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immunity to hantavirus challenge in meriones unguiculatus induced by vaccinia-vectored viral proteins.vaccinia virus recombinants were constructed that incorporated genomic sequences coding for the nucleoprotein (n) and glycoproteins (g1 and g2) of the hantavirus r22 strain isolated from a rat in china, and designated as rnv and rmv9, respectively. the proteins expressed by rnv and rmv9 were identified by radioimmunoprecipitation and indirect immunofluorescence assay using a panel of monoclonal antibodies and polyclonal immune sera, and were found to be antigenically indistinguishable from authe ...19921359802
vaccinia virus: a versatile tool for molecular biologists.continued advances in genetic engineering have made possible the high-level expression of correctly processed cellular, viral and bacterial polypeptides. this article focuses on viral expression vectors and, more specifically, the vaccinia virus expression system. vaccinia virus has been used to express a variety of proteins with useful immunogenic, catalytic or pharmaceutical properties. we discuss briefly the biology of vaccinia and its significance in the use of vaccinia as an expression vect ...19901366568
a modern vaccine for an ancient plague: rinderpest. 19901366928
propagation of recombinant vaccinia virus in hela cells: adsorption kinetics and replication in batch cultures.the influence of various culture parameters on infection and replication of recombinant vaccinia virus in hela cells was examined during various phases of viral replication. a modified form of the model of valentine and allison (biochim. biophys. acta 1960, 40, 393-399) model was used to predict successfully the viral adsorption rates in cell suspensions. an experimentally determined aggregation factor, epsilon, was included in the model to account for deviations of the observed adsorption rates ...19911367173
vaccinia virus vectors for gene expression.improvements to vaccinia virus expression vectors continue to be made. in particular, there are new methods for the construction of recombinant viruses, ways of increasing the level of gene expression, and vectors that allow the inducible expression of selected genes.19911367723
biological control of wild animal infections.until recently, there were few examples of biological control of wild animal infections. the most significant development in this field is the use of a vaccinia--rabies recombinant virus or other recombinants for the control of rabies by oral vaccination of vectors, both in europe and in north america.19911367833
vaccinia and other poxvirus expression vectors.over the past year improvements have been made in recombinant vaccinia virus gene expression and a new method for inserting dna into the poxvirus genome has been developed, along with alternative methods for selecting recombinant viruses. attenuated and non-replicating vaccinia virus and avian poxvirus vectors are now being used successfully. field trials of an oral, wild-life rabies vaccine and phase 1 testing of human vaccines derived from vaccinia virus are underway.19921368937
cytokine expression by recombinant viruses--a new vaccine strategy. 19921369132
a cd4+ cytotoxic t-lymphocyte clone to a conserved epitope on human immunodeficiency virus type 1 p24: cytotoxic activity and secretion of interleukin-2 and interleukin-6.a cd4+ cytotoxic t-lymphocyte (ctl) clone, established from the peripheral blood of a human immunodeficiency virus (hiv)-seropositive donor, lysed autologous target cells that were infected with a recombinant vaccinia virus containing the gag gene of hiv type 1 and target cells pulsed with p24gag construct expressed in escherichia coli. the recognition of the hla-dq-restricted epitope by this clone was further defined by using overlapping synthetic peptides. the epitope recognized by this cd4+ c ...19921370094
expression of the p-protein of the human hepatitis b virus in a vaccinia virus system and detection of the nucleocapsid-associated p-gene product by radiolabelling at newly introduced phosphorylation sites.hepatitis b virus (hbv) contains a particle-associated dna polymerase/reverse transcriptase activity encoded by the p (pol) open reading frame. due to its low abundance, the corresponding protein has so far escaped direct detection and structural analysis. as a first step to overcome these difficulties, a series of recombinant vaccinia viruses was constructed and used for the synthesis in human hepatoma cells of both the authentic full length protein and of its functional domains. pulse chase ex ...19921371344
volume-regulated chloride channels associated with the human multidrug-resistance p-glycoprotein.expression of p-glycoprotein, the product of the mdr1 gene, confers multidrug resistance on cell lines and human tumours (reviewed in refs 1,2). p-glycoprotein (relative molecular mass 170,000) is an atp-dependent, active transporter which pumps hydrophobic drugs out of cells, but its normal physiological role is unknown. it is a member of the abc (atp-binding cassette) superfamily of transporters, which includes many bacterial transport systems, the putative peptide transporter from the major h ...19921371598
an antigenic structure of the trans-activator protein encoded by human t-cell leukemia virus type-i (htlv-i), as defined by a panel of monoclonal antibodies.in order to study an antigenic structure of the trans-activator protein encoded by human t-cell leukemia virus type-i (htlv-i), tax1 antigen, we generated and characterized a panel of rat anti-tax1 monoclonal antibodies (mabs) designated watm-1, watm-2, watm-3, and watm-4. these mabs were derived from wka rats immunized with htlv-i-transformed (htlv-i+) syngeneic t cells. immunoblot assays showed that: (1) all the mabs reacted with the tax1 antigen in htlv-i+ cell lines and a recombinant tax1 an ...19921371693
interaction between viruses and monoclonal antibodies studied by surface plasmon resonance.an automated biosensor system designed for measuring molecular interactions in real time and without any labelling of the reactants has been used to study the interaction of two animal viruses (vaccinia virus and poliovirus) and two plant viruses (cowpea mosaic virus and tobacco mosaic virus) with monoclonal antibodies. using monoclonal antibodies specific for different conformational states of viral protein, it was found that the virus particles retained their conformational integrity when immo ...19921372280
