Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0002871 (anemia)
 52,094 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We describe here a one step HPLC technique for purifying the four gag proteins (p26, p15, p11 and p9) and two env glycoproteins (gp90 and gp45) from purified equine infectious anemia virus (EIAV), a member of the lentivirus subfamily of retroviruses. The purification procedure employs a reverse-phase phenyl Radial-pak cartridge contained in a high pressure radial compression chamber in which a shallow, multistep acetonitrile gradient is applied at ambient temperatures. The purified proteins are recovered at an efficiency of 60-70%. Moreover, the isolated components retain their antigenicity and are suitable for a variety of biochemical analyses including protein sequencing. The purification of EIAV gp90 and gp45 represents the first successful isolation of a lentivirus glycoprotein from purified virus preparations. The availability of these separated proteins permitted direct protein sequencing which confirmed the previously reported env gene sequence and provides important antigens for the development of diagnostic immunoassays and subunit vaccines. The procedures described appear applicable to other lentiviruses, including human immunodeficiency virus (HIV), and perhaps to hydrophobic membrane proteins in general.
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PMID:Lentivirus antigen purification and characterization: isolation of equine infectious anemia virus gag and env proteins in one step by reverse phase HPLC and application to human immunodeficiency virus glycoproteins. 283 62

Monoclonal antibodies (MAbs) against the major core protein p26 of equine infectious anaemia virus (EIAV) were produced and characterized. Sensitive enzyme-linked immunosorbent assay and Western blot immunoassay were employed to confirm the specificity of these MAbs. Western blot analysis also indicated that MAbs to p26 reacted with another EIAV protein of 55,000 apparent Mr (designated here as Pr55gag) present in density gradient-purified virus preparations. Rabbit antiserum prepared against p26 as well as MAbs to p26 detected Pr55gag and several other intermediate clevage products in detergent-soluble lysates of virus-infected cells in Western blot and immunoprecipitation assays. The results suggest that Pr55gag is the gag polyprotein of EIAV.
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PMID:Identification of gag precursor of equine infectious anaemia virus with monoclonal antibodies to the major viral core protein, p26. 283 4

Previous results from our laboratory have demonstrated that equine infectious anemia virus displays structural variations in its surface glycoproteins and RNA genome during passage and chronic infections in experimentally infected Shetland ponies (Montelaro et al., J. Biol. Chem. 259:10539-10544, 1984; Payne et al., J. Gen. Virol. 65:1395-1399, 1984). The present study was undertaken to obtain an antigenic and biochemical characterization of equine infectious anemia virus isolates recovered from an experimentally infected pony during sequential disease episodes, each separated by intervals of only 4 to 8 weeks. The virus isolates could be distinguished antigenically by neutralization assays with serum from the infected pony and by Western blot analysis with a monoclonal antibody against the major surface glycoprotein gp90, thus demonstrating that novel antigenic variants of equine infectious anemia virus predominate during each clinical episode. The respective virion glycoproteins displayed different electrophoretic mobilities on sodium dodecyl sulfate-polyacrylamide gels, indicating structural variation. Tryptic peptide and glycopeptide maps of the viral proteins of each virus isolate revealed biochemical alterations involving amino acid sequence and glycosylation patterns in the virion surface glycoproteins gp90 and gp45. In contrast, no structural variation was observed in the internal viral proteins pp15, p26, and p9 from any of the four virus isolates. Oligonucleotide mapping experiments revealed similar but unique RNase T1-resistant oligonucleotide fingerprints of the RNA genomes of each of the virus isolates. Localization of altered oligonucleotides for one virus isolate placed two of three unique oligonucleotides within the predicted env gene region of the genome, perhaps correlating with the structural variation observed in the envelope glycoproteins. Thus these results support the concept that equine infectious anemia virus is indeed capable of relatively rapid genomic variations during replication, some of which result in altered glycoprotein structures and antigenic variants which are responsible for the unique periodic disease nature observed in persistently infected animals. The findings of envelope specific differences in isolates of visna virus and of human T-cell lymphotropic virus III (acquired immune deficiency syndrome-related virus) suggest that this variation may be a common characteristic of the subfamily Lentivirinae.
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PMID:Rapid emergence of novel antigenic and genetic variants of equine infectious anemia virus during persistent infection. 300 67

