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Sample records for purified envelope glycoprotein

  1. Envelope glycoproteins of HIV-1, HIV-2, and SIV purified with Galanthus nivalis agglutinin induce strong immune responses.

    PubMed

    Gilljam, G

    1993-05-01

    Lectin affinity chromatography was used to purify in a single step the envelope glycoproteins of HIV-1, HIV-2, and SIV. Envelope glycoproteins carry the major determinants essential for protection by the humoral immune response. The purification of these proteins has previously been a laborious procedure. The glycoproteins were purified by a one-step procedure to a high level of purity by using Galanthus nivalis agglutinin (GNA). The purified glycoprotein had CD4-binding and antigenic reactivities. Strong immune responses to envelope proteins and peptides were seen in mice and primates after immunization with these preparations.

  2. Purified envelope glycoproteins from human immunodeficiency virus type 1 variants induce individual, type-specific neutralizing antibodies.

    PubMed Central

    Nara, P L; Robey, W G; Pyle, S W; Hatch, W C; Dunlop, N M; Bess, J W; Kelliher, J C; Arthur, L O; Fischinger, P J

    1988-01-01

    Repeated immunizations of goats, horses, or chimpanzees with envelope glycoprotein gp120 isolated from human immunodeficiency virus type 1 (HIV-1) resulted in type-specific neutralizing-antibody responses, which began to decay approximately 20 days following the administration of antigen. This was true repeatedly for serum samples from animals hyperimmunized with gp120s from either the HTLV-IIIB (IIIB) or the envelope-divergent HTLV-IIIRF (RF) HIV-1 isolates. Animals previously immunized with the IIIB gp120 were then inoculated with purified RF gp120. The first response in these animals was an anamnestic resurgence of neutralizing antibody to IIIB without detectable neutralizing antibody for RF. However, with later RF gp120 boosts, the IIIB neutralizing-antibody titers fell and an RF type-specific neutralizing-antibody response developed. When assessed with other HIV-1 variants, no group-specific neutralizing antibody was seen in any of the vaccination protocols evaluated. These results will pose real obstacles in the development of an effective vaccine for HIV. PMID:3392769

  3. Purifying selection in Porcine reproductive and respiratory syndrome virus ORF5a protein influences variation in envelope glycoprotein 5 glycosylation

    PubMed Central

    Robinson, Sally R.; Abrahante, Juan E.; Johnson, Craig R.; Murtaugh, Michael P.

    2013-01-01

    Porcine Reproductive and Respiratory Syndrome Virus ORF5a protein is encoded in an alternate open reading frame upstream of the major envelope glycoprotein (GP5) in subgenomic mRNA5. Bioinformatic analysis of 3,466 Type 2 PRRSV sequences showed that the two proteins have co-evolved through a fine balance of purifying codon usage to maintain a conserved RQ-rich motif in ORF5a protein, while eliciting a variable N-linked glycosylation motif in the alternative GP5 reading frame. Conservation of the ORF5a protein RQ-motif also explains an anomalous uracil desert in GP5 hypervariable glycosylation region. The N-terminus of the mature GP5 protein was confirmed to start with amino acid 32, the hypervariable region of the ectodomain. Since GP5 glycosylation variability is assumed to result from immunological selection against neutralizing antibodies, these findings show that an alternative possibility unrelated to immunological selection not only exists, but provides a foundation for investigating previously unsuspected aspects of PRRSV biology. Understanding functional consequences of subtle nucleotide sequence modifications in the region responsible for critical function in ORF5a protein and GP5 glycosylation is essential for rational design of new vaccines against PRRS. PMID:24084290

  4. Solubilization of glycoproteins of envelope viruses by detergents

    SciTech Connect

    Berezin, V.E.; Zaides, V.M.; Artamsnov, A.F.; Isaeva, E.S.; Zhdanov, V.M.

    1986-11-20

    The action of a number of known ionic and nonionic detergents, as well as the new nonionic detergent MESK, on envelope viruses was investigated. It was shown that the nonionic detergents MESK, Triton X-100, and octyl-..beta..-D-glucopyranoside selectively solubilize the outer glycoproteins of the virus particles. The nonionic detergent MESK has the mildest action. Using MESK, purified glycoproteins of influenza, parainfluenza, Venezuelan equine encephalomyelitis, vesicular stomatitis, rabies, and herpes viruses were obtained. The procedure for obtaining glycoproteins includes incubation of the virus suspension with the detergent MESK, removal of subvirus structures by centrifuging, and purification of glycoproteins from detergents by dialysis. Isolated glycoproteins retain a native structure and biological activity and possess high immunogenicity. The detergent MESK is promising for laboratory tests and with respect to the production of subunit vaccines.

  5. Absence of cytotoxic antibody to human immunodeficiency virus-infected cells in humans and its induction in animals after infection or immunization with purified envelope glycoprotein gp120

    SciTech Connect

    Nara, P.L.; Robey, W.G.; Gonda, M.A.; Carter, S.G.; Fischinger, P.J.

    1987-06-01

    The presence of antibody-dependent complement-mediated cytotoxicity (ACC) was assessed in humans and chimpanzees, which are capable of infection with human immunodeficiency virus isolate HTLV-IIIb, and examined in the goat after immunization with the major viral glycoprotein (gp120) of HTLV-IIIb. In infected humans no antibody mediating ACC was observed regardless of the status of disease. Even healthy individuals with high-titer, broadly reactive, neutralizing antibodies has no ACC. In contrast, chimpanzees infected with HTLV-IIIb, from whom virus could be isolated, not only had neutralizing antibody but also antibodies broadly reactive in ACC, even against distantly related human immunodeficiency virus isolates, as well as against their own reisolated virus. In the goat, the gp120 of HTLV-IIIb induced a highly type-specific response as measured by both ACC and flow cytofluorometry of live infected H9 cells. Normal human cells were not subject to ACC by animal anti-HTLV-III gp120-specific sera. Induction of ACC and neutralizing antibody were closely correlated in the animal experimental models but not in humans. The presence of ACC in gp120-inoculated goats and HTLV-III-infected chimpanzees represent a qualitative difference that may be important in the quest for the elicitation of a protective immunity in humans.

  6. Immunological responses to envelope glycoprotein 120 from subtypes of human immunodeficiency virus type 1.

    PubMed

    Gilljam, G; Svensson, A; Ekström, A; Wahren, B

    1999-07-01

    The outer envelope glycoprotein (gp120) from subtypes A-E of HIV-1 was purified using a specific high mannose-binding lectin, Galanthus nivalis agglutinin. All isolates were grown in peripheral blood lymphocyte cells in order to avoid selection in cell lines. A comparison of the reactivities of the envelope proteins was made using sera from patients infected with the different subtypes. In this study, the B and C subtype envelope glycoproteins showed the strongest immunological reactivity, when reacted with sera from patients infected with the same subtype of virus. On the other hand, sera of patients infected with subtype A or C virus had the strongest and broadest reactivities, to envelope glycoproteins of many subtypes. The purified gp120 proteins from all five subtypes stimulated mononuclear cells from HIV-1 (subtype B)-infected patients, indicating conserved T cell-activating epitopes. The immunological reactivities indicate that strong antigenicity does not always predict the broadest immunogenicity of an envelope glycoprotein. Glycoprotein 120 from foreign subtypes may serve to induce strong cross-reactive immune responses.

  7. Localization of P-glycoprotein at the nuclear envelope of rat brain cells

    SciTech Connect

    Babakhanian, Karlo; Bendayan, Moise; Bendayan, Reina . E-mail: r.bendayan@utoronto.ca

    2007-09-21

    P-Glycoprotein is a plasma membrane drug efflux protein implicated in extrusion of cytotoxic compounds out of a cell. There is now evidence that suggests expression of this transporter at several subcellular sites, including the nucleus, mitochondria, and Golgi apparatus. This study investigated the localization and expression of P-glycoprotein at the nuclear membrane of rat brain microvessel endothelial (RBE4) and microglial (MLS-9) cell lines. Immunocytochemistry at the light and electron microscope levels using P-glycoprotein monoclonals antibodies demonstrated the localization of the protein at the nuclear envelope of RBE4 and MLS-9 cells. Western blot analysis revealed a single band of 170-kDa in purified nuclear membranes prepared from isolated nuclei of RBE4 and MLS-9 cells. These findings indicate that P-glycoprotein is expressed at the nuclear envelope of rat brain cells and suggest a role in multidrug resistance at this subcellular site.

  8. Respiratory syncytial virus envelope glycoprotein (G) has a novel structure.

    PubMed Central

    Satake, M; Coligan, J E; Elango, N; Norrby, E; Venkatesan, S

    1985-01-01

    Amino acid sequence of human respiratory syncytial virus envelope glycoprotein (G) was deduced from the DNA sequence of a recombinant plasmid and confirmed by limited amino acid microsequencing of purified 90K G protein. The calculated molecular mass of the protein encoded by the only long open reading frame of 298 amino acids was 32,588 daltons and was somewhat smaller than the 36K polypeptide translated in vitro from mRNA selected by this plasmid. Inspection of the sequence revealed a single hydrophobic domain of 23 amino acids capable of membrane insertion at 41 residues from the N-terminus. There was no N-terminal signal sequence and the hydrophilic N-terminal 20 residues probably represent the cytoplasmic tail of the protein. The N-terminally oriented membrane insertion was somewhat analogous to paramyxovirus hemagglutinin-neuraminidase (HN) and influenza neuraminidase (NA). The protein was moderately hydrophilic and rich in hydroxy-amino acids. It was both N- and O-glycosylated with the latter contributing significantly to the net molecular mass 90K. Images PMID:4069997

  9. HIV Entry and Envelope Glycoprotein-mediated Fusion*

    PubMed Central

    Blumenthal, Robert; Durell, Stewart; Viard, Mathias

    2012-01-01

    HIV entry involves binding of the trimeric viral envelope glycoprotein (Env) gp120/gp41 to cell surface receptors, which triggers conformational changes in Env that drive the membrane fusion reaction. The conformational landscape that the lipids and Env navigate en route to fusion has been examined by biophysical measurements on the microscale, whereas electron tomography, x-rays, and NMR have provided insights into the process on the nanoscale and atomic scale. However, the coupling between the lipid and protein pathways that give rise to fusion has not been resolved. Here, we discuss the known and unknown about the overall HIV Env-mediated fusion process. PMID:23043104

  10. Hepatitis C Virus E2 Envelope Glycoprotein Core Structure

    SciTech Connect

    Kong, Leopold; Giang, Erick; Nieusma, Travis; Kadam, Rameshwar U.; Cogburn, Kristin E.; Hua, Yuanzi; Dai, Xiaoping; Stanfield, Robyn L.; Burton, Dennis R.; Ward, Andrew B.; Wilson, Ian A.; Law, Mansun

    2014-08-26

    Hepatitis C virus (HCV), a Hepacivirus, is a major cause of viral hepatitis, liver cirrhosis, and hepatocellular carcinoma. HCV envelope glycoproteins E1 and E2 mediate fusion and entry into host cells and are the primary targets of the humoral immune response. The crystal structure of the E2 core bound to broadly neutralizing antibody AR3C at 2.65 angstroms reveals a compact architecture composed of a central immunoglobulin-fold β sandwich flanked by two additional protein layers. The CD81 receptor binding site was identified by electron microscopy and site-directed mutagenesis and overlaps with the AR3C epitope. The x-ray and electron microscopy E2 structures differ markedly from predictions of an extended, three-domain, class II fusion protein fold and therefore provide valuable information for HCV drug and vaccine design.

  11. N-Glycosylation Profiling of Porcine Reproductive and Respiratory Syndrome Virus Envelope Glycoprotein 5

    PubMed Central

    Li, Juan; Tao, Shujuan; Orlando, Ron; Murtaugh, Michael P.

    2015-01-01

    Porcine reproductive and respiratory syndrome virus (PRRSV) is a positive-sense ssRNA virus whose envelope contains four glycoproteins and three nonglycosylated proteins. Glycans of major envelope glycoprotein 5 (GP5) are proposed as important for virus assembly and entry into permissive cells. Structural characterization of GP5 glycans would facilitate the mechanistic understanding of these processes. Thus, we purified the PRRSV type 2 prototype strain, VR2332, and analyzed the virion-associated glycans by both biochemical and mass spectrometric methods. Endoglycosidase digestion showed that GP5 was the primary protein substrate, and that the carbohydrate moieties were primarily complex-type N-glycans. Mass spectrometric analysis (HPLC-ESI-MS/MS) of GP5 N-glycans revealed an abundance of N-acetylglucosamine (GlcNAc) and N-acetyllactosamine (LacNAc) oligomers in addition to sialic acids. GlcNAc and LacNAc accessibility to ligands was confirmed by lectin co-precipitation. Our findings help to explain PRRSV infection of cells lacking sialoadhesin and provide a glycan database to facilitate molecular structural studies of PRRSV. PMID:25726973

  12. Morphology and Molecular Composition of Purified Bovine Viral Diarrhea Virus Envelope

    PubMed Central

    Callens, Nathalie; Brügger, Britta; Bonnafous, Pierre; Drobecq, Hervé; Gerl, Mathias J.; Krey, Thomas; Roman-Sosa, Gleyder; Rümenapf, Till; Lambert, Olivier; Dubuisson, Jean; Rouillé, Yves

    2016-01-01

    The family Flaviviridae includes viruses that have different virion structures and morphogenesis mechanisms. Most cellular and molecular studies have been so far performed with viruses of the Hepacivirus and Flavivirus genera. Here, we studied bovine viral diarrhea virus (BVDV), a member of the Pestivirus genus. We set up a method to purify BVDV virions and analyzed their morphology by electron microscopy and their protein and lipid composition by mass spectrometry. Cryo-electron microscopy showed near spherical viral particles displaying an electron-dense capsid surrounded by a phospholipid bilayer with no visible spikes. Most particles had a diameter of 50 nm and about 2% were larger with a diameter of up to 65 nm, suggesting some size flexibility during BVDV morphogenesis. Morphological and biochemical data suggested a low envelope glycoprotein content of BVDV particles, E1 and E2 being apparently less abundant than Erns. Lipid content of BVDV particles displayed a ~2.3 to 3.5-fold enrichment in cholesterol, sphingomyelin and hexosyl-ceramide, concomitant with a 1.5 to 5-fold reduction of all glycerophospholipid classes, as compared to lipid content of MDBK cells. Although BVDV buds in the endoplasmic reticulum, its lipid content differs from a typical endoplasmic reticulum membrane composition. This suggests that BVDV morphogenesis includes a mechanism of lipid sorting. Functional analyses confirmed the importance of cholesterol and sphingomyelin for BVDV entry. Surprisingly, despite a high cholesterol and sphingolipid content of BVDV envelope, E2 was not found in detergent-resistant membranes. Our results indicate that there are differences between the structure and molecular composition of viral particles of Flaviviruses, Pestiviruses and Hepaciviruses within the Flaviviridae family. PMID:26939061

  13. Morphology and Molecular Composition of Purified Bovine Viral Diarrhea Virus Envelope.

    PubMed

    Callens, Nathalie; Brügger, Britta; Bonnafous, Pierre; Drobecq, Hervé; Gerl, Mathias J; Krey, Thomas; Roman-Sosa, Gleyder; Rümenapf, Till; Lambert, Olivier; Dubuisson, Jean; Rouillé, Yves

    2016-03-01

    The family Flaviviridae includes viruses that have different virion structures and morphogenesis mechanisms. Most cellular and molecular studies have been so far performed with viruses of the Hepacivirus and Flavivirus genera. Here, we studied bovine viral diarrhea virus (BVDV), a member of the Pestivirus genus. We set up a method to purify BVDV virions and analyzed their morphology by electron microscopy and their protein and lipid composition by mass spectrometry. Cryo-electron microscopy showed near spherical viral particles displaying an electron-dense capsid surrounded by a phospholipid bilayer with no visible spikes. Most particles had a diameter of 50 nm and about 2% were larger with a diameter of up to 65 nm, suggesting some size flexibility during BVDV morphogenesis. Morphological and biochemical data suggested a low envelope glycoprotein content of BVDV particles, E1 and E2 being apparently less abundant than Erns. Lipid content of BVDV particles displayed a ~2.3 to 3.5-fold enrichment in cholesterol, sphingomyelin and hexosyl-ceramide, concomitant with a 1.5 to 5-fold reduction of all glycerophospholipid classes, as compared to lipid content of MDBK cells. Although BVDV buds in the endoplasmic reticulum, its lipid content differs from a typical endoplasmic reticulum membrane composition. This suggests that BVDV morphogenesis includes a mechanism of lipid sorting. Functional analyses confirmed the importance of cholesterol and sphingomyelin for BVDV entry. Surprisingly, despite a high cholesterol and sphingolipid content of BVDV envelope, E2 was not found in detergent-resistant membranes. Our results indicate that there are differences between the structure and molecular composition of viral particles of Flaviviruses, Pestiviruses and Hepaciviruses within the Flaviviridae family.

  14. Phenylalanines at positions 88 and 159 of Ebolavirus envelope glycoprotein differentially impact envelope function

    SciTech Connect

    Ou Wu; King, Harlan; Delisle, Josie; Shi Dashuang; Wilson, Carolyn A.

    2010-01-05

    The envelope glycoprotein (GP) of Ebolavirus (EBOV) mediates viral entry into host cells. Through mutagenesis, we and other groups reported that two phenylalanines at positions 88 and 159 of GP are critical for viral entry. However, it remains elusive which steps of viral entry are impaired by F88 or F159 mutations and how. In this study, we further characterized these two phenylalanines through mutagenesis and examined the impact on GP expression, function, and structure. Our data suggest that F159 plays an indirect role in viral entry by maintaining EBOV GP's overall structure. In contrast, we did not detect any evidence for conformational differences in GP with F88 mutations. The data suggest that F88 influences viral entry during a step after cathepsin processing, presumably impacting viral fusion.

  15. Identification of a human immunodeficiency virus type 1 envelope glycoprotein variant resistant to cold inactivation.

    PubMed

    Kassa, Aemro; Finzi, Andrés; Pancera, Marie; Courter, Joel R; Smith, Amos B; Sodroski, Joseph

    2009-05-01

    The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein trimer consists of gp120 and gp41 subunits and undergoes a series of conformational changes upon binding to the receptors, CD4 and CCR5/CXCR4, that promote virus entry. Surprisingly, we found that the envelope glycoproteins of some HIV-1 strains are functionally inactivated by prolonged incubation on ice. Serial exposure of HIV-1 to extremes of temperature, followed by expansion of replication-competent viruses, allowed selection of a temperature-resistant virus. The envelope glycoproteins of this virus resisted cold inactivation due to a single passage-associated change, H66N, in the gp120 exterior envelope glycoprotein. Histidine 66 is located within the gp41-interactive inner domain of gp120 and, in other studies, has been shown to decrease the sampling of the CD4-bound conformation by unliganded gp120. Substituting asparagine or other amino acid residues for histidine 66 in cold-sensitive HIV-1 envelope glycoproteins resulted in cold-stable phenotypes. Cold inactivation of the HIV-1 envelope glycoproteins occurred even at high pH, indicating that protonation of histidine 66 is not necessary for this process. Increased exposure of epitopes in the ectodomain of the gp41 transmembrane envelope glycoprotein accompanied cold inactivation, but shedding of gp120 did not. An amino acid change in gp120 (S375W) that promotes the CD4-bound state or treatment with soluble CD4 or a small-molecule CD4 mimic resulted in increased cold sensitivity. These results indicate that the CD4-bound intermediate of the HIV-1 envelope glycoproteins is cold labile; avoiding the CD4-bound state increases temperature stability.

  16. Envelope glycoproteins of human immunodeficiency virus type 1: profound influences on immune functions.

    PubMed Central

    Chirmule, N; Pahwa, S

    1996-01-01

    Infection by human immunodeficiency virus type 1 (HIV-1) leads to progressive destruction of the CD4+ T-cell subset, resulting in immune deficiency and AIDS. The specific binding of the viral external envelope glycoprotein of HIV-1, gp120, to the CD4 molecules initiates viral entry. In the past few years, several studies have indicated that the interaction of HIV-1 envelope glycoprotein with cells and molecules of the immune system leads to pleiotropic biological effects on immune functions, which include effects on differentiation of CD34+ lymphoid progenitor cells and thymocytes, aberrant activation and cytokine secretion patterns of mature T cells, induction of apoptosis, B-cell hyperactivity, inhibition of T-cell dependent B-cell differentiation, modulation of macrophage functions, interactions with components of complement, and effects on neuronal cells. The amino acid sequence homologies of the envelope glycoproteins with several cellular proteins have suggested that molecular mimicry may play a role in the pathogenesis of the disease. This review summarizes work done by several investigators demonstrating the profound biological effects of envelope glycoproteins of HIV-1 on immune system cells. Extensive studies have also been done on interactions of the viral envelope proteins with components of the immune system which may be important for eliciting a "protective immune response." Understanding the influences of HIV-1 envelope glycoproteins on the immune system may provide valuable insights into HIV-1 disease pathogenesis and carries implications for the trials of HIV-1 envelope protein vaccines and immunotherapeutics. PMID:8801439

  17. The transmembrane protein of HIV-1 primary isolates modulates cell surface expression of their envelope glycoproteins.

    PubMed

    Lebigot, S; Roingeard, P; Thibault, G; Lemiale, F; Verrier, B; Barin, F; Brand, D

    2001-11-10

    We have recently shown that the level of cell surface expression of envelope glycoproteins derived from various human immunodeficiency virus type 1 (HIV-1) primary isolates (PI) was lower than those of envelope glycoproteins derived from T-cell laboratory-adapted (TCLA) HIV-1 (D. Brand et al., 2000, Virology 271, 350-362). We investigated this phenomenon by comparing the cell surface expression of chimeric envelope glycoproteins constructed by swapping the gp120 surface and gp41 transmembrane glycoproteins of the TCLA HIV-1MN and the PI HIV-1(133), HIV-1G365, or HIV-1EFRA. We found that each chimeric envelope construct had a cell surface-specific pattern of expression similar to that of the parental envelope glycoproteins corresponding to the gp41. Thus, the difference in cell surface expression observed between TCLA viruses and various PI is probably due to a signal located in gp41. Identification of this signal may be important for the design of PI envelope-derived immunogens and may increase our understanding of the mechanisms by which HIV-1 escapes from the immune system.

  18. Role of envelope glycoproteins in intracellular virus maturation

    SciTech Connect

    Matsuoka, Y.

    1988-01-01

    The possible role viral glycoproteins in intracellular maturation was studied by using two different viruses, avian infectious bronchitis virus (IBV), a coronavirus, and Punta Toro virus (PTV), a bunyavirus. Using the antibiotic tunicamycin, which inhibits glycosylation of N-linked glycoproteins, it was shown that coronavirus particles are formed in the absence of glycosylation. Analysis of the protein composition of these particles indicated that they contain an unglycosylated form of the membrane-associated E1 glycoprotein but lack the E2 spike glycoprotein. A cDNA clone derived from the PTV M RNA genome segment, which encodes the G1 and G2 glycoproteins, was cloned into vaccinia virus. Studies by indirect immunofluorescence microscopy revealed that the glycoproteins synthesized from this recombinant were found to accumulate intracellularly at the Golgi complex, where virus budding usually takes place. Surface immunoprecipitation and {sup 125}I-protein A binding assays also demonstrated that a majority of the glycoproteins are retained intracellularly and are not transported to the cellular surface. The sequences which encode the G1 and G2 glycoproteins were independently cloned into vaccinia virus as well.

  19. A Con A- purified hydatid glycoprotein fraction effectively diagnoses human hydatidosis.

    PubMed

    Kamel, Manal M; Maher, Kesmat M; Rabia, Ibrahim; Helmy, Ahmed H; El-Adawi, Azza I; Mousa, Mousa A; Mahgoub, Abeer M

    2006-12-01

    Diagnosis and quantification of Echinococcus granulosus infection in man and animal hosts are centralized to feasible control. This study included 93 serum samples, 25 sure positive hydatid cases confirmed surgically, 7 suspected cases diagnosed by indirect haemagglutination IHA and 41 cases other parasitic infections (15 S. mansoni, 8 Fasciola, 7 Ascaris, 5 H. nana & 6 Ancylostoma) diagnosed by microscopic examination and were negative by ELISA and/or IHA for anti-hydatid antibody. Twenty negative serum samples served as healthy controls. Six types of hydatid fluid antigens (crude, host-free & Con-A purified) of human and camel origin were subjected to electrophoretic separation (SDS-PAGE) and immunoblotting (EITB). The anti-hydatid IgG was detected in sera of the different groups for evaluation of sensitivity, specificity and diagnostic efficacy of each type of antigens. Detection of circulating hydatid antigen (CAg) was performed using anti rabbit hyperimmune sera raised against Con-A purified either human or camel hydatid antigen. SDS-PAGE revealed several bands ranging from 55-185 kDa with 10 kDa band shared by all antigens. The specific bands revealed by EITB for Con-A purified camel and human antigens were at 80, 110 & 55, 110 kDa respectively. ELISA highest sensitivity (96.9%) was by using host-free Con-A purified glycoprotein fraction of human hydatid antigen. Highest specificity (98.4%) was recorded upon use of either Con-A purified camel or human antigen with 94.5% & 97.7% diagnostic efficacy respectively. Detection of circulating antigen by polyclonal antibodies against Con-A purified human hydatid antigen revealed 91.8% specificity.

  20. Pseudotyping of vesicular stomatitis virus with the envelope glycoproteins of highly pathogenic avian influenza viruses.

    PubMed

    Zimmer, Gert; Locher, Samira; Berger Rentsch, Marianne; Halbherr, Stefan J

    2014-08-01

    Pseudotype viruses are useful for studying the envelope proteins of harmful viruses. This work describes the pseudotyping of vesicular stomatitis virus (VSV) with the envelope glycoproteins of highly pathogenic avian influenza viruses. VSV lacking the homotypic glycoprotein (G) gene (VSVΔG) was used to express haemagglutinin (HA), neuraminidase (NA) or the combination of both. Propagation-competent pseudotype viruses were only obtained when HA and NA were expressed from the same vector genome. Pseudotype viruses containing HA from different H5 clades were neutralized specifically by immune sera directed against the corresponding clade. Fast and sensitive reading of test results was achieved by vector-mediated expression of GFP. Pseudotype viruses expressing a mutant VSV matrix protein showed restricted spread in IFN-competent cells. This pseudotype system will facilitate the detection of neutralizing antibodies against virulent influenza viruses, circumventing the need for high-level biosafety containment.

  1. Biological properties of purified recombinant HCV particles with an epitope-tagged envelope

    SciTech Connect

    Takahashi, Hitoshi; Akazawa, Daisuke; Kato, Takanobu; Date, Tomoko; Shirakura, Masayuki; Nakamura, Noriko; Mochizuki, Hidenori; Tanaka-Kaneko, Keiko; Sata, Tetsutaro; Tanaka, Yasuhito; Mizokami, Masashi; Suzuki, Tetsuro; Wakita, Takaji

    2010-05-14

    To establish a simple system for purification of recombinant infectious hepatitis C virus (HCV) particles, we designed a chimeric J6/JFH-1 virus with a FLAG (FL)-epitope-tagged sequence at the N-terminal region of the E2 hypervariable region-1 (HVR1) gene (J6/JFH-1/1FL). We found that introduction of an adaptive mutation at the potential N-glycosylation site (E2N151K) leads to efficient production of the chimeric virus. This finding suggests the involvement of glycosylation at Asn within the envelope protein(s) in HCV morphogenesis. To further analyze the biological properties of the purified recombinant HCV particles, we developed a strategy for large-scale production and purification of recombinant J6/JFH-1/1FL/E2N151K. Infectious particles were purified from the culture medium of J6/JFH-1/1FL/E2N151K-infected Huh-7 cells using anti-FLAG affinity chromatography in combination with ultrafiltration. Electron microscopy of the purified particles using negative staining showed spherical particle structures with a diameter of 40-60 nm and spike-like projections. Purified HCV particle-immunization induced both an anti-E2 and an anti-FLAG antibody response in immunized mice. This strategy may contribute to future detailed analysis of HCV particle structure and to HCV vaccine development.

  2. Interactions of HIV-1 and HIV-2 envelope glycoproteins with sulphated polysaccharides and mannose-6-phosphate.

    PubMed

    Mbemba, E; Gluckman, J C; Gattegno, L

    1994-02-01

    Envelope glycoproteins of human immunodeficiency viruses (HIV-1 and HIV-2) can interact with high-mannose glycans and with the mannosyl or N-acetylglucosaminyl core of complex-type oligosaccharidic structures. HIV-1 glycoproteins also specifically bind sulphated polysaccharides such as dextran sulphate (DS) and heparin. Here, we show that the latter property is shared by HIV-2 recombinant gp140 (rgp140) precursor glycoprotein. Binding of rgp140 and of corresponding rgp160 of HIV-1 to heparin- and DS-substituted (sulphated dextran beads; SDB) affinity matrices was inhibited by the soluble specific ligand and also by fetuin, asialofetuin or the anionic simple carbohydrate derivative mannose-6-phosphate (M6P). Interaction of HIV-1 rgp120 subunit with the two affinity matrices was also inhibited by M6P, but only rgp120 binding to heparin-agarose, and not that to SDB, was affected by fetuin and asialofetuin. These results suggest that HIV-1 and HIV-2 envelope glycoproteins presumably display different sulphated polysaccharide and carbohydrate recognition sites. Some of these may be common or in close proximity: with respect to rgp160, for example, the sites may be common on the gp41 moiety and/or in a region of gp120 which would be more accessible when expressed on rgp160 than on processed gp120, while they may be distinct on the cleaved gp120 subunit. Finally, because M6P is a marker of lysosomal enzymes, we verified that HIV-1 and HIV-2 envelope glycoproteins could specifically bind in a M6P-inhibitable manner to a representative lysosomal enzyme, bovine liver beta-glucuronidase coupled to agarose, suggesting that they may possibly interfere with lysosomal enzyme sorting in HIV-infected cells.

  3. Roles of bovine viral diarrhea virus envelope glycoproteins in inducing autophagy in MDBK cells.

    PubMed

    Fu, Qiang; Shi, Huijun; Shi, Mengting; Meng, Luping; Bao, Haiyang; Zhang, Guoqi; Ren, Yan; Zhang, Hui; Guo, Fei; Qiao, Jun; Jia, Bin; Wang, Pengyan; Ni, Wei; Sheng, Jinliang; Chen, Chuangfu

    2014-11-01

    Macroautophagy (autophagy) is an evolutionarily conserved control process that maintains cellular homeostasis in eukaryotic cells. Autophagy principally serves an adaptive role to degrade dysfunctional proteins and to clean damaged organelles in response to pathogenic, viral, or microbial infection, nutrient deprivation and endoplasmic reticulum (ER) stress. In previous study, we showed bovine viral diarrhea virus (BVDV) NADL infection induced autophagy and significantly elevated the expression levels of autophagy-related genes, Beclin1 and ATG14, at 12 h post-infection in MDBK cells. However, the specific mechanisms involved in controlling autophagic activity remain unclear. Here, we investigate the effects of BVDV NADL envelope glycoproteins overexpression on inducing autophagy. The results show that viral envelope glycoproteins E(rns) and E2 overexpression mediated by lentivirus increase the formation of autophagosome, the percentage of GFP-LC3 puncta-positive cells and the expression levels of Beclin1 and ATG14. Whereas E1 overexpression doesn't affect autophagic activity. Collectively, these findings suggest that the viral envelope glycoproteins E(rns) and E2 are involved in inducing autophagy, and provide a mechanistic insight into the regulation of autophagy in viral infected cells.

  4. Averaging of viral envelope glycoprotein spikes from electron cryotomography reconstructions using Jsubtomo.

    PubMed

    Huiskonen, Juha T; Parsy, Marie-Laure; Li, Sai; Bitto, David; Renner, Max; Bowden, Thomas A

    2014-01-01

    Enveloped viruses utilize membrane glycoproteins on their surface to mediate entry into host cells. Three-dimensional structural analysis of these glycoprotein 'spikes' is often technically challenging but important for understanding viral pathogenesis and in drug design. Here, a protocol is presented for viral spike structure determination through computational averaging of electron cryo-tomography data. Electron cryo-tomography is a technique in electron microscopy used to derive three-dimensional tomographic volume reconstructions, or tomograms, of pleomorphic biological specimens such as membrane viruses in a near-native, frozen-hydrated state. These tomograms reveal structures of interest in three dimensions, albeit at low resolution. Computational averaging of sub-volumes, or sub-tomograms, is necessary to obtain higher resolution detail of repeating structural motifs, such as viral glycoprotein spikes. A detailed computational approach for aligning and averaging sub-tomograms using the Jsubtomo software package is outlined. This approach enables visualization of the structure of viral glycoprotein spikes to a resolution in the range of 20-40 Å and study of the study of higher order spike-to-spike interactions on the virion membrane. Typical results are presented for Bunyamwera virus, an enveloped virus from the family Bunyaviridae. This family is a structurally diverse group of pathogens posing a threat to human and animal health. PMID:25350719

  5. Altering Entry Site Preference of Lentiviral Vectors into Neuronal Cells by Pseudotyping with Envelope Glycoproteins.

    PubMed

    Kobayashi, Kenta; Kato, Shigeki; Inoue, Ken-Ichi; Takada, Masahiko; Kobayashi, Kazuto

    2016-01-01

    A lentiviral vector system provides a powerful strategy for gene therapy trials against a variety of neurological and neurodegenerative disorders. Pseudotyping of lentiviral vectors with different envelope glycoproteins not only confers the neurotropism to the vectors, but also alters the preference of sites of vector entry into neuronal cells. One major group of lentiviral vectors is a pseudotype with vesicular stomatitis virus glycoprotein (VSV-G) that enters preferentially cell body areas (somata/dendrites) of neurons and transduces them. Another group contains lentiviral vectors pseudotyped with fusion envelope glycoproteins composed of different sets of rabies virus glycoprotein and VSV-G segments that enter predominantly axon terminals of neurons and are transported through axons retrogradely to their cell bodies, resulting in enhanced retrograde gene transfer. This retrograde gene transfer takes a considerable advantage of delivering the transgene into neuronal cell bodies situated in regions distant from the injection site of the vectors. The rational use of these two vector groups characterized by different entry mechanisms will further extend the strategy for gene therapy of neurological and neurodegenerative disorders.

  6. Averaging of Viral Envelope Glycoprotein Spikes from Electron Cryotomography Reconstructions using Jsubtomo

    PubMed Central

    Huiskonen, Juha T.; Parsy, Marie-Laure; Li, Sai; Bitto, David; Renner, Max; Bowden, Thomas A.

    2014-01-01

    Enveloped viruses utilize membrane glycoproteins on their surface to mediate entry into host cells. Three-dimensional structural analysis of these glycoprotein ‘spikes’ is often technically challenging but important for understanding viral pathogenesis and in drug design. Here, a protocol is presented for viral spike structure determination through computational averaging of electron cryo-tomography data. Electron cryo-tomography is a technique in electron microscopy used to derive three-dimensional tomographic volume reconstructions, or tomograms, of pleomorphic biological specimens such as membrane viruses in a near-native, frozen-hydrated state. These tomograms reveal structures of interest in three dimensions, albeit at low resolution. Computational averaging of sub-volumes, or sub-tomograms, is necessary to obtain higher resolution detail of repeating structural motifs, such as viral glycoprotein spikes. A detailed computational approach for aligning and averaging sub-tomograms using the Jsubtomo software package is outlined. This approach enables visualization of the structure of viral glycoprotein spikes to a resolution in the range of 20-40 Å and study of the study of higher order spike-to-spike interactions on the virion membrane. Typical results are presented for Bunyamwera virus, an enveloped virus from the family Bunyaviridae. This family is a structurally diverse group of pathogens posing a threat to human and animal health. PMID:25350719

  7. Antioxidant activity of glycoprotein purified from Undaria pinnatifida measured by an in vitro digestion model.

    PubMed

    Rafiquzzaman, S M; Kim, Eun-Young; Kim, Yu-Ri; Nam, Taek-Jeong; Kong, In-Soo

    2013-11-01

    The present study was performed to investigate the chemical composition and antioxidant activity of glycoprotein purified from Undaria pinnatifida Harvey (UPGP). On SDS-PAGE, UPGP migrated as a single band with a molecular weight of approximately 10 kDa and confirmed by staining with Schiff's reagent as glycoprotein. It consists of a carbohydrate component (42.53%) and protein component (57.47%). Amino acid profile, FT-IR spectrum and enzymatic glycosylation analysis suggested that protein is linked with carbohydrate by O-glycosylation. UPGP showed dose-dependent antioxidant activities as detected by different assays before and after in vitro digestion. The IC50 values of undigested UPGP were 0.25 ± 0.03, 0.08 ± 0.005, 0.69 ± 0.12, and 0.25 ± 0.08 mg/mL for DPPH, ABTS, FRAP, and NO, respectively. Following in vitro digestion, the antioxidant activities of UPGP were decreased during the gastric phase compared to those of undigested UPGP, with an increase occurring during the duodenal phase in all assays. However, the reducing power was unchanged after in vitro digestion. Furthermore, UPGP showed protective activity against oxidative DNA damage both undigested, after saliva and duodenal phase of digestion. These results indicate that the antioxidant and DNA protection activities of UPGP may be pH-dependent and assay specific.

  8. Specialization of Hepatitis C Virus Envelope Glycoproteins for B Lymphocytes in Chronically Infected Patients

    PubMed Central

    Douam, Florian; Bobay, Louis-Marie; Maurin, Guillemette; Fresquet, Judith; Calland, Noémie; Maisse, Carine; Durand, Tony; Cosset, François-Loïc; Féray, Cyrille

    2015-01-01

    ABSTRACT Hepatitis C virus (HCV) productively infects hepatocytes. Virion surface glycoproteins E1 and E2 play a major role in this restricted cell tropism by mediating virus entry into particular cell types. However, several pieces of evidence have suggested the ability of patient-derived HCV particles to infect peripheral blood mononuclear cells. The viral determinants and mechanisms mediating such events remain poorly understood. Here, we aimed at isolating viral determinants of HCV entry into B lymphocytes. For this purpose, we constructed a library of full E1E2 sequences isolated from serum and B lymphocytes of four chronically infected patients. We observed a strong phylogenetic compartmentalization of E1E2 sequences isolated from B lymphocytes in one patient, indicating that E1E2 glycoproteins can represent important mediators of the strong segregation of two specialized populations in some patients. Most of the E1E2 envelope glycoproteins were functional and allowed transduction of hepatocyte cell lines using HCV-derived pseudoparticles. Strikingly, introduction of envelope glycoproteins isolated from B lymphocytes into the HCV JFH-1 replicating virus switched the entry tropism of this nonlymphotropic virus from hepatotropism to lymphotropism. Significant detection of viral RNA and viral proteins within B cells was restricted to infections with JFH-1 harboring E1E2 from lymphocytes and depended on an endocytic, pH-dependent entry pathway. Here, we achieved for the first time the isolation of HCV viral proteins carrying entry-related lymphotropism determinants. The identification of genetic determinants within E1E2 represents a first step for a better understanding of the complex relationship between HCV infection, viral persistence, and extrahepatic disorders. IMPORTANCE Hepatitis C virus (HCV) mainly replicates within the liver. However, it has been shown that patient-derived HCV particles can slightly infect lymphocytes in vitro and in vivo, highlighting

  9. Comparative immunoprophylactic efficacy of Haemonchus contortus recombinant enolase (rHcENO) and Con A purified native glycoproteins in sheep.