target epitope in the tax protein of human t-cell leukemia virus type i recognized by class i major histocompatibility complex-restricted cytotoxic t cells.a trans-acting regulatory gene product p40tax (tax) of human t-cell leukemia virus type i (htlv-i) is one of the main target antigens recognized by cytotoxic t lymphocytes (ctl) specific for htlv-i. a ctl epitope within the tax protein was identified in this report. htlv-i-specific cd8+ ctl lines established from two htlv-i carriers with htlv-i-associated myelopathy or sjögren syndrome were previously demonstrated to kill predominantly the target cells expressing htlv-i tax. the ctl from two pat ...19921373197
ternary complex formation by vaccinia virus rna polymerase at an early viral promoter: analysis by native gel electrophoresis.we have resolved, by native gel electrophoresis, two intermediates in the transcription of a vaccinia virus early gene by the virus-encoded rna polymerase. polymerase holoenzyme containing the vaccinia virus early transcription factor (vetf) forms a complex of vetf bound to the promoter as the first step in a pathway leading to establishment of a committed ternary elongation complex. formation of the vetf-dna complex is stimulated by magnesium but is uninfluenced by nucleoside triphosphates. a s ...19921373199
mutation of human immunodeficiency virus type 1 at amino acid 585 on gp41 results in loss of killing by cd8+ a24-restricted cytotoxic t lymphocytes.a human leukocyte antigen a24-restricted cd8+ cytotoxic t-cell clone specific for gp41 of human immunodeficiency virus type 1 was isolated from an infected individual. the epitope was localized to amino acids 584 to 591 (ylkdqqll, nl43 env sequence) of gp41 by using a panel of recombinant vaccinia viruses that contain truncated env genes and synthetic peptides. the clone killed autologous b-lymphoblastoid cell lines pulsed with a synthetic peptide reflecting the sequence of the iiib and mn strai ...19921373204
expression of authentic vaccinia virus-specific and inserted viral and cellular genes under control of an early vaccinia virus promoter is regulated post-transcriptionally in interferon-treated chick embryo fibroblasts.the interferon sensitivity of the expression of an influenza-virus hemagglutinin (ha) gene cloned into the thymidine kinase (tk) gene of vaccinia virus was studied in chick embryo fibroblasts (cef) and madin-darby bovine kidney (mdbk) cells. in cef, the expression of the ha gene is inhibited by pretreatment of cells with homologous interferon. in mdbk cells, on the other hand, expression of the ha is not impaired by pretreatment with human interferon-alpha, and the synthesis of early vaccinia vi ...19921373550
mechanism of interferon action: cdna structure, expression, and regulation of the interferon-induced, rna-dependent p1/eif-2 alpha protein kinase from human cells.a molecular cdna clone (p1 kin) was isolated that encodes the human rna-dependent p1/eif-2 alpha protein kinase. the complete cdna sequence of the p1 kin cdna was determined; the longest open reading frame (orf) encoded a 551 amino acid protein with a deduced molecular weight of 62055 da. transcripts prepared from the p1 kin cdna by transcription in vitro with t7 rna polymerase programmed the cell-free synthesis of a protein indistinguishable by immunoprecipitation and immunoblot gel analyses fr ...19921373553
mechanism of interferon action: identification of a rna binding domain within the n-terminal region of the human rna-dependent p1/eif-2 alpha protein kinase.a molecular cdna clone of the human rna-dependent p1/eif-2 alpha protein kinase was expressed in escherichia coli. mutant p1 proteins were examined for rna binding activity by northwestern blot analysis using the reovirus s1 mrna, an activator of the kinase; the adenovirus vai rna, an inhibitor of kinase activation; or human immunodeficiency virus (hiv) tar rna as probe. analysis of trpe-p1 deletion mutant fusion proteins revealed that the 11-kda n-terminal region of the p1 protein bound reoviru ...19921373554
disulfide-bonded discontinuous epitopes on the glycoprotein of vesicular stomatitis virus (new jersey serotype).intrachain disulfide bonds between paired cysteines in the glycoprotein (g) of vesicular stomatitis virus (vsv) are required for the recognition of discontinuous epitopes by specific monoclonal antibodies (mabs) (w. keil and r. r. wagner, virology 170:392-407, 1989). cleavage by staphylococcus aureus v8 protease of the 517-amino-acid vsv-new jersey g protein, limited to the glutamic acid at residue 110, resulted in a protein (designated gv8) with greatly retarded migration by polyacrylamide gel ...19921374811
broad cross-reactivity with marked fine specificity in the cytotoxic t cell response to flaviviruses.cytotoxic t (tc) cells were generated in mice of five h-2 haplotypes against the flaviviruses kunjin and west nile (wnv). a panel of recombinant vaccinia viruses which between them expressed cdna of the entire kunjin virus genome were used to infect targets. anti-kunjin virus responses to determinants derived from non-structural proteins, especially ns3, ns4a and ns4b, were dominant in most mouse strains; usually only one class i major histocompatibility complex (mhc) restriction element was inv ...19921375278
intracellular processing of the human respiratory syncytial virus fusion glycoprotein: amino acid substitutions affecting folding, transport and cleavage.the intracellular processing and transport of the respiratory syncytial virus (rsv) fusion (f) glycoprotein was examined by comparing the maturation and stability of wild-type f, uncleaved mutant f and chimeric f glycoproteins expressed by recombinant vaccinia viruses to that of f protein expressed by rsv. one of the recombinant viruses, vf317, expressed f protein (f317) that was processed like the rsv f glycoprotein. f317 was synthesized initially as f0, the uncleaved glycosylated precursor of ...19921375280
hiv-1 env, nef, and gag-specific t-cell immunity in mice: conserved epitopes in nef p27 and gag p25 proteins.cellular immunogenicity of env gp160, nef p27, and gag p55 proteins of human immunodeficiency virus type 1 (hiv-1) was studied in mice immunized with vaccinia virus recombinants. proliferative responses of spleen cells were comparable against env gp160, nef p27, and gag p25 recombinant proteins. no specific activity was observed against gag p18 protein. env, nef, and gag-specific t-cell lines were generated by repeated stimulation of immune spleen cells with recombinant hiv-1 proteins. they were ...19921376136
induction of cytotoxic t cells to a cross-reactive epitope in the hepatitis c virus nonstructural rna polymerase-like protein.cytotoxic t lymphocytes (ctl) have been found to mediate protection in vivo against certain virus infections. ctl also may play an important role in control of infection by hepatitis c virus (hcv), but no ctl epitopes have yet been defined in any hcv protein. the nonstructural protein with homology to rna polymerase should be a relatively conserved target protein for ctl. to investigate the epitope specificity of ctl specific for this protein, we used 28 peptides from this sequence to study muri ...19921376366