The viral core proteins (p15, p26, p11, and p9) of equine infectious anemia virus (EIAV) (Wyoming strain) were purified by reverse-phase high-pressure liquid chromatography. Each purified protein was analyzed for amino acid content, N-terminal amino acid sequence, C-terminal amino acid sequence, and phosphoamino acid content. The results of N- and C-terminal amino acid sequence analysis of each gag protein, taken together with the nucleotide sequence of the EIAV gag gene (R. M. Stephens, J. W. Casey, and N. R. Rice, Science 231:589-594, 1986), show that the order of the proteins in the precursor is p15-p26-*-p11-p9, where a pentapeptide also found in the virus is represented by the asterisk. The data are in complete agreement with the predicted structure of the gag polyprotein and show the peptide bonds cleaved during proteolytic processing. The N-terminus of p15 is blocked to Edman degradation. The p11 protein is identical to the nucleic acid-binding protein of EIAV previously isolated (C. W. Long, L. E. Henderson, and S. Oroszlan, Virology 104:491-496, 1980). High-titer rabbit antiserum was prepared against each purified protein. These antisera were used to detect the putative gag precursor (Pr55gag) and intermediate cleavage products designated Pr49 (p15-p26-*-p11), Pr40 (p15-p26), and Pr35 (p26-*-p11) in the virus and in virus-infected cells. High-titer antisera to EIAV p15 and p26 showed cross-reactivity with the homologous protein of human T-cell lymphotropic virus type III/lymphadenopathy-associated virus.
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PMID:Chemical and immunological characterizations of equine infectious anemia virus gag-encoded proteins. 302 6

We describe procedures for the large-scale production of equine infectious anemia virus (EIAV) and for the isolation of the four major non-glycosylated virion proteins, designated p26, p15, p11, and p9. Comparisons of the purified proteins by peptide mapping procedures and by enzyme-linked immunosorbent assays demonstrated the unrelatedness of the four proteins. The characteristic properties of each purified protein were examined by determining isoelectric points and amino acid compositions. We found that EIAV p26 and p9 focus at pI values of 6.2 and 5.0, respectively, and that these proteins contain no unusual amino acids. In contrast, EIAV p15 reproducibly displayed a heterogeneous isoelectric focusing pattern, with major pI values ranging from 5.7 to 8.3. This charge variation evidently correlated with different levels of phosphorylated serine or threonine or both, which could be detected by an amino acid analysis of purified p15. EIAV p11 apparently focused at a pI of greater than 10, reflecting its high content of basic amino acids. Moreover, localization experiments indicated that all four nonglycosylated proteins constitute the internal components of the virus, with all of the virion p11 closely associated with the viral RNA genome. Thus, our results demonstrated that EIAV, a lentivirus, contains structural polypeptides which are analogous to the structural polypeptides described previously in prototype C oncoviruses.
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PMID:Isolation and comparative biochemical properties of the major internal polypeptides of equine infectious anemia virus. 617 43

A sensitive specific enzyme-linked immunosorbent assay utilizing purified p26 antigen was developed for the detection of antibodies to equine infectious anemia virus in naturally and experimentally infected horses. Generally, antibodies to the virus could be detected by the enzyme-linked immunosorbent assay 3 to 4 days earlier than by the standard agar gel immunodiffusion test, and they could be detected more reliably in horses with weak or equivocal agar gel immunodiffusion test reactions. The enzyme-linked immunosorbent assay was also successfully applied to the detection of p26 antigen in tissue culture fluids.
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PMID:Enzyme-linked immunosorbent assay for detection of equine infectious anemia virus p26 antigen and antibody. 632 88

Cells infected with vaccinia viruses expressing the equine infectious anaemia virus (EIAV) gag gene (VGag) or gag plus the 5' pol encoding protease (VGag/PR) were evaluated with monoclonal antibody to a p26 capsid protein linear epitope (QEISKFLTD). Both recombinant viruses expressed Gag precursor protein (55K) whereas only VGag/PR expressed a detectable Gag-Pol fusion protein (82K) with a functional protease, shown by subviral particles containing processed p26. Horses inoculated with VGag/PR produced antibodies reactive with EIAV Gag proteins.
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PMID:Expression of functional protease and subviral particles by vaccinia virus containing equine infectious anaemia virus gag and 5' pol genes. 815 2