    PubMed

    Kalyanasundaram, Aravindan; Jawahar, Shabnam; Ilangopathy, Manikkavasagan; Palavesam, Azahahianambi; Raman, Muthusamy

    2015-07-01

    Haemonchus contortus is the most economically important blood feeding nematode parasite of sheep and goats all over the world. Enolase in helminth parasites is a multi-functional enzyme which involves in glycolysis and host tissue invasion. In this study, the recombinant H. contortus enolase (rHcENO) was evaluated for its immunoprophylactic efficacy in sheep along with Con A purified native glycoproteins in a vaccine challenge trial. Group I and Group II experimental sheep were immunized thrice with rHcENO and Con A purified native glycoproteins along with Montanide ISA 61 VG adjuvant. The animals were challenged with 5000 L3 stage active H. contortus larvae after 21 days of third immunization. A significant increase in the IgG titre was observed in rHcENO and Con A purified native glycoproteins immunized animals as compared to the control animals. Immunoprotective efficacy of Con A purified native glycoproteins was comparatively higher than rHcENO antigen.

  10. Structural characteristics correlate with immune responses induced by HIV envelope glycoprotein vaccines.

    PubMed

    Sharma, Victoria A; Kan, Elaine; Sun, Yide; Lian, Ying; Cisto, Jimna; Frasca, Verna; Hilt, Susan; Stamatatos, Leonidas; Donnelly, John J; Ulmer, Jeffrey B; Barnett, Susan W; Srivastava, Indresh K

    2006-08-15

    HIV envelope glycoprotein (Env) is the target for inducing neutralizing antibodies. Env is present on the virus surface as a trimer, and, upon binding to CD4, a cascade of events leads to structural rearrangement exposing the co-receptor binding site and entry into the CD4+ host target cells. We have designed monomeric and trimeric Env constructs with and without deletion of the variable loop 2 (ΔV2) from SF162, a subtype B primary isolate, and performed biophysical, biochemical and immunological studies to establish a potential structure–functional relationship. We expressed these Envs in CHO cells, purified the proteins to homogeneity and performed biophysical studies to define the binding properties to CD4, structural characteristics and exposure of epitopes recognized by b12 and CD4i mAb (17B) on both full-length and mutant HIV Env proteins. Parameters evaluated include oligomerization state, number and affinity of CD4 binding sites, enthalpy and entropy of the Env–CD4 interaction and affinity for b12 and 17b mAbs. We observed one CD4 binding site per monomer and three active CD4 binding sites per trimer. A40-fold difference in affinity of the gp120 monomer vs. the o-gp140 trimer towards CD4 was observed (Kd = 58 nM and 1.5 nM, respectively),whereas only a 2-fold difference was observed for the V2 deleted Envs (Kd of gp120ΔV2 = 19 nM, Kd of o-gp140DV2 = 9.3 nM). Monomers had 3-fold higher affinity to the mAb 17b and at least 3-fold weaker affinity to b12 compared to trimers, with gp120DV2 having the weakest affinity for b12 (Kd = 446 nM). Affinity of CD4 binding correlated with proportion of the antibodies induced against the conformational epitopes by the corresponding Envs, and changes in mAb binding correlated with the induction of antibodies directed against linear epitopes. Furthermore,biophysical analysis reveals that the V2 deletion has broad structural implications in the monomer not shared by the trimer, and these changes are reflected in the

  11. Glycoprotein from the liver constitutes the inner layer of the egg envelope (zona pellucida interna) of the fish, Oryzias latipes

    SciTech Connect

    Hamazaki, T.S.; Nagahama, Y.; Iuchi, I.; Yamagami, K.

    1989-05-01

    A glycoprotein from the liver, which shares epitopes with chorion (egg envelope or zona pellucida) glycoproteins, is present only in the spawning female fish, Oryzias latipes, under natural conditions. This spawning female-specific (SF) substance is distinct from vitellogenin but closely resembles a major glycoprotein component, ZI-3, of the inner layer (zona radiata interna) of the ovarian egg envelope with respect to some biochemical and immunochemical characteristics. Here we report that the (/sup 125/I)SF substance, injected into the abdominal cavity of the spawning female fish, was rapidly transported by the blood circulation into the ovary and incorporated into the inner layer of egg envelope of the growing oocytes. The result strongly suggests that the SF substance from the liver is a precursor substance of the major component, ZI-3, of the inner layer of egg envelope in the fish.

  12. Palmitoylation of the feline immunodeficiency virus envelope glycoprotein and its effect on fusion activity and envelope incorporation into virions

    SciTech Connect

    Gonzalez, Silvia A.; Paladino, Monica G.; Affranchino, Jose L.

    2012-06-20

    The feline immunodeficiency virus (FIV) envelope glycoprotein (Env) possesses a short cytoplasmic domain of 53 amino acids containing four highly conserved cysteines at Env positions 804, 811, 815 and 848. Since palmitoylation of transmembrane proteins occurs at or near the membrane anchor, we investigated whether cysteines 804, 811 and 815 are acylated and analyzed the relevance of these residues for Env functions. Replacement of cysteines 804, 811 and 815 individually or in combination by serine residues resulted in Env glycoproteins that were efficiently expressed and processed. However, mutations C804S and C811S reduced Env fusogenicity by 93% and 84%, respectively, compared with wild-type Env. By contrast, mutant C815S exhibited a fusogenic capacity representing 50% of the wild-type value. Remarkably, the double mutation C804S/C811S abrogated both Env fusion activity and Env incorporation into virions. Finally, by means of Click chemistry assays we demonstrated that the four FIV Env cytoplasmic cysteines are palmitoylated.

  13. Proteolysis of Xenopus laevis egg envelope ZPA triggers envelope hardening.

    PubMed

    Lindsay, Leann L; Hedrick, Jerry L

    2004-11-12

    The egg envelope of most animal eggs is modified following fertilization, resulting in the prevention of polyspermy and hardening of the egg envelope. In frogs and mammals a prominent feature of envelope modification is N-terminal proteolysis of the envelope glycoprotein ZPA. We have purified the ZPA protease from Xenopus laevis eggs and characterized it as a zinc metalloprotease. Proteolysis of isolated egg envelopes by the isolated protease resulted in envelope hardening. The N-terminal peptide fragment of ZPA remained disulfide bond linked to the ZPA glycoprotein moiety following proteolysis. We propose a mechanism for egg envelope hardening involving ZPA proteolysis by an egg metalloprotease as a triggering event followed by induction of global conformational changes in egg envelope glycoproteins. PMID:15474476

  14. Naturally occurring variability in the envelope glycoprotein of HIV-1 and development of cell entry inhibitors.

    PubMed

    Brower, Evan T; Schön, Arne; Freire, Ernesto

    2010-03-23

    Naturally occurring genetic variability across HIV-1 subtypes causes amino acid polymorphisms in encoded HIV-1 proteins including the envelope glycoproteins associated with viral entry. The effects of amino acid polymorphisms on the mechanism of HIV-1 entry into cells, a process initiated by the binding of the viral envelope glycoprotein gp120 to the cellular CD4 receptor, are largely unknown. In this study, we demonstrate that amino acid polymorphisms affect the structural stability and domain cooperativity of gp120 and that those differences are reflected in the binding mechanism of the viral envelope glycoprotein to the cell surface receptor and coreceptor. Moreover, subtype differences also affect the binding behavior of experimental HIV cell entry inhibitors. While gp120-A has a slightly lower denaturation temperature than gp120-B, the most notable stability difference is that for gp120-B the van't Hoff to calorimetric enthalpy ratio (DeltaH(vH)/DeltaH) is 0.95 whereas for gp120-A is 0.6, indicative of more cooperative domain/domain interactions in gp120-B, as this protein more closely approaches a two-state transition. Isothermal titration calorimetry demonstrates that CD4 and 17b (a surrogate antibody for the chemokine coreceptor) exhibit 7- and 3-fold weaker binding affinities for gp120-A. The binding of these proteins as well as that of the experimental entry inhibitor NBD-556 induces smaller conformational changes in gp120-A as evidenced by significantly smaller binding enthalpies and binding entropies. Together, these results describe the effects of gp120 polymorphisms on binding to host cell receptors and emphasize that guidelines for developing future entry inhibitors must recognize and deal with genomic differences between HIV strains.

  15. Reduction of cerebral glucose utilization by the HIV envelope glycoprotein Gp-120

    SciTech Connect

    Kimes, A.S.; London, E.D.; Szabo, G.; Raymon, L.; Tabakoff, B. )

    1991-05-01

    Gp-120 is a glycoprotein constituent of the human immunodeficiency virus (HIV) envelope. The effects of gp-120 on cerebral glucose utilization in rats were studied by the quantitative 2-deoxy-D-(1-14C) glucose method. Intracerebroventricular injection of gp-120 significantly reduced glucose utilization in the lateral habenula and the suprachiasmatic nucleus and decreased the global cerebral metabolic rate for glucose. The findings suggest that gp-120 and closely related peptides can alter neuronal function, thereby contributing to the sequelae of HIV infection.

  16. Pre-fusion structure of trimeric HIV-1 envelope glycoprotein determined by cryo-electron microscopy

    PubMed Central

    Bartesaghi, Alberto; Merk, Alan; Borgnia, Mario J.; Milne, Jacqueline L. S.; Subramaniam, Sriram

    2014-01-01

    The activation of trimeric HIV-1 envelope glycoprotein (Env) by its binding to the cell surface receptor CD4 and co-receptors (CCR5 or CXCR4) represents the first of a series of events that lead to fusion between viral and target cell membranes. Here, we present the cryo-electron microscopic structure, at ~ 6 Å resolution, of the closed, pre-fusion state of trimeric HIV-1 Env in complex with the broadly neutralizing antibody VRC03. We show that three gp41 helices at the core of the trimer serve as an anchor around which the rest of Env is reorganized upon activation to the open quaternary conformation. The architecture of trimeric HIV-1 Env in pre-fusion and activated intermediate states resembles the corresponding states of influenza hemagglutinin trimers, providing direct evidence for the similarity in entry mechanisms employed by HIV-1, influenza and related enveloped viruses. PMID:24154805

  17. Mutagenesis of the palmitoylation site in vaccinia virus envelope glycoprotein B5.

    PubMed

    Lorenzo, María M; Sánchez-Puig, Juana M; Blasco, Rafael

    2012-04-01

    The outer envelope of vaccinia virus extracellular virions is derived from intracellular membranes that, at late times in infection, are enriched in several virus-encoded proteins. Although palmitoylation is common in vaccinia virus envelope proteins, little is known about the role of palmitoylation in the biogenesis of the enveloped virus. We have studied the palmitoylation of B5, a 42 kDa type I transmembrane glycoprotein comprising a large ectodomain and a short (17 aa) cytoplasmic tail. Mutation of two cysteine residues located in the cytoplasmic tail in close proximity to the transmembrane domain abrogated palmitoylation of the protein. Virus mutants expressing non-palmitoylated versions of B5 and/or lacking most of the cytoplasmic tail were isolated and characterized. Cell-to-cell virus transmission and extracellular virus formation were only slightly affected by those mutations. Notably, B5 versions lacking palmitate showed decreased interactions with proteins A33 and F13, but were still incorporated into the virus envelope. Expression of mutated B5 by transfection into uninfected cells showed that both the cytoplasmic tail and palmitate have a role in the intracellular transport of B5. These results indicate that the C-terminal portion of protein B5, while involved in protein transport and in protein-protein interactions, is broadly dispensable for the formation and egress of infectious extracellular virus and for virus transmission.

  18. Determining the Structure of an Unliganded and Fully Glycosylated SIV gp120 Envelope Glycoprotein

    SciTech Connect

    Chen, Bing; Vogan, Erik M.; Gong, Haiyun; Skehel, John J.; Wiley, Don C.; Harrison, Stephen C.

    2010-07-13

    HIV/SIV envelope glycoproteins mediate the first steps in viral infection. They are trimers of a membrane-anchored polypeptide chain, cleaved into two fragments known as gp120 and gp41. The structure of HIV gp120 bound with receptor (CD4) has been known for some time. We have now determined the structure of a fully glycosylated SIV gp120 envelope glycoprotein in an unliganded conformation by X-ray crystallography at 4.0 {angstrom} resolution. We describe here our experimental and computational approaches, which may be relevant to other resolution-limited crystallographic problems. Key issues were attention to details of beam geometry mandated by small, weakly diffracting crystals, and choice of strategies for phase improvement, starting with two isomorphous derivatives and including multicrystal averaging. We validated the structure by analyzing composite omit maps, averaged among three distinct crystal lattices, and by calculating model-based, SeMet anomalous difference maps. There are at least four ordered sugars on many of the thirteen oligosaccharides.

  19. Expression of the human immunodeficiency virus envelope glycoprotein is restricted to basolateral surfaces of polarized epithelial cells

    SciTech Connect

    Owens, R.J.; Compans, R.W.

    1989-02-01

    Polarized epithelial cells exhibit apical (lumenal) and basolateral (serosal) membrane domains that are separated by circumferential tight junctions. In such cells, enveloped viruses that mature by budding at cell surfaces are released at particular membrane domains. The authors have used a vaccinia virus recombinant to investigate the site of surface expression of the human immunodeficiency virus type 1 envelope glycoprotein in Madin-Darby canine kidney cells. Cells were infected with the vaccinia virus recombinant, and surface expression of the glycoprotein was analyzed by indirect immunofluorescence, /sup 125/I-protein A binding, and immunoelectron microscopy. The glycoprotein appeared exclusively at the basolateral surface as early as 2 h postinfection and reached a maximum level at 8 h postinfection. The gp120 glycoprotein was found to be secreted efficiently into culture medium, and this secretion occurred exclusively at the basolateral surface.

  20. Structure of a flavivirus envelope glycoprotein in its low-pH-induced membrane fusion conformation

    PubMed Central

    Bressanelli, Stéphane; Stiasny, Karin; Allison, Steven L; Stura, Enrico A; Duquerroy, Stéphane; Lescar, Julien; Heinz, Franz X; Rey, Félix A

    2004-01-01

    Enveloped viruses enter cells via a membrane fusion reaction driven by conformational changes of specific viral envelope proteins. We report here the structure of the ectodomain of the tick-borne encephalitis virus envelope glycoprotein, E, a prototypical class II fusion protein, in its trimeric low-pH-induced conformation. We show that, in the conformational transition, the three domains of the neutral-pH form are maintained but their relative orientation is altered. Similar to the postfusion class I proteins, the subunits rearrange such that the fusion peptide loops cluster at one end of an elongated molecule and the C-terminal segments, connecting to the viral transmembrane region, run along the sides of the trimer pointing toward the fusion peptide loops. Comparison with the low-pH-induced form of the alphavirus class II fusion protein reveals striking differences at the end of the molecule bearing the fusion peptides, suggesting an important conformational effect of the missing membrane connecting segment. PMID:14963486

  1. Mutations altering the gammaretrovirus endoproteolytic motif affect glycosylation of the envelope glycoprotein and early events of the virus life cycle.

    PubMed

    Argaw, Takele; Wilson, Carolyn A

    2015-01-15

    Previously, we found that mutation of glutamine to proline in the endoproteolytic cleavage signal of the PERV-C envelope (RQKK to RPKK) resulted in non-infectious vectors. Here, we show that RPKK results in a non-infectious vector when placed in not only a PERV envelope, but also the envelope of a related gammaretrovirus, FeLV-B. The amino acid substitutions do not prevent envelope precursor cleavage, viral core and genome assembly, or receptor binding. Rather, the mutations result in the formation of hyperglycosylated glycoprotein and a reduction in the reverse transcribed minus strand synthesis and undetectable 2-LTR circular DNA in cells exposed to vectors with these mutated envelopes. Our findings suggest novel functions associated with the cleavage signal sequence that may affect trafficking through the glycosylation machinery of the cell. Further, the glycosylation status of the envelope appears to impact post-binding events of the viral life cycle, either membrane fusion, internalization, or reverse transcription.

  2. Characterization of virulence-associated determinants in the envelope glycoprotein of Pichinde virus.

    PubMed

    Kumar, Naveen; Wang, Jialong; Lan, Shuiyun; Danzy, Shamika; McLay Schelde, Lisa; Seladi-Schulman, Jill; Ly, Hinh; Liang, Yuying

    2012-11-10

    We use a small animal model, based on guinea pigs infected with a non-pathogenic Pichinde virus (PICV), to understand the virulence mechanisms of arenavirus infections in the hosts. PICV P2 strain causes a mild febrile reaction in guinea pigs, while P18 causes severe disease with clinical and pathological features reminiscent of Lassa hemorrhagic fever in humans. The envelope glycoproteins (GPC) of P2 and P18 viruses differ at positions 119, 140, and 164, all localized to the receptor-binding G1 subunit. We found that lentiviral pseudotyped virions (VLPs) bearing P18 GPC show more efficient cell entry than those with P2 GPC, and that the E140 residue plays a critical role in this process. Infection of guinea pigs with the recombinant viruses containing the E140K change demonstrated that E140 of GPC is a necessary virulence determinant of P18 infections, possibly by enhancing the ability of virus to enter target cells.

  3. Development of Global Consensus of Dengue Virus Envelope Glycoprotein for Epitopes Based Vaccine Design.

    PubMed

    Hussain, Mazhar; Idrees, Muhammad; Afzal, Samia

    2015-01-01

    Dengue virus (DENV) is the member of Flaviviridae and causative agent of Dengue Haemorrhagic Fever and Dengue Shock Syndrome. Every year, around 70% of the world population is at risk, due to epidemic episodes orchestrated by one or more of its serotypes. So, a tetravalent DENV vaccine is needed which may induce the immune response against all four DENV serotypes. In this study, B-cell and T-cell epitopes have been predicted from the DENV envelope glycoprotein (Eg) using a consensus based approach in complement with the physico-chemical property (PCP) conservancy analysis. Through DENV-Eg analysis, a total of 7 PCP conserved, water soluble, in vitro and in vivo stable epitopes were predicted which may induce the B-cell and T-cell mediated anti-viral immune response.

  4. Opposite polarity of virus budding and of viral envelope glycoprotein distribution in epithelial cells derived from different tissues

    PubMed Central

    1992-01-01

    We compared the surface envelope glycoprotein distribution and the budding polarity of four RNA viruses in Fischer rat thyroid (FRT) cells and in CaCo-2 cells derived from a human colon carcinoma. Whereas both FRT and CaCo-2 cells sort similarly influenza hemagglutinin and vesicular stomatitis virus (VSV) G protein, respectively, to apical and basolateral membrane domains, they differ in their handling of two togaviruses, Sindbis and Semliki Forest virus (SFV). By conventional EM Sindbis virus and SFV were shown to bud apically in FRT cells and basolaterally in CaCo-2 cells. Consistent with this finding, the distribution of the p62/E2 envelope glycoprotein of SFV, assayed by immunoelectronmicroscopy and by domain-selective surface biotinylation was predominantly apical on FRT cells and basolateral on CaCo-2 cells. We conclude that a given virus and its envelope glycoprotein can be delivered to opposite membrane domains in epithelial cells derived from different tissues. The tissue specificity in the polarity of virus budding and viral envelope glycoprotein distribution indicate that the sorting machinery varies considerably between different epithelial cell types. PMID:1572895

  5. Conformational Evaluation of HIV-1 Trimeric Envelope Glycoproteins Using a Cell-based ELISA Assay

    PubMed Central

    Veillette, Maxime; Désormeaux, Anik; Roger, Michel; Finzi, Andrés

    2014-01-01

    HIV-1 envelope glycoproteins (Env) mediate viral entry into target cells and are essential to the infectious cycle. Understanding how those glycoproteins are able to fuel the fusion process through their conformational changes could lead to the design of better, more effective immunogens for vaccine strategies. Here we describe a cell-based ELISA assay that allows studying the recognition of trimeric HIV-1 Env by monoclonal antibodies. Following expression of HIV-1 trimeric Env at the surface of transfected cells, conformation specific anti-Env antibodies are incubated with the cells. A horseradish peroxidase-conjugated secondary antibody and a simple chemiluminescence reaction are then used to detect bound antibodies. This system is highly flexible and can detect Env conformational changes induced by soluble CD4 or cellular proteins. It requires minimal amount of material and no highly-specialized equipment or know-how. Thus, this technique can be established for medium to high throughput screening of antigens and antibodies, such as newly-isolated antibodies. PMID:25286159

  6. Immunogenicity and functional characterization of Leishmania-derived hepatitis C virus envelope glycoprotein complex

    PubMed Central

    Grzyb, Katarzyna; Czarnota, Anna; Brzozowska, Agnieszka; Cieślik, Anna; Rąbalski, Łukasz; Tyborowska, Jolanta; Bieńkowska-Szewczyk, Krystyna

    2016-01-01

    Hepatitis C virus (HCV) envelope glycoproteins E1 and E2 are the main inducers of a cross-neutralizing antibody response which plays an important role in the early phase of viral infection. Correctly folded and immunologically active E1E2 complex can be expressed in mammalian cells, though the production process might still prove restrictive, even if the immunological response of a vaccine candidate is positive. Here, we report a characterization and immunogenicity study of a full-length (fE1E2) and soluble version of the E1E2 complex (tE1E2) from genotype 1a, successfully expressed in the cells of Leishmania tarentolae. In a functional study, we confirmed the binding of both Leishmania-derived E1E2 complexes to the CD-81 receptor and the presence of the major epitopes participating in a neutralizing antibody response. Both complexes were proved to be highly immunogenic in mice and elicited neutralizing antibody response. Moreover, cross-reactivity of the mouse sera was detected for all tested HCV genotypes with the highest signal intensity observed for genotypes 1a, 1b, 5 and 6. Since the development of a prophylactic vaccine against HCV is still needed to control the global infection, our Leishmania-derived E1E2 glycoproteins could be considered a potential cost-effective vaccine candidate. PMID:27481352

  7. Immunogenicity and functional characterization of Leishmania-derived hepatitis C virus envelope glycoprotein complex.

    PubMed

    Grzyb, Katarzyna; Czarnota, Anna; Brzozowska, Agnieszka; Cieślik, Anna; Rąbalski, Łukasz; Tyborowska, Jolanta; Bieńkowska-Szewczyk, Krystyna

    2016-01-01

    Hepatitis C virus (HCV) envelope glycoproteins E1 and E2 are the main inducers of a cross-neutralizing antibody response which plays an important role in the early phase of viral infection. Correctly folded and immunologically active E1E2 complex can be expressed in mammalian cells, though the production process might still prove restrictive, even if the immunological response of a vaccine candidate is positive. Here, we report a characterization and immunogenicity study of a full-length (fE1E2) and soluble version of the E1E2 complex (tE1E2) from genotype 1a, successfully expressed in the cells of Leishmania tarentolae. In a functional study, we confirmed the binding of both Leishmania-derived E1E2 complexes to the CD-81 receptor and the presence of the major epitopes participating in a neutralizing antibody response. Both complexes were proved to be highly immunogenic in mice and elicited neutralizing antibody response. Moreover, cross-reactivity of the mouse sera was detected for all tested HCV genotypes with the highest signal intensity observed for genotypes 1a, 1b, 5 and 6. Since the development of a prophylactic vaccine against HCV is still needed to control the global infection, our Leishmania-derived E1E2 glycoproteins could be considered a potential cost-effective vaccine candidate. PMID:27481352

  8. [Hsp70 Fused with the Envelope Glycoprotein E0 of Classical Swine Fever Virus Enhances Immune Responses in Balb/c Mice].

    PubMed

    Xu, Qianqian; Zhang, Xiaomin; Jing, Jiao; Shi, Baojun; Wang, Shiqi; Zhou, Bin; Chen, Puyan

    2015-07-01

    Heat-shock protein (Hsp) 70 potentiates specific immune responses to some antigenic peptides fused to it. Here, the prokaryotic plasmids harboring the envelope glycoprotein E0 gene of classical swine fever virus (CSFV) and/or the Hsp70 gene of Haemophilus parasuis were constructed and expressed in Escherichia coli Rosseta 2(R2). The fusion proteins were then purified. Groups of Balb/c mice were immunized with these fusion proteins, respectively, and sera collected 7 days after the third immunization. Immune effects were determined via an enzyme-linked immunosorbent assay and flow cytometric analyses. E0-Hsp70 fusion protein and E0+Hsp70 mixture significantly improved the titer of E-specific antibody, levels of CD4+ and CD8+ T cells, and release of interferon-γ. These findings suggested that Hsp70 can significantly enhance the immune effects of the envelope glycoprotein E0 of CSFV, thereby laying the foundation of further application in pigs. PMID:26524908

  9. Production and neurotropism of lentivirus vectors pseudotyped with lyssavirus envelope glycoproteins.

    PubMed

    Desmaris, N; Bosch, A; Salaün, C; Petit, C; Prévost, M C; Tordo, N; Perrin, P; Schwartz, O; de Rocquigny, H; Heard, J M

    2001-08-01

    We investigated the production efficiency and the gene transfer capacity in the central nervous system of HIV-1-based vectors pseudotyped with either the G protein of the Mokola lyssaviruses (MK-G), a neurotropic virus causing rabies disease, or the vesiculo-stomatitis G protein (VSV-G). Both envelopes induced syncitia in cell cultures. They were incorporated into vector particles and mature virions were observed by electron microscopy. Vector production was two- to sixfold more efficient with VSV-G than with MK-G. For equivalent amounts of physical particles, vector titration was 5- to 25-fold higher with VSV-G than with MK-G pseudotypes on cultured cells, and in vivo gene expression in mouse brain was more intense. Thus, VSV-G pseudotypes were produced more efficiently and were more infectious than MK-G pseudotypes. Tropism for brain cells was analyzed by intrastriatal injections in rats. Both pseudotypes preferentially transduced neurons (70-90% of transduced cells). Retrograde axonal transport was investigated by instilling vector suspensions in the rat nasal cavity. Both pseudotypes were efficiently transported to olfactive neuron bodies. Thus, although coating HIV-1 particles with rabdhovirus envelope glycoproteins enables them to enter neuronal cells efficiently, pseudotyping is not sufficient to confer the powerful neurotropism of lyssaviruses to lentivirus vectors.

  10. Inflammatory response of endothelial cells to hepatitis C virus recombinant envelope glycoprotein 2 protein exposure

    PubMed Central

    Urbaczek, Ana Carolina; Ribeiro, Lívia Carolina de Abreu; Ximenes, Valdecir Farias; Afonso, Ana; Nogueira, Camila Tita; Generoso, Wesley Cardoso; Alberice, Juliana Vieira; Rudnicki, Martina; Ferrer, Renila; da Fonseca, Luiz Marcos; da Costa, Paulo Inácio

    2014-01-01

    The hepatitis C virus (HCV) encodes approximately 10 different structural and non-structural proteins, including the envelope glycoprotein 2 (E2). HCV proteins, especially the envelope proteins, bind to cell receptors and can damage tissues. Endothelial inflammation is the most important determinant of fibrosis progression and, consequently, cirrhosis. The aim of this study was to evaluate and compare the inflammatory response of endothelial cells to two recombinant forms of the HCV E2 protein produced in different expression systems (Escherichia coli and Pichia pastoris). We observed the induction of cell death and the production of nitric oxide, hydrogen peroxide, interleukin-8 and vascular endothelial growth factor A in human umbilical vein endothelial cells (HUVECs) stimulated by the two recombinant E2 proteins. The E2-induced apoptosis of HUVECs was confirmed using the molecular marker PARP. The apoptosis rescue observed when the antioxidant N-acetylcysteine was used suggests that reactive oxygen species are involved in E2-induced apoptosis. We propose that these proteins are involved in the chronic inflammation caused by HCV. PMID:25317702

  11. Human herpesvirus 8 envelope glycoprotein K8.1A interaction with the target cells involves heparan sulfate.

    PubMed

    Wang, F Z; Akula, S M; Pramod, N P; Zeng, L; Chandran, B

    2001-08-01

    Human herpesvirus-8 (HHV-8) or Kaposi's sarcoma-associated herpesvirus K8.1 gene encodes for two immunogenic glycoproteins, gpK8.1A and gpK8.1B, originating from spliced messages. The 228-amino-acid (aa) gpK8.1A is the predominant form associated with the virion envelope, consisting of a 167-aa region identical to gpK8.1B and a 61-aa unique region (L. Zhu, V. Puri, and B. Chandran, Virology 262:237-249, 1999). HHV-8 has a broad in vivo and in vitro cellular tropism, and our studies showed that this may be in part due to HHV-8's interaction with the ubiquitous host cell surface molecule, heparan sulfate (HS). Since HHV-8 K8.1 gene is positionally colinear to the Epstein-Barr virus (EBV) gene encoding the gp350/gp220 protein involved in EBV binding to the target cells, gpK8.1A's ability to interact with the target cells was examined. The gpK8.1A without the transmembrane and carboxyl domains (DeltaTMgpK8.1A) was expressed in a baculovirus system and purified. Radiolabeled purified DeltaTMgpK8.1A protein bound to the target cells, which was blocked by unlabeled DeltaTMgpK8.1A. Unlabeled DeltaTMgpK8.1A blocked the binding of [(3)H]thymidine-labeled purified HHV-8 to the target cells. Binding of radiolabeled DeltaTMgpK8.1A to the target cells was inhibited in a dose-dependent manner by soluble heparin, a glycosaminoglycan (GAG) closely related to HS, but not by other GAGs such as chondroitin sulfate A and C, N-acetyl heparin and de-N-sulfated heparin. Cell surface absorbed DeltaTMgpK8.1A was displaced by soluble heparin. Radiolabeled DeltaTMgpK8.1A also bound to HS expressing Chinese hamster ovary (CHO-K1) cells, and binding to mutant CHO cell lines deficient in HS was significantly reduced. The DeltaTMgpK8.1A specifically bound to heparin-agarose beads, which was inhibited by HS and heparin, but not by other GAGs. Virion envelope-associated gpK8.1A was specifically precipitated by heparin-agarose beads. These findings suggest that gpK8.1A interaction with target cells

  12. Two distinct oncornaviruses harbor an intracytoplasmic tyrosine-based basolateral targeting signal in their viral envelope glycoprotein.

    PubMed Central

    Lodge, R; Delamarre, L; Lalonde, J P; Alvarado, J; Sanders, D A; Dokhélar, M C; Cohen, E A; Lemay, G

    1997-01-01

    It has been clearly established that the budding of the human immunodeficiency virus (HIV-1), a lentivirus, occurs specifically through the basolateral membrane in polarized epithelial cells. More recently, the signal was assigned to a tyrosine-based motif located in the intracytoplasmic domain of the envelope glycoprotein, as previously observed on various other viral and cellular basolateral proteins. In the present study, expression of human T-cell leukemia virus type 1 (HTLV-1) or Moloney murine leukemia virus envelope glycoproteins was used for trans-complementation of an envelope-negative HIV-1. This demonstrated the potential of oncornaviral retrovirus envelope glycoproteins to confer polarized basolateral budding in epithelial Madin-Darby canine kidney cells (MDCK cells). Site-directed mutagenesis confirmed the importance of a common motif encompassing at least one crucial membrane-proximal intracytoplasmic tyrosine residue. The conservation of a similar basolateral maturation signal in different retroviruses further supports its importance in the biology of this group of viruses. PMID:9188652

  13. Hepatitis C Virus Envelope Glycoprotein E1 Forms Trimers at the Surface of the Virion

    PubMed Central

    Falson, Pierre; Bartosch, Birke; Alsaleh, Khaled; Tews, Birke Andrea; Loquet, Antoine; Ciczora, Yann; Riva, Laura; Montigny, Cédric; Montpellier, Claire; Duverlie, Gilles; Pécheur, Eve-Isabelle; le Maire, Marc; Cosset, François-Loïc

    2015-01-01

    ABSTRACT In hepatitis C virus (HCV)-infected cells, the envelope glycoproteins E1 and E2 assemble as a heterodimer. To investigate potential changes in the oligomerization of virion-associated envelope proteins, we performed SDS-PAGE under reducing conditions but without thermal denaturation. This revealed the presence of SDS-resistant trimers of E1 in the context of cell-cultured HCV (HCVcc) as well as in the context of HCV pseudoparticles (HCVpp). The formation of E1 trimers was found to depend on the coexpression of E2. To further understand the origin of E1 trimer formation, we coexpressed in bacteria the transmembrane (TM) domains of E1 (TME1) and E2 (TME2) fused to reporter proteins and analyzed the fusion proteins by SDS-PAGE and Western blotting. As expected for strongly interacting TM domains, TME1–TME2 heterodimers resistant to SDS were observed. These analyses also revealed homodimers and homotrimers of TME1, indicating that such complexes are stable species. The N-terminal segment of TME1 exhibits a highly conserved GxxxG sequence, a motif that is well documented to be involved in intramembrane protein-protein interactions. Single or double mutations of the glycine residues (Gly354 and Gly358) in this motif markedly decreased or abrogated the formation of TME1 homotrimers in bacteria, as well as homotrimers of E1 in both HCVpp and HCVcc systems. A concomitant loss of infectivity was observed, indicating that the trimeric form of E1 is essential for virus infectivity. Taken together, these results indicate that E1E2 heterodimers form trimers on HCV particles, and they support the hypothesis that E1 could be a fusion protein. IMPORTANCE HCV glycoproteins E1 and E2 play an essential role in virus entry into liver cells as well as in virion morphogenesis. In infected cells, these two proteins form a complex in which E2 interacts with cellular receptors, whereas the function of E1 remains poorly understood. However, recent structural data suggest that E1

  14. Chemical Cross-Linking Stabilizes Native-Like HIV-1 Envelope Glycoprotein Trimer Antigens

    PubMed Central

    Schiffner, Torben; de Val, Natalia; Russell, Rebecca A.; de Taeye, Steven W.; de la Peña, Alba Torrents; Ozorowski, Gabriel; Kim, Helen J.; Nieusma, Travis; Brod, Florian; Cupo, Albert; Sanders, Rogier W.; Moore, John P.; Ward, Andrew B.

    2015-01-01

    ABSTRACT Major neutralizing antibody immune evasion strategies of the HIV-1 envelope glycoprotein (Env) trimer include conformational and structural instability. Stabilized soluble trimers such as BG505 SOSIP.664 mimic the structure of virion-associated Env but nevertheless sample different conformational states. Here we demonstrate that treating BG505 SOSIP.664 trimers with glutaraldehyde or a heterobifunctional cross-linker introduces additional stability with relatively modest effects on antigenicity. Thus, most broadly neutralizing antibody (bNAb) epitopes were preserved after cross-linking, whereas the binding of most weakly or nonneutralizing antibodies (non-NAb) was reduced. Cross-linking stabilized all Env conformers present within a mixed population, and individual conformers could be isolated by bNAb affinity chromatography. Both positive selection of cross-linked conformers using the quaternary epitope-specific bNAbs PGT145, PGT151, and 3BC315 and negative selection with non-NAbs against the V3 region enriched for trimer populations with improved antigenicity for bNAbs. Similar results were obtained using the clade B B41 SOSIP.664 trimer. The cross-linking method may, therefore, be useful for countering the natural conformational heterogeneity of some HIV-1 Env proteins and, by extrapolation, also vaccine immunogens from other pathogens. IMPORTANCE The development of a vaccine to induce protective antibodies against HIV-1 is of primary public health importance. Recent advances in immunogen design have provided soluble recombinant envelope glycoprotein trimers with near-native morphology and antigenicity. However, these trimers are conformationally flexible, potentially reducing B-cell recognition of neutralizing antibody epitopes. Here we show that chemical cross-linking increases trimer stability, reducing binding of nonneutralizing antibodies while largely maintaining neutralizing antibody binding. Cross-linking followed by positive or negative

  15. Antigenicity and immunogenicity of HIV-1 consensus subtype B envelope glycoproteins

    SciTech Connect

    Kothe, Denise L.; Decker, Julie M.; Li Yingying; Weng Zhiping; Bibollet-Ruche, Frederic; Zammit, Kenneth P.; Salazar, Maria G.; Chen, Yalu; Salazar-Gonzalez, Jesus F.; Moldoveanu, Zina; Mestecky, Jiri; Gao Feng; Haynes, Barton F.; Shaw, George M. ||; Muldoon, Mark; Korber, Bette T.M. |; Hahn, Beatrice H. |. E-mail: bhahn@uab.edu

    2007-03-30

    'Centralized' (ancestral and consensus) HIV-1 envelope immunogens induce broadly cross-reactive T cell responses in laboratory animals; however, their potential to elicit cross-reactive neutralizing antibodies has not been fully explored. Here, we report the construction of a panel of consensus subtype B (ConB) envelopes and compare their biologic, antigenic, and immunogenic properties to those of two wild-type Env controls from individuals with early and acute HIV-1 infection. Glycoprotein expressed from full-length (gp160), uncleaved (gp160-UNC), truncated (gp145), and N-linked glycosylation site deleted (gp160-201N/S) versions of the ConB env gene were packaged into virions and, except for the fusion defective gp160-UNC, mediated infection via the CCR5 co-receptor. Pseudovirions containing ConB Envs were sensitive to neutralization by patient plasma and monoclonal antibodies, indicating the preservation of neutralizing epitopes found in contemporary subtype B viruses. When used as DNA vaccines in guinea pigs, ConB and wild-type env immunogens induced appreciable binding, but overall only low level neutralizing antibodies. However, all four ConB immunogens were significantly more potent than one wild-type vaccine at eliciting neutralizing antibodies against a panel of tier 1 and tier 2 viruses, and ConB gp145 and gp160 were significantly more potent than both wild-type vaccines at inducing neutralizing antibodies against tier 1 viruses. Thus, consensus subtype B env immunogens appear to be at least as good as, and in some instances better than, wild-type B env immunogens at inducing a neutralizing antibody response, and are amenable to further improvement by specific gene modifications.

  16. The influence of the type of immunosorbent on rabies antibody EIA; advantages of purified glycoprotein over whole virus.

    PubMed

    Perrin, P; Versmisse, P; Delagneau, J F; Lucas, G; Rollin, P E; Sureau, P

    1986-04-01

    Two types of in vitro assay (enzyme immunoassay and sero-neutralization test) for the titration of rabies antibodies were used to assay sera from mice and humans immunized with cell culture vaccines or neural tissue vaccines. Enzyme immunoassays (EIA) were performed in plates sensitized with whole virus, purified glycoprotein or purified nucleocapsid. Neutralizing antibody titres were determined by the rapid fluorescent focus inhibition test (REFIT) and by an in vitro seroneutralization test including a rapid enzyme immunotitration of intracellular antigens (REITICA). The results obtained with sera of immunized mice and humans showed that (1) cell culture vaccines mainly induced the synthesis of antiglycoprotein neutralizing antibodies; and (2) neural tissue vaccines induced a high synthesis of antinucleocapsid non-neutralizing antibodies and a more or less important synthesis of antiglycoprotein antibodies depending on the origin of the tissue used for their preparation. Consequently, it was emphasized that when using EIA, the antibody titration must be run in glycoprotein-coated plates rather than in whole virus-coated plates to appreciate correctly the immunizing potency of a rabies vaccine, especially neural tissue vaccine.

  17. Mutations altering the gammaretrovirus endoproteolytic motif affect glycosylation of the envelope glycoprotein and early events of the virus life cycle

    SciTech Connect

    Argaw, Takele; Wilson, Carolyn A.