mouse hepatitis virus nucleocapsid protein-specific cytotoxic t lymphocytes are ld restricted and specific for the carboxy terminus.infection of mice with the jhm strain of mouse hepatitis virus (mhv) results in an acute encephalomyelitis associated with primary demyelination of the central nervous system. efforts at understanding the components of the immune response in the development of chronic mhv-induced demyelination have implicated the antibody response and both the cd4+ and cd8+ t cell responses. in this report, we demonstrate that balb/c (h-2d) mice immunized with the jhm (jhmv) strain of mhv develop a cd8+ cytotoxi ...19921376538
protection from herpes simplex virus type 2 is associated with t cells involved in delayed type hypersensitivity that recognize glycosylation-related epitopes on glycoprotein d.immunization of mice with a vaccinia recombinant (vp176) that expresses a fully glycosylated herpes simplex virus (hsv) glycoprotein d (gd) induces long-term (greater than or equal to 50 days) hsv-specific lymphoproliferation and delayed type hypersensitivity (dth) responses, the ability to eliminate a high challenge dose of hsv-2 from the epidermis and protection from fatal disease due to hsv replication in the nervous system. adoptive transfer studies indicate that protection is mediated by th ...19921376951
immunocytochemical analysis reveals differences between the subcellular localization of normal and delta phe508 recombinant cystic fibrosis transmembrane conductance regulator.cystic fibrosis (cf) is caused by mutations in the gene coding for the cystic fibrosis transmembrane conductance regulator (cftr). the most common mutation responsible for cf is the deletion of amino acid residue phe508, with an average allelic frequency of 70%. we have isolated an anti-cftr monoclonal antibody which specifically recognizes recombinant normal and delta phe508-cftr produced by a vaccinia virus expression system. immunocytochemical analysis of l cells expressing either normal or d ...19921377133
localization of epstein-barr virus cytotoxic t cell epitopes using recombinant vaccinia: implications for vaccine development.there is considerable interest in designing an effective vaccine to the ubiquitous epstein-barr virus (ebv). an important role for ebv-specific cytotoxic t lymphocytes (ctls) in eliminating virus-infected cells is well established. limited studies using a small number of immune donors have defined target epitopes within the latent antigens of ebv. the present study provides an extensive analysis of the distribution of class i-restricted ctl epitopes within ebv-encoded proteins. using recombinant ...19921377222
restoration of the lfa-3 adhesion pathway in burkitt's lymphoma cells using an lfa-3 recombinant vaccinia virus: consequences for t cell recognition.conjugate formation between cytotoxic t lymphocytes (ctl) and target b cells, as observed in vitro, is mediated by interactions between adhesion molecules on the two cell surfaces rather than involving immune recognition through the t cell receptor. it is still not clear to what extent such adhesive contacts facilitate the process of immune recognition and target cell lysis. however, work on the epstein-barr virus (ebv)-associated malignancy burkitt's lymphoma (bl) has suggested that down-regula ...19921378014
definition of linear antigenic regions of the hpv16 l1 capsid protein using synthetic virion-like particles.mice of three haplotypes (h-2d, h-2b, and h-2d/b) were immunized with synthetic hpv16 virus-like particles (vlps), produced using a vaccinia virus doubly recombinant for the l1 and l2 proteins of hpv16. the resultant anti-vlp antisera recognized hpv16 capsids by elisa assay and baculovirus recombinant hpv16 l1 and l2 protein on immunoblot. overlapping peptides corresponding to the hpv16 l1 amino acid sequence were used to define the immunoreactive regions of the l1 protein. the majority of the l ...19921379392
recombinant dna approaches for the development of metabolic systems used in in vitro toxicology.in the past few years there has been considerable progress in the development of mammalian cell systems for use in genetic toxicology by the stable transfer of genes/cdnas coding for drug metabolizing enzymes directly into the target cell. alternative approaches have also been developed in which mammalian cells are transiently transfected with cdnas coding for drug-metabolizing enzymes and s9 preparations expressing a single metabolizing enzyme isolated and used for metabolic activation. progres ...19921381053
the influence of n-linked glycosylation on the antigenicity and immunogenicity of rubella virus e1 glycoprotein.rubella virus e1 glycoprotein contains three functional n-linked glycosylation sites. the role of n-linked glycosylation on the antigenicity and immunogenicity of e1 glycoprotein was studied using vaccinia recombinants expressing e1 glycosylation mutants. expressed e1 glycosylation mutant proteins were recognized by a panel of e1-specific monoclonal antibodies in radioimmunoprecipitation, immunofluorescence, and immunoblotting, indicating that carbohydrate side chains on e1 are not involved in t ...19921381541
an o-linked carbohydrate neutralization epitope of hiv-1 gp 120 is expressed by hiv-1 env gene recombinant vaccinia virus.previous studies have disagreed about the presence of o-linked carbohydrate epitopes on gp 120 of hiv, although antibodies against short-chain o-linked glycans neutralize hiv infection and block syncytium formation in vitro. to settle this question, we analysed the o-linked glycans of gp 120 by chemical methods using purified hiv-1 gp 120 from cells infected with recombinant vaccinia virus solely expressing gp 160 or gp 120. alkaline borohydride degradation of recombinant gp 120 released monosac ...19921381907
poliovirus-specific major histocompatibility complex class i-restricted cytolytic t-cell epitopes in mice localize to neutralizing antigenic regions.a major histocompatibility complex (mhc) class i-restricted cytotoxic t-lymphocyte (ctl) response is induced in balb/c mice upon immunization with poliovirus serotype 1 (mahoney strain). a similar class i-restricted response is also induced upon immunization with purified vp1 capsid proteins. thus, poliovirus-specific mhc class i ctl responses can be induced independently of viral infection in murine hosts. in experiments using recombinant vaccinia virus vectors expressing different segments of ...19921382144
vaccinia virus as vector to express ion channel genes. 19921382194