The gene coding for the major core protein (p26) of the lentivirus equine infectious anemia virus (EIAV) was cloned from EIAV infected serum, expressed in E. coli, and the resultant protein purified to electrophoretic homogeneity. The protein was expressed in a soluble form and was purified by conventional protein separation methods. When analyzed by SDS-PAGE, under both reducing and non-reducing conditions, the purified protein migrated as a 26 kDa monomer. Recombinant p26 (rp26), therefore, does not contain any intermolecular disulfide bond. Gel filtration chromatography also indicated that the protein occurs as a monomer in solution. Labeling of free sulphydryl groups with [1-14C]iodoacetamide suggests that none of the three cysteine residues of rp26 is involved in intramolecular disulfide bonds. The circular dichroism spectrum of rp26 was consistent with the following assignment of secondary structure elements: 51% a-helix, 15% beta-turn, and 34% aperiodic. Fluorescencespectroscopy revealed that the three tryptophan residues in rp26 occupy two different environments. These data support the conclusion that the recombinant protein is folded into an ordered and probably native conformation. Immunoblotting and enzyme immunoassay with EIAV infected sera demonstrated that recombinant p26 protein may be useful for diagnostic purposes.
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PMID:Cloning, expression, purification, and characterization of the major core protein (p26) from equine infectious anemia virus. 916

Cardinal features of equine infectious anemia (EIA) include fever, hemolytic anemia and thrombocytopenia during the acute phase of the disease, and cachexia and anemia seen during the chronic phase. These signs are thought to result from the release of inflammatory cytokines such as TNF-alpha. In order to determine if TNF-alpha has a role in the pathogenesis of acute EIA and vaccine-induced disease enhancement, we measured plasma concentrations of TNF-alpha in ponies immunized with virus enriched major core protein-p26 and/or experimentally infected with EIAV. Naturally infected inapparent EIAV carriers were also studied. TNF-alpha levels were determined by means of a WEHI 164, clone 13 cytotoxicity assay. We show a significant positive temporal correlation between TNF-alpha levels, severity of symptoms (fever and thrombocytopenia) and viremia. Furthermore, TNF-alpha levels also correlate with strain virulence and the disease enhancement seen in vaccinated ponies. Of this group of animals, those challenged with a heterologous virulent strain presented the most unfavorable outcome as well as the highest levels of TNF-alpha and viremia. The TNF-alpha activity observed in the bioassay was completely abrogated by a polyclonal rabbit anti-human TNF-alpha antiserum, thus confirming the specificity of the plasma cytotoxicity. Our observations indicate that TNF-alpha production correlates with the outcome of infection with EIAV.
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PMID:Tumor necrosis factor-alpha production and disease severity after immunization with enriched major core protein (p26) and/or infection with equine infectious anemia virus. 923 36

Most equine infectious anemia virus (EIAV)-infected horses have acute clinical disease, but they eventually control the disease and become lifelong carriers. Cytotoxic T lymphocytes (CTL) are considered an important immune component in the control of infections with lentiviruses including EIAV, but definitive evidence for CTL in the control of disease in carrier horses is lacking. By using retroviral vector-transduced target cells expressing different Gag proteins and overlapping synthetic peptides of 16 to 25 amino acids, peptides containing at least 12 Gag CTL epitopes recognized by virus-stimulated PBMC from six long-term EIAV-infected horses were identified. All identified peptides were located within Gag matrix (p15) and capsid (p26) proteins, as no killing of target cells expressing p11 and p9 occurred. Each of the six horses had CTL recognizing at least one Gag epitope, while CTL from one horse recognized at least eight different Gag epitopes. None of the identified peptides were recognized by CTL from all six horses. Two nonamer peptide epitopes were defined from Gag p26; one (18a) was likely restricted by class I equine leukocyte alloantigen A5.1 (ELA-A5.1) molecules, and the other (28b-1) was likely restricted by ELA-A9 molecules. Sensitization of equine kidney target cells for CTLm killing required 10 nM peptide 18a and 1 nM 28b-1. The results demonstrated that diverse CTL responses against Gag epitopes were generated in long-term EIAV-infected horses and indicated that ELA-A class I molecules were responsible for the diversity of CTL epitopes recognized. This information indicates that multiple epitopes or whole proteins will be needed to induce CTL in horses with different ELA-A alleles in order to evaluate their role in controlling EIAV.
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PMID:Gag protein epitopes recognized by ELA-A-restricted cytotoxic T lymphocytes from horses with long-term equine infectious anemia virus infection. 981 94


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