    2015-01-15

    Previously, we found that mutation of glutamine to proline in the endoproteolytic cleavage signal of the PERV-C envelope (RQKK to RPKK) resulted in non-infectious vectors. Here, we show that RPKK results in a non-infectious vector when placed in not only a PERV envelope, but also the envelope of a related gammaretrovirus, FeLV-B. The amino acid substitutions do not prevent envelope precursor cleavage, viral core and genome assembly, or receptor binding. Rather, the mutations result in the formation of hyperglycosylated glycoprotein and a reduction in the reverse transcribed minus strand synthesis and undetectable 2-LTR circular DNA in cells exposed to vectors with these mutated envelopes. Our findings suggest novel functions associated with the cleavage signal sequence that may affect trafficking through the glycosylation machinery of the cell. Further, the glycosylation status of the envelope appears to impact post-binding events of the viral life cycle, either membrane fusion, internalization, or reverse transcription. - Highlights: • Env cleavage signal impacts infectivity of gammaretroviruses. • Non-infectious mutants have hyper-glycosylated envelope that bind target cells. • Non-infectious mutants have defects in the formation of the double-stranded DNA. • Env cleavage motif has functions beyond cleavage of the env precursor.

  18. Crystal Structure of West Nile Virus Envelope Glycoprotein Reveals Viral Surface Epitopes

    SciTech Connect

    Kanai,R.; Kar, K.; Anthony, K.; Gould, L.; Ledizet, M.; Fikrig, E.; Marasco, W.; Koski, R.; Modis, Y.

    2006-01-01

    West Nile virus, a member of the Flavivirus genus, causes fever that can progress to life-threatening encephalitis. The major envelope glycoprotein, E, of these viruses mediates viral attachment and entry by membrane fusion. We have determined the crystal structure of a soluble fragment of West Nile virus E. The structure adopts the same overall fold as that of the E proteins from dengue and tick-borne encephalitis viruses. The conformation of domain II is different from that in other prefusion E structures, however, and resembles the conformation of domain II in postfusion E structures. The epitopes of neutralizing West Nile virus-specific antibodies map to a region of domain III that is exposed on the viral surface and has been implicated in receptor binding. In contrast, we show that certain recombinant therapeutic antibodies, which cross-neutralize West Nile and dengue viruses, bind a peptide from domain I that is exposed only during the membrane fusion transition. By revealing the details of the molecular landscape of the West Nile virus surface, our structure will assist the design of antiviral vaccines and therapeutics.

  19. Influences on Trimerization and Aggregation of Soluble, Cleaved HIV-1 SOSIP Envelope Glycoprotein

    PubMed Central

    Klasse, Per Johan; Depetris, Rafael S.; Pejchal, Robert; Julien, Jean-Philippe; Khayat, Reza; Lee, Jeong Hyun; Marozsan, Andre J.; Cupo, Albert; Cocco, Nicolette; Korzun, Jacob; Yasmeen, Anila; Ward, Andrew B.; Wilson, Ian A.

    2013-01-01

    We describe methods to improve the properties of soluble, cleaved gp140 trimers of the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins (Env) for use in structural studies and as immunogens. In the absence of nonionic detergents, gp140 of the KNH1144 genotype, terminating at residue 681 in gp41 (SOSIP.681), has a tendency to form higher-order complexes or aggregates, which is particularly undesirable for structure-based research. We found that this aggregation in the absence of detergent does not involve the V1, V2, or V3 variable regions of gp120. Moreover, we observed that detergent forms micelles around the membrane-proximal external region (MPER) of the SOSIP.681 gp140 trimers, whereas deletion of most of the MPER residues by terminating the gp140 at residue 664 (SOSIP.664) prevented the aggregation that otherwise occurs in SOSIP.681 in the absence of detergent. Although the MPER can contribute to trimer formation, truncation of most of it only modestly reduced trimerization and lacked global adverse effects on antigenicity. Thus, the MPER deletion minimally influenced the kinetics of the binding of soluble CD4 and a CD4-binding site antibody to immobilized trimers, as detected by surface plasmon resonance. Furthermore, the MPER deletion did not alter the overall three-dimensional structure of the trimers, as viewed by negative-stain electron microscopy. Homogeneous and aggregate-free MPER-truncated SOSIP Env trimers are therefore useful for immunogenicity and structural studies. PMID:23824824

  20. Bloch spin waves and emergent structure in protein folding with HIV envelope glycoprotein as an example.

    PubMed

    Dai, Jin; Niemi, Antti J; He, Jianfeng; Sieradzan, Adam; Ilieva, Nevena

    2016-03-01

    We inquire how structure emerges during the process of protein folding. For this we scrutinize collective many-atom motions during all-atom molecular dynamics simulations. We introduce, develop, and employ various topological techniques, in combination with analytic tools that we deduce from the concept of integrable models and structure of discrete nonlinear Schrödinger equation. The example we consider is an α-helical subunit of the HIV envelope glycoprotein gp41. The helical structure is stable when the subunit is part of the biological oligomer. But in isolation, the helix becomes unstable, and the monomer starts deforming. We follow the process computationally. We interpret the evolving structure both in terms of a backbone based Heisenberg spin chain and in terms of a side chain based XY spin chain. We find that in both cases the formation of protein supersecondary structure is akin the formation of a topological Bloch domain wall along a spin chain. During the process we identify three individual Bloch walls and we show that each of them can be modelled with a precision of tenths to several angstroms in terms of a soliton solution to a discrete nonlinear Schrödinger equation. PMID:27078392

  1. The membranotropic regions of the endo and ecto domains of HIV gp41 envelope glycoprotein.

    PubMed

    Moreno, Miguel R; Giudici, Marcela; Villalaín, José

    2006-01-01

    We have identified the membranotropic regions of the full sequence of the HIV gp41 envelope glycoprotein by performing an exhaustive study of membrane rupture, phospholipid-mixing and fusion induced by two 15-mer gp41-derived peptide libraries from HIV strains HIV_MN and HIV_consensus_B on model membranes having different phospholipid compositions. The data obtained for the two strains and its comparison have led us to identify different gp41 membranotropic segments in both ecto- and endodomains which might be implicated in viral membrane fusion and/or membrane interaction. The membranotropic segments corresponding to the gp41 ectodomain were the fusion domain, a stretch located on the N-heptad repeat region adjacent to the fusion domain, part of the immunodominant loop, the pre-transmembrane domain and the transmembrane domain. The membranotropic segments corresponding to the gp41 endodomain were mainly located at some specific parts of the previously described lentivirus lytic sequences. Significantly, the C-heptad repeat region and the Kennedy sequence located in the ectodomain and in the endodomain, respectively, presented no membranotropic activity in any model membrane assayed. The identification of these gp41 segments as well as their membranotropic propensity sustain the notion that different segments of gp41 provide the driving force for the merging of the viral and target cell membranes as well as they help us to define those segments as attractive targets for further development of new anti-viral compounds. PMID:16483537

  2. Bloch spin waves and emergent structure in protein folding with HIV envelope glycoprotein as an example

    NASA Astrophysics Data System (ADS)

    Dai, Jin; Niemi, Antti J.; He, Jianfeng; Sieradzan, Adam; Ilieva, Nevena

    2016-03-01

    We inquire how structure emerges during the process of protein folding. For this we scrutinize collective many-atom motions during all-atom molecular dynamics simulations. We introduce, develop, and employ various topological techniques, in combination with analytic tools that we deduce from the concept of integrable models and structure of discrete nonlinear Schrödinger equation. The example we consider is an α -helical subunit of the HIV envelope glycoprotein gp41. The helical structure is stable when the subunit is part of the biological oligomer. But in isolation, the helix becomes unstable, and the monomer starts deforming. We follow the process computationally. We interpret the evolving structure both in terms of a backbone based Heisenberg spin chain and in terms of a side chain based XY spin chain. We find that in both cases the formation of protein supersecondary structure is akin the formation of a topological Bloch domain wall along a spin chain. During the process we identify three individual Bloch walls and we show that each of them can be modelled with a precision of tenths to several angstroms in terms of a soliton solution to a discrete nonlinear Schrödinger equation.

  3. Engineering and Characterization of a Fluorescent Native-Like HIV-1 Envelope Glycoprotein Trimer

    PubMed Central

    Sliepen, Kwinten; van Montfort, Thijs; Ozorowski, Gabriel; Pritchard, Laura K.; Crispin, Max; Ward, Andrew B.; Sanders, Rogier W.

    2015-01-01

    Generation of a stable, soluble mimic of the HIV-1 envelope glycoprotein (Env) trimer on the virion surface has been considered an important first step for developing a successful HIV-1 vaccine. Recently, a soluble native-like Env trimer (BG505 SOSIP.664) has been described. This protein has facilitated major advances in the HIV-1 vaccine field, since it was the first Env immunogen that induced consistent neutralizing antibodies against a neutralization-resistant (tier 2) virus. Moreover, BG505 SOSIP.664 enabled elucidation of the atomic resolution structure of the Env trimer and facilitated the isolation and characterization of new broadly neutralizing antibodies against HIV-1. Here, we designed and characterized the BG505 SOSIP.664 trimer fused to fluorescent superfolder GFP (sfGFP), a GFP variant that allows efficient folding (BG505 SOSIP.664-sfGFP). Despite the presence of the sfGFP, the Env protein largely retained its morphology, antigenicity, glycan composition, and thermostability. In addition, we show that BG505 SOSIP.664-sfGFP can be used for fluorescence-based assays, such as flow cytometry. PMID:26512709

  4. A broad HIV-1 inhibitor blocks envelope glycoprotein transitions critical for entry

    PubMed Central

    Herschhorn, Alon; Gu, Christopher; Espy, Nicole; Richard, Jonathan; Finzi, Andrés; Sodroski, Joseph G.

    2014-01-01

    Binding to the primary receptor, CD4, triggers conformational changes in the metastable envelope glycoprotein (Env) trimer (gp1203/gp413) of human immunodeficiency virus (HIV-1) that are important for virus entry into host cells. These changes include an “opening” of the trimer, creation of a binding site for the CCR5 coreceptor, and formation/exposure of a gp41 coiled coil. Here we identify a new compound, 18A (1), that specifically inhibits the entry of a wide range of HIV-1 isolates. 18A does not interfere with CD4 or CCR5 binding, but inhibits the CD4-induced disruption of quaternary structures at the trimer apex and the formation/exposure of the gp41 HR1 coiled coil. Analysis of HIV-1 variants exhibiting increased or reduced sensitivity to 18A suggests that the inhibitor can distinguish distinct conformational states of gp120 in the unliganded Env trimer. The broad-range activity and observed hypersensitivity of resistant mutants to antibody neutralization support further investigation of 18A. PMID:25174000

  5. Crystal structure of west nile virus envelope glycoprotein reveals viral surface epitopes.

    PubMed

    Kanai, Ryuta; Kar, Kalipada; Anthony, Karen; Gould, L Hannah; Ledizet, Michel; Fikrig, Erol; Marasco, Wayne A; Koski, Raymond A; Modis, Yorgo

    2006-11-01

    West Nile virus, a member of the Flavivirus genus, causes fever that can progress to life-threatening encephalitis. The major envelope glycoprotein, E, of these viruses mediates viral attachment and entry by membrane fusion. We have determined the crystal structure of a soluble fragment of West Nile virus E. The structure adopts the same overall fold as that of the E proteins from dengue and tick-borne encephalitis viruses. The conformation of domain II is different from that in other prefusion E structures, however, and resembles the conformation of domain II in postfusion E structures. The epitopes of neutralizing West Nile virus-specific antibodies map to a region of domain III that is exposed on the viral surface and has been implicated in receptor binding. In contrast, we show that certain recombinant therapeutic antibodies, which cross-neutralize West Nile and dengue viruses, bind a peptide from domain I that is exposed only during the membrane fusion transition. By revealing the details of the molecular landscape of the West Nile virus surface, our structure will assist the design of antiviral vaccines and therapeutics.

  6. Two-dimensional polyacrylamide-gel electrophoresis of the proteins and glycoproteins of purified human platelet surface and intracellular membranes.

    PubMed

    Hack, N; Crawford, N

    1984-08-15

    By using highly purified surface and intracellular membrane fractions prepared from human platelets by free-flow electrophoresis, the polypeptide and glycopeptides of these membranes have been characterized by high-resolution gel electrophoresis under reducing and non-reducing conditions. Silver staining and a variety of glycoprotein-staining procedures have been applied to identify the major components. The principal finding was the clear disparity between the distribution patterns for these two membrane fractions. There are proportionately more low-Mr acidic components present in the intracellular membrane than in the surface-derived membrane. Of the major platelet surface glycoproteins GPIb, IIb, IIIa and IIIb (or IV) well expressed in the surface membrane only, GPIIb and IIIa appear as trace components in the intracellular membrane. The cytoskeleton proteins, actin, myosin, tropomyosin, actin-binding protein and alpha-actinin are prominent features of the surface membrane and essentially absent from the intracellular membrane. Neuraminidase treatment at the whole-cell level, before homogenization, which is an essential requirement for good resolution of the two membrane subfractions, modifies a number of the glycoprotein subunits with respect to their pI characteristics, suggesting much molecular micro-heterogeneity with respect to sialic acid content. A comparison of the staining characteristics of the major glycoproteins with periodic acid/Schiff's reagent and concanavalin A/peroxidase detection and a combined procedure revealed significant differences in associated carbohydrate structures, and the major concanavalin A-binding component was shown to be GPIIIa. These observations are discussed in the context of functional activities of both membrane systems in the physiological behaviour of the platelet.

  7. Importance of the short cytoplasmic domain of the feline immunodeficiency virus transmembrane glycoprotein for fusion activity and envelope glycoprotein incorporation into virions

    SciTech Connect

    Celma, Cristina C.P.; Paladino, Monica G.; Gonzalez, Silvia A.; Affranchino, Jose L.

    2007-09-30

    The mature form of the envelope (Env) glycoprotein of lentiviruses is a heterodimer composed of the surface (SU) and transmembrane (TM) subunits. Feline immunodeficiency virus (FIV) possesses a TM glycoprotein with a cytoplasmic tail of approximately 53 amino acids which is unusually short compared with that of the other lentiviral glycoproteins (more than 100 residues). To investigate the relevance of the FIV TM cytoplasmic domain to Env-mediated viral functions, we characterized the biological properties of a series of Env glycoproteins progressively shortened from the carboxyl terminus. All the mutant Env proteins were efficiently expressed in feline cells and processed into the SU and TM subunits. Deletion of 5 or 11 amino acids from the TM C-terminus did not significantly affect Env surface expression, fusogenic activity or Env incorporation into virions, whereas removal of 17 or 23 residues impaired Env-mediated cell-to-cell fusion. Further truncation of the FIV TM by 29 residues resulted in an Env glycoprotein that was poorly expressed at the cell surface, exhibited only 20% of the wild-type Env fusogenic capacity and was inefficiently incorporated into virions. Remarkably, deletion of the TM C-terminal 35 or 41 amino acids restored or even enhanced Env biological functions. Indeed, these mutant Env glycoproteins bearing cytoplasmic domains of 18 or 12 amino acids were found to be significantly more fusogenic than the wild-type Env and were efficiently incorporated into virions. Interestingly, truncation of the TM cytoplasmic domain to only 6 amino acids did not affect Env incorporation into virions but abrogated Env fusogenicity. Finally, removal of the entire TM cytoplasmic tail or deletion of as many as 6 amino acids into the membrane-spanning domain led to a complete loss of Env functions. Our results demonstrate that despite its relatively short length, the FIV TM cytoplasmic domain plays an important role in modulating Env-mediated viral functions.

  8. Detection of Receptor-Induced Glycoprotein Conformational Changes on Enveloped Virions by Using Confocal Micro-Raman Spectroscopy

    PubMed Central

    Lu, Xiaonan; Liu, Qian; Benavides-Montano, Javier A.; Nicola, Anthony V.; Aston, D. Eric; Rasco, Barbara A.

    2013-01-01

    Conformational changes in the glycoproteins of enveloped viruses are critical for membrane fusion, which enables viral entry into cells and the pathological cell-cell fusion (syncytia) associated with some viral infections. However, technological capabilities for identifying viral glycoproteins and their conformational changes on actual enveloped virus surfaces are generally scarce, challenging, and time-consuming. Our model, Nipah virus (NiV), is a syncytium-forming biosafety level 4 pathogen with a high mortality rate (40 to 75%) in humans. Once the NiV attachment glycoprotein (G) (NiV-G) binds the cell receptor ephrinB2 or -B3, G triggers conformational changes in the fusion glycoprotein (F) that result in membrane fusion and viral entry. We demonstrate that confocal micro-Raman spectroscopy can, within minutes, simultaneously identify specific G and F glycoprotein signals and receptor-induced conformational changes in NiV-F on NiV virus-like particles (VLPs). First, we identified reproducible G- and F-specific Raman spectral features on NiV VLPs containing M (assembly matrix protein), G, and/or F or on NiV/vesicular stomatitis virus (VSV) pseudotyped virions via second-derivative transformations and principal component analysis (PCA). Statistical analyses validated our PCA models. Dynamic temperature-induced conformational changes in F and G or receptor-induced target membrane-dependent conformational changes in F were monitored in NiV pseudovirions in situ in real time by confocal micro-Raman spectroscopy. Advantageously, Raman spectroscopy can identify specific protein signals in relatively impure samples. Thus, this proof-of-principle technological development has implications for the rapid identification and biostability characterization of viruses in medical, veterinary, and food samples and for the analysis of virion glycoprotein conformational changes in situ during viral entry. PMID:23283947

  9. Apolipoprotein E Likely Contributes to a Maturation Step of Infectious Hepatitis C Virus Particles and Interacts with Viral Envelope Glycoproteins

    PubMed Central

    Lee, Ji-Young; Acosta, Eliana G.; Stoeck, Ina Karen; Long, Gang; Hiet, Marie-Sophie; Mueller, Birthe; Fackler, Oliver T.; Kallis, Stephanie

    2014-01-01

    ABSTRACT The assembly of infectious hepatitis C virus (HCV) particles is tightly linked to components of the very-low-density lipoprotein (VLDL) pathway. We and others have shown that apolipoprotein E (ApoE) plays a major role in production of infectious HCV particles. However, the mechanism by which ApoE contributes to virion assembly/release and how it gets associated with the HCV particle is poorly understood. We found that knockdown of ApoE reduces titers of infectious intra- and extracellular HCV but not of the related dengue virus. ApoE depletion also reduced amounts of extracellular HCV core protein without affecting intracellular core amounts. Moreover, we found that ApoE depletion affected neither formation of nucleocapsids nor their envelopment, suggesting that ApoE acts at a late step of assembly, such as particle maturation and infectivity. Importantly, we demonstrate that ApoE interacts with the HCV envelope glycoproteins, most notably E2. This interaction did not require any other viral proteins and depended on the transmembrane domain of E2 that also was required for recruitment of HCV envelope glycoproteins to detergent-resistant membrane fractions. These results suggest that ApoE plays an important role in HCV particle maturation, presumably by direct interaction with viral envelope glycoproteins. IMPORTANCE The HCV replication cycle is tightly linked to host cell lipid pathways and components. This is best illustrated by the dependency of HCV assembly on lipid droplets and the VLDL component ApoE. Although the role of ApoE for production of infectious HCV particles is well established, it is still poorly understood how ApoE contributes to virion formation and how it gets associated with HCV particles. Here, we provide experimental evidence that ApoE likely is required for an intracellular maturation step of HCV particles. Moreover, we demonstrate that ApoE associates with the viral envelope glycoproteins. This interaction appears to be dispensable

  10. Affinity-purified antibodies of defined specificity for use in a solid-phase microplate radioimmunoassay of human Tamm-Horsfall glycoprotein in urine.

    PubMed

    Hunt, J S; McGiven, A R; Groufsky, A; Lynn, K L; Taylor, M C

    1985-05-01

    Rabbit antibodies to human Tamm-Horsfall glycoprotein (prepared by salt precipitation from normal urine) were purified by affinity chromatography using columns containing Tamm-Horsfall glycoprotein linked to CNBr-activated Sepharose 4B. The specificity of these antibodies was determined by analysis of their binding characteristics on Western blots of Tamm-Horsfall protein from sodium dodecyl sulphate/polyacrylamide gradient gels and comparison with the reactivity of monoclonal antibodies to this glycoprotein. Optimal conditions of adsorption to poly(vinyl chloride) microtitre plates were established such that these purified antibodies could be used in a solid-phase radioimmunoassay for the determination of urinary Tamm-Horsfall-glycoprotein concentration. The specificity of the immunoassay was confirmed by competitive inhibition of the urinary Tamm-Horsfall glycoprotein by purified freeze-dried material in solution. A standard curve obtained with this material showed the radioimmunoassay to have a sensitivity of at least 5 ng/ml, with linearity between 30 and 600 ng/ml. The mean coefficient of variation over the linear section of the curve was 11.3 +/- 2.2% (n = 13). The effects of dialysis and freezing of urine samples before determination of Tamm-Horsfall-glycoprotein concentrations were investigated and the mean 24 h urinary excretion rate in 60 normal donors was shown to be 84.9 +/- 44.1 mg.

  11. Genetic signatures in the envelope glycoproteins of HIV-1 that associate with broadly neutralizing antibodies.

    PubMed

    Gnanakaran, S; Daniels, Marcus G; Bhattacharya, Tanmoy; Lapedes, Alan S; Sethi, Anurag; Li, Ming; Tang, Haili; Greene, Kelli; Gao, Hongmei; Haynes, Barton F; Cohen, Myron S; Shaw, George M; Seaman, Michael S; Kumar, Amit; Gao, Feng; Montefiori, David C; Korber, Bette

    2010-01-01

    A steady increase in knowledge of the molecular and antigenic structure of the gp120 and gp41 HIV-1 envelope glycoproteins (Env) is yielding important new insights for vaccine design, but it has been difficult to translate this information to an immunogen that elicits broadly neutralizing antibodies. To help bridge this gap, we used phylogenetically corrected statistical methods to identify amino acid signature patterns in Envs derived from people who have made potently neutralizing antibodies, with the hypothesis that these Envs may share common features that would be useful for incorporation in a vaccine immunogen. Before attempting this, essentially as a control, we explored the utility of our computational methods for defining signatures of complex neutralization phenotypes by analyzing Env sequences from 251 clonal viruses that were differentially sensitive to neutralization by the well-characterized gp120-specific monoclonal antibody, b12. We identified ten b12-neutralization signatures, including seven either in the b12-binding surface of gp120 or in the V2 region of gp120 that have been previously shown to impact b12 sensitivity. A simple algorithm based on the b12 signature pattern was predictive of b12 sensitivity/resistance in an additional blinded panel of 57 viruses. Upon obtaining these reassuring outcomes, we went on to apply these same computational methods to define signature patterns in Env from HIV-1 infected individuals who had potent, broadly neutralizing responses. We analyzed a checkerboard-style neutralization dataset with sera from 69 HIV-1-infected individuals tested against a panel of 25 different Envs. Distinct clusters of sera with high and low neutralization potencies were identified. Six signature positions in Env sequences obtained from the 69 samples were found to be strongly associated with either the high or low potency responses. Five sites were in the CD4-induced coreceptor binding site of gp120, suggesting an important role for

  12. Genomic and antigenic variations of porcine reproductive and respiratory syndrome virus major envelope GP5 glycoprotein.

    PubMed Central

    Pirzadeh, B; Gagnon, C A; Dea, S

    1998-01-01

    The objective of the present study was to evaluate the importance of genomic and antigenic variations which may have affected the major envelope glycoprotein GP5 of porcine reproductive and respiratory syndrome virus (PRRSV) isolates responsible for outbreaks in Quebec and Ontario, in comparison with the modified-live U.S. vaccine strain (MLV) and the European prototype strain from Lelystad (LV). Nucleotide sequence analyses of the open reading frame (ORF)5 genes showed that all of the isolates studied were heterogenous, amino acid (aa) identities varied from 88 to 99% with the MLV strain, and between 51 and 54% with the LV strain. The aa substitutions were randomly scattered across the protein, although one region between residues 26 and 39 was found to correspond to a hypervariable region which involved 0 to 3 potential N-glycosylation sites. The ORF5 encoded products of 5 of these isolates, including the MLV and LV strains, were expressed in E. coli as recombinant proteins fused to the glutathione S-transferase (GST) protein and used to raise hyperimmune anti-ORF5 sera in rabbits. The reactivity patterns of strain-specific hyperimmune anti-ORF5 sera and a panel of 4 monoclonal antibodies directed against the ORF5 gene product of the Quebec IAF-Klop strain of PRRSV, indicated that GP5 of field isolates also underwent antigenic variations. The data suggest that neutralizing epitopes, independent of conformation and glycosylation, are also associated with antigenic variability of the GP5 of PRRSV. PMID:9684045

  13. Development and Characterization of Rabbit and Mouse Antibodies Against Ebolavirus Envelope Glycoproteins

    PubMed Central

    Ou, Wu; Delisle, Josie; Konduru, Krishnamurthy; Bradfute, Steven; Radoshitzky, Sheli R.; Retterer, Cary; Kota, Krishna; Bavari, Sina; Kuhn, Jens H.; Jahrling, Peter B.; Kaplan, Gerardo; Wilson, Carolyn A.

    2011-01-01

    Ebolaviruses are the etiologic agents of severe viral hemorrhagic fevers in primates, including humans, and could be misused for the development of biological weapons. The ability to rapidly detect and differentiate these viruses is therefore crucial. Antibodies that can detect reliably the ebolavirus surface envelope glycoprotein GP1,2 or a truncated variant that is secreted from infected cells (sGP) are required for advanced development of diagnostic assays such as sandwich ELISAs or Western blots (WB). We used a GP1,2 peptide conserved among Bundibugyo, Ebola, Reston, Sudan, and Taï Forest viruses and a mucin-like domain-deleted Sudan virus GP1,2 (SudanGPΔMuc) to immunize mice or rabbits, and developed a panel of antibodies that either cross-react or are virus-specific. These antibodies detected full-length GP1,2 and sGP in different assays such as ELISA, FACS, or WB. In addition, some of the antibodies were shown to have potential clinical relevance, as they detected ebolavirus-infected cells by immunofluorescence assay and gave a specific increase in signal by sandwich ELISA against sera from mouse-adapted Ebola virus-infected mice over uninfected mouse sera. Rabbit anti-SudanGPΔMuc polyclonal antibody neutralized gammaretroviral particles pseudotyped with Sudan virus GP1,2, but not particles pseudotyped with other ebolavirus GP1,2. Together, our results suggest that this panel of antibodies may prove useful for both in vitro analyses of ebolavirus GP1,2, as well as analysis of clinically relevant samples. PMID:21513741

  14. The use of aqueous two-phase systems to concentrate and purify bovine leukemia virus outer envelope protein gp51.

    PubMed

    Hammar, L; Merza, M; Malm, K; Eriksson, S; Morein, B

    1989-06-01

    Enzootic bovine leucosis is a chronic lymphoproliferative disease of cattle. The causative agent, bovine leukemia virus (BLV), is related to the human retroviruses HTLV-I and -II. The external env-protein of BLV, a glycoprotein of 51 kDa, carries neutralizing epitopes and should be an essential component in a vaccine against the virus. Problems have been encountered with the concentration and purification of intact virions of BLV and other retroviruses. During centrifugation procedures the external env-proteins are to a great extent detached and consequently poorly recovered with the virion particles. Therefore, other methods are sought to obtain a high yield of the external glycoproteins. The use of two-phase systems based on water soluble polymers is described for the extraction of BLV-gp51 from culture medium. Several polymer systems were tested and the results showed that some were attractive for large scale application. The classical combination dextran-polyethylene glycol gave promising results; a partition coefficient of about 0.02 was obtained for the distribution of the gp51 between the top and combined inter- and bottom phases. In a single extraction step it was possible to obtain 45% of the glycoprotein in a small volume bottom phase and at the same time about 15-fold purified. That should be compared with a recovery of less than 20% with the conventional centrifugation procedures. It is concluded that extraction in phase systems based on water soluble polymers is a methodology well suited for the concentration and purification of BLV-gp51. PMID:2474306

  15. Proteomics computational analyses suggest that hepatitis C virus E1 and pestivirus E2 envelope glycoproteins are truncated class II fusion proteins.

    PubMed

    Garry, Robert F; Dash, Srikanta

    2003-03-15

    Class II fusion proteins encoded by tick-borne encephalitis virus (TBEV), dengue virus, and Semliki Forest virus have a fusion peptide located at the end of a rod-like molecule comprised of three antiparallel beta sheet domains. Proteomics computational analyses suggest that hepatitis C virus (HCV) envelope glycoprotein E1 and pestivirus envelope glycoprotein E2 are truncated class II fusion proteins. Similarities were also detected between the receptor-binding portion of TBEV E and HCV E2, and between TBEV small membrane protein precursor prM and pestivirus E1. The proposed models of Flaviviridae envelope proteins can facilitate drug and vaccine development.

  16. Comparative evaluation of trimeric envelope glycoproteins derived from subtype C and B HIV-1 R5 isolates.

    PubMed

    Srivastava, Indresh K; Kan, Elaine; Sun, Yide; Sharma, Victoria A; Cisto, Jimna; Burke, Brian; Lian, Ying; Hilt, Susan; Biron, Zohar; Hartog, Karin; Stamatatos, Leonidas; Diaz-Avalos, Ruben; Cheng, R Holland; Ulmer, Jeffrey B; Barnett, Susan W

    2008-03-15

    We previously reported that an envelope (Env) glycoprotein immunogen (o-gp140DeltaV2SF162) containing a partial deletion in the second variable loop (V2) derived from the R5-tropic HIV-1 isolate SF162 partially protected vaccinated rhesus macaques against pathogenic SHIV(SF162P4) virus. Extending our studies to subtype C isolate TV1, we have purified o-gp140DeltaV2TV1 (subtype C DeltaV2 trimer) to homogeneity, performed glycosylation analysis, and determined its ability to bind CD4, as well as a panel of well-characterized neutralizing monoclonal antibodies (mAb). In general, critical epitopes are preserved on the subtype C DeltaV2 trimer; however, we did not observe significant binding for the b12 mAb. The molecular mass of subtype C DeltaV2 trimer was found to be 450 kDa, and the hydrodynamic radius was found to be 10.87 nm. Our data suggest that subtype C DeltaV2 trimer binds to CD4 with an affinity comparable to o-gp140DeltaV2SF162 (subtype B DeltaV2 trimer). Using isothermal titration calorimetric (ITC) analysis, we demonstrated that all three CD4 binding sites (CD4-BS) in both subtype C and B trimers are exposed and accessible. However, compared to subtype B trimer, the three CD4-BS in subtype C trimer have different affinities for CD4, suggesting a cooperativity of CD4 binding in subtype C trimer but not in subtype B trimer. Negative staining electron microscopy of the subtype C DeltaV2 trimer has demonstrated that it is in fact a trimer. These results highlight the importance of studying subtype C Env, and also of developing appropriate subtype C-specific reagents that may be used for better immunological characterization of subtype C Env for developing an AIDS vaccine.

  17. Comparative evaluation of trimeric envelope glycoproteins derived from subtype C and B HIV-1 R5 isolates

    SciTech Connect

    Srivastava, Indresh K. Kan, Elaine; Sun Yide; Sharma, Victoria A.; Cisto, Jimna; Burke, Brian; Lian Ying; Hilt, Susan; Biron, Zohar; Hartog, Karin; Stamatatos, Leonidas; Cheng, R. Holland; Ulmer, Jeffrey B.; Barnett, Susan W.

    2008-03-15

    We previously reported that an envelope (Env) glycoprotein immunogen (o-gp140{delta}V2SF162) containing a partial deletion in the second variable loop (V2) derived from the R5-tropic HIV-1 isolate SF162 partially protected vaccinated rhesus macaques against pathogenic SHIV{sub SF162P4} virus. Extending our studies to subtype C isolate TV1, we have purified o-gp140{delta}V2TV1 (subtype C {delta}V2 trimer) to homogeneity, performed glycosylation analysis, and determined its ability to bind CD4, as well as a panel of well-characterized neutralizing monoclonal antibodies (mAb). In general, critical epitopes are preserved on the subtype C {delta}V2 trimer; however, we did not observe significant binding for the b12 mAb. The molecular mass of subtype C {delta}V2 trimer was found to be 450 kDa, and the hydrodynamic radius was found to be 10.87 nm. Our data suggest that subtype C {delta}V2 trimer binds to CD4 with an affinity comparable to o-gp140{delta}V2SF162 (subtype B {delta}V2 trimer). Using isothermal titration calorimetric (ITC) analysis, we demonstrated that all three CD4 binding sites (CD4-BS) in both subtype C and B trimers are exposed and accessible. However, compared to subtype B trimer, the three CD4-BS in subtype C trimer have different affinities for CD4, suggesting a cooperativity of CD4 binding in subtype C trimer but not in subtype B trimer. Negative staining electron microscopy of the subtype C {delta}V2 trimer has demonstrated that it is in fact a trimer. These results highlight the importance of studying subtype C Env, and also of developing appropriate subtype C-specific reagents that may be used for better immunological characterization of subtype C Env for developing an AIDS vaccine.

  18. Genetic Diversity Underlying the Envelope Glycoproteins of Hepatitis C Virus: Structural and Functional Consequences and the Implications for Vaccine Design

    PubMed Central

    Tarr, Alexander W.; Khera, Tanvi; Hueging, Kathrin; Sheldon, Julie; Steinmann, Eike; Pietschmann, Thomas; Brown, Richard J. P.

    2015-01-01

    In the 26 years since the discovery of Hepatitis C virus (HCV) a major global research effort has illuminated many aspects of the viral life cycle, facilitating the development of targeted antivirals. Recently, effective direct-acting antiviral (DAA) regimens with >90% cure rates have become available for treatment of chronic HCV infection in developed nations, representing a significant advance towards global eradication. However, the high cost of these treatments results in highly restricted access in developing nations, where the disease burden is greatest. Additionally, the largely asymptomatic nature of infection facilitates continued transmission in at risk groups and resource constrained settings due to limited surveillance. Consequently a prophylactic vaccine is much needed. The HCV envelope glycoproteins E1 and E2 are located on the surface of viral lipid envelope, facilitate viral entry and are the targets for host immunity, in addition to other functions. Unfortunately, the extreme global genetic and antigenic diversity exhibited by the HCV glycoproteins represents a significant obstacle to vaccine development. Here we review current knowledge of HCV envelope protein structure, integrating knowledge of genetic, antigenic and functional diversity to inform rational immunogen design. PMID:26193307

  19. Genetic Diversity Underlying the Envelope Glycoproteins of Hepatitis C Virus: Structural and Functional Consequences and the Implications for Vaccine Design.

    PubMed

    Tarr, Alexander W; Khera, Tanvi; Hueging, Kathrin; Sheldon, Julie; Steinmann, Eike; Pietschmann, Thomas; Brown, Richard J P

    2015-07-01

    In the 26 years since the discovery of Hepatitis C virus (HCV) a major global research effort has illuminated many aspects of the viral life cycle, facilitating the development of targeted antivirals. Recently, effective direct-acting antiviral (DAA) regimens with >90% cure rates have become available for treatment of chronic HCV infection in developed nations, representing a significant advance towards global eradication. However, the high cost of these treatments results in highly restricted access in developing nations, where the disease burden is greatest. Additionally, the largely asymptomatic nature of infection facilitates continued transmission in at risk groups and resource constrained settings due to limited surveillance. Consequently a prophylactic vaccine is much needed. The HCV envelope glycoproteins E1 and E2 are located on the surface of viral lipid envelope, facilitate viral entry and are the targets for host immunity, in addition to other functions. Unfortunately, the extreme global genetic and antigenic diversity exhibited by the HCV glycoproteins represents a significant obstacle to vaccine development. Here we review current knowledge of HCV envelope protein structure, integrating knowledge of genetic, antigenic and functional diversity to inform rational immunogen design. PMID:26193307

  20. Mass spectrometric characterization of glycosylation of Hepatitis C virus E2 envelope glycoprotein reveals extended microheterogeneity of N-glycans

    PubMed Central

    Iacob, Roxana E.; Perdivara, Irina; Przybylski, Michael; Tomer, Kenneth B.

    2008-01-01

    Hepatitis C virus (HCV) causes acute and chronic liver disease in humans, including chronic hepatitis, cirrhosis, and hepatocellular carcinoma. The polyprotein encoded in the HCV genome is co- and posttranslationally processed by host and viral peptidases, generating the structural proteins Core, E1, E2 and p7, and five non-structural proteins. The two envelope proteins E1 and E2 are heavily glycosylated. Studying the glycan moieties attached to the envelope E2 glycoprotein is important because the N-linked glycans on E2 envelope protein are involved in the interaction with some human neutralizing antibodies, and may also have a direct or indirect effect on protein folding. In the present study we report the mass spectrometric characterization of the glycan moieties attached to the E2 glycoprotein. The mass spectrometric analysis clearly identified the nature, composition and microheterogeneity of the sugars attached to the E2 glycopeptides. All 11 sites of glycosylation on E2 protein were characterized, and the majority of these sites proved to be occupied by high mannose glycans. However, complex type oligosaccharides, which have not been previously identified, were exclusively observed at two N-linked sites and their identity and heterogeneity were determined. PMID:18187336

  1. Influences on the Design and Purification of Soluble, Recombinant Native-Like HIV-1 Envelope Glycoprotein Trimers

    PubMed Central

    Ringe, Rajesh P.; Yasmeen, Anila; Ozorowski, Gabriel; Go, Eden P.; Pritchard, Laura K.; Guttman, Miklos; Ketas, Thomas A.; Cottrell, Christopher A.; Wilson, Ian A.; Sanders, Rogier W.; Cupo, Albert; Crispin, Max; Lee, Kelly K.; Desaire, Heather; Ward, Andrew B.; Klasse, P. J.

    2015-01-01

    . IMPORTANCE Soluble, recombinant multimeric proteins based on the HIV-1 env gene are current candidate immunogens for vaccine trials in humans. These proteins are generally designed to mimic the native trimeric envelope glycoprotein (Env) that is the target of virus-neutralizing antibodies on the surfaces of virions. The underlying hypothesis is that an Env-mimetic protein may be able to induce antibodies that can neutralize the virus broadly and potently enough for a vaccine to be protective. Multiple different designs for Env-mimetic trimers have been put forth. Here, we used the CZA97.012 and 92UG037.8 env genes to compare some of these designs and determine which ones best mimic virus-associated Env trimers. We conclude that the most widely used versions of CZA97.012 and 92UG037.8 oligomeric Env proteins do not resemble the trimeric Env glycoprotein on HIV-1 viruses, which has implications for the design and interpretation of ongoing or proposed clinical trials of these proteins. PMID:26311893

  2. Naturally Occurring Variability in the Envelope Glycoprotein of HIV-1 and the Development of Cell Entry Inhibitors

    PubMed Central

    Brower, Evan T.; Schön, Arne; Freire, Ernesto

    2010-01-01

    Naturally occurring genetic variability across HIV-1 subtypes causes amino acid polymorphisms in encoded HIV-1 proteins including the envelope glycoproteins associated with viral entry. The effects of amino acid polymorphisms on the mechanism of HIV-1 entry into cells, a process initiated by the binding of the viral envelope glycoprotein gp120 to the cellular CD4 receptor, are largely unknown. In this study, we demonstrate that amino acid polymorphisms affect the structural stability and domain cooperativity of gp120 and that those differences are reflected in the binding mechanism of the viral envelope glycoprotein to the cell surface receptor and coreceptor. Moreover, subtype differences also affect the binding behavior of experimental HIV cell entry inhibitors. While gp120-A has a slightly lower denaturation temperature than gp120-B, the most notable stability difference is that for gp120-B the van't Hoff to calorimetric enthalpy ratio (ΔHvH/ΔH) is 0.95 whereas for gp120-A is 0.6, indicative of more cooperative domain/domain interactions in gp120-B, as this protein more closely approaches a two-state transition. Isothermal titration calorimetry demonstrates that CD4 and 17b (a surrogate antibody for the chemokine coreceptor) exhibit 7 and 3-fold weaker binding affinities for gp120-A. The binding of these proteins as well as that of the experimental entry inhibitor NBD-556 induce smaller conformational changes in gp120-A as evidenced by significantly smaller binding enthalpies and binding entropies. Together, these results describe the effects of gp120 polymorphisms on binding to host cell receptors and emphasize that guidelines for developing future entry inhibitors must recognize and deal with genomic differences between HIV strains. PMID:20166763

  3. Activation of Cannabinoid Type 2 Receptors Inhibits HIV-1 Envelope Glycoprotein gp120-Induced Synapse Loss

    PubMed Central

    Kim, Hee Jung; Shin, Angela H.