cellular and humoral immune responses to rubella virus structural proteins e1, e2, and c.better understanding of cell-mediated immune responses to rubella virus would provide the basis for the development of safe and effective vaccines against rubella and would aid in analysis of the pathophysiology of congenital rubella syndrome. we have expressed individual rubella virus structural proteins, e1, e2 and c, via vaccinia virus recombinants. using the expressed recombinant proteins as antigens, we were able to demonstrate antigen-specific lymphocyte proliferative responses in control ...19921383269
immunization of mice with lipopeptides bypasses the prerequisite for adjuvant. immune response of balb/c mice to human immunodeficiency virus envelope glycoprotein.in vivo priming of ctl requires the association with mhc class i molecules of peptides derived from the processing of endogenously produced proteins. immunization with exogenous proteins or peptides rarely induces mhc class i-restricted ctl unless they are associated with lipidic compounds. the capacity to induce ctl was compared in synthetic peptides and simple lipopeptides containing the immunodominant mhc class i h-2dd-restricted t-cell epitope of hiv-1 gp160. in contrast with free peptides i ...19921385524
attenuation of autoimmune disease and lymphocyte accumulation in mrl/lpr mice by treatment with anti-v beta 8 antibodies.mrl-mp-lpr/lpr mice are afflicted by a severe systemic autoimmune disease that is aggravated by the lpr mutation resulting in the accumulation of phenotypically abnormal lpr cells (cd3+cd4-cd8-) in all lymphoid issues including hyperplastic lymph nodes. given that products of the t cell receptor v beta 8 gene family are overrepresented among lpr cells, different schedules aimed at selectively decreasing the frequency of lpr cells were designed. first, continuous administration of the monoclonal ...19921386316
in vitro excision and replication of 5' telomeres of minute virus of mice dna from cloned palindromic concatemer junctions.hela cell extracts containing wild-type copies of the minute virus of mice ns-1 polypeptide produced from a recombinant vaccinia virus vector could support the excision and replication of viral 5' telomeres from cloned concatemer junction fragments. resolution did not occur if wild-type ns-1 was omitted from the extract or if the substrate dna contained palindromic sequences without specific viral resolution sites. in the presence of ns-1, [32p]dgtp incorporation into all templates was slightly ...19921388310
an ovarian tumor marker with homology to vaccinia virus contains an igv-like region and multiple transmembrane domains.the monoclonal antibody ovtl3 has a highly restricted reactivity with ovarian carcinomas and defines a surface glycoprotein, oa3, which has been used for immunotargeting. to understand why oa3 is found on ovarian tumors we isolated a complementary dna by expression cloning. the clone encodes a 323-amino acid protein with 5 putative membrane spanning domains, reminiscent of a membrane receptor or channel, but of a new type with little or no sequence similarity with these families of proteins. int ...19921394148
a soluble receptor for interleukin-1 beta encoded by vaccinia virus: a novel mechanism of virus modulation of the host response to infection.vaccinia virus gene b15r is shown to encode an abundant, secretory glycoprotein that functions as a soluble interleukin-1 (il-1) receptor. this il-1 receptor has novel specificity since, in contrast with cellular counterparts, it binds only il-1 beta and not il-1 alpha or the natural competitor il-1 receptor antagonist. the vaccinia il-1 beta receptor is secreted when expressed in a baculovirus system and competitively inhibited binding of il-1 beta to the natural receptor on t cells. deletion o ...19921394428
the cdna structure of the porcine pro-hormone convertase pc2 and the comparative processing by pc1 and pc2 of the n-terminal glycopeptide segment of porcine pomc.the complete cdna structure of the porcine (p) pro-protein and pro-hormone convertase pc2 (ppc2) was obtained from a cdna library of pituitary neurointermediate lobes mrna. the deduced amino acid sequence revealed that ppc2 exhibits a 99-97% sequence identity to the human, mouse and rat homologues. the 3' end of the 2.1 kb cdna is the least conserved segment. on northern blots of pars intermedia poly a+ rna two transcripts of 3 and 5 kb were detected. molecular analysis of the n-terminal glycope ...19921397279
a transient transfection system for identifying biosynthesized proteins processed and presented to class i mhc restricted t lymphocytes.cd8+ cytotoxic t lymphocytes (ctl) constitute a major portion of immune responses to foreign and self antigens. ctl recognize class i major histocompatibility complex molecules complexed to peptides of 8-10 residues derived from cytosolic proteins. to understand ctl responses to these antigens and to manipulate ctl responses optimally, it is necessary to identify the specific peptides recognized by ctl. the methods currently used for this purpose have significant drawbacks. we describe a plasmid ...19921401939
ultra-high-resolution scanning electron microscopy of vaccinia virus and its recombinant carrying the gag gene of human immunodeficiency virus type 1.fl cells infected with vaccinia virus or its recombinant carrying the gag gene of human immunodeficiency virus type 1 (hiv-1) were examined by ultra-high-resolution scanning electron microscopy. virions, whether located extracellularly or intracellularly, had a brick-shaped or watermelon appearance as a whole. extracellular virions observed on the surface of infected cells had variable surface ultrastructures depending on the manner in which particular virions were wrapped in cell membranes. mos ...19921402523
antivirally protective cytotoxic t cell memory to lymphocytic choriomeningitis virus is governed by persisting antigen.the basis of antiviral protection by memory cytotoxic t lymphocytes (ctl) was investigated in vivo and in vitro using lymphocytic choriomeningitis virus (lcmv) and recombinant vaccinia viruses expressing the lcmv-glycoprotein (vacc-gp) or -nucleoprotein (vacc-np). the widely replicating lcmv with a tendency to persist induced solid long-term protective memory. the poorly replicating vaccinia recombinant viruses revealed in the vaccinated host that the antiviral capacity of the secondary immune t ...19921402673
mutagenesis of the l protein encoded by bunyamwera virus and production of monospecific antibodies.bacterial fusion proteins containing portions of the bunyamwera virus l protein were used as immunogens to prepare antisera in rabbits. of five fusion proteins injected into rabbits, three yielded sera that reacted with the bunyamwera virus l protein, detected by western blotting or immunoprecipitation. two of these antisera were specific for either the amino- or carboxy-terminal regions of the l protein. the specificity of these antisera was confirmed by their pattern of reactivity with full-le ...19921402814
