virology 152:241-251, 1986). Synthetic oligonucleotides, designed from nucleotide sequences of the cDNAs, were used to direct dideoxynucleotide sequencing of gene junctions in PF3 genomic RNA (vRNA). From sequencing of vRNA, a sixth viral gene was detected and identified as the large nucleocapsid protein (L) gene by hybridization of a synthetic oligonucleotide to intracellular PF3 mRNAs separated by gel electrophoresis. The order of the six PF3 genes on vRNA was 3'-NP-P+C-M-F-HN-L-5'. The five intergenic regions consisted of the trinucleotide 3'-GAA. The PF3 genes initiated with semiconserved 10-nucleotide gene-start sequences and terminated with semiconserved 12-nucleotide gene-end sequences. The M gene terminated with an aberrant gene-end sequence; analysis of intracellular mRNA showed that this aberrant sequence correlated with a disproportionately high accumulation of readthrough mRNA. These studies showed that PF3 encodes six unique mRNAs (NP, P+C, M, F, HN, and L) that encode seven proteins (NP, P, C, M, F, HN, and L) and provided evidence of a close relationship between PF3 and Sendai (murine parainfluenza type 1) viruses.
Assuntos
Genes Virais , Vírus da Parainfluenza 3 Humana/genética , RNA Mensageiro/genética , RNA Viral/genética , Respirovirus/genética , Proteínas Virais/genética , Sequência de Bases , Capsídeo/genética , DNA , Peptídeos/genética , Proteínas do Core Viral/genética , Proteínas da Matriz ViralRESUMO
The genes encoding the 1C and 1B mRNAs of human respiratory syncytial (RS) virus are first in the order of viral transcription and encode nonstructural (NS) proteins of approximate molecular weights 14,000 and 11,000, respectively, estimated by gel electrophoresis. The complete nucleotide sequences of the 1C and 1B mRNAs determined from several full-length cDNA clones are described. The 1C and 1B mRNAs contain 528 and 499 nucleotides, respectively, exclusive of poly(A), and encode proteins of 139 and 124 amino acids. The calculated molecular weights of the predicted NS1C and NS1B proteins are 15,567 and 14,674, respectively. Both mRNA sequences contain the 5'-terminal sequence, 5' GGGGCAAAU . . . , and the 3'-terminal sequence, 5' . . . AGUAUA(N)1-4-poly(A), that were identified previously as conserved among six other RS viral mRNAs. In addition, a dicistronic readthrough RNA having the general structure 5' 1C mRNA-intergenic sequence-1B mRNA 3' was identified by dideoxynucleotide sequencing of intracellular poly(A)+ RNA using a DNA primer derived from a 1B-cDNA clone. In the dicistronic RNA, the nucleotide sequences of the 1C and 1B cistrons are separated by, in mRNA sense, four A residues and the intergenic sequence 5' . . . CUUAACAGAAGACAAAAAN . . . 3' (N represents unidentified nucleotide). The significance of these sequences is discussed.