    2011-01-01

    HIV-1 infection of the central nervous system is associated with dendritic and synaptic damage that correlates with cognitive decline in patients with HIV-1-associated dementia (HAD). HAD is due in part to the release of viral proteins from infected cells. Because cannabinoids modulate neurotoxic and inflammatory processes, we investigated their effects on changes in synaptic connections induced by the HIV-1 envelope glycoprotein gp120. Morphology and synapses between cultured hippocampal neurons were visualized by confocal imaging of neurons expressing DsRed2 and postsynaptic density protein 95 fused to green fluorescent protein (PSD95-GFP). Twenty-four-hour treatment with gp120 IIIB decreased the number of PSD95-GFP puncta by 37 ± 4%. The decrease was concentration-dependent (EC50 = 153 ± 50 pM). Synapse loss preceded cell death as defined by retention of DsRed2 fluorescence gp120 activated CXCR4 on microglia to evoke interleukin-1β (IL-1β) release. Pharmacological studies determined that sequential activation of CXCR4, the IL-1β receptor, and the N-methyl-d-aspartate receptor was required. Expression of alternative reading frame polypeptide, which inhibits the ubiquitin ligase murine double minute 2, protected synapses, implicating the ubiquitin-proteasome pathway. Cannabimimetic drugs are of particular relevance to HAD because of their clinical and illicit use in patients with AIDS. The cannabinoid receptor full agonist [(R)-(+)-[2,3-dihydro-5-methyl-3[(4-morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazinyl]-(1-naphthalenyl) methanone mesylate salt] (Win55,212-2) inhibited gp120-induced IL-1β production and synapse in a manner reversed by a cannabinoid type 2 receptor antagonist. In contrast, Win55,212-2 did not inhibit synapse loss elicited by exposure to the HIV-1 protein Tat. These results indicate that cannabinoids prevent the impairment of network function produced by gp120 and, thus, might have therapeutic potential in HAD. PMID:21670103

  4. An alternative conformation of the gp41 heptad repeat 1 region coiled coil exists in the human immunodeficiency virus (HIV-1) envelope glycoprotein precursor

    SciTech Connect

    Mische, Claudia C.; Yuan Wen; Strack, Bettina; Craig, Stewart; Farzan, Michael; Sodroski, Joseph . E-mail: joseph_sodroski@dfci.harvard.edu

    2005-07-20

    The human immunodeficiency virus (HIV-1) transmembrane envelope glycoprotein, gp41, which mediates virus-cell fusion, exists in at least three different conformations within the trimeric envelope glycoprotein complex. The structures of the prefusogenic and intermediate states are unknown; structures representing the postfusion state have been solved. In the postfusion conformation, three helical heptad repeat 2 (HR2) regions pack in an antiparallel fashion into the hydrophobic grooves on the surface of a triple-helical coiled coil formed by the heptad repeat 1 (HR1) regions. We studied the prefusogenic conformation of gp41 by mutagenic alteration of membrane-anchored and soluble forms of the HIV-1 envelope glycoproteins. Our results indicate that, in the HIV-1 envelope glycoprotein precursor, the gp41 HR1 region is in a conformation distinct from that of a trimeric coiled coil. Thus, the central gp41 coiled coil is formed during the transition of the HIV-1 envelope glycoproteins from the precursor state to the receptor-bound intermediate.

  5. Truncation of the cytoplasmic domain of the simian immunodeficiency virus envelope glycoprotein increases env incorporation into particles and fusogenicity and infectivity.

    PubMed Central

    Zingler, K; Littman, D R

    1993-01-01

    Growth of macaque simian immunodeficiency virus (SIVmac) in certain cloned human T-cell lines, such as HUT.78, selects for isolates containing a premature stop codon within the cytoplasmic domain of the transmembrane envelope glycoprotein. In contrast, propagation of virus in macaques or in their cultured T cells favors replication of virus containing the full-length envelope glycoprotein. To elucidate the causes of this phenomenon, we used a human immunodeficiency virus pseudotyping system to assess the effects on infectivity of the cytoplasmic domains of envelope glycoproteins obtained from SIVmac1A11 and SIVmac239. These envelopes contain truncated and full-length cytoplasmic domains, respectively. By analyzing human immunodeficiency virus particles containing selectable genes pseudotyped with each glycoprotein or with chimeric derivatives, we found that truncation of the cytoplasmic domain resulted in a significant advantage in viral entry into HUT.78 T cells and CD4+ U87.MG glial cells. Truncation of the cytoplasmic domain significantly enhanced both envelope density on particles and envelope-mediated cell-to-cell fusion. It is likely that one or both of these effects contribute to the observed differences in infectivity and to the selection of virions with short cytoplasmic tails in human T cells. Images PMID:8474176

  6. Structure of Hepatitis C Virus Envelope Glycoprotein E1 Antigenic Site 314-324 in Complex with Antibody IGH526.

    PubMed

    Kong, Leopold; Kadam, Rameshwar U; Giang, Erick; Ruwona, Tinashe B; Nieusma, Travis; Culhane, Jeffrey C; Stanfield, Robyn L; Dawson, Philip E; Wilson, Ian A; Law, Mansun

    2015-08-14

    Hepatitis C virus (HCV) is a positive-strand RNA virus within the Flaviviridae family. The viral "spike" of HCV is formed by two envelope glycoproteins, E1 and E2, which together mediate viral entry by engaging host receptors and undergoing conformational changes to facilitate membrane fusion. While E2 can be readily produced in the absence of E1, E1 cannot be expressed without E2 and few reagents, including monoclonal antibodies (mAbs), are available for study of this essential HCV glycoprotein. A human mAb to E1, IGH526, was previously reported to cross-neutralize different HCV isolates, and therefore, we sought to further characterize the IGH526 neutralizing epitope to obtain information for vaccine design. We found that mAb IGH526 bound to a discontinuous epitope, but with a major component corresponding to E1 residues 314-324. The crystal structure of IGH526 Fab with this E1 glycopeptide at 1.75Å resolution revealed that the antibody binds to one face of an α-helical peptide. Single mutations on the helix substantially lowered IGH526 binding but did not affect neutralization, indicating either that multiple mutations are required or that additional regions are recognized by the antibody in the context of the membrane-associated envelope oligomer. Molecular dynamics simulations indicate that the free peptide is flexible in solution, suggesting that it requires stabilization for use as a candidate vaccine immunogen. PMID:26135247

  7. Feline immunodeficiency virus envelope glycoprotein mediates apoptosis in activated PBMC by a mechanism dependent on gp41 function

    SciTech Connect

    Garg, Himanshu; Joshi, Anjali; Tompkins, Wayne A. . E-mail: Wayne_Tompkins@ncsu.edu

    2004-12-20

    Feline Immunodeficiency Virus (FIV) is a lentivirus that causes immunodeficiency in cats, which parallels HIV-1-induced immunodeficiency in humans. It has been established that HIV envelope (Env) glycoprotein mediates T cell loss via a mechanism that requires CXCR4 binding. The Env glycoprotein of FIV, similar to HIV, requires CXCR4 binding for viral entry, as well as inducing membrane fusion leading to syncytia formation. However, the role of FIV Env in T cell loss and the molecular mechanisms governing this process have not been elucidated. We studied the role of Env glycoprotein in FIV-mediated T cell apoptosis in an in vitro model. Our studies demonstrate that membrane-expressed FIV Env induces apoptosis in activated feline peripheral blood mononuclear cells (PBMC) by a mechanism that requires CXCR4 binding, as the process was inhibited by CXCR4 antagonist AMD3100 in a dose-dependent manner. Interestingly, studies regarding the role of CD134, the recently identified primary receptor of FIV, suggest that binding to CD134 may not be important for induction of apoptosis in PBMC. However, inhibiting Env-mediated fusion post CXCR4 binding by FIV gp41-specific fusion inhibitor also inhibited apoptosis. Under similar conditions, a fusion-defective gp41 mutant was unable to induce apoptosis in activated PBMC. Our findings are the first report suggesting the potential of FIV Env to mediate apoptosis in bystander cells by a process that is dependent on gp41 function.

  8. Genetic analysis of heptad-repeat regions in the G2 fusion subunit of the Junin arenavirus envelope glycoprotein

    SciTech Connect

    York, Joanne . E-mail: joanne.york@umontana.edu; Agnihothram, Sudhakar S. . E-mail: sudhakar.agnihothram@umontana.edu; Romanowski, Victor . E-mail: victor@biol.unlp.edu.ar; Nunberg, Jack H. . E-mail: jack.nunberg@umontana.edu

    2005-12-20

    The G2 fusion subunit of the Junin virus envelope glycoprotein GP-C contains two hydrophobic heptad-repeat regions that are postulated to form a six-helix bundle structure required for the membrane fusion activity of Class I viral fusion proteins. We have investigated the role of these heptad-repeat regions and, specifically, the importance of the putative interhelical a and d position sidechains by using alanine-scanning mutagenesis. All the mutant glycoproteins were expressed and transported to the cell surface. Proteolytic maturation at the subtilisin kexin isozyme-1/site-1-protease (SKI-1/S1P) cleavage site was observed in all but two of the mutants. Among the adequately cleaved mutant glycoproteins, four positions in the N-terminal region (I333, L336, L347 and L350) and two positions in the C-terminal region (R392 and W395) were shown to be important determinants of cell-cell fusion. Taken together, our results indicate that {alpha}-helical coiled-coil structures are likely critical in promoting arenavirus membrane fusion. These findings support the inclusion of the arenavirus GP-C among the Class I viral fusion proteins and suggest pharmacologic and immunologic strategies for targeting arenavirus infection and hemorrhagic fever.

  9. Effect of trimerization motifs on quaternary structure, antigenicity, and immunogenicity of a noncleavable HIV-1 gp140 envelope glycoprotein

    SciTech Connect

    Du, Sean X.; Idiart, Rebecca J.; Mariano, Ellaine B.; Chen, Helen; Jiang Peifeng; Xu Li; Ostrow, Kristin M.; Wrin, Terri; Phung, Pham; Binley, James M.; Petropoulos, Christos J.; Ballantyne, John A.; Whalen, Robert G.

    2009-12-05

    The external domains of the HIV-1 envelope glycoprotein (gp120 and the gp41 ectodomain, collectively known as gp140) contain all known viral neutralization epitopes. Various strategies have been used to create soluble trimers of the envelope to mimic the structure of the native viral protein, including mutation of the gp120-gp41 cleavage site, introduction of disulfide bonds, and fusion to heterologous trimerization motifs. We compared the effects on quaternary structure, antigenicity, and immunogenicity of three such motifs: T4 fibritin, a GCN4 variant, and the Escherichia coli aspartate transcarbamoylase catalytic subunit. Fusion of each motif to the C-terminus of a noncleavable JRCSF gp140(-) envelope protein led to enhanced trimerization but had limited effects on the antigenic profile and CD4-binding ability of the trimers. Immunization of rabbits provided no evidence that the trimerized gp140(-) constructs induced significantly improved neutralizing antibodies to several HIV-1 pseudoviruses, compared to gp140 lacking a trimerization motif. However, modest differences in both binding specificity and neutralizing antibody responses were observed among the various immunogens.

  10. A Specific Interaction of Small Molecule Entry Inhibitors with the Envelope Glycoprotein Complex of the Junín Hemorrhagic Fever Arenavirus*

    PubMed Central

    Thomas, Celestine J.; Casquilho-Gray, Hedi E.; York, Joanne; DeCamp, Dianne L.; Dai, Dongcheng; Petrilli, Erin B.; Boger, Dale L.; Slayden, Richard A.; Amberg, Sean M.; Sprang, Stephen R.; Nunberg, Jack H.

    2011-01-01

    Arenaviruses are responsible for acute hemorrhagic fevers worldwide and are recognized to pose significant threats to public health and biodefense. Small molecule compounds have recently been discovered that inhibit arenavirus entry and protect against lethal infection in animal models. These chemically distinct inhibitors act on the tripartite envelope glycoprotein (GPC) through its unusual stable signal peptide subunit to stabilize the complex against pH-induced activation of membrane fusion in the endosome. Here, we report the production and characterization of the intact transmembrane GPC complex of Junín arenavirus and its interaction with these inhibitors. The solubilized GPC is antigenically indistinguishable from the native protein and forms a homogeneous trimer in solution. When reconstituted into a lipid bilayer, the purified complex interacts specifically with its cell-surface receptor transferrin receptor-1. We show that small molecule entry inhibitors specific to New World or Old World arenaviruses bind to the membrane-associated GPC complex in accordance with their respective species selectivities and with dissociation constants comparable with concentrations that inhibit GPC-mediated membrane fusion. Furthermore, competitive binding studies reveal that these chemically distinct inhibitors share a common binding pocket on GPC. In conjunction with previous genetic studies, these findings identify the pH-sensing interface of GPC as a highly vulnerable target for antiviral intervention. This work expands our mechanistic understanding of arenavirus entry and provides a foundation to guide the development of small molecule compounds for the treatment of arenavirus hemorrhagic fevers. PMID:21159779

  11. Localization and Characterization of Flavivirus Envelope Glycoprotein Cross-Reactive Epitopes

    PubMed Central

    Crill, Wayne D.; Chang, Gwong-Jen J.

    2004-01-01

    The flavivirus E glycoprotein, the primary antigen that induces protective immunity, is essential for membrane fusion and mediates binding to cellular receptors. Human flavivirus infections stimulate virus species-specific as well as flavivirus cross-reactive immune responses. Flavivirus cross-reactive antibodies in human sera create a serious problem for serodiagnosis, especially for secondary flavivirus infections, due to the difficulty of differentiating primary from secondary cross-reactive serum antibodies. The presence of subneutralizing levels of flavivirus cross-reactive serum antibodies may result in a dramatic increase in the severity of secondary flavivirus infections via antibody-dependent enhancement. An understanding of flavivirus E-glycoprotein cross-reactive epitopes is therefore critical for improving public health responses to these serious diseases. We identified six E-glycoprotein residues that are incorporated into three distinct flavivirus cross-reactive epitopes. Two of these epitopes which are recognized by distinct monoclonal antibodies contain overlapping continuous residues located within the highly conserved fusion peptide. The third epitope consists of discontinuous residues that are structurally related to the strictly conserved tryptophan at dengue virus serotype 2 E-glycoprotein position 231. PMID:15564505

  12. Basic Residues in Hypervariable Region 1 of Hepatitis C Virus Envelope Glycoprotein E2 Contribute to Virus Entry

    PubMed Central

    Callens, Nathalie; Ciczora, Yann; Bartosch, Birke; Vu-Dac, Ngoc; Cosset, François-Loïc; Pawlotsky, Jean-Michel; Penin, François; Dubuisson, Jean

    2005-01-01

    The N terminus of hepatitis C virus (HCV) envelope glycoprotein E2 contains a hypervariable region (HVR1) which has been proposed to play a role in viral entry. Despite strong amino acid variability, HVR1 is globally basic, with basic residues located at specific sequence positions. Here we show by analyzing a large number of HVR1 sequences that the frequency of basic residues at each position is genotype dependent. We also used retroviral pseudotyped particles (HCVpp) harboring genotype 1a envelope glycoproteins to study the role of HVR1 basic residues in entry. Interestingly, HCVpp infectivity globally increased with the number of basic residues in HVR1. However, a shift in position of some charged residues also modulated HCVpp infectivity. In the absence of basic residues, infectivity was reduced to the same level as that of a mutant deleted of HVR1. We also analyzed the effect of these mutations on interactions with some potential HCV receptors. Recognition of CD81 was not affected by changes in the number of charged residues, and we did not find a role for heparan sulfates in HCVpp entry. The involvement of the scavenger receptor class B type I (SR-BI) was indirectly analyzed by measuring the enhancement of infectivity of the mutants in the presence of the natural ligand of SR-BI, high-density lipoproteins (HDL). However, no correlation between the number of basic residues within HVR1 and HDL enhancement effect was observed. Despite the lack of evidence of the involvement of known potential receptors, our results demonstrate that the presence of basic residues in HVR1 facilitates virus entry. PMID:16306604

  13. Basic residues in hypervariable region 1 of hepatitis C virus envelope glycoprotein e2 contribute to virus entry.

    PubMed

    Callens, Nathalie; Ciczora, Yann; Bartosch, Birke; Vu-Dac, Ngoc; Cosset, François-Loïc; Pawlotsky, Jean-Michel; Penin, François; Dubuisson, Jean

    2005-12-01

    The N terminus of hepatitis C virus (HCV) envelope glycoprotein E2 contains a hypervariable region (HVR1) which has been proposed to play a role in viral entry. Despite strong amino acid variability, HVR1 is globally basic, with basic residues located at specific sequence positions. Here we show by analyzing a large number of HVR1 sequences that the frequency of basic residues at each position is genotype dependent. We also used retroviral pseudotyped particles (HCVpp) harboring genotype 1a envelope glycoproteins to study the role of HVR1 basic residues in entry. Interestingly, HCVpp infectivity globally increased with the number of basic residues in HVR1. However, a shift in position of some charged residues also modulated HCVpp infectivity. In the absence of basic residues, infectivity was reduced to the same level as that of a mutant deleted of HVR1. We also analyzed the effect of these mutations on interactions with some potential HCV receptors. Recognition of CD81 was not affected by changes in the number of charged residues, and we did not find a role for heparan sulfates in HCVpp entry. The involvement of the scavenger receptor class B type I (SR-BI) was indirectly analyzed by measuring the enhancement of infectivity of the mutants in the presence of the natural ligand of SR-BI, high-density lipoproteins (HDL). However, no correlation between the number of basic residues within HVR1 and HDL enhancement effect was observed. Despite the lack of evidence of the involvement of known potential receptors, our results demonstrate that the presence of basic residues in HVR1 facilitates virus entry.

  14. Dicalcin Inhibits Fertilization through Its Binding to a Glycoprotein in the Egg Envelope in Xenopus laevis*

    PubMed Central

    Miwa, Naofumi; Ogawa, Motoyuki; Shinmyo, Yukiko; Hiraoka, Yoshiki; Takamatsu, Ken; Kawamura, Satoru

    2010-01-01

    Fertilization comprises oligosaccharide-mediated sperm-egg interactions, including sperm binding to an extracellular egg envelope, sperm penetration through the envelope, and fusion with an egg plasma membrane. We show that Xenopus dicalcin, an S100-like Ca2+-binding protein, present in the extracellular egg envelope (vitelline envelope (VE)), is a suppressive mediator of sperm-egg interaction. Preincubation with specific antibody greatly increased the efficiency of in vitro fertilization, whereas prior application of exogenous dicalcin substantially inhibited fertilization as well as sperm binding to an egg and in vitro sperm penetration through the VE protein layer. Dicalcin showed binding to protein cores of gp41 and gp37, constituents of VE, in a Ca2+-dependent manner and increased in vivo reactivity of VE with a lectin, Ricinus communis agglutinin I, which was accounted for by increased binding ability of gp41 to the lectin and greater exposure of gp41 to an external environment. Our findings strongly suggest that dicalcin regulates the distribution of oligosaccharides within the VE through its binding to the protein core of gp41, probably by modulating configuration of oligosaccharides on gp41 and the three-dimensional structure of VE framework, and thereby plays a pivotal role in sperm-egg interactions during fertilization. PMID:20299459

  15. Chemical inactivation of recombinant vaccinia viruses and the effects on antigenicity and immunogenicity of recombinant simian immunodeficiency virus envelope glycoproteins.

    PubMed

    Hulskotte, E G; Dings, M E; Norley, S G; Osterhaus, A D

    1997-12-01

    The efficiency of paraformaldehyde (PFA) and binary ethylenimine (BEI) in inactivating recombinant vaccinia virus (rVV), present in baby hamster kidney cells expressing simian immunodeficiency virus envelope glycoproteins (SIV-Env), was measured in a series of inactivation studies. Both compounds were shown to be effective in reducing rVV titres. The use of standard 3-day titration assays proved to be inadequate to measure PFA inactivation, since upon prolonged incubation, residual rVV infectivity was detected in cultures negative at 3 days. Different procedures using PFA or BEI were selected to assess their influence on the antigenicity and immunogenicity or rVV expressed SIV-Env. Antigenicity, as defined by the ability to react with a panel of monoclonal antibodies recognizing major antigenic sites, and immunogenicity, as defined by the ability to induce SIV envelope specific and virus neutralizing serum antibodies in rats, proved to be preserved after either inactivation procedure. These data show that both protocols using PFA or BEI can be used successfully as part of the procedures to remove residual rVV infectivity.

  16. A Molecular Sensor To Characterize Arenavirus Envelope Glycoprotein Cleavage by Subtilisin Kexin Isozyme 1/Site 1 Protease

    PubMed Central

    Oppliger, Joel; da Palma, Joel Ramos; Burri, Dominique J.; Khatib, Abdel-Majid; Spiropoulou, Christina F.

    2015-01-01

    ABSTRACT Arenaviruses are emerging viruses including several causative agents of severe hemorrhagic fevers in humans. The advent of next-generation sequencing technology has greatly accelerated the discovery of novel arenavirus species. However, for many of these viruses, only genetic information is available, and their zoonotic disease potential remains unknown. During the arenavirus life cycle, processing of the viral envelope glycoprotein precursor (GPC) by the cellular subtilisin kexin isozyme 1 (SKI-1)/site 1 protease (S1P) is crucial for productive infection. The ability of newly emerging arenaviruses to hijack human SKI-1/S1P appears, therefore, to be a requirement for efficient zoonotic transmission and human disease potential. Here we implement a newly developed cell-based molecular sensor for SKI-1/S1P to characterize the processing of arenavirus GPC-derived target sequences by human SKI-1/S1P in a quantitative manner. We show that only nine amino acids flanking the putative cleavage site are necessary and sufficient to accurately recapitulate the efficiency and subcellular location of arenavirus GPC processing. In a proof of concept, our sensor correctly predicts efficient processing of the GPC of the newly emergent pathogenic Lujo virus by human SKI-1/S1P and defines the exact cleavage site. Lastly, we employed our sensor to show efficient GPC processing of a panel of pathogenic and nonpathogenic New World arenaviruses, suggesting that GPC cleavage represents no barrier for zoonotic transmission of these pathogens. Our SKI-1/S1P sensor thus represents a rapid and robust test system for assessment of the processing of putative cleavage sites derived from the GPCs of newly discovered arenavirus by the SKI-1/S1P of humans or any other species, based solely on sequence information. IMPORTANCE Arenaviruses are important emerging human pathogens that can cause severe hemorrhagic fevers with high mortality in humans. A crucial step in productive arenavirus

  17. Human herpesvirus 8 envelope-associated glycoprotein B interacts with heparan sulfate-like moieties.

    PubMed

    Akula, S M; Pramod, N P; Wang, F Z; Chandran, B

    2001-06-01

    Cell-surface heparan sulfate (HS) serves as an initial attachment receptor for several herpesviruses. The gamma2-human herpesvirus-8 (HHV-8) or Kaposi's sarcoma associated herpesvirus DNA and transcripts have been detected in B cells, endothelial cells, macrophages, and epithelial cells. HHV-8 infects a variety of human and animal cell lines leading to latent or abortive infection. Our studies showed that this broad cellular tropism may be in part due to HHV-8's interaction with the ubiquitous host cell-surface HS-like molecules. HHV-8 binding to the target cells and the infection were inhibited by soluble heparan, a glycosaminoglycan (GAG) closely related to HS. Since HHV-8 gB possess a putative heparan-binding domain (HBD) in the extracellular domain, the interaction of gB with HS-like moieties was examined. Unlike gB of gamma1-Epstein-Barr virus and gamma2-murine herpesvirus 68, HHV-8 gB was expressed on the surface of the infected cell membranes and virion envelopes. Envelope-associated gB was made up of 75 and 54 kDa polypeptides forming disulfide-linked heterodimers and multimers. Rabbit anti-gB antibodies neutralized HHV-8 infection. Virion envelope-associated gB specifically bound to heparan-agarose, which was eluted by high concentration of soluble heparan, but not by chondroitin sulfates. In vitro transcribed and translated products of gB gene specifically bound to heparan-agarose beads, which was blocked by HS and heparan, but not by other GAGs such as chondroitin sulfates (A, B, and C), N-acetyl heparan, and de-N-sulfated heparan. Biotinylated gB peptide corresponding to the putative HBD also bound to heparan. These results suggest that gB plays an important role in the infectious process of HHV-8 and virus interaction with cell-surface HS-like moieties could be in part mediated by the envelope-associated gB.

  18. Structural models of the membrane anchors of envelope glycoproteins E1 and E2 from pestiviruses

    PubMed Central

    Wang, Jimin; Li, Yue; Modis, Yorgo

    2014-01-01

    The membrane anchors of viral envelope proteins play essential roles in cell entry. Recent crystal structures of the ectodomain of envelope protein E2 from a pestivirus suggest that E2 belongs to a novel structural class of membrane fusion machinery. Based on geometric constraints from the E2 structures, we generated atomic models of the E1 and E2 membrane anchors using computational approaches. The E1 anchor contains two amphipathic perimembrane helices and one transmembrane helix; the E2 anchor contains a short helical hairpin stabilized in the membrane by an arginine residue, similar to flaviviruses. A pair of histidine residues in the E2 ectodomain may participate in pH sensing. The proposed atomic models point to Cys987 in E2 as the site of disulfide bond linkage with E1 to form E1–E2 heterodimers. The membrane anchor models provide structural constraints for the disulfide bonding pattern and overall backbone conformation of the E1 ectodomain. PMID:24725935

  19. Structural models of the membrane anchors of envelope glycoproteins E1 and E2 from pestiviruses.

    PubMed

    Wang, Jimin; Li, Yue; Modis, Yorgo

    2014-04-01

    The membrane anchors of viral envelope proteins play essential roles in cell entry. Recent crystal structures of the ectodomain of envelope protein E2 from a pestivirus suggest that E2 belongs to a novel structural class of membrane fusion machinery. Based on geometric constraints from the E2 structures, we generated atomic models of the E1 and E2 membrane anchors using computational approaches. The E1 anchor contains two amphipathic perimembrane helices and one transmembrane helix; the E2 anchor contains a short helical hairpin stabilized in the membrane by an arginine residue, similar to flaviviruses. A pair of histidine residues in the E2 ectodomain may participate in pH sensing. The proposed atomic models point to Cys987 in E2 as the site of disulfide bond linkage with E1 to form E1-E2 heterodimers. The membrane anchor models provide structural constraints for the disulfide bonding pattern and overall backbone conformation of the E1 ectodomain.

  20. Structural models of the membrane anchors of envelope glycoproteins E1 and E2 from pestiviruses

    SciTech Connect

    Wang, Jimin Li, Yue; Modis, Yorgo

    2014-04-15

    The membrane anchors of viral envelope proteins play essential roles in cell entry. Recent crystal structures of the ectodomain of envelope protein E2 from a pestivirus suggest that E2 belongs to a novel structural class of membrane fusion machinery. Based on geometric constraints from the E2 structures, we generated atomic models of the E1 and E2 membrane anchors using computational approaches. The E1 anchor contains two amphipathic perimembrane helices and one transmembrane helix; the E2 anchor contains a short helical hairpin stabilized in the membrane by an arginine residue, similar to flaviviruses. A pair of histidine residues in the E2 ectodomain may participate in pH sensing. The proposed atomic models point to Cys987 in E2 as the site of disulfide bond linkage with E1 to form E1–E2 heterodimers. The membrane anchor models provide structural constraints for the disulfide bonding pattern and overall backbone conformation of the E1 ectodomain. - Highlights: • Structures of pestivirus E2 proteins impose constraints on E1, E2 membrane anchors. • Atomic models of the E1 and E2 membrane anchors were generated in silico. • A “snorkeling” arginine completes the short helical hairpin in the E2 membrane anchor. • Roles in pH sensing and E1–E2 disulfide bond formation are proposed for E1 residues. • Implications for E1 ectodomain structure and disulfide bonding pattern are discussed.

  1. A single amino acid change in the cytoplasmic domain of the simian immunodeficiency virus transmembrane molecule increases envelope glycoprotein expression on infected cells.

    PubMed Central

    LaBranche, C C; Sauter, M M; Haggarty, B S; Vance, P J; Romano, J; Hart, T K; Bugelski, P J; Marsh, M; Hoxie, J A

    1995-01-01

    We have described a virus termed CP-MAC, derived from the BK28 molecular clone of simian immunodeficiency virus, that was remarkable for its ability to infect Sup-T1 cells with rapid kinetics, cell fusion, and CD4 down-modulation (C. C. LaBranche, M. M. Sauter, B. S. Haggarty, P. J. Vance, J. Romano, T. K. Hart, P. J. Bugelski, and J. A. Hoxie, J. Virol. 68:5509-5522, 1994 [Erratum 68:7665-7667]). Compared with BK28, CP-MAC exhibited a number of changes in its envelope glycoproteins, including a highly stable association between the external (SU) and transmembrane (TM) molecules, a more rapid electrophoretic mobility of TM, and, of particular interest, a marked increase in the level of envelope protein expression on the surface of infected cells. These changes were shown to be associated with 11 coding mutations in the env gene (5 in SU and 6 in TM). In this report, we demonstrate that a single amino acid mutation of a Tyr to a Cys at position 723 (Y723C) in the TM cytoplasmic domain of CP-MAC is the principal determinant for the increased expression of envelope glycoproteins on the cell surface. When introduced into the env gene of BK28, the Y723C mutation produced up to a 25-fold increase in the levels of SU and TM on chronically infected cells, as determined by fluorescence-activated cell sorter analysis with monoclonal and polyclonal antibodies. A similar effect was observed when a Tyr-to-Cys change was introduced at the analogous position (amino acid 721) in the SIVmac239 molecular clone, which, unlike BK28 does not contain a premature stop codon in its TM cytoplasmic tail. Substituting other amino acids, including Ala, Ile, and Ser, at this position produced increases in surface envelope glycoproteins that were similar to that observed for the Cys substitution, while a Tyr-to-Phe mutation produced a smaller increase. These results could not be accounted for by differences in the kinetics or efficiency of envelope glycoprotein processing or by shedding of SU

  2. A 45,000-M(r) glycoprotein in the Sendai virus envelope triggers virus-cell fusion.

    PubMed Central

    Kumar, M; Hassan, M Q; Tyagi, S K; Sarkar, D P

    1997-01-01

    Sendai virus envelopes devoid of hemagglutinin-neuraminidase but containing the fusion protein (F-virosomes) were prepared. F-virosomes exhibited discernible serine protease activity at neutral pH. Electrophoretic analysis of the protein profile of the F-virosomes under nonreducing conditions, by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing, led to the identification of a previously unknown glycoprotein with a relative molecular weight of 45,000 (45K protein) associated with the F protein. The identity of the 45K protein, as distinct from F protein, was established by Western blot analysis with F- and 45K-specific antibodies. This 45K protein forms a nexus with the F protein through noncovalent hydrophobic interactions, as proved by its sensitivity to urea treatment, and it is essential for the proteolytic activity of the F-virosomes as well as for the fusion of the viral envelope with host cell membrane. N-terminal sequence analysis (first 11 amino acids) of this protein showed strong homology (> 90%) to flavivirus NS3 serine proteases but no similarity to any of the Sendai viral proteins. On the basis of the N-terminal sequence, oligonucleotides were designed corresponding to the sense and antisense DNA sequences. Dot blot hybridization and primer extension with these oligonucleotides with the viral and the host genome confirmed the host origin of this protein. Further, the limited proteolytic digestion of the target membrane resulted in significant inhibition of viral fusion with it. On the basis of these results, we postulate a model for the molecular mechanism of F protein-induced membrane fusion, which may provide a rationale for other paramyxoviruses. PMID:9261357

  3. Structure of a phleboviral envelope glycoprotein reveals a consolidated model of membrane fusion.

    PubMed

    Halldorsson, Steinar; Behrens, Anna-Janina; Harlos, Karl; Huiskonen, Juha T; Elliott, Richard M; Crispin, Max; Brennan, Benjamin; Bowden, Thomas A

    2016-06-28

    An emergent viral pathogen termed severe fever with thrombocytopenia syndrome virus (SFTSV) is responsible for thousands of clinical cases and associated fatalities in China, Japan, and South Korea. Akin to other phleboviruses, SFTSV relies on a viral glycoprotein, Gc, to catalyze the merger of endosomal host and viral membranes during cell entry. Here, we describe the postfusion structure of SFTSV Gc, revealing that the molecular transformations the phleboviral Gc undergoes upon host cell entry are conserved with otherwise unrelated alpha- and flaviviruses. By comparison of SFTSV Gc with that of the prefusion structure of the related Rift Valley fever virus, we show that these changes involve refolding of the protein into a trimeric state. Reverse genetics and rescue of site-directed histidine mutants enabled localization of histidines likely to be important for triggering this pH-dependent process. These data provide structural and functional evidence that the mechanism of phlebovirus-host cell fusion is conserved among genetically and patho-physiologically distinct viral pathogens.

  4. Structure of a phleboviral envelope glycoprotein reveals a consolidated model of membrane fusion.

    PubMed

    Halldorsson, Steinar; Behrens, Anna-Janina; Harlos, Karl; Huiskonen, Juha T; Elliott, Richard M; Crispin, Max; Brennan, Benjamin; Bowden, Thomas A

    2016-06-28

    An emergent viral pathogen termed severe fever with thrombocytopenia syndrome virus (SFTSV) is responsible for thousands of clinical cases and associated fatalities in China, Japan, and South Korea. Akin to other phleboviruses, SFTSV relies on a viral glycoprotein, Gc, to catalyze the merger of endosomal host and viral membranes during cell entry. Here, we describe the postfusion structure of SFTSV Gc, revealing that the molecular transformations the phleboviral Gc undergoes upon host cell entry are conserved with otherwise unrelated alpha- and flaviviruses. By comparison of SFTSV Gc with that of the prefusion structure of the related Rift Valley fever virus, we show that these changes involve refolding of the protein into a trimeric state. Reverse genetics and rescue of site-directed histidine mutants enabled localization of histidines likely to be important for triggering this pH-dependent process. These data provide structural and functional evidence that the mechanism of phlebovirus-host cell fusion is conserved among genetically and patho-physiologically distinct viral pathogens. PMID:27325770

  5. Residues in the membrane-spanning domain core modulate conformation and fusogenicity of the HIV-1 envelope glycoprotein

    SciTech Connect

    Shang Liang; Hunter, Eric

    2010-09-01

    The membrane-spanning domain (MSD) of human immunodeficiency virus type I (HIV-1) envelope glycoprotein (Env) is critical for its biological activity. Initial studies have defined an almost invariant 'core' structure in the MSD and demonstrated that it is crucial for anchoring Env in the membrane and virus entry. We show here that amino acid substitutions in the MSD 'core' do not influence specific virus-cell attachment, nor CD4 receptor and CXCR4 coreceptor recognition by Env. However, substitutions within the MSD 'core' delayed the kinetics and reduced the efficiency of cell-cell fusion mediated by Env. Although we observed no evidence that membrane fusion mediated by the MSD core mutants was arrested at a hemifusion stage, impaired Env fusogenicity was correlated with minor conformational changes in the V2, C1, and C5 regions in gp120 and the immunodominant loop in gp41. These changes could delay initiation of the conformational changes required in the fusion process.

  6. Depression of Rauscher leukemia virus envelope glycoprotein gp71 binding by lymphoid cells during leukemogenesis in mice.

    PubMed Central

    Fowler, A K; Reed, C D; Riggs, C W; Twardzik, D R; Weislow, O S; Hellman, A

    1979-01-01

    The availability of membrane receptors for the 71,000-dalton envelope glycoprotein (gp71) of Rauscher murine leukemia virus on splenic and thymic cells from BALB/c mice during Rauscher murine leukemia virus-induced leukemogenesis was determined utilizing a radiolabeled gp71 binding assay. Shortly after infection, the relative cellular [125I]gp71 binding level decreased, first with splenic cells (at day 7 to 10 after infection) and later with thymic cells (at day 10 to 20 after infection). The dependency of the reduction of binding on the replication of the inoculated virus was demonstrated by regression analyses using cellular gp71 binding level as the dependent variable and infectious virus titer, as well as viral gp71 and p30 levels, of spleens and thymuses from infected mice as independent variables. With each independent variable, the reduction of gp71 binding for both cell types was highly dependent (P less than 0.01) on the level of virus detected in their respective organ. In the early stages of leukemogenesis, the [125I]gp71 binding level declined to approximately 20 to 30% of control values. During this period the rate of reduction of binding was very rapid and, in general was similar for both splenic and thymic cells. Further progression of the disease resulted in little or no further reduction in binding. The application of this technique to monitor host ecotropic virus synthesis and to study cell surface virus receptor control mechanisms in vivo is discussed. PMID:468372

  7. Early intermediates in HIV-1 envelope glycoprotein-mediated fusion triggered by CD4 and co-receptor complexes.

    PubMed

    Dimitrov, A S; Xiao, X; Dimitrov, D S; Blumenthal, R

    2001-08-10

    An early step in the process of HIV-1 entry into target cells is the activation of its envelope glycoprotein (GP120-GP41) to a fusogenic state upon binding to target cell CD4 and cognate co-receptor. Incubation of human immunodeficiency virus (HIV)-1 Env-expressing cells with an excess of CD4 and co-recepeptor-bearing target cells resulted in an influx of an impermeant nucleic acid-staining fluorescent dye into the Env-expressing cells. The dye influx occurred concomitant with cell fusion. No influx of dye into target cells was observed if they were incubated with an excess of Env-expressing cells. The permeabilization of Env-expressing cells was also triggered by CD4.co-receptor complexes attached to Protein G-Sepharose beads in the absence of target cells. The CD4 and co-receptor-induced permeabilization of Env-expressing cells occurred with the same specificity with respect to co-receptor usage as cell fusion. Natural ligands for the co-receptors and C-terminal GP41 peptide inhibitors of HIV-1 fusion blocked this effect. Our results indicate that the process of HIV-1 Env-mediated fusion is initiated by the destabilization of HIV-1 Env-expressing membranes. Further elucidation of these early intermediates may help identify and develop potential inhibitors of HIV-1 entry into cells.

  8. Envelope glycoprotein gp120 of human immunodeficiency virus type 1 alters ion transport in astrocytes: implications for AIDS dementia complex.