transcription of viral late genes is dependent on expression of the viral intermediate gene g8r in cells infected with an inducible conditional-lethal mutant vaccinia virus.there are three temporal classes of vaccinia virus genes: early, intermediate, and late. the object of this study was to determine the effects on virus replication of regulating the expression of g8r, an intermediate gene that encodes a late transcription factor. we inserted the lac operator adjacent to the rna start site of the g8r gene in a recombinant vaccinia virus that constitutively expresses the escherichia coli lac repressor to make expression of the g8r gene dependent on the inducer iso ...19921404599
r339h and p453s: cyp21 mutations associated with nonclassic steroid 21-hydroxylase deficiency that are not apparent gene conversions.steroid 21-hydroxylase deficiency is the most common enzymatic defect causing congenital adrenal hyperplasia, an inherited disorder of cortisol biosynthesis. all mutations thus far characterized that cause this disorder appear to result from recombinations between the gene encoding the enzyme, cyp21b (cyp21), and the adjacent pseudogene, cyp21a (cyp21p). these are either deletions caused by unequal crossing-over during meiosis or apparent transfers of deleterious sequences from cyp21a to cyp21b, ...19921406709
oxidative stress and heat shock induce a human gene encoding a protein-tyrosine phosphatase.reactive oxygen species have been implicated both in the ageing process and in degenerative diseases, including arthritis and cancer. bacteria adapt to the lethal effects of oxidants such as hydrogen peroxide by inducing the expression of protective stress genes. analogous responses have been identified in human cells. for example, haem oxygenase is a major stress protein in human cells treated with oxidants, and reactive oxygen intermediates activate nf-kappa b, a transcriptional regulator of g ...19921406996
thyroid hormone alters the dna binding properties of chicken thyroid hormone receptors alpha and beta.the effects of thyroid hormone agonists on thyroid hormone receptor (tr)/dna complex formation was investigated to elucidate the mechanism by which trs transactivate genes in response to ligand. the data, obtained from gel shift experiments, indicate that thyroid hormones alter the conformation of trs bound to dna, irrespective of if the element is occupied by monomeric tr, homodimeric tr/tr, or heterodimeric complexes with the retinoid receptors rar or rxr. furthermore, triiodo-thyronine (t3) p ...19921408794
high level expression of active human prothrombin in a vaccinia virus expression system.we have worked out an efficient and time saving procedure for the expression of recombinant human prothrombin. the glycoprotein was expressed in the vaccinia virus expression system in several mammalian cell lines. the kidney cell lines vero and bhk and the human cell line hela were found to efficiently secrete prothrombin. expression level of 3-4 micrograms of factor ii per 10(6) cells per day corresponding to 18-23 mu per 10(6) cells per day were achieved. since the expression levels obtained ...19921412155
in situ investigations of single living cells infected by viruses.in this paper we report the direct observation of biological processes on living cells. the experiments were done in situ by scanning force microscopy on a scale inaccessible by other techniques under physiological conditions. living monkey-kidney cultured cells were imaged under normal growth conditions and showed reproducible features on the 10 nm scale. upon adding a suspension of pox viruses, characteristic changes of the cell membrane were repeatedly observed in different experiments on dif ...19921413253
risks connected with the use of conventional and genetically engineered vaccines.a review is given of real and potential risks connected with the use of conventional and genetically engineered live and dead vaccines. special attention is given to live carrier vaccines expressing one or more heterologous genes of other microorganisms. because most carrier vaccines are still in an experimental phase, there is only limited experience with the risks of carrier vaccines. there are three potential risks of live carrier vaccines which will be discussed: 1. changes in cell, tissue, ...19921413441
evidence for multiple species of vaccinia virus-encoded palmitylated proteins.when cells were infected with vaccinia virus in the presence of [3h]palmitic acid, radiolabel was incorporated into six viral proteins with apparent molecular weights of 92, 41, 37, 26, 17, and 14 kda, all of which are expressed at late times during the infection cycle. the [3h]palmitate-labeled fatty acid moieties from the modified proteins were isolated, converted to p-nitrophenacyl derivatives, and subjected to reverse phase hplc analysis which confirmed the identity of the fatty acid group a ...19921413506
expression of functional parvoviral ns1 from recombinant vaccinia virus: effects of mutations in the nucleotide-binding motif.the gene encoding the major replicative protein, ns1, of minute virus of mice (mvm) was transferred into a recombinant vaccinia virus vector in place of the vaccinia thymidine kinase gene. the ns1 gene was placed under control of a bacteriophage t7 promoter and expressed in cells coinfected with another recombinant vaccinia virus, vtf7-3, which encodes the t7 rna polymerase. expression of ns1 was further enhanced by the presence of a 5' untranslated region, derived from encephalomyocarditis viru ...19921413512
polylactosaminoglycan modification of the respiratory syncytial virus small hydrophobic (sh) protein: a conserved feature among human and bovine respiratory syncytial viruses.we investigated the nature of the oligosaccharide modification of the glycosylated forms of the small hydrophobic integral membrane protein, sh (previously designated 1a), of respiratory syncytial (rs) virus. analysis of sh protein expressed in cells infected with rs virus or with a recombinant vaccinia virus revealed two glycosylated sh protein species, shg and shp, which contained n-linked carbohydrate residues. shp migrated diffusely on polyacrylamide gels, which suggested modification by pol ...19921413513
characterization of the antibody response to three different versions of the htlv-i envelope protein expressed by recombinant vaccinia viruses: induction of neutralizing antibody.recombinant vaccinia viruses (rvv) designated rvv e1, rvv e2, and rvv e3, were constructed to express three different versions of the human t cell leukemia virus type i (htlv-i) envelope proteins to determine which configuration elicits an optimal antibody response. rvv e1 expressed the native htlv-i envelope proteins gp46 (surface protein) and gp21 (transmembrane protein), while rvv e2 expressed the envelope precursor with the proteolytic cleavage site deleted. the rvv e3 construct expressed on ...19921413516