    PubMed Central

    Benos, D J; Hahn, B H; Bubien, J K; Ghosh, S K; Mashburn, N A; Chaikin, M A; Shaw, G M; Benveniste, E N

    1994-01-01

    Infection by human immunodeficiency virus type 1 (HIV-1) is often complicated by a variety of neurological abnormalities. The most common clinical syndrome, termed acquired immunodeficiency syndrome (AIDS) dementia complex, presents as a subcortical dementia with cognitive, motor, and behavioral disturbances and is unique to HIV-1 infection. The pathogenesis of this syndrome is poorly understood but is believed to involve interactions among virally infected macrophages/microglia, astrocytes, and neurons. In this study, we show that exposure of primary rat and human astrocytes to heat-activated HIV-1 virions, or to eukaryotically expressed HIV-1 and HIV-2 envelope glycoproteins (gp120) stimulates amiloride-sensitive Na+/H+ antiport, potassium conductance, and glutamate efflux. These effects are blocked specifically by amiloride, an inhibitor of Na+/H+ antiport and by the selective removal of gp120 with immobilized monoclonal antibody. As a result of modulation of astrocytic function by gp120, the ensuing neuronal depolarization and glutamate exposure could activate both voltage-gated and N-methyl-D-aspartate-regulated Ca2+ channels, leading to increases in intraneuronal Ca2+ and neuronal death. These findings implicate the astrocyte directly in the pathogenesis of AIDS dementia complex. PMID:8290553

  9. Distinct requirements for signal peptidase processing and function in the stable signal peptide subunit of the Junin virus envelope glycoprotein

    SciTech Connect

    York, Joanne; Nunberg, Jack H. . E-mail: jack.nunberg@umontana.edu

    2007-03-01

    The arenavirus envelope glycoprotein (GP-C) retains a cleaved and stable signal peptide (SSP) as an essential subunit of the mature complex. This 58-amino-acid residue peptide serves as a signal sequence and is additionally required to enable transit of the assembled GP-C complex to the Golgi, and for pH-dependent membrane fusion activity. We have investigated the C-terminal region of the Junin virus SSP to study the role of the cellular signal peptidase (SPase) in generating SSP. Site-directed mutagenesis at the cleavage site (positions - 1 and - 3) reveals a pattern of side-chain preferences consistent with those of SPase. Although position - 2 is degenerate for SPase cleavage, this residue in the arenavirus SSP is invariably a cysteine. In the Junin virus, this cysteine is not involved in disulfide bonding. We show that replacement with alanine or serine is tolerated for SPase cleavage but prevents the mutant SSP from associating with GP-C and enabling transport to the cell surface. Conversely, an arginine mutation at position - 1 that prevents SPase cleavage is fully compatible with GP-C-mediated membrane fusion activity when the mutant SSP is provided in trans. These results point to distinct roles of SSP sequences in SPase cleavage and GP-C biogenesis. Further studies of the unique structural organization of the GP-C complex will be important in identifying novel opportunities for antiviral intervention against arenaviral hemorrhagic disease.

  10. Dissection of the Role of the Stable Signal Peptide of the Arenavirus Envelope Glycoprotein in Membrane Fusion

    PubMed Central

    Messina, Emily L.; York, Joanne

    2012-01-01

    The arenavirus envelope glycoprotein (GPC) retains a stable signal peptide (SSP) as an essential subunit in the mature complex. The 58-amino-acid residue SSP comprises two membrane-spanning hydrophobic regions separated by a short ectodomain loop that interacts with the G2 fusion subunit to promote pH-dependent membrane fusion. Small-molecule compounds that target this unique SSP-G2 interaction prevent arenavirus entry and infection. The interaction between SSP and G2 is sensitive to the phylogenetic distance between New World (Junín) and Old World (Lassa) arenaviruses. For example, heterotypic GPC complexes are unable to support virion entry. In this report, we demonstrate that the hybrid GPC complexes are properly assembled, proteolytically cleaved, and transported to the cell surface but are specifically defective in their membrane fusion activity. Chimeric SSP constructs reveal that this incompatibility is localized to the first transmembrane segment of SSP (TM1). Genetic changes in TM1 also affect sensitivity to small-molecule fusion inhibitors, generating resistance in some cases and inhibitor dependence in others. Our studies suggest that interactions of SSP TM1 with the transmembrane domain of G2 may be important for GPC-mediated membrane fusion and its inhibition. PMID:22438561

  11. Release of gp120 Restraints Leads to an Entry-Competent Intermediate State of the HIV-1 Envelope Glycoproteins

    PubMed Central

    Ma, Xiaochu; Gu, Christopher; Ventura, John D.; Castillo-Menendez, Luis; Melillo, Bruno; Terry, Daniel S.; Smith, Amos B.; Blanchard, Scott C.; Munro, James B.; Mothes, Walther; Finzi, Andrés

    2016-01-01

    ABSTRACT Primary human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) trimers [(gp120/gp41)3] typically exist in a metastable closed conformation (state 1). Binding the CD4 receptor triggers Env to undergo extensive conformational changes to mediate virus entry. We identified specific gp120 residues that restrain Env in state 1. Alteration of these restraining residues destabilized state 1, allowing Env to populate a functional conformation (state 2) intermediate between state 1 and the full CD4-bound state (state 3). Increased state 2 occupancy was associated with lower energy barriers between the states. State 2 was an obligate intermediate for all transitions between state 1 and state 3. State 2-enriched Envs required lower CD4 concentrations to trigger virus entry and more efficiently infected cells expressing low levels of CD4. These Envs were resistant to several broadly neutralizing antibodies and small-molecule inhibitors. Thus, state 2 is an Env conformation on the virus entry pathway; sampling state 2 increases the adaptability of HIV-1 to different host cell receptor levels and immune environments. Our results provide new insights into the conformational regulation of HIV-1 entry. PMID:27795397

  12. ASPP2 involvement in p53-mediated HIV-1 envelope glycoprotein gp120 neurotoxicity in mice cerebrocortical neurons

    PubMed Central

    Liu, Zhiying; Zang, Yunjin; Qiao, Luxin; Liu, Kai; Ouyang, Yabo; Zhang, Yulin; Chen, Dexi

    2016-01-01

    The mechanisms behind HIV-1-associated neurocognitive disorders are still unclear. Apoptosis-stimulating protein 2 of p53 (ASPP2) is a damage-inducible p53-binding protein that stimulates p53-mediated apoptosis and transactivates proapoptotic and cell cycle regulatory genes. It has been reported that ASPP2 has a specific regulatory function in the death of retinal ganglion cells and the development of Alzheimer’s disease. In this study, we used p53 and ASPP2 knockout mice and primary cerebrocortical neuron culture to analyze the role of the interaction between ASPP2 with p53 in HIV-1 envelope glycoprotein gp120-induced neurotoxicity. The results showed that 10 ng/mL gp120 protein might stimulate p53 overexpression and translocation to the nucleus, and 30 ng/mL gp120 protein could stimulate both p53 and ASPP2 translocation to the nucleus, but only with p53 overexpression. The primary cultured neurons of p53−/−ASPP2+/− mice had a higher survival rate than p53−/− mice under gp120 protein stress. The interaction of ASPP2 with p53 induced by a high dose of gp120 stimulated Bax transcription and contributed to caspase-3 cleavage, and ASPP2-siRNA attenuated gp120 induced neuron death through inhibition of Bax expression. These results suggest that ASPP2 plays an important role in p53-mediated neuronal apoptosis under gp120 stress. PMID:27625111

  13. ASPP2 involvement in p53-mediated HIV-1 envelope glycoprotein gp120 neurotoxicity in mice cerebrocortical neurons.

    PubMed

    Liu, Zhiying; Zang, Yunjin; Qiao, Luxin; Liu, Kai; Ouyang, Yabo; Zhang, Yulin; Chen, Dexi

    2016-01-01

    The mechanisms behind HIV-1-associated neurocognitive disorders are still unclear. Apoptosis-stimulating protein 2 of p53 (ASPP2) is a damage-inducible p53-binding protein that stimulates p53-mediated apoptosis and transactivates proapoptotic and cell cycle regulatory genes. It has been reported that ASPP2 has a specific regulatory function in the death of retinal ganglion cells and the development of Alzheimer's disease. In this study, we used p53 and ASPP2 knockout mice and primary cerebrocortical neuron culture to analyze the role of the interaction between ASPP2 with p53 in HIV-1 envelope glycoprotein gp120-induced neurotoxicity. The results showed that 10 ng/mL gp120 protein might stimulate p53 overexpression and translocation to the nucleus, and 30 ng/mL gp120 protein could stimulate both p53 and ASPP2 translocation to the nucleus, but only with p53 overexpression. The primary cultured neurons of p53(-/-)ASPP2(+/-) mice had a higher survival rate than p53(-/-) mice under gp120 protein stress. The interaction of ASPP2 with p53 induced by a high dose of gp120 stimulated Bax transcription and contributed to caspase-3 cleavage, and ASPP2-siRNA attenuated gp120 induced neuron death through inhibition of Bax expression. These results suggest that ASPP2 plays an important role in p53-mediated neuronal apoptosis under gp120 stress. PMID:27625111

  14. Agar gel immunodiffusion analysis using baculovirus-expressed recombinant bovine leukemia virus envelope glycoprotein (gp51/gp30(T-)).

    PubMed

    Lim, Seong In; Jeong, Wooseog; Tark, Dong Seob; Yang, Dong Kun; Kweon, Chang Hee

    2009-12-01

    Bovine leukemia virus (BLV) envelope glycoprotein (gp51/ gp30(T-)), consisting of BLV gp51 and BLV gp30 that lacked its C-terminal transmembrane domain, was expressed in insect cells under the control of the baculovirus polyhedron promoter. Recombinant BLV gp51/gp30(T-) secreted from insect cells was determined by immunofluorescence, enzyme-linked immunosorbent and western blot assays using a BLV-specific monoclonal antibody and BLV-positive bovine antibodies. An agar gel immunodiffusion (AGID) test using gp51/gp30(T-) as the antigen for the detection of BLV antibodies in serum was developed and compared to traditional AGID, which uses wild type BLV antigen derived from fetal lamb kidney cells. AGID with the recombinant BLV gp51/gp30(T-) was relatively more sensitive than traditional AGID. When the two methods were tested with bovine sera from the field, the recombinant BLV gp51/gp30(T-) and traditional antigen had a relative sensitivity of 69.8% and 67.4%, respectively, and a relative specificity of 93.3% and 92.3%. These results indicated that the recombinant BLV gp51/gp30(T-) is an effective alternative antigen for the diagnosis of BLV infection in cattle.

  15. Generation and evaluation of avian leukosis virus subgroup J envelope glycoprotein recombinant pseudovirions.

    PubMed

    Zhang, Zhenjie; Cui, Lina; Wang, Liang; Yang, Zhikun; Cui, Zhizhong; Chang, Weishan

    2014-06-01

    Retroviral and lentiviral vector pseudotypes (based on human immunodeficiency virus type 1, HIV-1) have been used for stable and safe gene transfer because of their broad host ranges and high mechanical strength. In the present study, a recombinant avian leukosis virus subgroup J (ALV-J) polypeptide pseudotyped with lentivirus membrane glycoproteins gp85 and gp37, HIV/env-ALV, was generated, characterized in vitro and evaluated for its ability to infect natural host cells. We optimized the newly developed micro-neutralization (MN) assay using recombinant pseudovirion HIV/env-ALV expressing enhanced green fluorescent protein and well-characterized sera from chickens with confirmed ALV-J disease or virus-free controls. HIV/env-ALV could infect CEF and DF-1 but not pk15, 293FT, MDCK or VERO E6 cells, therefore demonstrating a cellular tropism similar to the wild-type ALV-J. The MN assay indicated that the IC50 values of positive sera offered a considerable advantage in both speed and accuracy. These results suggest that this pseudotyped lentivirus is a good model for studying the functions of ALV-J env and that the MN assay is a reliable serological method for assessing antibody levels in investigating the actual status of the current ALV-J epidemic. These recombinant pseudovirions may prove to be useful for studying ALV-J biology in lower biosafety level laboratory environments, and also for the detection and quantification of neutralizing antibodies to ALV-J in a manner akin to ELISA assays, but that would also be applicable to other viruses.

  16. Novel innate immune functions for galectin-1: galectin-1 inhibits cell fusion by Nipah virus envelope glycoproteins and augments dendritic cell secretion of proinflammatory cytokines.

    PubMed

    Levroney, Ernest L; Aguilar, Hector C; Fulcher, Jennifer A; Kohatsu, Luciana; Pace, Karen E; Pang, Mabel; Gurney, Kevin B; Baum, Linda G; Lee, Benhur

    2005-07-01

    Galectin-1 (gal-1), an endogenous lectin secreted by a variety of cell types, has pleiotropic immunomodulatory functions, including regulation of lymphocyte survival and cytokine secretion in autoimmune, transplant disease, and parasitic infection models. However, the role of gal-1 in viral infections is unknown. Nipah virus (NiV) is an emerging pathogen that causes severe, often fatal, febrile encephalitis. The primary targets of NiV are endothelial cells. NiV infection of endothelial cells results in cell-cell fusion and syncytia formation triggered by the fusion (F) and attachment (G) envelope glycoproteins of NiV that bear glycan structures recognized by gal-1. In the present study, we report that NiV envelope-mediated cell-cell fusion is blocked by gal-1. This inhibition is specific to the Paramyxoviridae family because gal-1 did not inhibit fusion triggered by envelope glycoproteins of other viruses, including two retroviruses and a pox virus, but inhibited fusion triggered by envelope glycoproteins of the related Hendra virus and another paramyxovirus. The physiologic dimeric form of gal-1 is required for fusion inhibition because a monomeric gal-1 mutant had no inhibitory effect on cell fusion. gal-1 binds to specific N-glycans on NiV glycoproteins and aberrantly oligomerizes NiV-F and NiV-G, indicating a mechanism for fusion inhibition. gal-1 also increases dendritic cell production of proinflammatory cytokines such as IL-6, known to be protective in the setting of other viral diseases such as Ebola infections. Thus, gal-1 may have direct antiviral effects and may also augment the innate immune response against this emerging pathogen.

  17. Comparison of dengue-1 virus envelope glycoprotein gene sequences from French Polynesia.

    PubMed

    Laille, Manola; Roche, Claudine

    2004-10-01

    Dengue (DEN) is the leading arboviral infection of humans, with 100 million cases annually in the tropical areas of the world. The recent severe DEN-1 epidemic in French Polynesia in 2001, with an incidence rate of 16% and more than 45% of the cases with dengue hemorrhagic fever/dengue shock syndrome among 1,400 hospitalized children and eight fatalities, led us to study this new circulating strain. The entire envelope (E) gene of two French Polynesian DEN-1 virus isolates from the two epidemics of 1988-1989 (FP89) and 2001 (FP01) were sequenced and compared with 29 published DEN-1 virus E gene sequences. Phylogenetic relationships showed that the FP89 strain belonged to genotype V and the FP01 strain to genotype IV based on studies on the same region of DEN-1 virus genome (1,485 nucleotides). The recent dengue epidemic in French Polynesia in 2001 was probably due to the introduction of a new DEN-1 virus from Southeast Asia, since the minimum nucleotide divergence was 3.3% with A88, the Indonesian strain isolated in 1988 in Jakarta.

  18. Challenge of chimpanzees (Pan troglodytes) immunized with human immunodeficiency virus envelope glycoprotein gp120.

    PubMed Central

    Arthur, L O; Bess, J W; Waters, D J; Pyle, S W; Kelliher, J C; Nara, P L; Krohn, K; Robey, W G; Langlois, A J; Gallo, R C

    1989-01-01

    The human immunodeficiency virus type 1 (HIV-1), the causative agent of acquired immunodeficiency syndrome, infects humans and chimpanzees. To determine the efficacy of immunization for preventing infection, chimpanzees were immunized with gp120 purified from human T-cell lymphotrophic virus type IIIB (HTLV-IIIB)-infected cell membranes and challenged with the homologous virus, HTLV-IIIB. A challenge stock of HTLV-IIIB was prepared by using unconcentrated HTLV-IIIB produced in H9 cells. The titer of the virus from this stock on human and chimpanzee peripheral blood mononuclear cells and in human lymphoid cell lines was determined; a cell culture infectivity of 10(4) was assigned. All chimpanzees inoculated intravenously with 40 cell culture infectious units or more became infected, as demonstrated by virus isolation and seroconversion. One of two chimpanzees inoculated with 4 cell culture infectious units became infected. Chimpanzees immunized with gp120 formulated in alum developed antibodies which precipitated gp120 and neutralized HTLV-IIIB. Peripheral blood mononuclear cells from gp120-vaccinated and HIV-infected animals showed a significantly greater response in proliferation assays with HIV proteins than did peripheral blood mononuclear cells from nonvaccinated and non-HIV-infected chimpanzees. Two of the gp120-alum-immunized chimpanzees were challenged with virus from the HTLV-IIIB stock. One animal received 400 cell culture infectious units, and one received 40 infectious units. Both animals became infected with HIV, indicating that the immune response elicited by immunization with gp120 formulated in alum was not effective in preventing infection with HIV-1. PMID:2555541

  19. Expression and characterization of genetically engineered human immunodeficiency virus-like particles containing modified envelope glycoproteins: implications for development of a cross-protective AIDS vaccine.

    PubMed

    Rovinski, B; Haynes, J R; Cao, S X; James, O; Sia, C; Zolla-Pazner, S; Matthews, T J; Klein, M H

    1992-07-01

    Noninfectious human immunodeficiency virus type 1 (HIV-1) viruslike particles containing chimeric envelope glycoproteins were expressed in mammalian cells by using inducible promoters. We engineered four expression vectors in which a synthetic oligomer encoding gp120 residues 306 to 328 (amino acids YNKRKRIHIGP GRAFYTTKNIIG) from the V3 loop of the MN viral isolate was inserted at various positions within the endogenous HIV-1LAI env gene. Expression studies revealed that insertion of the heterologous V3(MN) loop segment at two different locations within the conserved region 2 (C2) of gp120, either 173 or 242 residues away from the N terminus of the mature subunit, resulted in the secretion of fully assembled HIV-like particles containing chimeric LAI/MN envelope glycoproteins. Both V3 loop epitopes were recognized by loop-specific neutralizing antibodies. However, insertion of the V3(MN) loop segment into other regions of gp120 led to the production of envelope-deficient viruslike particles. Immunization with HIV-like particles containing chimeric envelope proteins induced specific antibody responses against both the autologous and heterologous V3 loop epitopes, including cross-neutralizing antibodies against the HIV-1LAI and HIV-1MN isolates. This study, therefore, demonstrates the feasibility of genetically engineering optimized HIV-like particles capable of eliciting cross-neutralizing antibodies. PMID:1602531

  20. HIV-1 tropism for the central nervous system: Brain-derived envelope glycoproteins with lower CD4 dependence and reduced sensitivity to a fusion inhibitor

    SciTech Connect

    Martin-Garcia, Julio . E-mail: julio.martin-garcia@drexelmed.edu; Cao, Wei; Varela-Rohena, Angel; Plassmeyer, Matthew L.; Gonzalez-Scarano, Francisco

    2006-03-01

    We previously described envelope glycoproteins of an HIV-1 isolate adapted in vitro for growth in microglia that acquired a highly fusogenic phenotype and lower CD4 dependence, as well as resistance to inhibition by anti-CD4 antibodies. Here, we investigated whether similar phenotypic changes are present in vivo. Envelope clones from the brain and spleen of an HIV-1-infected individual with neurological disease were amplified, cloned, and sequenced. Phylogenetic analysis demonstrated clustering of sequences according to the tissue of origin, as expected. Functional clones were then used in cell-to-cell fusion assays to test for CD4 and co-receptor utilization and for sensitivity to various antibodies and inhibitors. Both brain- and spleen-derived envelope clones mediated fusion in cells expressing both CD4 and CCR5 and brain envelopes also used CCR3 as co-receptor. We found that the brain envelopes had a lower CD4 dependence, since they efficiently mediated fusion in the presence of low levels of CD4 on the target cell membrane, and they were significantly more resistant to blocking by anti-CD4 antibodies than the spleen-derived envelopes. In contrast, we observed no difference in sensitivity to the CCR5 antagonist TAK-779. However, brain-derived envelopes were significantly more resistant than those from spleen to the fusion inhibitor T-1249 and concurrently showed slightly greater fusogenicity. Our results suggest an increased affinity for CD4 of brain-derived envelopes that may have originated from in vivo adaptation to replication in microglial cells. Interestingly, we note the presence of envelopes more resistant to a fusion inhibitor in the brain of an untreated, HIV-1-infected individual.

  1. Herpes simplex virus glycoproteins gB and gH function in fusion between the virion envelope and the outer nuclear membrane.

    PubMed

    Farnsworth, Aaron; Wisner, Todd W; Webb, Michael; Roller, Richard; Cohen, Gary; Eisenberg, Roselyn; Johnson, David C

    2007-06-12

    Herpesviruses must traverse the nuclear envelope to gain access to the cytoplasm and, ultimately, to exit cells. It is believed that herpesvirus nucleocapsids enter the perinuclear space by budding through the inner nuclear membrane (NM). To reach the cytoplasm these enveloped particles must fuse with the outer NM and the unenveloped capsids then acquire a second envelope in the trans-Golgi network. Little is known about the process by which herpesviruses virions fuse with the outer NM. Here we show that a herpes simplex virus (HSV) mutant lacking both the two putative fusion glycoproteins gB and gH failed to cross the nuclear envelope. Enveloped virions accumulated in the perinuclear space or in membrane vesicles that bulged into the nucleoplasm (herniations). By contrast, mutants lacking just gB or gH showed only minor or no defects in nuclear egress. We concluded that either HSV gB or gH can promote fusion between the virion envelope and the outer NM. It is noteworthy that fusion associated with HSV entry requires the cooperative action of both gB and gH, suggesting that the two types of fusion (egress versus entry) are dissimilar processes.

  2. Diverse specificity and effector function among human antibodies to HIV-1 envelope glycoprotein epitopes exposed by CD4 binding

    SciTech Connect

    Guan, Yongjun; Pazgier, Marzena; Sajadi, Mohammad M.; Kamin-Lewis, Roberta; Al-Darmarki, Salma; Flinko, Robin; Lovo, Elena; Wu, Xueji; Robinson, James E.; Seaman, Michael S.; Fouts, Timothy R.; Gallo, Robert C.; DeVico, Anthony L.; Lewis, George K.

    2012-12-13

    The HIV-1 envelope glycoprotein (Env) undergoes conformational transitions consequent to CD4 binding and coreceptor engagement during viral entry. The physical steps in this process are becoming defined, but less is known about their significance as targets of antibodies potentially protective against HIV-1 infection. Here we probe the functional significance of transitional epitope exposure by characterizing 41 human mAbs specific for epitopes exposed on trimeric Env after CD4 engagement. These mAbs recognize three epitope clusters: cluster A, the gp120 face occluded by gp41 in trimeric Env; cluster B, a region proximal to the coreceptor-binding site (CoRBS) and involving the V1/V2 domain; and cluster C, the coreceptor-binding site. The mAbs were evaluated functionally by antibody-dependent, cell-mediated cytotoxicity (ADCC) and for neutralization of Tiers 1 and 2 pseudoviruses. All three clusters included mAbs mediating ADCC. However, there was a strong potency bias for cluster A, which harbors at least three potent ADCC epitopes whose cognate mAbs have electropositive paratopes. Cluster A epitopes are functional ADCC targets during viral entry in an assay format using virion-sensitized target cells. In contrast, only cluster C contained epitopes that were recognized by neutralizing mAbs. There was significant diversity in breadth and potency that correlated with epitope fine specificity. In contrast, ADCC potency had no relationship with neutralization potency or breadth for any epitope cluster. In conclusion, Fc-mediated effector function and neutralization coselect with specificity in anti-Env antibody responses, but the nature of selection is distinct for these two antiviral activities.

  3. Diverse specificity and effector function among human antibodies to HIV-1 envelope glycoprotein epitopes exposed by CD4 binding

    DOE PAGESBeta

    Guan, Yongjun; Pazgier, Marzena; Sajadi, Mohammad M.; Kamin-Lewis, Roberta; Al-Darmarki, Salma; Flinko, Robin; Lovo, Elena; Wu, Xueji; Robinson, James E.; Seaman, Michael S.; et al

    2012-12-13

    The HIV-1 envelope glycoprotein (Env) undergoes conformational transitions consequent to CD4 binding and coreceptor engagement during viral entry. The physical steps in this process are becoming defined, but less is known about their significance as targets of antibodies potentially protective against HIV-1 infection. Here we probe the functional significance of transitional epitope exposure by characterizing 41 human mAbs specific for epitopes exposed on trimeric Env after CD4 engagement. These mAbs recognize three epitope clusters: cluster A, the gp120 face occluded by gp41 in trimeric Env; cluster B, a region proximal to the coreceptor-binding site (CoRBS) and involving the V1/V2 domain;more » and cluster C, the coreceptor-binding site. The mAbs were evaluated functionally by antibody-dependent, cell-mediated cytotoxicity (ADCC) and for neutralization of Tiers 1 and 2 pseudoviruses. All three clusters included mAbs mediating ADCC. However, there was a strong potency bias for cluster A, which harbors at least three potent ADCC epitopes whose cognate mAbs have electropositive paratopes. Cluster A epitopes are functional ADCC targets during viral entry in an assay format using virion-sensitized target cells. In contrast, only cluster C contained epitopes that were recognized by neutralizing mAbs. There was significant diversity in breadth and potency that correlated with epitope fine specificity. In contrast, ADCC potency had no relationship with neutralization potency or breadth for any epitope cluster. In conclusion, Fc-mediated effector function and neutralization coselect with specificity in anti-Env antibody responses, but the nature of selection is distinct for these two antiviral activities.« less

  4. Evidence for immune selection of hepatitis C virus (HCV) putative envelope glycoprotein variants: potential role in chronic HCV infections.

    PubMed Central

    Weiner, A J; Geysen, H M; Christopherson, C; Hall, J E; Mason, T J; Saracco, G; Bonino, F; Crawford, K; Marion, C D; Crawford, K A

    1992-01-01

    E2/nonstructural protein 1, the putative envelope glycoprotein (gp72) of HCV, possesses an N-terminal hypervariable (E2 HV) domain from amino acids 384 to 414 of unknown significance. The high degree of amino acid sequence variation in the E2 HV domain appears to be comparable to that observed in the human immunodeficiency virus type 1 gp120 V3 domain. This observation and the observation that the HCV E2 HV domain lacks conserved secondary structure imply that, like the V3 loop of human immunodeficiency virus 1 gp120, the N-terminal E2 region may encode protective epitopes that are subject to immune selection. Antibody-epitope binding studies revealed five isolate-specific linear epitopes located in the E2 HV region. These results suggest that the E2 HV domain is a target for the human immune response and that, in addition to the three major groups of HCV, defined by nucleotide and amino acid sequence identity among HCV isolates, E2 HV-specific subgroups also exist. Analysis of the partial or complete E2 sequences of two individuals indicated that E2 HV variants can either coexist simultaneously in a single individual or that a particular variant may predominate during different episodes of disease. In the latter situation, we found one individual who developed antibodies to a subregion of the E2 HV domain (amino acids 396-407) specific to a variant that was predominant during one major episode of hepatitis but who lacked detectable antibodies to the corresponding region of a second variant that was predominant during a later episode of disease. The data suggest that the variability in the E2 HV domain may result from immune selection. The findings of this report could impact vaccine strategies and drug therapy programs designed to control and eliminate HCV. PMID:1314389

  5. Stabilizing Exposure of Conserved Epitopes by Structure Guided Insertion of Disulfide Bond in HIV-1 Envelope Glycoprotein

    PubMed Central

    Sarkar, Pampi; Labranche, Celia; Go, Eden P.; Clark, Daniel F.; Sun, Yide; Nandi, Avishek; Hartog, Karin; Desaire, Heather; Montefiori, David; Carfi, Andrea; Srivastava, Indresh K.; Barnett, Susan W.

    2013-01-01

    Entry of HIV-1 into target cells requires binding of the viral envelope glycoprotein (Env) to cellular receptors and subsequent conformational changes that culminates in fusion of viral and target cell membranes. Recent structural information has revealed that these conformational transitions are regulated by three conserved but potentially flexible layers stacked between the receptor-binding domain (gp120) and the fusion arm (gp41) of Env. We hypothesized that artificial insertion of a covalent bond will ‘snap’ Env into a conformation that is less mobile and stably expose conserved sites. Therefore, we analyzed the interface between these gp120 layers (layers 1, 2 and 3) and identified residues that may form disulfide bonds when substituted with cysteines. We subsequently probed the structures of the resultant mutant gp120 proteins by assaying their binding to a variety of ligands using Surface Plasmon Resonance (SPR) assay. We found that a single disulfide bond strategically inserted between the highly conserved layers 1 and 2 (C65-C115) is able to ‘lock’ gp120 in a CD4 receptor bound conformation (in the absence of CD4), as indicated by the lower dissociation constant (Kd) for the CD4-induced (CD4i) epitope binding 17b antibody. When disulfide-stabilized monomeric (gp120) and trimeric (gp140) Envs were used to immunize rabbits, they were found to elicit a higher proportion of antibodies directed against both CD4i and CD4 binding site epitopes than the wild-type proteins. These results demonstrate that structure-guided stabilization of inter-layer interactions within HIV-1 Env can be used to expose conserved epitopes and potentially overcome the sequence diversity of these molecules. PMID:24146829

  6. Epstein-Barr virus envelope glycoprotein gp350 induces NF-kappaB activation and IL-1beta synthesis in human monocytes-macrophages involving PKC and PI3-K.

    PubMed

    D'Addario, M; Ahmad, A; Xu, J W; Menezes, J

    1999-12-01

    Epstein-Barr virus (EBV) is a highly immunotropic human herpesvirus with oncogenic potential and is involved in numerous pathologies. EBV utilizes its major envelope glycoprotein gp350 to bind to its receptor CR2/CD21 on target cells for initiating the infection. We have previously shown that EBV is able to modulate transcription and translation of a number of cytokine genes via its gp350-mediated binding to this receptor. However, the effects of the binding of purified gp350 to CR2/CD21 on plastic-adherent monocyte-macrophages (AMM) have not been investigated. These cells are a rich source of potent proinflammatory and immune-modulating cytokines, and express low levels of CR2/CD21. We show here for the first time that recombinant gp350 (rgp350) causes production of the potent proinflammatory cytokine IL-1beta in human AMM. Surprisingly, rgp350 is comparable in this capacity to the phorbol ester 12-0-tetradecanoylphorbol 13-acetate. This induction of IL-1beta production was accompanied by increased steady-state levels of its mRNA in gp350-treated AMM, and was dependent on the specific binding of rgp350 to the EBV receptor CR2/CD21. We also show that the signaling pathways resulting in the induction of IL-1beta synthesis by rgp350 required protein kinase C and phosphatidylinositol 3,4,5 triphosphate kinase activities and occurred via activation of the NF-kappaB family of transcription factors.-D'Addario, M., Ahmad, A., Xu, J. W., Menezes, J. Epstein-Barr virus envelope glycoprotein gp350 induces NF-kappaB activation and IL-1beta synthesis in human monocytes-macrophages involving PKC and PI3-K. PMID:10593868

  7. Primary sequence of the envelope glycoprotein of a dengue type 2 virus isolated from patient with dengue hemorrhagic fever and encephalopathy.

    PubMed

    Sistayanarain, A; Maneekarn, N; Polprasert, B; Sirisanthana, V; Makino, Y; Fukunaga, T; Sittisombut, N

    1996-06-01

    Dengue viruses exist in nature as a collection of highly similar but not identical members (quasispecies). In order to correlate the presence of viral quasispecies with rare occurrence of unusual clinical manifestations in dengue-infected individuals, a dengue type 2 virus was isolated from the peripheral blood of a 12-year-old boy who presented with fever, headache, drowsiness and tonic seizure of the left arm, and subsequently manifested symptoms and signs of dengue hemorrhagic fever. Analysis of the envelope glycoprotein sequence of the encephalopathy-associated virus and two other dengue type 2 viruses from the same epidemic season in Chiang Mai, Thailand revealed that all three viruses belonged to the subtype IIIa of the five-subtype phylogenetic nomenclature system for dengue type 2 virus. The encephalopathy-associated dengue virus was more divergent from the others and was characterized by an Ala-->Val substitution at the position 173 of the envelope glycoprotein. This substitution mapped to the central domain 1 which was not known to be involved directly in envelope-receptor interaction.

  8. Structural and Functional Analysis of Interhelical Interactions in the Human Immunodeficiency Virus Type 1 gp41 Envelope Glycoprotein by Alanine-Scanning Mutagenesis

    PubMed Central

    Lu, Min; Stoller, Marisa O.; Wang, Shilong; Liu, Jie; Fagan, Melinda B.; Nunberg, Jack H.

    2001-01-01

    Membrane fusion by human immunodeficiency virus type 1 (HIV-1) is promoted by the refolding of the viral envelope glycoprotein into a fusion-active conformation. The structure of the gp41 ectodomain core in its fusion-active state is a trimer of hairpins in which three antiparallel carboxyl-terminal helices pack into hydrophobic grooves on the surface of an amino-terminal trimeric coiled coil. In an effort to identify amino acid residues in these grooves that are critical for gp41 activation, we have used alanine-scanning mutagenesis to investigate the importance of individual side chains in determining the biophysical properties of the gp41 core and the membrane fusion activity of the gp120-gp41 complex. Alanine substitutions at Leu-556, Leu-565, Val-570, Gly-572, and Arg-579 positions severely impaired membrane fusion activity in envelope glycoproteins that were for the most part normally expressed. Whereas alanine mutations at Leu-565 and Val-570 destabilized the trimer-of-hairpins structure, mutations at Gly-572 and Arg-579 led to the formation of a stable gp41 core. Our results suggest that the Leu-565 and Val-570 residues are important determinants of conserved packing interactions between the amino- and carboxyl-terminal helices of gp41. We propose that the high degree of sequence conservation at Gly-572 and Arg-579 may result from selective pressures imposed by prefusogenic conformations of the HIV-1 envelope glycoprotein. Further analysis of the gp41 activation process may elucidate targets for antiviral intervention. PMID:11602754

  9. Learning the Relationship between the Primary Structure of HIV Envelope Glycoproteins and Neutralization Activity of Particular Antibodies by Using Artificial Neural Networks

    PubMed Central

    Buiu, Cătălin; Putz, Mihai V.; Avram, Speranta

    2016-01-01

    The dependency between the primary structure of HIV envelope glycoproteins (ENV) and the neutralization data for given antibodies is very complicated and depends on a large number of factors, such as the binding affinity of a given antibody for a given ENV protein, and the intrinsic infection kinetics of the viral strain. This paper presents a first approach to learning these dependencies using an artificial feedforward neural network which is trained to learn from experimental data. The results presented here demonstrate that the trained neural network is able to generalize on new viral strains and to predict reliable values of neutralizing activities of given antibodies against HIV-1. PMID:27727189

  10. Tryptophan dendrimers that inhibit HIV replication, prevent virus entry and bind to the HIV envelope glycoproteins gp120 and gp41.

    PubMed

    Rivero-Buceta, Eva; Doyagüez, Elisa G; Colomer, Ignacio; Quesada, Ernesto; Mathys, Leen; Noppen, Sam; Liekens, Sandra; Camarasa, María-José; Pérez-Pérez, María-Jesús; Balzarini, Jan; San-Félix, Ana

    2015-12-01

    Dendrimers containing from 9 to 18 tryptophan residues at the peryphery have been efficiently synthesized and tested against HIV replication. These compounds inhibit an early step of the replicative cycle of HIV, presumably virus entry into its target cell. Our data suggest that HIV inhibition can be achieved by the preferred interaction of the compounds herein described with glycoproteins gp120 and gp41 of the HIV envelope preventing interaction between HIV and the (co)receptors present on the host cells. The results obtained so far indicate that 9 tryptophan residues on the periphery are sufficient for efficient gp120/gp41 binding and anti-HIV activity.

  11. A quantitative test to estimate neutralizing antibodies to the hepatitis C virus: cytofluorimetric assessment of envelope glycoprotein 2 binding to target cells.

    PubMed Central

    Rosa, D; Campagnoli, S; Moretto, C; Guenzi, E; Cousens, L; Chin, M; Dong, C; Weiner, A J; Lau, J Y; Choo, Q L; Chien, D; Pileri, P; Houghton, M; Abrignani, S

    1996-01-01

    Hepatitis C virus (HCV) is a major cause of chronic hepatitis. The virus does not replicate efficiently in cell cultures, and it is therefore difficult to assess infection-neutralizing antibodies and to evaluate protective immunity in vitro. To study the binding of the HCV envelope to cell-surface receptors, we developed an assay to assess specific binding of recombinant envelope proteins to human cells and neutralization thereof. HCV recombinant envelope proteins expressed in various systems were incubated with human cells, and binding was assessed by flow cytometry using anti-envelope antibodies. Envelope glycoprotein 2 (E2) expressed in mammalian cells, but not in yeast or insect cells, binds human cells with high affinity (Kd approximately 10(-8) M). We then assessed antibodies able to neutralize E2 binding in the sera of both vaccinated and carrier chimpanzees, as well as in the sera of humans infected with various HCV genotypes. Vaccination with recombinant envelope proteins expressed in mammalian cells elicited high titers of neutralizing antibodies that correlated with protection from HCV challenge. HCV infection does not elicit neutralizing antibodies in most chimpanzees and humans, although low titers of neutralizing antibodies were detectable in a minority of infections. The ability to neutralize binding of E2 derived from the HCV-1 genotype was equally distributed among sera from patients infected with HCV genotypes 1, 2, and 3, demonstrating that binding of E2 is partly independent of E2 hypervariable regions. However, a mouse monoclonal antibody raised against the E2 hypervariable region 1 can partially neutralize binding of E2, indicating that at least two neutralizing epitopes, one of which is hypervariable, should exist on the E2 protein. The neutralization-of-binding assay described will be useful to study protective immunity to HCV infection and for vaccine development. PMID:8700831

  12. Effect of Trimerization Motifs on Quaternary Structure, Antigenicity, and Immunogenicity of a Non-cleavable HIV-1 gp140 Envelope Glycoprotein

    PubMed Central

    Du, Sean X.; Idiart, Rebecca J.; Mariano, Ellaine B.; Chen, Helen; Jiang, Peifeng; Xu, Li; Ostrow, Kristin M.; Wrin, Terri; Phung, Pham; Binley, James M.; Petropoulos, Christos J.; Ballantyne, John A.; Whalen, Robert G.

    2009-01-01

    The external domains of the HIV-1 envelope glycoprotein (gp120 and the gp41 ectodomain, collectively known as gp140) contain all known viral neutralization epitopes. Various strategies have been used to create soluble trimers of the envelope to mimic the structure of the native viral protein, including mutation of the gp120-gp41 cleavage site, introduction of disulfide bonds, and fusion to heterologous trimerization motifs. We compared the effects on quaternary structure, antigenicity, and immunogenicity of three such motifs: T4 fibritin, a GCN4 variant, and the E. coli aspartate transcarbamoylase catalytic subunit. Fusion of each motif to the C-terminus of a non-cleavable JRCSF gp140(-) envelope protein led to enhanced trimerization but had limited effects on the antigenic profile and CD4 binding ability of the trimers. Immunization of rabbits provided no evidence that the trimerized gp140(-) constructs induced significantly improved neutralizing antibodies to several HIV-1 pseudoviruses, compared to gp140 lacking a trimerization motif. However, modest differences in both binding specificity and neutralizing antibody responses were observed among the various immunogens. PMID:19815247

  13. Naphthalenedisulfonic acid derivatives inhibit HIV-1-induced cytopathogenesis, syncytia formation and virus-cell binding by interaction with the viral envelope glycoprotein

    SciTech Connect

    Mohan, P.; Schols, D.; De Clercq, E.; Shigeta, S.; Baba, M.

    1993-12-31

    Bis naphthalenedisulfonic acid analogs with biphenyl spacers have exhibited potent and selective inhibition of HIV-1 replication and giant cell formation. FACS analysis has revealed that these agents also inhibit viral binding to the target cell. Further mechanism of action studies by the FACA method demonstrate that the sulfonic acid analogs inhibit binding of anti-gp120 monoclonal antibody to the viral envelope of glycoprotein, gp120. Binding of OKT4A/Leu3a monoclonal antibody to the target cell CD4 receptor is not affected by these compounds. This investigation suggests that these naphthalenedisulfonic acid derivatives exert their anti-HIV-1 activity by inhibiting the gp120-CD4 interaction through binding of these agents to the viral gp120 antigen.

  14. Evolution Rescues Folding of Human Immunodeficiency Virus-1 Envelope Glycoprotein GP120 Lacking a Conserved Disulfide Bond

    PubMed Central

    Hsu, Shang-Te D.; van Anken, Eelco; Liscaljet, I. Marije; Dankers, Martijn; Bontjer, Ilja; Land, Aafke; Braakman, Ineke; Bonvin, Alexandre M.J.J.; Berkhout, Ben

    2008-01-01

    The majority of eukaryotic secretory and membrane proteins contain disulfide bonds, which are strongly conserved within protein families because of their crucial role in folding or function. The exact role of these disulfide bonds during folding is unclear. Using virus-driven evolution we generated a viral glycoprotein variant, which is functional despite the lack of an absolutely conserved disulfide bond that links two antiparallel β-strands in a six-stranded β-barrel. Molecular dynamics simulations revealed that improved hydrogen bonding and side chain packing led to stabilization of the β-barrel fold, implying that β-sheet preference codirects glycoprotein folding in vivo. Our results show that the interactions between two β-strands that are important for the formation and/or integrity of the β-barrel can be supported by either a disulfide bond or β-sheet favoring residues. PMID:18753405

  15. N-terminal substitutions in HIV-1 gp41 reduce the expression of non-trimeric envelope glycoproteins on the virus

    SciTech Connect

    Dey, Antu K.; David, Kathryn B.; Ray, Neelanjana; Ketas, Thomas J.; Klasse, Per J.; Doms, Robert W.; Moore, John P.

    2008-03-01

    The native, functional HIV-1 envelope glycoprotein (Env) complex is a trimer of two non-covalently associated subunits: the gp120 surface glycoprotein and the gp41 transmembrane glycoprotein. However, various non-functional forms of Env are present on virus particles and HIV-1-infected cells, some of which probably arise as the native complex decays. The aberrant forms include gp120-gp41 monomers and oligomers, as well as gp41 subunits from which gp120 has dissociated. The presence of non-functional Env creates binding sites for antibodies that do not recognize native Env complexes and that are, therefore, non-neutralizing. Non-native Env forms (monomers, dimers, tetramers and aggregates) can also arise when soluble gp140 proteins, lacking the cytoplasmic and transmembrane domains of gp41, are expressed for vaccine studies. We recently identified five amino acids in the gp41 N-terminal region (I535, Q543, S553, K567 and R588) that promote gp140 trimerization. We have now studied their influence on the function and antigenic properties of JR-FL Env expressed on the surfaces of pseudoviruses and Env-transfected cells. The 5 substitutions in gp41 reduce the expression of non-trimeric gp160s, without affecting trimer levels. Pseudovirions bearing the mutant Env are fully infectious with similar kinetics of Env-mediated fusion. Various non-neutralizing antibodies bind less strongly to the Env mutant, but neutralizing antibody binding is unaffected. Hence the gp41 substitutions do not adversely affect Env structure, supporting their use for making new Env-based vaccines. The mutant Env might also help in studies intended to correlate antibody binding to virus neutralization. Of note is that the 5 residues are much more frequent, individually or collectively, in viruses from subtypes other than B.

  16. Ninety-five- and 25-kDa fragments of the human immunodeficiency virus envelope glycoprotein gp120 bind to the CD4 receptor

    SciTech Connect

    Nygren, A.; Bergman, T.; Matthews, T.; Joernvall, H.; Wigzell, H.

    1988-09-01

    Iodine-125-labeled gp120 (120-kDa envelope glycoprotein) from the BH10 isolate of human immunodeficiency virus is cleaved to a limited extend with the glutamate-specific protease from Staphylococcus aureus. After disulfide bond reduction, fragments with approximate molecular masses of 95, 60, 50, and 25 kDa are produced. Tests for binding to CD4-positive cells show that only two fragments, the 95- and 25- kDa peptides, are observed in cleavage products that retain the selective binding capacity of gp120. Radiosequence analysis of the fragments after sodium dodecyl sulfate/polyacrylamide gel electrophoresis and electroblotting demonstrates that the 95-kDa fragment lacks the N-terminal region of gp120 and starts at position 143 of the mature envelope protein. The 50-kDa fragment starts at the same position. The 25-kDa binding fragment was similarly deduced to be generated as a small fragment from a cleavage site in the C-terminal part of gp120. The identifications of these fragments demonstrate that radiosequence analysis utilizing /sup 125/I-labeled tyrosine residues can function as a useful and reliable method for small-scale determination of cleavage sites in proteins. Combined, the data suggest domain-like subdivisions of gp120, define at least two intervening segments especially sensitive to proteolytic cleavage, and demonstrate the presence of a functional region for receptor binding in the C-terminal part of the molecule.

  17. Several N-Glycans on the HIV Envelope Glycoprotein gp120 Preferentially Locate Near Disulphide Bridges and Are Required for Efficient Infectivity and Virus Transmission.

    PubMed

    Mathys, Leen; Balzarini, Jan

    2015-01-01

    The HIV envelope glycoprotein gp120 contains nine disulphide bridges and is highly glycosylated, carrying on average 24 N-linked glycans. Using a probability calculation, we here demonstrate that there is a co-localization of disulphide bridges and N-linked glycans in HIV-1 gp120, with a predominance of N-linked glycans in close proximity to disulphide bridges, at the C-terminal side of the involved cysteines. Also, N-glycans are frequently found immediately adjacent to disulphide bridges in gp120 at the N-terminal side of the involved cysteines. In contrast, N-glycans at positions close to, but not immediately neighboring disulphide bridges seem to be disfavored at the N-terminal side of the involved cysteines. Such a pronounced co-localization of disulphide bridges and N-glycans was also found for the N-glycans on glycoprotein E1 of the hepatitis C virus (HCV) but not for other heavily glycosylated proteins such as E2 from HCV and the surface GP from Ebola virus. The potential functional role of the presence of N-glycans near disulphide bridges in HIV-1 gp120 was studied using site-directed mutagenesis, either by deleting conserved N-glycans or by inserting new N-glycosylation sites near disulphide bridges. The generated HIV-1NL4.3 mutants were subjected to an array of assays, determining the envelope glycoprotein levels in mutant viral particles, their infectivity and the capture and transmission efficiencies of mutant virus particles by DC-SIGN. Three N-glycans located nearby disulphide bridges were found to be crucial for the preservation of several of these functions of gp120. In addition, introduction of new N-glycans upstream of several disulphide bridges, at locations where there was a significant absence of N-glycans in a broad variety of virus strains, was found to result in a complete loss of viral infectivity. It was shown that the N-glycan environment around well-defined disulphide bridges of gp120 is highly critical to allow efficient viral infection

  18. Several N-Glycans on the HIV Envelope Glycoprotein gp120 Preferentially Locate Near Disulphide Bridges and Are Required for Efficient Infectivity and Virus Transmission

    PubMed Central

    Mathys, Leen; Balzarini, Jan

    2015-01-01

    The HIV envelope glycoprotein gp120 contains nine disulphide bridges and is highly glycosylated, carrying on average 24 N-linked glycans. Using a probability calculation, we here demonstrate that there is a co-localization of disulphide bridges and N-linked glycans in HIV-1 gp120, with a predominance of N-linked glycans in close proximity to disulphide bridges, at the C-terminal side of the involved cysteines. Also, N-glycans are frequently found immediately adjacent to disulphide bridges in gp120 at the N-terminal side of the involved cysteines. In contrast, N-glycans at positions close to, but not immediately neighboring disulphide bridges seem to be disfavored at the N-terminal side of the involved cysteines. Such a pronounced co-localization of disulphide bridges and N-glycans was also found for the N-glycans on glycoprotein E1 of the hepatitis C virus (HCV) but not for other heavily glycosylated proteins such as E2 from HCV and the surface GP from Ebola virus. The potential functional role of the presence of N-glycans near disulphide bridges in HIV-1 gp120 was studied using site-directed mutagenesis, either by deleting conserved N-glycans or by inserting new N-glycosylation sites near disulphide bridges. The generated HIV-1NL4.3 mutants were subjected to an array of assays, determining the envelope glycoprotein levels in mutant viral particles, their infectivity and the capture and transmission efficiencies of mutant virus particles by DC-SIGN. Three N-glycans located nearby disulphide bridges were found to be crucial for the preservation of several of these functions of gp120. In addition, introduction of new N-glycans upstream of several disulphide bridges, at locations where there was a significant absence of N-glycans in a broad variety of virus strains, was found to result in a complete loss of viral infectivity. It was shown that the N-glycan environment around well-defined disulphide bridges of gp120 is highly critical to allow efficient viral infection

  19. Characterization of Envelope Glycoprotein Mutants for Human T-Cell Leukemia Virus Type 1 Infectivity and Immortalization

    PubMed Central

    Tsukahara, Tomonori; Wielgosz, Matthew M.; Ratner, Lee

    2001-01-01

    The human T-cell leukemia virus type 1 (HTLV-1) envelope protein is required for virus spread. This study further characterizes the role of the envelope protein in HTLV-1 immortalization. Viruses with single amino acid substitutions within the SU protein at residue 75, 81, 95, 101, 105, or 195 or with a C-terminal cytoplasmic domain truncation (CT), as well as an envelope-null (EN) virus, were generated within an infectious molecular clone, ACH. Transfection of 293T cells resulted in the release of similar amounts of virus particles from all of the mutants as determined by p19 enzyme-linked immunosorbent assay and immunoblot analysis of Gag in cell lysates and supernatants. The virus particles from all mutants except ACH-101, ACH-CT, and ACH-EN were infectious for B5 macaque cells in cell-free and cell-to-cell transmission assays and were capable of immortalizing transfected CD4+ lymphocytes. These results indicate that HTLV-1 spread is required for immortalization. PMID:11533220

  20. Identification of Conserved Residues in Hepatitis C Virus Envelope Glycoprotein E2 That Modulate Virus Dependence on CD81 and SRB1 Entry Factors

    PubMed Central

    Lavie, Muriel; Sarrazin, Stéphane; Montserret, Roland; Descamps, Véronique; Baumert, Thomas F.; Duverlie, Gilles; Séron, Karin; Penin, François

    2014-01-01

    ABSTRACT In spite of the high variability of its sequence, hepatitis C virus (HCV) envelope glycoprotein E2 contains several conserved regions. In this study, we explored the structural and functional features of the highly conserved E2 segment from amino acid (aa) 502 to 520, which had been proposed as a fusion peptide and shown to strongly overlap a potential conserved neutralizing epitope. For this purpose, we used reverse genetics to introduce point mutations within this region, and we characterized the phenotypes of these mutants in the light of the recently published structure of E2. The functional analyses showed that their phenotypes are in agreement with the positions of the corresponding residues in the E2 crystal structure. In contrast, our data ruled out the involvement of this region in membrane fusion, and they indicate that alternative conformations would be necessary to expose the potential neutralizing epitope present in this segment. Of particular interest, we identified three specific mutations (Y507L, V514A, and V515A) located within this neutralizing epitope which only mildly reduced infectivity and showed no assembly defect. These mutations modulated HCV dependence on the viral receptor SRB1, and/or they also modulated virion sensitivity to neutralizing antibodies. Importantly, their characterization also showed that amino acids Y507, V514, and V515 contribute to E2 interaction with HCV receptor CD81. In conclusion, our data show that the highly conserved E2 segment from aa 502 to 520 plays a key role in cell entry by influencing the association of the viral particle with coreceptors and neutralizing antibodies. IMPORTANCE Hepatitis C virus (HCV) envelope proteins E1 and E2 exhibit sequence variability. However, some segments of the envelope proteins are highly conserved, suggesting that these sequences play a key role at some steps of the HCV life cycle. In this work, we characterized the function and structure of a highly conserved E2 region

  1. Identification of amino acid changes in the envelope glycoproteins of bovine viral diarrhea viruses isolated from alpaca that may be involved in host adaptation.

    PubMed

    Neill, John D; Dubovi, Edward J; Ridpath, Julia F

    2015-09-30

    Bovine viral diarrhea viruses (BVDV) are most commonly associated with infections of cattle. However, BVDV are often isolated from closely related ruminants with a number of BVDV-1b viruses being isolated from alpacas that were both acutely and persistently infected. The complete nucleotide sequence of the open reading frame of eleven alpaca-adapted BVDV isolates and the region encoding the envelope glycoproteins of an additional three isolates were determined. With the exception of one, all alpaca isolates were >99.2% similar at the nucleotide level. The Hercules isolate was more divergent, with 95.7% sequence identity to the other viruses. Sequence similarity of the 14 viruses indicated they were isolates of a single BVDV strain that had adapted to and were circulating through alpaca herds. Hercules was a more distantly related strain that has been isolated only once in Canada and represented a separate adaptation event that possessed the same adaptive changes. Comparison of amino acid sequences of alpaca and bovine-derived BVDV strains revealed three regions with amino acid sequences unique to all alpaca isolates. The first contained two small in-frame deletions near the N-terminus of the E2 glycoprotein. The second was found near the C-terminus of the E2 protein where four altered amino acids were located within a 30 amino acid domain that participates in E2 homodimerization. The third region contained three variable amino acids in the C-terminus of the E(rns) within the amphipathic helix membrane anchor. These changes were found in the polar side of the amphipathic helix and resulted in an increased charge within the polar face. Titration of bovine and alpaca viruses in both bovine and alpaca cells indicated that with increased charge in the amphipathic helix, the ability to infect alpaca cells also increased.

  2. Single Amino Acid Substitution N659D in HIV-2 Envelope Glycoprotein (Env) Impairs Viral Release and Hampers BST-2 Antagonism

    PubMed Central

    Dufrasne, François E.; Lombard, Catherine; Goubau, Patrick; Ruelle, Jean

    2016-01-01

    BST-2 or tetherin is a host cell restriction factor that prevents the budding of enveloped viruses at the cell surface, thus impairing the viral spread. Several countermeasures to evade this antiviral factor have been positively selected in retroviruses: the human immunodeficiency virus type 2 (HIV-2) relies on the envelope glycoprotein (Env) to overcome BST-2 restriction. The Env gp36 ectodomain seems involved in this anti-tetherin activity, however residues and regions interacting with BST-2 are not clearly defined. Among 32 HIV-2 ROD Env mutants tested, we demonstrated that the asparagine residue at position 659 located in the gp36 ectodomain is mandatory to exert the anti-tetherin function. Viral release assays in cell lines expressing BST-2 showed a loss of viral release ability for the HIV-2 N659D mutant virus compared to the HIV-2 wild type virus. In bst-2 inactivated H9 cells, those differences were lost. Subtilisin treatment of infected cells demonstrated that the N659D mutant was more tethered at the cell surface. Förster resonance energy transfer (FRET) experiments confirmed a direct molecular link between Env and BST-2 and highlighted an inability of the mutant to bind BST-2. We also tested a virus presenting a truncation of 109 amino acids at the C-terminal part of Env, a cytoplasmic tail partial deletion that is spontaneously selected in vitro. Interestingly, viral release assays and FRET experiments indicated that a full Env cytoplasmic tail was essential in BST-2 antagonism. In HIV-2 infected cells, an efficient Env-mediated antagonism of BST-2 is operated through an intermolecular link involving the asparagine 659 residue as well as the C-terminal part of the cytoplasmic tail. PMID:27754450

  3. Enhanced CD4+ cellular apoptosis by CCR5-restricted HIV-1 envelope glycoprotein variants from patients with progressive HIV-1 infection

    SciTech Connect

    Wade, Jessica; Sterjovski, Jasminka; Gray, Lachlan; Roche, Michael; Chiavaroli, Lisa; Ellett, Anne; Jakobsen, Martin R.; Cowley, Daniel; Fonseca Pereira, Candida da; Saksena, Nitin; Wang, Bin; Purcell, Damian F.J.; Karlsson, Ingrid; Fenyoe, Eva-Maria; Churchill, Melissa; Gorry, Paul R.

    2010-01-20

    CCR5-using (R5) human immunodeficiency virus type 1 (HIV-1) strains cause CD4+ T-cell loss in most infected individuals, but mechanisms underlying cytopathicity of R5 viruses are poorly understood. We investigated mechanisms contributing to R5 envelope glycoprotein (Env)-mediated cellular apoptosis by constructing a panel of retroviral vectors engineered to co-express GFP and R5 Envs derived from two HIV-1-infected subjects spanning asymptomatic (Early, E-R5 Envs) to late stages of infection (Late, L-R5 Envs). The L-R5 Envs induced significantly more cellular apoptosis than E-R5 Envs, but only in Env-expressing (GFP-positive) cells, and only in cells where CD4 and CCR5 levels were limiting. Studies with fusion-defective Env mutants showed induction of apoptosis required membrane-fusing events. Our results provide evidence for an intracellular mechanism of R5 Env-induced apoptosis of CD4+ cells that requires membrane fusion. Furthermore, they contribute to a better understanding of mechanisms involved in CD4+ T-cell loss in subjects experiencing progressive R5 HIV-1 infection.

  4. Design and evaluation of antiretroviral peptides corresponding to the C-terminal heptad repeat region (C-HR) of human immunodeficiency virus type 1 envelope glycoprotein gp41

    SciTech Connect

    Soonthornsata, Bongkot; Tian, Yu-Shi; Utachee, Piraporn; Sapsutthipas, Sompong; Isarangkura-na-ayuthaya, Panasda; Auwanit, Wattana; Takagi, Tatsuya; Ikuta, Kazuyoshi; Sawanpanyalert, Pathom; Kawashita, Norihito; Kameoka, Masanori

    2010-09-15

    Two {alpha}-helical heptad repeats, N-HR and C-HR, located in the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp41, play an important role in membrane fusion by forming a 6-helix bundle. C34, a peptide mimicking C-HR, inhibits the formation of the 6-helix bundle; thus, it has potential as a novel antiretroviral compound. In order to improve the inhibitory effect of C34 on HIV-1 replication, we designed new C34-derived peptides based on computational analysis of the stable conformation of the 6-helix bundle. Newly designed peptides showed a stronger inhibitory effect on the replication of recombinant viruses containing CRF01{sub A}E, subtype B or subtype C Env than C34 or a fusion inhibitor, T-20. In addition, these peptides inhibited the replication of a T-20-resistant virus. We propose that these peptides could be applied to develop novel antiretroviral compounds to inhibit the replication of various subtypes of HIV-1 as well as of T-20-resistant variants.

  5. A systematic analysis of a broadly neutralizing antibody AR3C epitopes on Hepatitis C virus E2 envelope glycoprotein and their cross-reactivity

    PubMed Central

    2015-01-01

    Background Hepatitis C virus (HCV) belongs to Flaviviridae family of viruses. HCV represents a major challenge to public health since its estimated global prevalence is 2.8% of the world's human population. The design and development of HCV vaccine has been hampered by rapid evolution of viral quasispecies resulting in antibody escape variants. HCV envelope glycoprotein E1 and E2 that mediate fusion and entry of the virus into host cells are primary targets of the host immune responses. Results Structural characterization of E2 core protein and a broadly neutralizing antibody AR3C together with E1E2 sequence information enabled the analysis of B-cell epitope variability. The E2 binding site by AR3C and its surrounding area were identified from the crystal structure of E2c-AR3C complex. We clustered HCV strains using the concept of "discontinuous motif/peptide" and classified B-cell epitopes based on their similarity. Conclusions The assessment of antibody neutralizing coverage provides insights into potential cross-reactivity of the AR3C neutralizing antibody across a large number of HCV variants. PMID:26681161

  6. Expression of Bovine Viral Diarrhea Virus Envelope Glycoprotein E2 in Yeast Pichia pastoris and its Application to an ELISA for Detection of BVDV Neutralizing Antibodies in Cattle.

    PubMed

    Behera, Sthita Pragnya; Mishra, Niranjan; Nema, Ram Kumar; Pandey, Pooja Dubey; Kalaiyarasu, Semmannan; Rajukumar, Katherukamem; Prakash, Anil

    2015-01-01

    The aim of this article is to express envelope glycoprotein E2 of bovine viral diarrhea virus (BVDV) in yeast Pichia pastoris and its utility as a diagnostic antigen in ELISA. The BVDV E2 gene was cloned into the pPICZαA vector followed by integration into the Pichia pastoris strain X-33 genome for methanol-induced expression. SDS-PAGE and Western blot results showed that the recombinant BVDV E2 protein (72 kDa) was expressed and secreted into the medium at a concentration of 40 mg/L of culture under optimized conditions. An indirect ELISA was then developed by using the yeast-expressed E2 protein. Preliminary testing of 300 field cattle serum samples showed that the E2 ELISA showed a sensitivity of 91.07% and a specificity of 92.02% compared to the reference virus neutralization test. The concordance between the E2 ELISA and VNT was 91.67%. This study demonstrates feasibility of BVDV E2 protein expression in yeast Pichia pastoris for the first time and its efficacy as an antigen in ELISA for detecting BVDV neutralizing antibodies in cattle.

  7. Computational prediction and analysis of envelop glycoprotein epitopes of DENV-2 and DENV-3 Pakistani isolates: a first step towards Dengue vaccine development.

    PubMed

    Amat-ur-Rasool, Hafsa; Saghir, Anam; Idrees, Muhammad

    2015-01-01

    Dengue fever of tropics is a mosquito transmitted devastating disease caused by dengue virus (DENV). There is no effective vaccine available, so far, against any of its four serotypes (DENV-1, DENV-2, DENV-3, and DENV-4). There is a need for the development of preventive and therapeutic vaccines against DENV to decrease the prevalence of dengue fever, especially in Pakistan. In this research, linear and conformational B-cell epitopes of envelope glycoprotein of DENV-2 and DENV-3 (the most prevalent serotypes in Pakistan) were predicted. We used Kolaskar and Tongaonkar method for linear epitope prediction, Emini's method for surface accessibility prediction and Karplus and Schulz's algorithm for flexibility determination. To propose three dimensional epitopes, the E proteins for both serotypes were homology modeled by using Phyre2 V 2.0 server, and ElliPro was used for the prediction of surface epitopes on their globular structure. Total 21 and 19 linear epitopes were predicted for DENV-2 and DENV-3 Pakistani isolates respectively. Whereas, 5 and 4 discontinuous epitopes were proposed for DENV-2 and DENV-3 Pakistani isolates respectively. Moreover, the values of surface accessibility, flexibility and solvent-accessibility can be helpful in analyzing vaccines against DENV-2 and DENV-3. In conclusion, the proposed continuous and discontinuous antigenic peptides can be valuable candidates for diagnostic and therapeutics of DENV.

  8. Comparative cellular processing of the human immunodeficiency virus (HIV-1) envelope glycoprotein gp160 by the mammalian subtilisin/kexin-like convertases.

    PubMed Central

    Vollenweider, F; Benjannet, S; Decroly, E; Savaria, D; Lazure, C; Thomas, G; Chrétien, M; Seidah, N G

    1996-01-01

    We present here the pulse and pulse-chase analysis of the biosynthesis of the envelope glycoprotein gp160 and its intracellular processing by the subtilisin/kexin-like convertases furin, PACE4, PC1, PC5 and its isoform PC5/6-B. We demonstrate that furin and to a much lesser extent PACE4, PC5/6-B and PC1 are candidate enzymes capable of processing gp160 intracellularly. Furthermore we show that furin can also process gp160/gp120 into gp77/gp53 products by cleavage at the sequence RIQR/GPGR just preceding the conserved GPGR structure found at the tip of the hypervariable V3 loop. The results show that processing into gp120 could occur at or before the trans-Golgi network (TGN) where sulphation of the oligosaccharide moieties of gp160 was detected. In contrast, the formation of gp77/gp53 by furin is a late event occurring after exit from the TGN. Our data also revealed that the alpha glucosidase I inhibitor N-butyldeoxynojirimycin, although affecting the oligosaccharide composition of gp160, does not impair the processing of either gp160 or gp120 by either furin or PACE4. Finally, the co-expression of the [Arg355, Arg358]-alpha-1-antitrypsin Portland variant was shown to potently inhibit the processing of both gp160 and gp120 by these convertases. PMID:8670066

  9. Human immunodeficiency virus type 1 envelope glycoprotein gp120 produces immune defects in CD4+ T lymphocytes by inhibiting interleukin 2 mRNA.

    PubMed Central

    Oyaizu, N; Chirmule, N; Kalyanaraman, V S; Hall, W W; Pahwa, R; Shuster, M; Pahwa, S

    1990-01-01

    Envelope glycoprotein gp120 of human immunodeficiency virus type 1 (HIV-1) is known to inhibit T-cell function, but little is known about the mechanisms of this immunosuppression. Pretreatment of a CD4+ tetanus toxoid-specific T-cell clone with soluble gp120 was found to exert a dose-dependent inhibition of soluble antigen-driven or anti-CD3 monoclonal antibody-driven proliferative response, interleukin 2 (IL-2) production, and surface IL-2 receptor (IL-2R) alpha-chain expression, all of which were reversed by the addition of exogenous IL-2. mRNA for the gene encoding IL-2 was suppressed by treatment with gp120, but IL-2R gene transcription was not inhibited. Bypass activation of the T-cell clone with phorbol 12-myristate 13-acetate plus ionomycin was unaffected by gp120 pretreatment. Thus, gp120-CD4 interaction interferes with an essential role of the CD4 molecule in signal transduction through the CD3-antigen receptor (Ti) complex. Such a mechanism of gp120-induced immunosuppression, if operative in vivo, could contribute to the depressed specific immune responses associated with HIV infection. Images PMID:2315327

  10. A conformational epitope mapped in the bovine herpesvirus type 1 envelope glycoprotein B by phage display and the HSV-1 3D structure.

    PubMed

    Almeida, Greyciele R; Goulart, Luiz Ricardo; Cunha-Junior, Jair P; Bataus, Luiz A M; Japolla, Greice; Brito, Wilia M E D; Campos, Ivan T N; Ribeiro, Cristina; Souza, Guilherme R L

    2015-08-01

    The selected dodecapeptide (1)DRALYGPTVIDH(12) from a phage-displayed peptide library and the crystal structure of the envelope glycoprotein B (Env gB) from Herpes Simplex Virus type 1 (HSV-1) led us to the identification of a new discontinuous epitope on the Bovine herpesvirus type 1 (BoHV-1) Env gB. In silico analysis revealed a short BoHV-1 gB motif ((338)YKRD(341)) within a epitope region, with a high similarity to the motifs shared by the dodecapeptide N-terminal region ((5)YxARD(1)) and HSV-1 Env gB ((326)YARD(329)), in which the (328)Arg residue is described to be a neutralizing antibody target. Besides the characterization of an antibody-binding site of the BoHV-1 Env gB, we have demonstrated that the phage-fused peptide has the potential to be used as a reagent for virus diagnosis by phage-ELISA assay, which discriminated BoHV-1 infected serum samples from negative ones. PMID:26267086

  11. Mechanistic Insight into Bunyavirus-Induced Membrane Fusion from Structure-Function Analyses of the Hantavirus Envelope Glycoprotein Gc

    PubMed Central

    Stettner, Eva; Jeffers, Scott Allen; Pérez-Vargas, Jimena; Pehau-Arnaudet, Gerard; Tortorici, M. Alejandra; Jestin, Jean-Luc; England, Patrick; Tischler, Nicole D.; Rey, Félix A.

    2016-01-01

    Hantaviruses are zoonotic viruses transmitted to humans by persistently infected rodents, giving rise to serious outbreaks of hemorrhagic fever with renal syndrome (HFRS) or of hantavirus pulmonary syndrome (HPS), depending on the virus, which are associated with high case fatality rates. There is only limited knowledge about the organization of the viral particles and in particular, about the hantavirus membrane fusion glycoprotein Gc, the function of which is essential for virus entry. We describe here the X-ray structures of Gc from Hantaan virus, the type species hantavirus and responsible for HFRS, both in its neutral pH, monomeric pre-fusion conformation, and in its acidic pH, trimeric post-fusion form. The structures confirm the prediction that Gc is a class II fusion protein, containing the characteristic β-sheet rich domains termed I, II and III as initially identified in the fusion proteins of arboviruses such as alpha- and flaviviruses. The structures also show a number of features of Gc that are distinct from arbovirus class II proteins. In particular, hantavirus Gc inserts residues from three different loops into the target membrane to drive fusion, as confirmed functionally by structure-guided mutagenesis on the HPS-inducing Andes virus, instead of having a single “fusion loop”. We further show that the membrane interacting region of Gc becomes structured only at acidic pH via a set of polar and electrostatic interactions. Furthermore, the structure reveals that hantavirus Gc has an additional N-terminal “tail” that is crucial in stabilizing the post-fusion trimer, accompanying the swapping of domain III in the quaternary arrangement of the trimer as compared to the standard class II fusion proteins. The mechanistic understandings derived from these data are likely to provide a unique handle for devising treatments against these human pathogens. PMID:27783711

  12. Production and characterization of high-affinity human monoclonal antibodies to human immunodeficiency virus type 1 envelope glycoproteins in a mouse model expressing human immunoglobulins.

    PubMed

    Sheppard, Neil C; Davies, Sarah L; Jeffs, Simon A; Vieira, Sueli M; Sattentau, Quentin J

    2007-02-01

    Human (Hu) monoclonal antibodies (MAbs) against the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins (Env) are useful tools in the structural and functional analysis of Env, are under development both as potential prophylaxis and as therapy for established HIV-1 infection, and have crucial roles in guiding the design of preventative vaccines. Despite representing more than 50% of infections globally, no MAbs have been generated in any species against C clade HIV-1 Env. To generate HuMAbs to a novel Chinese C clade Env vaccine candidate (primary isolate strain HIV-1(97CN54)), we used BAB5 mice that express a human immunoglobulin (Ig) M antibody repertoire in place of endogenous murine immunoglobulins. When immunized with HIV-1(97CN54) Env, these mice developed antigen-specific IgM antibodies. Hybridoma fusions using splenocytes from these mice enabled the isolation of two Env-specific IgM HuMAbs: N3C5 and N03B11. N3C5 bound to HIV-1 Env from clades A and C, whereas N03B11 bound two geographically distant clade C isolates but not Env from other clades. These HuMAbs bind conformational epitopes within the immunodominant region of the gp41 ectodomain. N3C5 weakly neutralized the autologous isolate in the absence of complement and weakly enhanced infection in the presence of complement. N03B11 has no effect on infectivity in either the presence or the absence of complement. These novel HuMAbs are useful reagents for the study of HIV-1 Env relevant to the global pandemic, and mice producing human immunoglobulin present a tool for the production of such antibodies.

  13. Comparative analysis of the fusion efficiency elicited by the envelope glycoprotein V1-V5 regions derived from human immunodeficiency virus type 1 transmitted perinatally.

    PubMed

    Guo, Hongyan; Abrahamyan, Levon G; Liu, Chang; Waltke, Mackenzie; Geng, Yunqi; Chen, Qimin; Wood, Charles; Kong, Xiaohong

    2012-12-01

    Understanding the properties of viruses preferentially establishing infection during perinatal transmission of human immunodeficiency virus type 1 (HIV-1) is critical for the development of effective measures to prevent transmission. A previous study demonstrated that the newly transmitted viruses (in infants) of chronically infected mother-infant pairs (MIPs) were fitter in terms of growth, which was imparted by their envelope (Env) glycoprotein V1-V5 regions, than those in the corresponding chronically infected mothers. In order to investigate whether the higher fitness of transmitted viruses was conferred by their higher entry efficiency directed by the V1-V5 regions during perinatal transmission, the fusogenicity of Env containing V1-V5 regions derived from transmitted and non-tranmsmitted viruses of five chronically infected MIPs and two acutely infected MIPs was analysed using two different cell-cell fusion assays. The results showed that, in one chronically infected MIP, a higher fusion efficiency was induced by the infant Env V1-V5 compared with that of the corresponding mother. Moreover, the V4-V5 regions played an important role in discriminating the transmitted and non-transmitted viruses in this pair. However, neither a consistent pattern nor significant differences in fusogenicity mediated by the V1-V5 regions between maternal and infant variants was observed in the other MIPs. This study suggests that there is no consistent and significant correlation between viral fitness selection and entry efficiency directed by the V1-V5 regions during perinatal transmission. Other factors such as the route and timing of transmission may also be involved.

  14. Dominant-negative effect of hetero-oligomerization on the function of the human immunodeficiency virus type 1 envelope glycoprotein complex

    SciTech Connect

    Herrera, Carolina; Klasse, Per Johan; Kibler, Christopher W.; Michael, Elizabeth; Moore, John P. . E-mail: jpm2003@med.cornell.edu; Beddows, Simon

    2006-07-20

    The human immunodeficiency virus type 1 (HIV-1) envelope (Env) glycoprotein forms trimers that mediate interactions with the CD4 receptor and a co-receptor on the target cell surface, thereby triggering viral fusion with the cell membrane. Cleavage of Env into its surface, gp120, and transmembrane, gp41, moieties is necessary for activation of its fusogenicity. Here, we produced pseudoviruses with phenotypically mixed wild-type (Wt) and mutant, cleavage-incompetent Env in order to quantify the effects of incorporating uncleaved Env on virion infectivity, antigenicity and neutralization sensitivity. We modeled the relative infectivity of three such phenotypically mixed viral strains, JR-FL, HXBc2 and a derivative of the latter, 3.2P, as a function of the relative amount of Wt Env. The data were fit very closely (R {sup 2} > 0.99) by models which assumed that only Wt homotrimers were functional, with different approximate thresholds of critical numbers of functional trimers per virion for the three strains. We also produced 3.2P pseudoviruses containing both a cleavage-competent Env that is defective for binding the neutralizing monoclonal antibody (NAb) 2G12, and a cleavage-incompetent Env that binds 2G12. The 2G12 NAb was not able to reduce the infectivity of these pseudoviruses detectably. Their neutralization by the CD4-binding site-directed agents CD4-IgG2 and NAb b12 was also unaffected by 2G12 binding to uncleaved Env. These results further strengthen the conclusion that only homotrimers consisting of cleaved Env are functional. They also imply that the function of a trimer is unaffected sterically by the binding of an antibody to an adjacent trimer.

  15. Fusion proteins of HIV-1 envelope glycoprotein gp120 with CD4-induced antibodies showed enhanced binding to CD4 and CD4 binding site antibodies

    SciTech Connect

    Chen, Weizao; Feng, Yang; Wang, Yanping; Zhu, Zhongyu; Dimitrov, Dimiter S.

    2012-09-07

    Highlights: Black-Right-Pointing-Pointer Some recombinant HIV-1 gp120s do not preserve their conformations on gp140s. Black-Right-Pointing-Pointer We hypothesize that CD4i antibodies could induce conformational changes in gp120. Black-Right-Pointing-Pointer CD4i antibodies enhance binding of CD4 and CD4bs antibodies to gp120. Black-Right-Pointing-Pointer CD4i antibody-gp120 fusion proteins could have potential as vaccine immunogens. -- Abstract: Development of successful AIDS vaccine immunogens continues to be a major challenge. One of the mechanisms by which HIV-1 evades antibody-mediated neutralizing responses is the remarkable conformational flexibility of its envelope glycoprotein (Env) gp120. Some recombinant gp120s do not preserve their conformations on gp140s and functional viral spikes, and exhibit decreased recognition by CD4 and neutralizing antibodies. CD4 binding induces conformational changes in gp120 leading to exposure of the coreceptor-binding site (CoRbs). In this study, we test our hypothesis that CD4-induced (CD4i) antibodies, which target the CoRbs, could also induce conformational changes in gp120 leading to better exposed conserved neutralizing antibody epitopes including the CD4-binding site (CD4bs). We found that a mixture of CD4i antibodies with gp120 only weakly enhanced CD4 binding. However, such interactions in single-chain fusion proteins resulted in gp120 conformations which bound to CD4 and CD4bs antibodies better than the original or mutagenically stabilized gp120s. Moreover, the two molecules in the fusion proteins synergized with each other in neutralizing HIV-1. Therefore, fusion proteins of gp120 with CD4i antibodies could have potential as components of HIV-1 vaccines and inhibitors of HIV-1 entry, and could be used as reagents to explore the conformational flexibility of gp120 and mechanisms of entry and immune evasion.

  16. The V4 and V5 Variable Loops of HIV-1 Envelope Glycoprotein Are Tolerant to Insertion of Green Fluorescent Protein and Are Useful Targets for Labeling.

    PubMed

    Nakane, Shuhei; Iwamoto, Aikichi; Matsuda, Zene

    2015-06-12

    The mature human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) comprises the non-covalently associated gp120 and gp41 subunits generated from the gp160 precursor. Recent structural analyses have provided quaternary structural models for gp120/gp41 trimers, including the variable loops (V1-V5) of gp120. In these models, the V3 loop is located under V1/V2 at the apical center of the Env trimer, and the V4 and V5 loops project outward from the trimeric protomers. In addition, the V4 and V5 loops are predicted to have less movement upon receptor binding during membrane fusion events. We performed insertional mutagenesis using a GFP variant, GFPOPT, placed into the variable loops of HXB2 gp120. This allowed us to evaluate the current structural models and to simultaneously generate a GFP-tagged HIV-1 Env, which was useful for image analyses. All GFP-inserted mutants showed similar levels of whole-cell expression, although certain mutants, particularly V3 mutants, showed lower levels of cell surface expression. Functional evaluation of their fusogenicities in cell-cell and virus-like particle-cell fusion assays revealed that V3 was the most sensitive to the insertion and that the V1/V2 loops were less sensitive than V3. The V4 and V5 loops were the most tolerant to insertion, and certain tag proteins other than GFPOPT could also be inserted without functional consequences. Our results support the current structural models and provide a GFPOPT-tagged Env construct for imaging studies.

  17. Immunogens Modeling a Fusion-Intermediate Conformation of gp41 Elicit Antibodies to the Membrane Proximal External Region of the HIV Envelope Glycoprotein

    PubMed Central

    Vassell, Russell; He, Yong; Vennakalanti, Prasad; Dey, Antu K.; Zhuang, Min; Wang, Wei; Sun, Yide; Biron-Sorek, Zohar; Srivastava, Indresh K.; LaBranche, Celia C.; Montefiori, David C.; Barnett, Susan W.; Weiss, Carol D.