a pcr-based method for manipulation of the vaccinia virus genome that eliminates the need for cloning.a general method is described for altering specific genes of vaccinia virus (vv). we demonstrate and evaluate the procedure by gene inactivation, using a dominant selectable marker in conjunction with recombinant polymerase chain reaction (pcr). primers based on the sequence of the target gene enable amplification of flanking arms and their subsequent attachment to the gpt cassette that confers resistance to mycophenolic acid. linear pcr constructs are transfected into cells infected with wild-t ...19921418979
window of vulnerability of vaccinia virus-infected cells to natural killer (nk) cell-mediated cytolysis correlates with enhanced nk cell triggering and is concomitant with a decrease in h-2 class i antigen expression.a time course study was performed in order to determine if vaccinia virus (vv)-infected targets were more susceptible to murine natural killer (nk) cell-mediated lysis during a discrete period of time postinfection. activated nk cells were used in short-term (e.g. 4 h) assays in order to avoid a further in vitro activation of the nk cells by interferon (ifn) and to test the innate susceptibility of target cells to lysis. the sensitivity of vv-infected l929 cells to lysis by nk cells increased as ...19921421956
vaccinia virus: a selectable eukaryotic cloning and expression vector. 1982. 19921422060
construction of poxviruses as cloning vectors: insertion of the thymidine kinase gene from herpes simplex virus into the dna of infectious vaccinia virus. 1982. 19921422062
protection from rabies by a vaccinia virus recombinant containing the rabies virus glycoprotein gene. 1984. 19921422063
[identification of dna-binding proteins of the intracellular virion of the vaccinia virus]. 19921425168
cytoplasmic delivery of ribozymes leads to efficient reduction in alpha-lactalbumin mrna levels in c127i mouse cells.ribozymes targeted to five sites along the alpha-lactalbumin (alpha-lac) mrna were delivered to the cytoplasm of mouse c127i mammary cells using the t7-vaccinia virus delivery system and the amount of alpha-lac mrna was monitored 24-48 h post-transfection. three target sites were selected in the alpha-lac coding region (nucleotides 15, 145 and 361) and two were located in the 3' non-coding region (nucleotides 442 and 694). acting in trans and at a target:ribozyme ratio of 1:1000, ribozymes targe ...19921425576
the role of conserved aspartate and serine residues in ligand binding and in function of the 5-ht1a receptor: a site-directed mutation study.wild-type and mutant serotonin 1a receptors were transiently expressed in cos-7 cells using the infection-transfection variant of the vaccinia virus/t7 polymerase vector system. the amino acid substitutions in the transmembrane regions, asp82-->asn82, asp116-->asn116, and ser198-->ala198 all resulted in a decrease in affinity for 5-ht by 60-100-fold, without affecting the affinity for the antagonist, pindolol. the binding of agonist to the additional mutant, thr199-->ala199, was too weak to be m ...19921426261
oral administration of immunomodulators and the mucosal immune system.orally administered antigens reach the lymphoid tissue in peyer's patches in the gut where they initiate an immune response with clonal expansion of antigen-specific t and b cells. activated t cells migrate through lymph and blood to intestinal epithelium (intra-epithelial leukocytes) whereas activated b cells migrate to the lamina propria, other mucosae and exocrine glands where they differentiate into plasma cells secreting polymeric iga1 or iga2. these antibodies are transported across the ep ...19921426671
regulation of complement activity by vaccinia virus complement-control protein.a major protein secreted by vaccinia virus-infected cells has structural similarity to the super-family of complement-control proteins. this vaccinia complement-control protein (vcp) was studied to determine how it regulates complement activation. vcp was bound by c4b and c3b and served as a cofactor with factor i in cleaving these two molecules. vcp inhibited the formation and accelerated the decay of the classical c3 convertase. it also accelerated decay of the alternative pathway convertase, ...19921431243
evaluation of protective efficacy of recombinant subunit vaccines against simian immunodeficiency virus infection of macaques.simian immunodeficiency virus (siv) was used as a model to study the protective efficacy of an immunization regimen currently being evaluated as candidate vaccines against hiv in human subjects. four macaca fascicularis were first immunized with recombinant vaccinia virus expressing the envelope glycoprotein gp160 of sivmne and then boosted with subunit gp160. both cell-mediated and humoral immune responses against siv, including neutralizing antibodies, were elicited. the macaques were shown to ...19921433262
sulfation of the human immunodeficiency virus envelope glycoprotein.sulfation is a posttranslational modification of proteins which occurs on either the tyrosine residues or the carbohydrate moieties of some glycoproteins. in the case of secretory proteins, sulfation has been hypothesized to act as a signal for export from the cell. we have shown that the human immunodeficiency virus type 1 (hiv-1) envelope glycoprotein precursor (gp160) as well as the surface (gp120) and transmembrane (gp41) subunits can be specifically labelled with 35so42-. sulfated hiv-1 env ...19921433500
importance of p12 protein in mason-pfizer monkey virus assembly and infectivity.mason-pfizer monkey virus (m-pmv) represents the prototype type d retrovirus, characterized by the assembly of intracytoplasmic a-type particles within the infected-cell cytoplasm. these immature particles migrate to the plasma membrane, where they are released by budding. the gag gene of m-pmv encodes a novel protein, p12, just 5' of the major capsid protein (ca) p27 on the polyprotein precursor. the function of p12 is not known, but an equivalent protein is found in mouse mammary tumor virus a ...19921433505
characterization of a vaccinia virus-encoded 42-kilodalton class i membrane glycoprotein component of the extracellular virus envelope.using a reverse genetic approach, we have demonstrated that the product of the b5r open reading frame (orf), which has homology with members of the family of complement control proteins, is a membrane glycoprotein present in the extracellular enveloped (eev) form of vaccinia virus but absent from the intracellular naked (inv) form. an antibody (c'-b5r) raised to a 15-amino-acid peptide from the translated b5r orf reacted with a 42-kda protein (gp42) found in vaccinia virus-infected cells and ces ...19921433514