    2015-01-01

    The membrane proximal external region (MPER) of the gp41 subunit of the HIV-1 envelope glycoprotein (Env) contains determinants for broadly neutralizing antibodies and has remained an important focus of vaccine design. However, creating an immunogen that elicits broadly neutralizing antibodies to this region has proven difficult in part due to the relative inaccessibility of the MPER in the native conformation of Env. Here, we describe the antigenicity and immunogenicity of a panel of oligomeric gp41 immunogens designed to model a fusion-intermediate conformation of Env in order to enhance MPER exposure in a relevant conformation. The immunogens contain segments of the gp41 N- and C-heptad repeats to mimic a trapped intermediate, followed by the MPER, with variations that include different N-heptad lengths, insertion of extra epitopes, and varying C-termini. These well-characterized immunogens were evaluated in two different immunization protocols involving gp41 and gp140 proteins, gp41 and gp160 DNA primes, and different immunization schedules and adjuvants. We found that the immunogens designed to reduce extension of helical structure into the MPER elicited the highest MPER antibody binding titers, but these antibodies lacked neutralizing activity. The gp41 protein immunogens also elicited higher MPER titers than the gp140 protein immunogen. In prime-boost studies, the best MPER responses were seen in the groups that received DNA priming with gp41 vectors followed by gp41 protein boosts. Finally, although titers to the entire protein immunogen were similar in the two immunization protocols, MPER-specific titers differed, suggesting that the immunization route, schedule, dose, or adjuvant may differentially influence MPER immunogenicity. These findings inform the design of future MPER immunogens and immunization protocols. PMID:26087072

  18. Mutations increasing exposure of a receptor binding site epitope in the soluble and oligomeric forms of the caprine arthritis-encephalitis lentivirus envelope glycoprotein

    SciTech Connect

    Hoetzel, Isidro . E-mail: ihotzel@gene.com; Cheevers, William P.

    2005-09-01

    The caprine arthritis-encephalitis (CAEV) and ovine maedi-visna (MVV) viruses are resistant to antibody neutralization, a feature shared with all other lentiviruses. Whether the CAEV gp135 receptor binding site(s) (RBS) in the functional surface envelope glycoprotein (Env) is protected from antibody binding, allowing the virus to resist neutralization, is not known. Two CAEV gp135 regions were identified by extrapolating a gp135 structural model that could affect binding of antibodies to the RBS: the V1 region and a short sequence analogous in position to the human immunodeficiency virus type 1 gp120 loop B postulated to be located between two major domains of CAEV gp135. Mutation of isoleucine-166 to alanine in the putative loop B of gp135 increased the affinity of soluble gp135 for the CAEV receptor(s) and goat monoclonal antibody (Mab) F7-299 which recognizes an epitope overlapping the gp135 RBS. The I166A mutation also stabilized or exposed the F7-299 epitope in anionic detergent buffers, indicating that the I166A mutation induces conformational changes and stabilizes the RBS of soluble gp135 and enhances Mab F7-299 binding. In contrast, the affinity of a V1 deletion mutant of gp135 for the receptor and Mab F7-299 and its structural stability did not differ from that of the wild-type gp135. However, both the I166A mutation and the V1 deletion of gp135 increased cell-to-cell fusion activity and binding of Mab F7-299 to the oligomeric Env. Therefore, the CAEV gp135 RBS is protected from antibody binding by mechanisms both dependent and independent of Env oligomerization which are disrupted by the V1 deletion and the I166A mutation, respectively. In addition, we found a correlation between side-chain {beta}-branching at amino acid position 166 and binding of Mab F7-299 to oligomeric Env and cell-to-cell fusion, suggesting local secondary structure constraints in the region around isoleucine-166 as one determinant of gp135 RBS exposure and antibody binding.

  19. Enhanced sensitivity to neutralizing antibodies in a variant of equine infectious anemia virus is linked to amino acid substitutions in the surface unit envelope glycoprotein.

    PubMed Central

    Cook, R F; Berger, S L; Rushlow, K E; McManus, J M; Cook, S J; Harrold, S; Raabe, M L; Montelaro, R C; Issel, C J

    1995-01-01

    Serial passage of the prototype (PR) cell-adapted Wyoming strain of equine infectious anemia virus (EIAV) in fetal donkey dermal (FDD) rather than fetal horse (designated fetal equine kidney [FEK]) cell cultures resulted in the generation of a variant virus strain which produced accelerated cytopathic effects in FDD cells and was 100- to 1,000-fold more sensitive to neutralizing antibodies than its parent. This neutralization-sensitive variant was designated the FDD strain. Although there were differences in glycosylation between the PR and FDD strains, passage of the FDD virus in FEK cells did not reduce its sensitivity to neutralizing antibody. Nucleotide sequencing of the region encoding the surface unit (SU) protein from the FDD strain revealed nine amino acid substitutions compared with the PR strain. Two of these substitutions resulted in changes in the polarity of charge, four caused the introduction of a charged residue, and three had no net change in charge. Nucleotide sequence analysis was extended to the region of the FDD virus genome encoding the extracellular domain of the transmembrane envelope glycoprotein (TM). Unlike the situation with the FDD virus coding region, there were minor variations in nucleotide sequence between individual molecular clones containing this region of the TM gene. Although each clone contained three nucleotide substitutions compared with the PR strain, only one of these was common to all, and this did not affect the amino acid content. Of the remaining two nucleotide substitutions, only one resulted in an amino acid change, and in each case, this change appeared to be conservative. To determine if amino acid substitutions in the SU protein of FDD cell-grown viruses were responsible for the enhanced sensitivity to neutralizing antibodies, chimeric viruses were constructed by using an infectious molecular clone of EIAV. These chimeric viruses contained all of the amino acid substitutions found in the FDD virus strain and were

  20. Effects of the I559P gp41 Change on the Conformation and Function of the Human Immunodeficiency Virus (HIV-1) Membrane Envelope Glycoprotein Trimer

    PubMed Central

    Sodroski, Joseph; Finzi, Andrés

    2015-01-01

    The mature human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) trimer is produced by proteolytic cleavage of a precursor and consists of three gp120 exterior and three gp41 transmembrane subunits. The metastable Env complex is induced to undergo conformational changes required for virus entry by the binding of gp120 to the receptors, CD4 and CCR5/CXCR4. An isoleucine-to-proline change (I559P) in the gp41 ectodomain has been used to stabilize soluble forms of HIV-1 Env trimers for structural characterization and for use as immunogens. In the native membrane-anchored HIV-1BG505 Env, the I559P change modestly decreased proteolytic maturation, increased the non-covalent association of gp120 with the Env trimer, and resulted in an Env conformation distinctly different from that of the wild-type HIV-1BG505 Env. Compared with the wild-type Env, the I559P Env was recognized inefficiently by polyclonal sera from HIV-1-infected individuals, by several gp41-directed antibodies, by some antibodies against the CD4-binding site of gp120, and by antibodies that preferentially recognize the CD4-bound Env. Some of the gp120-associated antigenic differences between the wild-type HIV-1BG505 Env and the I559P mutant were compensated by the SOS disulfide bond between gp120 and gp41, which has been used to stabilize cleaved soluble Env trimers. Nonetheless, regardless of the presence of the SOS changes, Envs with proline 559 were recognized less efficiently than Envs with isoleucine 559 by the VRC01 neutralizing antibody, which binds the CD4-binding site of gp120, and the PGT151 neutralizing antibody, which binds a hybrid gp120-gp41 epitope. The I559P change completely eliminated the ability of the HIV-1BG505 Env to mediate cell-cell fusion and virus entry, and abolished the capacity of the SOS Env to support virus infection in the presence of a reducing agent. These results suggest that differences exist between the quaternary structures of functional Env spikes and I559P

  1. Purified herpes simplex type 1 glycoprotein D (gD) genetically fused with the type 16 human papillomavirus E7 oncoprotein enhances antigen-specific CD8+ T cell responses and confers protective antitumor immunity.

    PubMed

    Porchia, Bruna F M M; Diniz, Mariana O; Cariri, Francisco A M O; Santana, Vinícius C; Amorim, Jaime H; Balan, Andrea; Braga, Catarina J M; Ferreira, Luís Carlos S

    2011-12-01

    Type 1 herpes virus (HSV-1) glycoprotein D (gD) enhances antigen-specific immune responses, particularly CD8(+) T cell responses, in mice immunized with DNA vaccines encoding hybrid proteins genetically fused with the target antigen at a site near the C-terminal end. These effects are attributed to the interaction of gD with the herpes virus entry mediator (HVEM) and the concomitant blockade of a coinhibitory mechanism mediated by the B- and T-lymphocyte attenuator (BTLA). However, questions concerning the requirement for endogenous synthesis of the antigen or the adjuvant/antigen fusion itself have not been addressed so far. In the present study, we investigated these points using purified recombinant gDs, genetically fused or not with type 16 papilloma virus (HPV-16) E7 oncoprotein. Soluble recombinant gDs, but not denatured forms, retained the ability to bind surface-exposed cellular receptors of HVEM-expressing U937 cells. In addition, in vivo administration of the recombinant proteins, particularly gD genetically fused with E7 (gDE7), promoted the activation of dendritic cells (DC) and antigen-specific cytotoxic CD8(+) T cells. More relevantly, mice immunized with the gDE7 protein developed complete preventive and partial therapeutic antitumor protection, as measured in mice following the implantation of TC-1 cells expressing HPV-16 oncoproteins. Collectively, these results demonstrate that the T cell adjuvant effects of the HSV-1 gD protein did not require endogenous synthesis and could be demonstrated in mice immunized with purified recombinant proteins.

  2. Protein of macaques against infection with simian type D retrovirus (SRV-1) by immunization with recombinant vaccinia virus expressing the envelope glycoproteins of either SRV-1 or Mason-Pfizer monkey virus (SRV-3).

    PubMed Central

    Brody, B A; Hunter, E; Kluge, J D; Lasarow, R; Gardner, M; Marx, P A

    1992-01-01

    Rhesus macaques were immunized with live vaccinia virus recombinants expressing the envelope glycoproteins (gp70 and gp22) of simian type D retrovirus (SRV), serotype 1 or 3. All of the animals immunized with either the SRV-1 env or the SRV-3 env vaccinia virus recombinant developed neutralizing antibodies against the homologous SRV. In addition, both groups developed cross-reactive antibodies and were protected against an intravenous live-virus challenge with SRV-1. The four control animals immunized with a vaccinia virus recombinant expressing the G protein of respiratory syncytial virus were not protected against the same SRV-1 challenge. Although SRV-1 and SRV-3 immune sera showed cross-neutralization, they failed to neutralize a separate, more distantly related serotype, SRV-2, in an in vitro assay. These findings are consistent with the known degree of serologic and genetic relatedness of these three SRV strains. Images PMID:1316495

  3. Purifying Nanomaterials

    NASA Technical Reports Server (NTRS)

    Hung, Ching-Cheh (Inventor); Hurst, Janet (Inventor)

    2014-01-01

    A method of purifying a nanomaterial and the resultant purified nanomaterial in which a salt, such as ferric chloride, at or near its liquid phase temperature, is used to penetrate and wet the internal surfaces of a nanomaterial to dissolve impurities that may be present, for example, from processes used in the manufacture of the nanomaterial.

  4. The NV Gene of Snakehead Rhabdovirus (SHRV) Is Not Required for Pathogenesis, and a Heterologous Glycoprotein Can Be Incorporated into the SHRV Envelope

    PubMed Central

    Alonso, Marta; Kim, Carol H.; Johnson, Marc C.; Pressley, Meagan; Leong, Jo-Ann

    2004-01-01

    Snakehead rhabdovirus (SHRV) affects warm-water fish in Southeast Asia and belongs to the genus Novirhabdovirus by virtue of its “nonvirion” (NV) gene. To examine the function of the NV gene, we used a recently developed reverse genetic system to produce a viable recombinant SHRV carrying an NV gene deletion. The recombinant virus was produced at the same rate and same final concentrations as the wild-type virus in cultured fish cells in spite of the NV gene deletion. The role of the NV protein in fish pathogenesis was also investigated. Zebra fish (Danio rerio) were infected with the NV deletion mutant or with a recombinant virus containing a copy of the SHRV genome, and similar mortality rates as well as final mortalities were recorded, suggesting no apparent role for the NV protein in fish pathogenesis. Interestingly, the unsuccessful rescue of fully viable recombinants with genomes containing deletions in the G/NV gene junction suggested a role for the gene junction in virus transcription and replication. Finally, we demonstrated that the SHRV glycoprotein can be replaced by the glycoprotein of infectious hematopoietic necrosis virus (IHNV) or by a hybrid protein composed of SHRV and IHNV sequences. PMID:15140985

  5. The NV gene of snakehead rhabdovirus (SHRV) is not required for pathogenesis, and a heterologous glycoprotein can be incorporated into the SHRV envelope.

    PubMed

    Alonso, Marta; Kim, Carol H; Johnson, Marc C; Pressley, Meagan; Leong, Jo-Ann

    2004-06-01

    Snakehead rhabdovirus (SHRV) affects warm-water fish in Southeast Asia and belongs to the genus Novirhabdovirus by virtue of its "nonvirion" (NV) gene. To examine the function of the NV gene, we used a recently developed reverse genetic system to produce a viable recombinant SHRV carrying an NV gene deletion. The recombinant virus was produced at the same rate and same final concentrations as the wild-type virus in cultured fish cells in spite of the NV gene deletion. The role of the NV protein in fish pathogenesis was also investigated. Zebra fish (Danio rerio) were infected with the NV deletion mutant or with a recombinant virus containing a copy of the SHRV genome, and similar mortality rates as well as final mortalities were recorded, suggesting no apparent role for the NV protein in fish pathogenesis. Interestingly, the unsuccessful rescue of fully viable recombinants with genomes containing deletions in the G/NV gene junction suggested a role for the gene junction in virus transcription and replication. Finally, we demonstrated that the SHRV glycoprotein can be replaced by the glycoprotein of infectious hematopoietic necrosis virus (IHNV) or by a hybrid protein composed of SHRV and IHNV sequences.

  6. N-Glycans on the Rift Valley Fever Virus Envelope Glycoproteins Gn and Gc Redundantly Support Viral Infection via DC-SIGN

    PubMed Central

    Phoenix, Inaia; Nishiyama, Shoko; Lokugamage, Nandadeva; Hill, Terence E.; Huante, Matthew B.; Slack, Olga A.L.; Carpio, Victor H.; Freiberg, Alexander N.; Ikegami, Tetsuro

    2016-01-01

    Rift Valley fever is a mosquito-transmitted, zoonotic disease that infects humans and ruminants. Dendritic cell specific intercellular adhesion molecule 3 (ICAM-3) grabbing non-integrin (DC-SIGN) acts as a receptor for members of the phlebovirus genus. The Rift Valley fever virus (RVFV) glycoproteins (Gn/Gc) encode five putative N-glycan sequons (asparagine (N)–any amino acid (X)–serine (S)/threonine (T)) at positions: N438 (Gn), and N794, N829, N1035, and N1077 (Gc). The N-glycosylation profile and significance in viral infection via DC-SIGN have not been elucidated. Gc N-glycosylation was first evaluated by using Gc asparagine (N) to glutamine (Q) mutants. Subsequently, we generated a series of recombinant RVFV MP-12 strain mutants, which encode N-to-Q mutations, and the infectivity of each mutant in Jurkat cells stably expressing DC-SIGN was evaluated. Results showed that Gc N794, N1035, and N1077 were N-glycosylated but N829 was not. Gc N1077 was heterogeneously N-glycosylated. RVFV Gc made two distinct N-glycoforms: “Gc-large” and “Gc-small”, and N1077 was responsible for “Gc-large” band. RVFV showed increased infection of cells expressing DC-SIGN compared to cells lacking DC-SIGN. Infection via DC-SIGN was increased in the presence of either Gn N438 or Gc N1077. Our study showed that N-glycans on the Gc and Gn surface glycoproteins redundantly support RVFV infection via DC-SIGN. PMID:27223297

  7. N-Glycans on the Rift Valley Fever Virus Envelope Glycoproteins Gn and Gc Redundantly Support Viral Infection via DC-SIGN.

    PubMed

    Phoenix, Inaia; Nishiyama, Shoko; Lokugamage, Nandadeva; Hill, Terence E; Huante, Matthew B; Slack, Olga A L; Carpio, Victor H; Freiberg, Alexander N; Ikegami, Tetsuro

    2016-01-01

    Rift Valley fever is a mosquito-transmitted, zoonotic disease that infects humans and ruminants. Dendritic cell specific intercellular adhesion molecule 3 (ICAM-3) grabbing non-integrin (DC-SIGN) acts as a receptor for members of the phlebovirus genus. The Rift Valley fever virus (RVFV) glycoproteins (Gn/Gc) encode five putative N-glycan sequons (asparagine (N)-any amino acid (X)-serine (S)/threonine (T)) at positions: N438 (Gn), and N794, N829, N1035, and N1077 (Gc). The N-glycosylation profile and significance in viral infection via DC-SIGN have not been elucidated. Gc N-glycosylation was first evaluated by using Gc asparagine (N) to glutamine (Q) mutants. Subsequently, we generated a series of recombinant RVFV MP-12 strain mutants, which encode N-to-Q mutations, and the infectivity of each mutant in Jurkat cells stably expressing DC-SIGN was evaluated. Results showed that Gc N794, N1035, and N1077 were N-glycosylated but N829 was not. Gc N1077 was heterogeneously N-glycosylated. RVFV Gc made two distinct N-glycoforms: "Gc-large" and "Gc-small", and N1077 was responsible for "Gc-large" band. RVFV showed increased infection of cells expressing DC-SIGN compared to cells lacking DC-SIGN. Infection via DC-SIGN was increased in the presence of either Gn N438 or Gc N1077. Our study showed that N-glycans on the Gc and Gn surface glycoproteins redundantly support RVFV infection via DC-SIGN. PMID:27223297

  8. Pushing the endogenous envelope

    PubMed Central

    Henzy, Jamie E.; Johnson, Welkin E.

    2013-01-01

    The majority of retroviral envelope glycoproteins characterized to date are typical of type I viral fusion proteins, having a receptor binding subunit associated with a fusion subunit. The fusion subunits of lentiviruses and alpha-, beta-, delta- and gammaretroviruses have a very conserved domain organization and conserved features of secondary structure, making them suitable for phylogenetic analyses. Such analyses, along with sequence comparisons, reveal evidence of numerous recombination events in which retroviruses have acquired envelope glycoproteins from heterologous sequences. Thus, the envelope gene (env) can have a history separate from that of the polymerase gene (pol), which is the most commonly used gene in phylogenetic analyses of retroviruses. Focusing on the fusion subunits of the genera listed above, we describe three distinct types of retroviral envelope glycoproteins, which we refer to as gamma-type, avian gamma-type and beta-type. By tracing these types within the ‘fossil record’ provided by endogenous retroviruses, we show that they have surprisingly distinct evolutionary histories and dynamics, with important implications for cross-species transmissions and the generation of novel lineages. These findings validate the utility of env sequences in contributing phylogenetic signal that enlarges our understanding of retrovirus evolution. PMID:23938755

  9. Water Purifier

    NASA Technical Reports Server (NTRS)

    1992-01-01

    The Floatron water purifier combines two space technologies - ionization for water purification and solar electric power generation. The water purification process involves introducing ionized minerals that kill microorganisms like algae and bacteria. The 12 inch unit floats in a pool while its solar panel collects sunlight that is converted to electricity. The resulting current energizes a specially alloyed mineral electrode below the waterline, causing release of metallic ions into the water. The electrode is the only part that needs replacing, and water purified by the system falls within EPA drinking water standards.

  10. The C-terminal tail of the gp41 transmembrane envelope glycoprotein of HIV-1 clades A, B, C, and D may exist in two conformations: an analysis of sequence, structure, and function

    SciTech Connect

    Hollier, Mark J.; Dimmock, Nigel J. . E-mail: n.j.dimmock@warwick.ac.uk

    2005-07-05

    In addition to the major ectodomain, the gp41 transmembrane glycoprotein of HIV-1 is now known to have a minor ectodomain that is part of the long C-terminal tail. Both ectodomains are highly antigenic, carry neutralizing and non-neutralizing epitopes, and are involved in virus-mediated fusion activity. However, data have so far been biologically based, and derived solely from T cell line-adapted (TCLA), B clade viruses. Here we have carried out sequence and theoretically based structural analyses of 357 gp41 C-terminal sequences of mainly primary isolates of HIV-1 clades A, B, C, and D. Data show that all these viruses have the potential to form a tail loop structure (the minor ectodomain) supported by three, {beta}-sheet, membrane-spanning domains (MSDs). This means that the first (N-terminal) tyrosine-based sorting signal of the gp41 tail is situated outside the cell membrane and is non-functional, and that gp41 that reaches the cell surface may be recycled back into the cytoplasm through the activity of the second tyrosine-sorting signal. However, we suggest that only a minority of cell-associated gp41 molecules - those destined for incorporation into virions - has 3 MSDs and the minor ectodomain. Most intracellular gp41 has the conventional single MSD, no minor ectodomain, a functional first tyrosine-based sorting signal, and in line with current thinking is degraded intracellularly. The gp41 structural diversity suggested here can be viewed as an evolutionary strategy to minimize HIV-1 envelope glycoprotein expression on the cell surface, and hence possible cytotoxicity and immune attack on the infected cell.

  11. High-Mannose Specific Lectin and Its Recombinants from a Carrageenophyta Kappaphycus alvarezii Represent a Potent Anti-HIV Activity Through High-Affinity Binding to the Viral Envelope Glycoprotein gp120.

    PubMed

    Hirayama, Makoto; Shibata, Hiromi; Imamura, Koji; Sakaguchi, Takemasa; Hori, Kanji

    2016-02-01

    We previously reported that a high-mannose binding lectin KAA-2 from the red alga Kappaphycus alvarezii, which is an economically important species and widely cultivated as a source of carrageenans, had a potent anti-influenza virus activity. In this study, the full-length sequences of two KAA isoforms, KAA-1 and KAA-2, were elucidated by a combination of peptide mapping and complementary DNA (cDNA) cloning. They consisted of four internal tandem-repeated domains, which are conserved in high-mannose specific lectins from lower organisms, including a cyanobacterium Oscillatoria agardhii and a red alga Eucheuma serra. Using an Escherichia coli expression system, an active recombinant form of KAA-1 (His-tagged rKAA-1) was successfully generated in the yield of 115 mg per liter of culture. In a detailed oligosaccharide binding analysis by a centrifugal ultrafiltration-HPLC method with 27 pyridylaminated oligosaccharides, His-tagged rKAA-1 and rKAA-1 specifically bound to high-mannose N-glycans with an exposed α1-3 mannose in the D2 arm as the native lectin did. Predicted from oligosaccharide binding specificity, a surface plasmon resonance analysis revealed that the recombinants exhibit strong interaction with gp120, a heavily glycosylated envelope glycoprotein of HIV with high association constants (1.48 - 1.61 × 10(9) M(-1)). Native KAAs and the recombinants inhibited the HIV-1 entry at IC50s of low nanomolar levels (7.3-12.9 nM). Thus, the recombinant proteins would be useful as antiviral reagents targeting the viral surface glycoproteins with high-mannose N-glycans, and the cultivated alga K. alvarezii could also be a good source of not only carrageenans but also this functional lectin(s).

  12. High-Mannose Specific Lectin and Its Recombinants from a Carrageenophyta Kappaphycus alvarezii Represent a Potent Anti-HIV Activity Through High-Affinity Binding to the Viral Envelope Glycoprotein gp120.

    PubMed

    Hirayama, Makoto; Shibata, Hiromi; Imamura, Koji; Sakaguchi, Takemasa; Hori, Kanji

    2016-02-01

    We previously reported that a high-mannose binding lectin KAA-2 from the red alga Kappaphycus alvarezii, which is an economically important species and widely cultivated as a source of carrageenans, had a potent anti-influenza virus activity. In this study, the full-length sequences of two KAA isoforms, KAA-1 and KAA-2, were elucidated by a combination of peptide mapping and complementary DNA (cDNA) cloning. They consisted of four internal tandem-repeated domains, which are conserved in high-mannose specific lectins from lower organisms, including a cyanobacterium Oscillatoria agardhii and a red alga Eucheuma serra. Using an Escherichia coli expression system, an active recombinant form of KAA-1 (His-tagged rKAA-1) was successfully generated in the yield of 115 mg per liter of culture. In a detailed oligosaccharide binding analysis by a centrifugal ultrafiltration-HPLC method with 27 pyridylaminated oligosaccharides, His-tagged rKAA-1 and rKAA-1 specifically bound to high-mannose N-glycans with an exposed α1-3 mannose in the D2 arm as the native lectin did. Predicted from oligosaccharide binding specificity, a surface plasmon resonance analysis revealed that the recombinants exhibit strong interaction with gp120, a heavily glycosylated envelope glycoprotein of HIV with high association constants (1.48 - 1.61 × 10(9) M(-1)). Native KAAs and the recombinants inhibited the HIV-1 entry at IC50s of low nanomolar levels (7.3-12.9 nM). Thus, the recombinant proteins would be useful as antiviral reagents targeting the viral surface glycoproteins with high-mannose N-glycans, and the cultivated alga K. alvarezii could also be a good source of not only carrageenans but also this functional lectin(s). PMID:26593063

  13. High-Mannose Specific Lectin and Its Recombinants from a Carrageenophyta Kappaphycus alvarezii Represent a Potent Anti-HIV Activity Through High-Affinity Binding to the Viral Envelope Glycoprotein gp120.

    PubMed

    Hirayama, Makoto; Shibata, Hiromi; Imamura, Koji; Sakaguchi, Takemasa; Hori, Kanji

    2016-04-01

    We previously reported that a high-mannose binding lectin KAA-2 from the red alga Kappaphycus alvarezii, which is an economically important species and widely cultivated as a source of carrageenans, had a potent anti-influenza virus activity. In this study, the full-length sequences of two KAA isoforms, KAA-1 and KAA-2, were elucidated by a combination of peptide mapping and cDNA cloning. They consisted of four internal tandem-repeated domains, which are conserved in high-mannose specific lectins from lower organisms, including a cyanobacterium Oscillatoria agardhii and a red alga Eucheuma serra. Using an Escherichia coli expression system, an active recombinant form of KAA-1 (His-tagged rKAA-1) was successfully generated in the yield of 115 mg per a litter of culture. In a detailed oligosaccharide binding analysis by a centrifugal ultrafiltration-HPLC method with 27 pyridylaminated oligosaccharides, His-tagged rKAA-1 and rKAA-1 specifically bound to high-mannose N-glycans with an exposed α1-3 mannose in the D2 arm as the native lectin did. Predicted from oligosaccharide-binding specificity, a surface plasmon resonance analysis revealed that the recombinants exhibit strong interaction with gp120, a heavily glycosylated envelope glycoprotein of HIV with high association constants (1.48-1.61 × 10(9) M(-1)). Native KAAs and the recombinants inhibited the HIV-1 entry at IC50s of low nanomolar levels (7.3-12.9 nM). Thus, the recombinant proteins would be useful as antiviral reagents targeting the viral surface glycoproteins with high-mannose N-glycans, and the cultivated alga K. alvarezii could also be a good source of not only carrageenans but also this functional lectin(s). PMID:26661793

  14. Selective killing of Kaposi's sarcoma-associated herpesvirus lytically infected cells with a recombinant immunotoxin targeting the viral gpK8.1A envelope glycoprotein.

    PubMed

    Chatterjee, Deboeeta; Chandran, Bala; Berger, Edward A

    2012-01-01

    Kaposi sarcoma-associated herpesvirus (KSHV, human herpesvirus 8) is etiologically associated with three neoplastic syndromes: Kaposi sarcoma and the uncommon HIV-associated B-cell lymphoproliferative disorders primary effusion lymphoma and multicentric Castleman disease. The incidence of the latter B-cell pathology has been increasing in spite of antiretroviral therapy; its association with lytic virus replication has prompted interest in therapeutic strategies aimed at this phase of the virus life cycle. We designed and expressed a recombinant immunotoxin (2014-PE38) targeting the gpK8.1A viral glycoprotein expressed on the surface of the virion and infected cells. We show that this immunotoxin selectively kills KSHV-infected cells in dose-dependent fashion, resulting in major reductions of infectious virus release. The immunotoxin and ganciclovir, an inhibitor of viral DNA replication, showed marked reciprocal potentiation of antiviral activities. These results suggest that the immunotoxin, alone or in combination, may represent a new approach to treat diseases associated with KSHV lytic replication. PMID:22377676

  15. Water Purifiers

    NASA Technical Reports Server (NTRS)

    1992-01-01

    Technology developed to purify the water aboard manned spacecraft has led to a number of spinoff applications. One of them is the Ambassador line of bacteriostatic water treatment systems, which employ high grade, high absorption media to inhibit bacteria growth and remove the medicinal taste and odor of chlorine. Company President, Ray Ward, originally became interested in the technology because of the "rusty" taste of his water supply.

  16. Specific interaction of CXCR4 with CD4 and CD8{alpha}: Functional analysis of the CD4/CXCR4 interaction in the context of HIV-1 envelope glycoprotein-mediated membrane fusion

    SciTech Connect

    Basmaciogullari, Stephane . E-mail: basmaciogullari@cochin.inserm.fr; Pacheco, Beatriz; Bour, Stephan; Sodroski, Joseph

    2006-09-15

    We investigated possible interactions between HIV-1 receptor (CD4) and the main coreceptors CXCR4 and CCR5. We found that CD4 and CXCR4 coexpressed in 293T cells form a complex that can be immunoprecipitated with antibodies directed against the extracellular domain of either protein. Mutagenesis revealed that the CD4/CXCR4 interaction maps to two previously uncharacterized basic motifs in the cytoplasmic domain of CD4. HIV-1 envelope glycoprotein-mediated membrane fusion was found to be independent of the ability of CD4 and CXCR4 to interact, whether fusion was studied in a virus-cell or a cell-cell model. However, this interaction might explain the adaptation of HIV-1 to CXCR4 as an alternative to CCR5. We found that CXCR4 also interacts with the cytoplasmic domain of CD8{alpha} in a way that is similar to the CD4/CXCR4 interaction. The CD4/CXCR4 and CD8{alpha}/CXCR4 interactions may thus be involved in cellular signaling pathways shared by the CD4 and CD8{alpha} molecules.

  17. A rev1-vpu polymorphism unique to HIV-1 subtype A and C strains impairs envelope glycoprotein expression from rev-vpu-env cassettes and reduces virion infectivity in pseudotyping assays

    SciTech Connect

    Kraus, Matthias H.; Parrish, Nicholas F.; Shaw, Katharina S.; Decker, Julie M.; Keele, Brandon F.; Salazar-Gonzalez, Jesus F.; Grayson, Truman; McPherson, David T.; Ping, Li-Hua; Anderson, Jeffrey A.; Swanstrom, Ronald; Williamson, Carolyn; Shaw, George M.; Hahn, Beatrice H.

    2010-02-20

    Functional studies of HIV-1 envelope glycoproteins (Envs) commonly include the generation of pseudoviruses, which are produced by co-transfection of rev-vpu-env cassettes with an env-deficient provirus. Here, we describe six Env constructs from transmitted/founder HIV-1 that were defective in the pseudotyping assay, although two produced infectious virions when expressed from their cognate proviruses. All of these constructs exhibited an unusual gene arrangement in which the first exon of rev (rev1) and vpu were in the same reading frame without an intervening stop codon. Disruption of the rev1-vpu fusion gene by frameshift mutation, stop codon, or abrogation of the rev initiation codon restored pseudovirion infectivity. Introduction of the fusion gene into wildtype Env cassettes severely compromised their function. The defect was not due to altered env and rev transcription or a dominant negative effect of the expressed fusion protein, but seemed to be caused by inefficient translation at the env initiation codon. Although the rev1-vpu polymorphism affects Env expression only in vitro, it can cause problems in studies requiring Env complementation, such as analyses of co-receptor usage and neutralization properties, since 3% of subtype A, 20% of subtype C and 5% of CRF01{sub A}/E viruses encode the fusion gene. A solution is to eliminate the rev initiation codon when amplifying rev-vpu-env cassettes since this increases Env expression irrespective of the presence of the polymorphism.

  18. A Mutation in the Ebola Virus Envelope Glycoprotein Restricts Viral Entry in a Host Species- and Cell-Type-Specific Manner

    PubMed Central

    Ndungo, Esther; Tantral, Lee; Miller, Emily Happy; Leung, Lawrence W.; Chandran, Kartik

    2013-01-01

    Zaire Ebola virus (EBOV) is a zoonotic pathogen that causes severe hemorrhagic fever in humans. A single viral glycoprotein (GP) mediates viral attachment and entry. Here, virus-like particle (VLP)-based entry assays demonstrate that a GP mutant, GP-F88A, which is defective for entry into a variety of human cell types, including antigen-presenting cells (APCs), such as macrophages and dendritic cells, can mediate viral entry into mouse CD11b+ APCs. Like that of wild-type GP (GP-wt), GP-F88A-mediated entry occurs via a macropinocytosis-related pathway and requires endosomal cysteine proteases and an intact fusion peptide. Several additional hydrophobic residues lie in close proximity to GP-F88, including L111, I113, L122, and F225. GP mutants in which these residues are mutated to alanine displayed preferential and often impaired entry into several cell types, although not in a species-specific manner. Niemann-Pick C1 (NPC1) protein is an essential filovirus receptor that binds directly to GP. Overexpression of NPC1 was recently demonstrated to rescue GP-F88A-mediated entry. A quantitative enzyme-linked immunosorbent assay (ELISA) demonstrated that while the F88A mutation impairs GP binding to human NPC1 by 10-fold, it has little impact on GP binding to mouse NPC1. Interestingly, not all mouse macrophage cell lines permit GP-F88A entry. The IC-21 cell line was permissive, whereas RAW 264.7 cells were not. Quantitative reverse transcription (RT)-PCR assays demonstrate higher NPC1 levels in GP-F88A permissive IC-21 cells and mouse peritoneal macrophages than in RAW 264.7 cells. Cumulatively, these studies suggest an important role for NPC1 in the differential entry of GP-F88A into mouse versus human APCs. PMID:23302883

  19. A mutation in the Ebola virus envelope glycoprotein restricts viral entry in a host species- and cell-type-specific manner.

    PubMed

    Martinez, Osvaldo; Ndungo, Esther; Tantral, Lee; Miller, Emily Happy; Leung, Lawrence W; Chandran, Kartik; Basler, Christopher F

    2013-03-01

    Zaire Ebola virus (EBOV) is a zoonotic pathogen that causes severe hemorrhagic fever in humans. A single viral glycoprotein (GP) mediates viral attachment and entry. Here, virus-like particle (VLP)-based entry assays demonstrate that a GP mutant, GP-F88A, which is defective for entry into a variety of human cell types, including antigen-presenting cells (APCs), such as macrophages and dendritic cells, can mediate viral entry into mouse CD11b(+) APCs. Like that of wild-type GP (GP-wt), GP-F88A-mediated entry occurs via a macropinocytosis-related pathway and requires endosomal cysteine proteases and an intact fusion peptide. Several additional hydrophobic residues lie in close proximity to GP-F88, including L111, I113, L122, and F225. GP mutants in which these residues are mutated to alanine displayed preferential and often impaired entry into several cell types, although not in a species-specific manner. Niemann-Pick C1 (NPC1) protein is an essential filovirus receptor that binds directly to GP. Overexpression of NPC1 was recently demonstrated to rescue GP-F88A-mediated entry. A quantitative enzyme-linked immunosorbent assay (ELISA) demonstrated that while the F88A mutation impairs GP binding to human NPC1 by 10-fold, it has little impact on GP binding to mouse NPC1. Interestingly, not all mouse macrophage cell lines permit GP-F88A entry. The IC-21 cell line was permissive, whereas RAW 264.7 cells were not. Quantitative reverse transcription (RT)-PCR assays demonstrate higher NPC1 levels in GP-F88A permissive IC-21 cells and mouse peritoneal macrophages than in RAW 264.7 cells. Cumulatively, these studies suggest an important role for NPC1 in the differential entry of GP-F88A into mouse versus human APCs. PMID:23302883

  20. Characterization of retrovirus-based reporter viruses pseudotyped with the precursor membrane and envelope glycoproteins of four serotypes of dengue viruses

    SciTech Connect

    Hu, H.-P.; Hsieh, S.-C.; King, C.-C.; Wang, W.-K.

    2007-11-25

    In this study, we successfully established retrovirus-based reporter viruses pseudotyped with the precursor membrane and envelope (PrM/E) proteins of each of the four serotypes of dengue viruses, which caused the most important arboviral diseases in this century. Co-sedimentation of the dengue E protein and HIV-1 core proteins by sucrose gradient analysis of the pseudotype reporter virus of dengue virus type 2, D2(HIVluc), and detection of HIV-1 core proteins by immunoprecipitation with anti-E monoclonal antibody suggested that dengue viral proteins were incorporated into the pseudotype viral particles. The infectivity in target cells, as assessed by the luciferase activity, can be inhibited by the lysosomotropic agents, suggesting a pH-dependent mechanism of entry. Amino acid substitutions of the leucine at position 107, a critical residue at the fusion loop of E protein, with lysine resulted in severe impairment in infectivity, suggesting that entry of the pseudotype reporter virus is mediated through the fusogenic properties of E protein. With more and more dengue viral sequences available from different outbreaks worldwide, this sensitive and convenient tool has the potential to facilitate molecular characterization of the PrM/E proteins of dengue field isolates.

  1. ERManI (Endoplasmic Reticulum Class I α-Mannosidase) Is Required for HIV-1 Envelope Glycoprotein Degradation via Endoplasmic Reticulum-associated Protein Degradation Pathway.

    PubMed

    Zhou, Tao; Frabutt, Dylan A; Moremen, Kelley W; Zheng, Yong-Hui

    2015-09-01

    Previously, we reported that the mitochondrial translocator protein (TSPO) induces HIV-1 envelope (Env) degradation via the endoplasmic reticulum (ER)-associated protein degradation (ERAD) pathway, but the mechanism was not clear. Here we investigated how the four ER-associated glycoside hydrolase family 47 (GH47) α-mannosidases, ERManI, and ER-degradation enhancing α-mannosidase-like (EDEM) proteins 1, 2, and 3, are involved in the Env degradation process. Ectopic expression of these four α-mannosidases uncovers that only ERManI inhibits HIV-1 Env expression in a dose-dependent manner. In addition, genetic knock-out of the ERManI gene MAN1B1 using CRISPR/Cas9 technology disrupts the TSPO-mediated Env degradation. Biochemical studies show that HIV-1 Env interacts with ERManI, and between the ERManI cytoplasmic, transmembrane, lumenal stem, and lumenal catalytic domains, the catalytic domain plays a critical role in the Env-ERManI interaction. In addition, functional studies show that inactivation of the catalytic sites by site-directed mutagenesis disrupts the ERManI activity. These studies identify ERManI as a critical GH47 α-mannosidase in the ER-associated protein degradation pathway that initiates the Env degradation and suggests that its catalytic domain and enzymatic activity play an important role in this process.

  2. The Single-Stranded DNA Genome of Novel Archaeal Virus Halorubrum Pleomorphic Virus 1 Is Enclosed in the Envelope Decorated with Glycoprotein Spikes▿ †

    PubMed Central

    Pietilä, Maija K.; Laurinavičius, Simonas; Sund, Jukka; Roine, Elina; Bamford, Dennis H.

    2010-01-01

    Only a few archaeal viruses have been subjected to detailed structural analyses. Major obstacles have been the extreme conditions such as high salinity or temperature needed for the propagation of these viruses. In addition, unusual morphotypes of many archaeal viruses have made it difficult to obtain further information on virion architectures. We used controlled virion dissociation to reveal the structural organization of Halorubrum pleomorphic virus 1 (HRPV-1) infecting an extremely halophilic archaeal host. The single-stranded DNA genome is enclosed in a pleomorphic membrane vesicle without detected nucleoproteins. VP4, the larger major structural protein of HRPV-1, forms glycosylated spikes on the virion surface and VP3, the smaller major structural protein, resides on the inner surface of the membrane vesicle. Together, these proteins organize the structure of the membrane vesicle. Quantitative lipid comparison of HRPV-1 and its host Halorubrum sp. revealed that HRPV-1 acquires lipids nonselectively from the host cell membrane, which is typical of pleomorphic enveloped viruses. PMID:19864380

  3. Pathogenic significance of alpha-N-acetylgalactosaminidase activity found in the envelope glycoprotein gp160 of human immunodeficiency virus Type 1.