mutational analysis of the octapeptide sequence motif at the ns1-ns2a cleavage junction of dengue type 4 virus.we have previously shown that proper processing of dengue type 4 virus ns1 from the ns1-ns2a region of the viral polyprotein requires a hydrophobic n-terminal signal and the downstream ns2a. results from deletion analysis indicate that a minimum length of eight amino acids at the c terminus of ns1 is required for cleavage at the ns1-ns2a junction. comparison of this eight-amino-acid sequence with the corresponding sequences of other flaviviruses suggests a consensus cleavage sequence of met/leu- ...19921433515
[function, molecular structure and gene expression of interleukin-1].recent cloning of human and murine il-1 receptor (il-1r) has revealed that there are at least two type of il-1r: type i il-1r is detected on t cells and fibroblasts and consists of 552 aas with a cytoplasmic domain of 213 aas, while type ii is detected on b cells and monocytic cell lines and consists of 398 aas with a short stretch intracytoplasmic domain of 29 aas. extracytoplasmic portion of il-1r has some homology with vaccinia virus b15 ag or fibroblast protein st-2, while cytoplasmic portio ...19921433966
[expression of orthopoxvirus dna sequences in escherichia coli cells].we observed the expression of recombinant plasmids genes containing ectromelia virus dna fragments in e. coli minicells. using plasmids with vaccinia or ectromelia viruses dna fragments inserted upstream of lacz gene we showed that certain orthopoxvirus genome fragments carry out a promoter-like function in bacterial cells.19921435772
molecular characterisation of a dna ligase gene of the extremely thermophilic archaeon desulfurolobus ambivalens shows close phylogenetic relationship to eukaryotic ligases.a 3382 bp fragment containing a gene for a dna ligase from the extremely thermophilic, acidophilic, and facultatively anaerobic archaeon (archaebacterium) desulfurolobus ambivalens was cloned and sequenced. the deduced amino acid sequence (600 amino acids, 67619 molecular weight) showed 30-34% sequence identity with the atp-dependent eucaryal (eukaryotic) dna ligases of schizosaccharomyces pombe, saccharomyces cerevisiae, the human dna ligase i, and with the vaccinia dna ligase. distant similari ...19921437556
structural analysis of ternary complexes of vaccinia rna polymerase.the structure of the elongation complex of vaccinia rna polymerase halted at discrete template positions was examined by dnase i footprinting. the leading edge of the footprint bore a constant relationship to the catalytic template position, being 22-24 nucleotides (nt) in advance on the nontemplate strand and 17 nt on the template strand. dnase hypersensitivity of the nontemplate strand at the leading edge suggested that the dna might be distorted as it entered the polymerase molecule. the regi ...19921438198
construction of chimeric vaccinia viruses by molecular cloning and packaging.foreign dna was inserted into unique restriction endonuclease cleavage sites (sma i or not i) of the 200,000-base-pair vaccinia virus genome by direct molecular cloning. the modified vaccinia virus dna was packaged in fowlpox virus-infected avian cells, and chimeric vaccinia virus was isolated from mammalian cells not supporting the growth of the fowlpox helper virus. in contrast to the classical "in vivo" recombination technique, chimeric viruses with inserts in both possible orientations and f ...19921438247
nonreplicating vaccinia vector efficiently expresses recombinant genes.modified vaccinia ankara (mva), a highly attenuated vaccinia virus strain that has been safety tested in humans, was evaluated for use as an expression vector. mva has multiple genomic deletions and is severely host cell restricted: it grows well in avian cells but is unable to multiply in human and most other mammalian cells tested. nevertheless, we found that replication of viral dna appeared normal and that both early and late viral proteins were synthesized in human cells. proteolytic proces ...19921438287
[processing of vaccinia genome terminal: initiation of dna synthesis and concatemer resolution]. 19921438814
[improvement and application of vaccinia virus vector]. 19921438843
[the quantitative detection of orthopoxvirus antibodies in studying sera in a single dilution in an immunoenzyme test system with a conjugate of protein a and peroxidase]. 19921441432
catalytic properties of the human cytochrome p450 2e1 produced by cdna expression in mammalian cells.a full-length cdna encoding human cytochrome p450 2e1 was expressed in mammalian cell lines using the vaccinia virus expression system. immunoblot analysis showed that the expressed protein reacted with a polyclonal antibody against rat 2e1 and comigrated with p450 2e1 from human liver microsomes. p450 2e1 expressed in hep g2 cells, a human cell line which contains both cytochrome b5 and nadph:p450 oxidoreductase, was able to metabolize several known p450 2e1 substrates: n-nitrosodimethylamine ( ...19921444447
vaccination or tolerance to prevent diabetes.experiments with transgenic mice expressing the glycoprotein (gp) of lymphocytic choriomeningitis virus (lcmv) under the control of the rat insulin promoter (rip) have demonstrated that potentially self-reactive t cells that normally ignore self peptides may nevertheless be induced by self peptides or "cross-reactive" foreign (e.g. viral) peptides that arise in the host in an immunogenic form; once activated these potentially self-reactive t cells may cause autoaggressive diseases (e.g. diabetes ...19921446706
vaccinia virus gene b1r encodes a 34-kda serine/threonine protein kinase that localizes in cytoplasmic factories and is packaged into virions.vaccinia virus open reading frame b1r was expressed in e. coli and shown to encode a serine/threonine protein kinase which phosphorylated casein and calf thymus histones in vitro. a polyclonal rabbit antiserum was raised against a trpe-b1r bacterial fusion protein and used to characterize the b1r gene product. immunoprecipitation and immunoblotting analyses detected a 34-kda polypeptide that was synthesized early during vaccinia virus infection and which was apparently stable since it was easily ...19921448924
expansion of the mammalian 3 beta-hydroxysteroid dehydrogenase/plant dihydroflavonol reductase superfamily to include a bacterial cholesterol dehydrogenase, a bacterial udp-galactose-4-epimerase, and open reading frames in vaccinia virus and fish lymphocystis disease virus.mammalian 3 beta-hydroxysteroid dehydrogenase and plant dihydroflavonol reductases are descended from a common ancestor. here we present evidence that nocardia cholesterol dehydrogenase, e. coli udp-galactose-4 epimerase, and open reading frames in vaccinia virus and fish lymphocystis disease virus are homologous to 3 beta-hydroxysteroid dehydrogenase and dihydroflavonol reductase. analysis of a multiple alignment of these sequences indicates that viral orfs are most closely related to the mamma ...19921451793
studies on processing, particle formation, and immunogenicity of the hiv-1 gag gene product: a possible component of a hiv vaccine.antigens in a particulate conformation were shown to be highly immunogenic in mammals. for this reason, the particle forming capacity of derivatives of the hiv-1 group specific core antigen p55 gag was assayed and compared dependent on various expression systems: recombinant bacteria, vaccinia- and baculoviruses were established encoding the entire core protein p55 either in its authentic sequence or lacking the myristylation consensus signal. moreover, p55 gag was expressed in combination with ...19921456888
immunological reactivity of a human immunodeficiency virus type i derived peptide representing a consensus sequence of the gp120 major neutralizing region v3.to reduce the opportunities for human immunodeficiency virus type 1 (hiv-1) to evade vaccine induced immunity, the development of subunit vaccines must focus on the characterization of immunogenic epitopes, which are major targets for the immune system. the most dominant site for elicitation of neutralising immune response is located on the external envelope glycoprotein gp120 within the third variable domain (v3). to overcome virus type specificity of antibodies directed to the v3-domain we des ...19921456889
genetic and functional analysis of a set of hiv-1 envelope genes obtained from biological clones with varying syncytium-inducing capacities.to study hiv-1 envelope-mediated syncytium formation we have amplified, cloned, expressed, and sequenced individual envelope genes from a set of eight biological hiv-1 clones. these clones were obtained from two patients and display either a syncytium-inducing (si) or nonsyncytium-inducing (nsi) phenotype. upon expression through recombinant vaccinia virus, individual envelope gene products display heterogeneous syncytium-inducing capacities which reflect the phenotype of the parental biological ...19921457193
sulfated polyester interactions with the cd4 molecule and with the third variable loop domain (v3) of gp120 are chemically distinct.sulfated polyesters (sp) that inhibit hiv infection interact with both the gp120 binding region of cd4 molecules and with the v3 domain loop of gp120 molecules (gp120/v3) but the contributions of these interactions to the inhibition of hiv env-mediated fusion are presently unclear. in order to characterize the molecular mechanisms by which sp inhibit hiv env-mediated fusion, we studied the effect of sp treatment on env-mediated fusion of cd4+ cells driven by recombinant vaccinia virus (vpe-16) a ...19921457206
immunogenicity and safety of a recombinant vaccinia virus vaccine expressing the carcinoembryonic antigen gene in a nonhuman primate.we have previously reported the development of a recombinant vaccinia virus vaccine expressing the human carcinoembryonic antigen (cea) gene, designated rv(nyc)-cea. this construct has been shown to elicit specific anti-cea immune responses and an antitumor effect in a murine tumor model. in the studies reported here, the safety and immunogenicity of this recombinant vaccinia virus were evaluated in a rhesus monkey model. human cea is a m(r) 180,000 glycoprotein expressed in approximately 90% of ...19921458480
inhibition of agonist-induced activation of phospholipase c following poxvirus infection.recent studies indicate that viruses may influence polyphosphoinositide levels. this study has examined the effects of vaccinia virus infection on phospholipase c activity. infection of bs-c-1 cells, an african green monkey kidney cell line, or a431 cells, a human carcinoma cell line, with vaccinia virus inhibits receptor-mediated phospholipase c activation. as a consequence, agonist-mediated ca2+ mobilization in bs-c-1 cells also was inhibited by vaccinia virus infection. alleviation of the inh ...19921460012
comparative clonal analysis of human immunodeficiency virus type 1 (hiv-1)-specific cd4+ and cd8+ cytolytic t lymphocytes isolated from seronegative humans immunized with candidate hiv-1 vaccines.the lysis of infected host cells by virus-specific cytolytic t lymphocytes (ctl) is an important factor in host resistance to viral infection. an optimal vaccine against human immunodeficiency virus type 1 (hiv-1) would elicit virus-specific ctl as well as neutralizing antibodies. the induction by a vaccine of hiv-1-specific cd8+ ctl in humans has not been previously reported. in this study, ctl responses were evaluated in hiv-1-seronegative human volunteers participating in a phase i acquired i ...19921460417
lineage-independent activation of immune system effector function by myeloid fc receptors.an emerging theme in immunology finds receptors which initiate cellular effector programs forming multichain complexes in which the ligand recognition elements associate with one or more 'trigger molecules' whose aggregation initiates a signal transduction cascade. the sequence motifs constituting the active sites of these trigger molecules are found in the t cell and b cell antigen receptors, and some fc receptors, and appear to be central to effector function activation. for example, of the ma ...19921464313
site-specific rna cleavage generates the 3' end of a poxvirus late mrna.the cowpox virus late mrnas encoding the major protein of the a-type inclusions have 3' ends corresponding to a single site in the dna template. the dna sequence of the alu i-xba i fragment at this position encodes an rna cis-acting signal, designated the ax element, which directs this rna 3' end formation. in cells infected with vaccinia virus the ax element functions independently of either the nature of the promoter element or the rna polymerase responsible for generating the primary rna. at ...19921465436
virus-specific cytotoxic t-lymphocytes in hiv-2-infected cynomolgus macaques.specific cytotoxic t-lymphocytes (ctl) are induced in humans or monkeys after infection with hiv-1 or sivmac, respectively. since, like hiv-1, hiv-2 causes aids, our objective was to determine the characteristics of the hiv-2-specific ctl response.19921466839
specific activation strategies for amplification of low ctl signals. 19921466958
evaluation of gp160 vaccinees in the hu-pbl-scid mouse model. 19921466962
an update on vaccine adjuvants in clinical trial. 19921466969
highly attenuated poxvirus vectors. 19921466978
use of lentivirus-like particles alone and in combination with live vaccinia-virus-based vaccines. 19921466979
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