    PubMed

    Yamamoto, Nobuto

    2006-03-01

    Serum vitamin D3-binding protein (Gc protein) is the precursor for the principal macrophage-activating factor (MAF). The precursor activity of serum Gc protein was lost or reduced in HIV-infected patients. These patient sera contained alpha-N-acetylgalactosaminidase (Nagalase), which deglycosylates serum Gc protein. Deglycosylated Gc protein cannot be converted to MAF and thus loses MAF precursor activity, leading to immunosuppression. Nagalase in the blood stream of HIV-infected patients was complexed with patient immunoglobulin G, suggesting that this enzyme is immunogenic, seemingly a viral gene product. In fact, Nagalase was inducible by treatment of cultures of HIV-infected patient peripheral blood mononuclear cells with a provirus-inducing agent. This enzyme was immunoprecipitable with polyclonal anti-HIV but not with anticellular constitutive enzyme or with antitumor Nagalase. The kinetic parameters (km value of 1.27 mM and pH optimum of 6.1), of the patient serum Nagalase were distinct from those of constitutive enzyme (km value of 4.83 mM and pH optimum of 4.3). This glycosidase should reside on an envelope protein capable of interacting with cellular membranous O-glycans. Although cloned gp160 exhibited no Nagalase activity, treatment of gp160 with trypsin expressed Nagalase activity, suggesting that proteolytic cleavage of gp160 to generate gp120 and gp41 is required for Nagalase activity. Cloned gp120 exhibited Nagalase activity while cloned gp41 showed no Nagalase activity. Since proteolytic cleavage of protein gp160 is required for expression of both fusion capacity and Nagalase activity, Nagalase seems to be an enzymatic basis for fusion in the infectious process. Therefore, Nagalase appears to play dual roles in viral infectivity and immunosuppression. PMID:16545013

  4. Evaluation of envelope glycoprotein E(rns) of an atypical bovine pestivirus as antigen in a microsphere immunoassay for the detection of antibodies against bovine viral diarrhea virus 1 and atypical bovine pestivirus.

    PubMed

    Vijayaraghavan, Balaje; Xia, Hongyan; Harimoorthy, Rajiv; Liu, Lihong; Belák, Sándor

    2012-11-01

    Atypical bovine pestiviruses are related antigenically and phylogenetically to bovine viral diarrhea viruses (BVDV-1 and BVDV-2), and may cause the same clinical manifestations in animals. Glycoprotein E(rns) of an atypical bovine pestivirus Th/04_KhonKaen was produced in a baculovirus expression system and was purified by affinity chromatography. The recombinant E(rns) protein was used as an antigen in a microsphere immunoassay for the detection of antibodies against BVDV-1 and atypical bovine pestivirus. The diagnostic performance of the new method was evaluated by testing a total of 596 serum samples, and the assay was compared with enzyme-linked immunosorbent assay (ELISA). Based on the negative/positive cut-off median fluorescence intensity (MFI) value of 2800, the microsphere immunoassay had a sensitivity of 100% and specificity of 100% compared to ELISA. The immunoassay was able to detect antibodies against both BVDV-1 and the atypical pestivirus. This novel microsphere immunoassay has the potential to be multiplexed for simultaneous detection of antibodies against different bovine pathogens in a high-throughput and economical way.

  5. A Particle-Associated Glycoprotein Signal Peptide Essential for Virus Maturation and Infectivity

    PubMed Central

    Lindemann, Dirk; Pietschmann, Thomas; Picard-Maureau, Marcus; Berg, Angelika; Heinkelein, Martin; Thurow, Jana; Knaus, Petra; Zentgraf, Hanswalter; Rethwilm, Axel

    2001-01-01

    Signal peptides (SP) are key determinants for targeting glycoproteins to the secretory pathway. Here we describe the involvement in particle maturation as an additional function of a viral glycoprotein SP. The SP of foamy virus (FV) envelope glycoprotein is predicted to be unusually long. Using an SP-specific antiserum, we demonstrate that its proteolytic removal occurs posttranslationally by a cellular protease and that the major N-terminal cleavage product, gp18, is found in purified viral particles. Analysis of mutants in proposed signal peptidase cleavage positions and N-glycosylation sites revealed an SP about 148 amino acids (aa) in length. FV particle release from infected cells requires the presence of cognate envelope protein and cleavage of its SP sequence. An N-terminal 15-aa SP domain with two conserved tryptophan residues was found to be essential for the egress of FV particles. While the SP N terminus was found to mediate the specificity of FV Env to interact with FV capsids, it was dispensable for Env targeting to the secretory pathway and FV envelope-mediated infectivity of murine leukemia virus pseudotypes. PMID:11390578

  6. Phosphorylation of the multidrug resistance associated glycoprotein

    SciTech Connect

    Mellado, W.; Horwitz, S.B.

    1987-11-03

    Drug-resistant cell lines derived from the mouse macrophage-like cell line J774.2 express the multidrug resistant phenotype which includes the overexpression of a membrane glycoprotein (130-140 kilodaltons). Phosphorylation of this resistant-specific glycoprotein (P-glycoprotein) in intact cells and in cell-free membrane fractions has been studied. The phosphorylated glycoprotein can be immunoprecipitated by a rabbit polyclonal antibody specific for the glycoprotein. Phosphorylation studies done with partially purified membrane fractions derived from colchicine-resistant cells indicated that (a) phosphorylation of the glycoprotein in 1 mM MgCl/sub 2/ was enhanced a minimum of 2-fold by 10 ..mu..M cAMP and (b) the purified catalytic subunit of the cAMP-dependent protein kinase (protein kinase A) phosphorylated partially purified glycoprotein that was not phosphorylated by (..gamma..-/sup 32/P)ATP alone, suggesting that autophosphorylation was not involved. These results indicate that the glycoprotein is a phosphoprotein and that at least one of the kinases responsible for its phosphorylation is a membrane-associated protein kinase A. The state of phosphorylation of the glycoprotein, which is a major component of the multidrug resistance phenotype, may be related to the role of the glycoprotein in maintaining drug resistance.

  7. Protection of sheep against bovine leukemia virus (BLV) infection by vaccination with recombinant vaccinia viruses expressing BLV envelope glycoproteins: correlation of protection with CD4 T-cell response to gp51 peptide 51-70.

    PubMed

    Gatei, M H; Naif, H M; Kumar, S; Boyle, D B; Daniel, R C; Good, M F; Lavin, M F

    1993-04-01

    We have previously constructed vaccinia virus (VV) recombinants containing a complete or truncated envelope (env) gene of bovine leukemia virus (BLV). Only recombinants carrying the complete env gene (VV-BLV2 and VV-BLV3) expressed env glycoprotein on the surface of virus-infected cells and produced an antibody response in rabbits. In the present study, these VV recombinants were used to immunize sheep prior to challenge with BLV-infected peripheral blood mononuclear cells. Both humoral and cell-mediated immunity were monitored in infected animals. Sheep inoculated with recombinants containing the complete env gene showed a CD4 response to a defined epitope of gp51, but this response was absent 4 months postchallenge. Anti-gp51 antibodies appeared in animals inoculated with complete env 2 weeks after challenge, reached a peak at 4 weeks, and subsequently declined over 16 months. No CD4 response was recorded in animals inoculated with recombinants containing truncated env gene (VV-BLV1). BLV-infected control animals and those animals receiving VV-BLV1 were slower to develop antibodies postchallenge, and the titers of anti-gp51 antibodies continued to increase over 16 months. Proviral DNA was detected by the polymerase chain reaction in the four groups at 6 weeks after challenge. However, it could not be detected 4 months postinfection in the VV groups inoculated with complete env. Provirus was present in the VV-BLV1 and control groups over the 16-month trial period. These results demonstrate that vaccination with VV recombinants containing the complete env gene of BLV protects sheep against infection and that protection correlated with a CD4 T-cell response to a defined epitope.

  8. Cross-linking of glycoprotein oligomers during herpes simplex virus type 1 entry.

    PubMed

    Handler, C G; Cohen, G H; Eisenberg, R J

    1996-09-01

    Herpes simplex virus (HSV) has 10 glycoproteins in its envelope. Glycoprotein B (gB), gC, gD, gH, and gL have been implicated in virus entry. We previously used chemical cross-linking to show that these five glycoproteins were close enough to each other to be cross-linked into homodimeric and hetero-oligomeric forms; hetero-oligomers of gB-gC, gC-gD, gD-gB, gH-gL, gC-gL and gD-gL were found in purified virions. To better understand the roles of these glycoproteins in viral entry, we have modified a standard HSV penetration assay to include cross-linkers. This allowed us to examine changes in associations of viral glycoproteins during the entry process. HSV-1(KOS) was adsorbed at 4 degrees C to human neuroblastoma cells (SY5Y). The temperature was raised to 37 degrees C and cells were treated with cross-linker at various times after the temperature shift. Cytoplasmic extracts were examined by Western blotting (immunoblotting) for viral glycoproteins. We found that (i) as in virus alone, the length and concentration of the cross-linking agent affected the number of specific complexes isolated; (ii) the same glycoprotein patterns found in purified virions were also present after attachment of virions to cells; and (iii) the ability to cross-link HSV glycoproteins changed as virus penetration proceeded, e.g., gB and gD complexes which were present during attachment disappeared with increasing time, and their disappearance paralleled the kinetics of penetration. However, this phenomenon appeared to be selective since it was not observed with gC oligomers. In addition, we examined the cross-linking patterns of gB and gD in null viruses K082 and KOSgD beta. Neither of these mutants, which attach but cannot penetrate, showed changes in glycoprotein cross-linking over time. We speculate that these changes are due to conformational changes which preclude cross-linking or spatial alterations which dissociate the glycoprotein interactions during the penetration events. PMID

  9. Separation and partial characterization of proteinases with substrate specificity for basic amino acids from human MOLT-4 T lymphocytes: identification of those inhibited by variable-loop-V3 peptides of HIV-1 (human immunodeficiency virus-1) envelope glycoprotein.

    PubMed Central

    Harvima, I T; Harvima, R J; Nilsson, G; Ivanoff, L; Schwartz, L B

    1993-01-01

    The V3 loop of the HIV (human immunodeficiency virus)-1 envelope glycoprotein gp120 likely plays a role in HIV-1 infectivity. Although the amino acid sequence of the V3 loop is hypervariable, it contains a conserved region, Gly-Pro-Gly-Arg, that shows similarity to the active-site Gly-Pro-Cys-Arg sequence of inter-alpha-trypsin and trypstatin proteinase inhibitors. The purpose of the present work was to identify proteinases recognizing substrates with basic amino acids in the P1 substrate site that are present in MOLT-4 cells, a human CD4-positive T helper lymphocyte cell line, and to characterize these enzymes in terms of substrate, pH and ionic-strength preferences, size and susceptibility to various inhibitors, including 24- and 36-amino-acid-long V3 loop peptides. Extraction of MOLT-4 cells at low ionic strength solubilized nearly all of the trypsin-like activity, which was separable into five peaks of activity by chromatography on Mono-Q: Peaks 1, 2a, 2b, 3 and 4. All showed a neutral pH optimum, and all except Peak 4 showed optimal activity at high ionic strength. Peak 1 preferred Tos-Gly-Pro-Arg, p-nitroanilide (-pNA) substrate; Peaks 2-4 preferred benzyloxycarbonyl-Val-Leu-Gly-Arg-pNA. Peak 1, a zinc-dependent enzyme with serine and histidine in the active site, exhibited an M(r) of 75,000 on Superose 12 and was poorly inhibited by V3 loop peptides. Peak 2 contained two overlapping peaks, called 2a and 2b, that exhibited properties of zinc-dependent metalloproteinases. Gel filtration of Peak 2 activities revealed a major peak of activity at 81 kDa and a shoulder centred at 240 kDa. Each was modestly inhibited by V3 loop peptides. Peak 3, a zinc-dependent proteinase, exhibited a molecular mass of 100 kDa by gel filtration and was particularly sensitive to inhibition by V3 loop peptides. Peak 4 exhibited a molecular mass of 1100 kDa by gel filtration and was not inhibited by V3 loop peptides. None of these enzymes could be classified as mast-cell tryptase

  10. Glycoprotein synthesis

    DOEpatents

    Schultz, Peter G.; Wang, Lei; Zhang, Zhiwen

    2007-05-15

    Methods for making glycoproteins, both in vitro and in vivo, are provided. One method involves incorporating an unnatural amino acid into a protein and attaching one or more saccharide moieties to the unnatural amino acid. Another method involves incorporating an unnatural amino acid that includes a saccharide moiety into a protein. Proteins made by both methods can be further modified with additional sugars.

  11. Glycoprotein synthesis

    DOEpatents

    Schultz, Peter G.; Wang, Lei; Zhang, Zhiwen

    2006-10-31

    Methods for making glycoproteins, both in vitro and in vivo, are provided. One method involves incorporating an unnatural amino acid into a protein and attaching one or more saccharide moieties to the unnatural amino acid. Another method involves incorporating an unnatural amino acid that includes a saccharide moiety into a protein. Proteins made by both methods can be further modified with additional sugars.

  12. Glycoprotein synthesis

    DOEpatents

    Schultz, Peter G.; Wang, Lei; Zhang, Zhiwen

    2007-08-28

    Methods for making glycoproteins, both in vitro and in vivo, are provided. One method involves incorporating an unnatural amino acid into a protein and attaching one or more saccharide moieties to the unnatural amino acid. Another method involves incorporating an unnatural amino acid that includes a saccharide moiety into a protein. Proteins made by both methods can be further modified with additional sugars.

  13. Glycoprotein synthesis

    DOEpatents

    Schultz, Peter G.; Wang, Lei; Zhang, Zhiwen

    2007-07-03

    Methods for making glycoproteins, both in vitro and in vivo, are provided. One method involves incorporating an unnatural amino acid into a protein and attaching one or more saccharide moieties to the unnatural amino acid. Another method involves incorporating an unnatural amino acid that includes a saccharide moiety into a protein. Proteins made by both methods can be further modified with additional sugars.

  14. Glycoprotein synthesis

    DOEpatents

    Schultz, Peter G.; Wang, Lei; Zhang, Zhiwen

    2005-08-09

    Methods for making glycoproteins, both in vitro and in vivo, are provided. One method involves incorporating an unnatural amino acid into a protein and attaching one or more saccharide moieties to the unnatural amino acid. Another method involves incorporating an unnatural amino acid that includes a saccharide moiety into a protein. Proteins made by both methods can be further modified with additional sugars.

  15. Glycoprotein synthesis

    DOEpatents

    Schultz, Peter G.; Wang, Lei; Zhang, Zhiwen

    2010-11-16

    Methods for making glycoproteins, both in vitro and in vivo, are provided. One method involves incorporating an unnatural amino acid into a protein and attaching one or more saccharide moieties to the unnatural amino acid. Another method involves incorporating an unnatural amino acid that includes a saccharide moiety into a protein. Proteins made by both methods can be further modified with additional sugars.

  16. Glycoprotein synthesis

    DOEpatents

    Schultz, Peter G.; Wang, Lei; Zhang, Zhiwen

    2010-11-02

    Methods for making glycoproteins, both in vitro and in vivo, are provided. One method involves incorporating an unnatural amino acid into a protein and attaching one or more saccharide moieties to the unnatural amino acid. Another method involves incorporating an unnatural amino acid that includes a saccharide moiety into a protein. Proteins made by both methods can be further modified with additional sugars.

  17. Glycoprotein synthesis

    DOEpatents

    Schultz, Peter G.; Wang, Lei; Zhang, Zhiwen

    2007-02-27

    Methods for making glycoproteins, both in vitro and in vivo, are provided. One method involves incorporating an unnatural amino acid into a protein and attaching one or more saccharide moieties to the unnatural amino acid. Another method involves incorporating an unnatural amino acid that includes a saccharide moiety into a protein. Proteins made by both methods can be further modified with additional sugars.

  18. Glycoprotein synthesis

    DOEpatents

    Methods for making glycoproteins, both in vitro and in vivo, are provided. One method involves incorporating an unnatural amino acid into a protein and attaching one or more saccharide moieties to the unnatural amino acid. Another method involves incorporating an unnatural amino acid that includes a saccharide moiety into a protein. Proteins made by both methods can be further modified with additional sugars.

    2009-07-14

    Methods for making glycoproteins, both in vitro and in vivo, are provided. One method involves incorporating an unnatural amino acid into a protein and attaching one or more saccharide moieties to the unnatural amino acid. Another method involves incorporating an unnatural amino acid that includes a saccharide moiety into a protein. Proteins made by both methods can be further modified with additional sugars.

  19. Glycoprotein synthesis

    DOEpatents

    Shultz, Peter G.; Wang, Lei; Zhang, Zhiwen

    2007-04-03

    Methods for making glycoproteins, both in vitro and in vivo, are provided. One method involves incorporating an unnatural amino acid into a protein and attaching one or more saccharide moieties to the unnatural amino acid. Another method involves incorporating an unnatural amino acid that includes a saccharide moiety into a protein. Proteins made by both methods can be further modified with additional sugars.

  20. Influenza Virus Assembly and Lipid Raft Microdomains: a Role for the Cytoplasmic Tails of the Spike Glycoproteins

    PubMed Central

    Zhang, Jie; Pekosz, Andrew; Lamb, Robert A.

    2000-01-01

    Influenza viruses encoding hemagglutinin (HA) and neuraminidase (NA) glycoproteins with deletions in one or both cytoplasmic tails (HAt− or NAt−) have a reduced association with detergent-insoluble glycolipids (DIGs). Mutations which eliminated various combinations of the three palmitoylation sites in HA exhibited reduced amounts of DIG-associated HA in virus-infected cells. The influenza virus matrix (M1) protein was also found to be associated with DIGs, but this association was decreased in cells infected with HAt− or NAt− virus. Regardless of the amount of DIG-associated protein, the HA and NA glycoproteins were targeted primarily to the apical surface of virus-infected, polarized cells. The uncoupling of DIG association and apical transport was augmented by the observation that the influenza A virus M2 protein as well as the influenza C virus HA-esterase-fusion glycoprotein were not associated with DIGs but were apically targeted. The reduced DIG association of HAt− and NAt− is an intrinsic property of the glycoproteins, as similar reductions in DIG association were observed when the proteins were expressed from cDNA. Examination of purified virions indicated reduced amounts of DIG-associated lipids in the envelope of HAt− and NAt− viruses. The data indicate that deletion of both the HA and NA cytoplasmic tails results in reduced DIG association and changes in both virus polypeptide and lipid composition. PMID:10775599

  1. Unusual molecular architecture of the machupo virus attachment glycoprotein.

    PubMed

    Bowden, Thomas A; Crispin, Max; Graham, Stephen C; Harvey, David J; Grimes, Jonathan M; Jones, E Yvonne; Stuart, David I

    2009-08-01

    New World arenaviruses, which cause severe hemorrhagic fever, rely upon their envelope glycoproteins for attachment and fusion into their host cell. Here we present the crystal structure of the Machupo virus GP1 attachment glycoprotein, which is responsible for high-affinity binding at the cell surface to the transferrin receptor. This first structure of an arenavirus glycoprotein shows that GP1 consists of a novel alpha/beta fold. This provides a blueprint of the New World arenavirus attachment glycoproteins and reveals a new architecture of viral attachment, using a protein fold of unknown origins.

  2. The cell surface glycoprotein layer of the extreme halophile Halobacterium salinarum and its relation to Haloferax volcanii: cryo-electron tomography of freeze-substituted cells and projection studies of negatively stained envelopes.

    PubMed

    Trachtenberg, S; Pinnick, B; Kessel, M

    2000-05-01

    We have studied the surface layer (S-layer) of Halobacterium salinarum (formerly Halobacterium halobium), an extreme halophile requiring high concentrations of sodium, by electron microscopy of (a) isolated, negatively stained, flattened envelopes and (b) cryo-fixation of intact cells in their high-salt growth medium followed by freeze substitution and tomography of thin sections. From the negatively stained isolated envelopes we have calculated a two-dimensional, projection map that is strikingly similar to that of Haloferax volcanii, an extreme halophile requiring high concentrations of magnesium; both projection maps show the hexagonal arrangement of the morphological units with an identical center-to-center spacing of 150 A; each of the morphological units of the two species has six subunits with a similar density distribution and apparent domain organization. In contrast to the two-dimensional map, the tomographic reconstruction of Halob. salinarum does not agree in a straightforward way with the three-dimensional, electron crystallographic map of negatively stained Halof. volcanii envelopes, although the main features of the lattice and the morphological units are evident. The tomographic reconstruction of sections from epoxy-embedded material suffers from directional compression due to sectioning stress and continuous dimensional changes and mass loss due to electron irradiation. This communication consists, therefore, of three parts: (a) a comparison of the projection maps of negatively stained envelopes of Halof. volcanii and Halob. salinarum; (b) a comparison of the three-dimensional maps obtained by electron crystallography (Halof. volcanii) and low-dose cryo-tomography (Halob. salinarum); and (c) a methodological study of mass loss and dimensional changes of plastic-embedded material under low-dose conditions at room and liquid nitrogen temperatures.

  3. Serological responses in chimpanzees inoculated with human immunodeficiency virus glycoprotein (gp120) subunit vaccine

    SciTech Connect

    Arthur, L.O.; Pyle, S.W.; Nara, P.L.; Bess, J.W. Jr.; Gonda, M.A.; Kelliher, J.C.; Gilden, R.V.; Robey, W.G.; Bolognesi, D.P.; Gallo, R.C.

    1987-12-01

    The major envelope glycoprotein of a human immunodeficiency virus (HIV) has been purified and was utilized as a prototype vaccine in chimpanzees. The 120,000-dalton glycoprotein (gp120) was purified from membranes of human T-lymphotropic virus (HTLV)-IIIB-infected cells and the final preparation contained low levels to no detectable HTLV-IIIB core antigen (p24) and low levels of endotoxin. Chimpanzees inoculated with gp120 responded by developing antibodies that precipitated radiolabeled gp120 and neutralized in vitro infection of HTLV-IIIB. Antibodies to HTLV-IIIB p24 were not detected in the gp120-immunized chimpanzees. Peripheral blood leukocytes from the vaccinated animals were examined for T4/sup +/ and T8/sup +/ cells, and no decrease in the T4/T8 ratio was found, indicating that immunization with a ligand (gp120) that binds to T4 has not detectable adverse effect on the population of T4/sup +/ cells. The only current animal model that can be reproducibly infected with HIV is the chimpanzee. Immunization of chimpanzees with HIV proteins will provide an experimental system for testing the effectiveness of prototype vaccines for preventing HIV infection in vivo.

  4. Cell entry of enveloped viruses.

    PubMed

    Cosset, François-Loic; Lavillette, Dimitri

    2011-01-01

    Enveloped viruses penetrate their cell targets following the merging of their membrane with that of the cell. This fusion process is catalyzed by one or several viral glycoproteins incorporated on the membrane of the virus. These envelope glycoproteins (EnvGP) evolved in order to combine two features. First, they acquired a domain to bind to a specific cellular protein, named "receptor." Second, they developed, with the help of cellular proteins, a function of finely controlled fusion to optimize the replication and preserve the integrity of the cell, specific to the genus of the virus. Following the activation of the EnvGP either by binding to their receptors and/or sometimes the acid pH of the endosomes, many changes of conformation permit ultimately the action of a specific hydrophobic domain, the fusion peptide, which destabilizes the cell membrane and leads to the opening of the lipidic membrane. The comprehension of these mechanisms is essential to develop medicines of the therapeutic class of entry inhibitor like enfuvirtide (Fuzeon) against human immunodeficiency virus (HIV). In this chapter, we will summarize the different envelope glycoprotein structures that viruses develop to achieve membrane fusion and the entry of the virus. We will describe the different entry pathways and cellular proteins that viruses have subverted to allow infection of the cell and the receptors that are used. Finally, we will illustrate more precisely the recent discoveries that have been made within the field of the entry process, with a focus on the use of pseudoparticles. These pseudoparticles are suitable for high-throughput screenings that help in the development of natural or artificial inhibitors as new therapeutics of the class of entry inhibitors.

  5. Pseudorabies virus mutants lacking the essential glycoprotein gII can be complemented by glycoprotein gI of bovine herpesvirus 1.

    PubMed Central

    Rauh, I; Weiland, F; Fehler, F; Keil, G M; Mettenleiter, T C

    1991-01-01

    The genome of pseudorabies virus (PrV) encodes at least seven glycoproteins. The glycoprotein complex gII consists of three related polypeptides, two of them derived by proteolytic cleavage from a common precursor and linked via disulfide bonds. It is homologous to herpes simplex virus (HSV) gB and is therefore thought to be essential for PrV replication, as is gB for HSV replication. To isolate PrV mutants deficient in gII expression, we established cell lines that stably carry the PrV gII gene. Line N7, of Vero cell origin, contains the gII gene under its own promoter and expresses gII after transactivation by herpesviral functions after infection. MDBK-derived line MT3 contains the gII gene under control of the mouse metallothionein promoter. However, it has essentially lost inducibility and constitutively produces high amounts of correctly processed glycoprotein gII. We used a beta-galactosidase expression cassette inserted into a partially deleted cloned copy of the gII gene for cotransfection with PrV DNA. gII- PrV mutants were isolated from viral progeny by taking advantage of their blue-plaque phenotype when incubated under an agarose overlay containing a chromogenic substrate. Analysis of these mutants proved that gII is indeed essential for PrV replication, since the gII- mutants grew normally on gII-complementing cells but were unable to produce plaques on noncomplementing cells. Surprisingly the PrV gII- mutants were also able to grow on a cell line constitutively expressing the gB-homologous glycoprotein gI from bovine herpesvirus 1 (BHV-1) to the same extent as on cells expressing PrV gII. gII- PrV propagated on cells expressing BHV-1 gI became susceptible to neutralization by anti-BHV-1 gI monoclonal antibodies. We also found that BHV-1 gI is present in the envelope of purified gII- pseudorabies virions grown on cells expressing BHV-1 gI, as judged by radioimmunoprecipitation and immunoelectron microscopy. These results prove that BHV-1 gI is

  6. Glycoprotein D protects mice against lethal challenge with herpes simplex virus types 1 and 2.

    PubMed Central

    Long, D; Madara, T J; Ponce de Leon, M; Cohen, G H; Montgomery, P C; Eisenberg, R J

    1984-01-01

    Glycoprotein D is a virion envelope component of herpes simplex virus types 1 and 2. Sets of mice were immunized with purified gD-1 or gD-2 and were challenged with a lethal dose of herpes simple virus, either type 1 or type 2. All or virtually all of the immunized mice survived challenge with either agent, whereas challenge of sham-immunized mice was almost always fatal. Serum samples taken before challenge contained gD-specific antibodies which had 50% neutralization titers ranging from 1:16 to 1:512 against homologous and heterologous virus types. We conclude that either gD-1 or gD-2 is a potential candidate for a subunit vaccine against herpetic infections. Images PMID:6319291

  7. The differences in short- and long-term varicella-zoster virus (VZV) immunoglobulin G levels following varicella vaccination of healthcare workers measured by VZV fluorescent-antibody-to-membrane-antigen assay (FAMA), VZV time-resolved fluorescence immunoassay and a VZV purified glycoprotein enzyme immunoassay.

    PubMed

    Maple, P A C; Haedicke, J; Quinlivan, M; Steinberg, S P; Gershon, A A; Brown, K E; Breuer, J

    2016-08-01

    Healthcare workers (HCWs) reporting no history of varicella frequently receive varicella vaccination (vOka) if they test varicella-zoster virus (VZV) immunoglobulin G (IgG) negative. In this study, the utilities of VZV-IgG time-resolved fluorescence immunoassay (VZV-TRFIA) and a commercial VZV-IgG purified glycoprotein enzyme immunoassay (gpEIA) currently used in England for confirming VZV immunity have been compared to the fluorescent-antibody-to-membrane-antigen assay (FAMA). A total of 110 HCWs received two doses of vOka vaccine spaced 6 weeks apart and sera collected pre-vaccination (n = 100), at 6 weeks post-completion of vaccination (n = 86) and at 12-18 months follow-up (n = 73) were analysed. Pre-vaccination, by FAMA, 61·0% sera were VZV IgG negative, and compared to FAMA the sensitivities of VZV-TRFIA and gpEIA were 74·4% [95% confidence interval (CI) 57·9-87·0] and 46·2% (95% CI 30·1-62·8), respectively. Post-completion of vaccination the seroconversion rate by FAMA was 93·7% compared to rates of 95·8% and 70·8% determined by VZV-TRFIA and gpEIA, respectively. At 12-18 months follow-up seropositivity rates by FAMA, VZV-TRFIA and gpEIA were 78·1%, 74·0% and 47·9%, respectively. Compared to FAMA the sensitivities of VZV-TRFIA and gpEIA for measuring VZV IgG following vaccination were 96·4% (95% CI 91·7-98·8) and 74·6% (95% CI 66·5-81·6), respectively. Using both FAMA and VZV-TRFIA to identify healthy adult VZV susceptibles and measure seroconversion showed that vOka vaccination of HCWs is highly immunogenic.

  8. Gas stream purifier

    NASA Technical Reports Server (NTRS)

    Adam, Steven J.

    1994-01-01

    A gas stream purifier has been developed that is capable of removing corrosive acid, base, solvent, organic, inorganic, and water vapors as well as particulates from an inert mixed gas stream using only solid scrubbing agents. This small, lightweight purifier has demonstrated the ability to remove contaminants from an inert gas stream with a greater than 99 percent removal efficiency. The Gas Stream Purifier has outstanding market and sales potential in manufacturing, laboratory and science industries, medical, automotive, or any commercial industry where pollution, contamination, or gas stream purification is a concern. The purifier was developed under NASA contract NAS9-18200 Schedule A for use in the international Space Station. A patent application for the Gas Stream Purifier is currently on file with the United States Patent and Trademark Office.

  9. SAFEGUARDS ENVELOPE

    SciTech Connect

    Duc Cao; Richard Metcalf

    2010-07-01

    The Safeguards Envelope is a strategy to determine a set of specific operating parameters within which nuclear facilities may operate to maximize safeguards effectiveness without sacrificing safety or plant efficiency. This paper details advanced statistical techniques that will be applied to real plant process monitoring (PM) data from the Idaho Chemical Processing Plant (ICPP). In a simulation based on this data, multi-tank and multi-attribute correlations were tested against synthetic diversion scenarios. Kernel regression smoothing was used to fit a curve to the historical data, and multivariable, residual analysis and cumulative sum techniques set parameters for operating conditions. Diversion scenarios were created and tested, showing improved results when compared with a previous study utilizing only one-variable Z-testing. A brief analysis of the impact of the safeguards optimization on the rest of plant efficiency, criticality concerns, and overall requirements is presented.

  10. METHOD FOR PURIFYING URANIUM

    DOEpatents

    Knighton, J.B.; Feder, H.M.

    1960-04-26

    A process is given for purifying a uranium-base nuclear material. The nuclear material is dissolved in zinc or a zinc-magnesium alloy and the concentration of magnesium is increased until uranium precipitates.

  11. Antibodies elicited by yeast glycoproteins recognize HIV-1 virions and potently neutralize virions with high mannose N-glycans.

    PubMed

    Zhang, Hong; Fu, Hu; Luallen, Robert J; Liu, Bingfen; Lee, Fang-Hua; Doms, Robert W; Geng, Yu

    2015-09-22

    The glycan shield on the human immunodeficiency virus 1 (HIV-1) envelope (Env) glycoprotein has drawn attention as a target for HIV-1 vaccine design given that an increasing number of potent and broadly neutralizing antibodies (bNAbs) recognize epitopes entirely or partially comprised of high mannose type N-linked glycans. In an attempt to generate immunogens that target the glycan shield of HIV-1, we previously engineered a triple mutant (TM) strain of Saccharomyces cerevisiae that results in exclusive presentation of high mannose type N-glycans, and identified five TM yeast glycoproteins that support strong binding of 2G12, a bNAb that targets a cluster of high mannose glycans on the gp120 subunit of Env. Here, we further analyzed the antigenicity and immunogenicity of these proteins in inducing anti-HIV responses. Our study demonstrated that the 2G12-reactive TM yeast glycoproteins efficiently bound to recently identified bNAbs including PGT125-130 and PGT135 that recognize high mannose glycan-dependent epitopes. Immunization of rabbits with a single TM yeast glycoprotein (Gp38 or Pst1), when conjugated to a promiscuous T-cell epitope peptide and coadministered with a Toll-like receptor 2 agonist, induced glycan-specific HIV-1 Env cross-reactive antibodies. The immune sera bound to both synthetic mannose oligosaccharides and gp120 proteins from a broad range of HIV-1 strains. The purified antibodies recognized and captured virions that contain both complex- and high mannose-type of N-glycans, and potently neutralized virions from different HIV-1 clades but only when the virions were enforced to retain high mannose N-glycans. This study provides insights into the elicitation of anti-carbohydrate, HIV-1 Env-cross reactive antibodies with a heterologous glycoprotein and may have applications in the design and administration of immunogens that target the viral glycan shield for development of an effective HIV-1 vaccine. PMID:26277072

  12. Bovine herpesvirus type 1 glycoprotein C expression in MDBK cells and its reactivity in enzyme-linked immunosorbent assay.

    PubMed

    Gupta, P K; Saini, M; Bandyopadhyay, S K; Garg, S K

    1998-12-01

    Glycoprotein C (gC) of bovine herpesvirus type 1 (BHV-1) is a major viral glycoprotein expressed at high level on the surface of infected cells and on the virion envelope. This glycoprotein is also a major target of immune response at both humoral and cellular levels. The plasmid pRSV-gC having complete coding gene for BHV-1 gC was transfected into MDBK cells and the expression of gC in these cells was detected by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE) and immunoblot analysis. Transcription of the gC gene was confirmed by reverse transcription-polymerase chain reaction (RT-PCR) of total RNA isolated from transfected cells. MDBK cells expressing BHV-1 gC were used as antigen in enzyme-linked immunosorbent assay (ELISA) of antibodies against BHV-1 in field sera. The results were found comparable (92.44%) with those obtained with BHV-1 purified antigen.

  13. Incorporation of Spike and Membrane Glycoproteins into Coronavirus Virions

    PubMed Central

    Ujike, Makoto; Taguchi, Fumihiro

    2015-01-01

    The envelopes of coronaviruses (CoVs) contain primarily three proteins; the two major glycoproteins spike (S) and membrane (M), and envelope (E), a non-glycosylated protein. Unlike other enveloped viruses, CoVs bud and assemble at the endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC). For efficient virion assembly, these proteins must be targeted to the budding site and to interact with each other or the ribonucleoprotein. Thus, the efficient incorporation of viral envelope proteins into CoV virions depends on protein trafficking and protein–protein interactions near the ERGIC. The goal of this review is to summarize recent findings on the mechanism of incorporation of the M and S glycoproteins into the CoV virion, focusing on protein trafficking and protein–protein interactions. PMID:25855243

  14. Chimeric Measles Viruses with a Foreign Envelope

    PubMed Central

    Spielhofer, Pius; Bächi, Thomas; Fehr, Thomas; Christiansen, Gudrun; Cattaneo, Roberto; Kaelin, Karin; Billeter, Martin A.; Naim, Hussein Y.

    1998-01-01

    Measles virus (MV) and vesicular stomatitis virus (VSV) are both members of the Mononegavirales but are only distantly related. We generated two genetically stable chimeric viruses. In MGV, the reading frames of the MV envelope glycoproteins H and F were substituted by a single reading frame encoding the VSV G glycoprotein; MG/FV is similar but encodes a G/F hybrid in which the VSV G cytoplasmic tail was replaced by that of MV F. In contrast to MG/FV, MGV virions do not contain the MV matrix (M) protein. This demonstrates that virus assembly is possible in the absence of M; conversely, the cytoplasmic domain of F allows incorporation of M and enhances assembly. The formation of chimeric viruses was substantially delayed and the titers obtained were reduced about 50-fold in comparison to standard MV. In the novel chimeras, transcription and replication are mediated by the MV ribonucleoproteins but the envelope glycoproteins dictate the host range. Mice immunized with the chimeric viruses were protected against lethal doses of wild-type VSV. These findings suggest that it is feasible to construct MV variants bearing a variety of different envelopes for use as vaccines or for gene therapeutic purposes. PMID:9499071

  15. Purified silicon production system

    DOEpatents

    Wang, Tihu; Ciszek, Theodore F.

    2004-03-30

    Method and apparatus for producing purified bulk silicon from highly impure metallurgical-grade silicon source material at atmospheric pressure. Method involves: (1) initially reacting iodine and metallurgical-grade silicon to create silicon tetraiodide and impurity iodide byproducts in a cold-wall reactor chamber; (2) isolating silicon tetraiodide from the impurity iodide byproducts and purifying it by distillation in a distillation chamber; and (3) transferring the purified silicon tetraiodide back to the cold-wall reactor chamber, reacting it with additional iodine and metallurgical-grade silicon to produce silicon diiodide and depositing the silicon diiodide onto a substrate within the cold-wall reactor chamber. The two chambers are at atmospheric pressure and the system is open to allow the introduction of additional source material and to remove and replace finished substrates.

  16. Variability of mammalian liver nuclear-envelope preparations.

    PubMed

    Agutter, P S; Gleed, C D

    1980-10-15

    The composition, density and enzymic activities of sheep liver nuclear-envelope preparations were found to vary markedly according to the concentrations of nuclei during the lysis stage. The effect of nuclear concentration on the properties of the purified envelopes could not be attributed to bound Mg2+ or to other ions, and appeared to result from some component of the nucleus which was not eluted during lysis. The implications of these findings for studies on the nuclear envelope are discussed.

  17. Preparation and Characterization of Envelope Membranes from Nongreen Plastids

    PubMed Central

    Alban, Claude; Joyard, Jacques; Douce, Roland

    1988-01-01

    We have developed a reliable procedure for the purification of envelope membranes from cauliflower (Brassica oleracea L.) bud plastids and sycamore (Acer pseudoplatanus L.) cell amyloplasts. After disruption of purified intact plastids, separation of envelope membranes was achieved by centrifugation on a linear sucrose gradient. A membrane fraction, having a density of 1.122 grams per cubic centimeter and containing carotenoids, was identified as the plastid envelope by the presence of monogalactosyldiacylglycerol synthase. Using antibodies raised against spinach chloroplast envelope polypeptides E24 and E30, we have demonstrated that both the outer and the inner envelope membranes were present in this envelope fraction. The major polypeptide in the envelope fractions from sycamore and cauliflower plastids was identified immunologically as the phosphate translocator. In the envelope membranes from cauliflower and sycamore plastids, the major glycerolipids were monogalactosyldiacylglycerol, digalactosyldiacylglycerol, and phosphatidylcholine. Purified envelope membranes from cauliflower bud plastids and sycamore amyloplasts also contained a galactolipid:galactolipid galactosyltransferase, enzymes for phosphatidic acid and diacylglycerol biosynthesis, acyl-coenzyme A thioesterase, and acyl-coenzyme A synthetase. These results demonstrate that envelope membranes from nongreen plastids present a high level of homology with chloroplasts envelope membranes. Images Fig. 4 Fig. 5 Fig. 6 Fig. 7 PMID:16666372

  18. Method of purifying isosaccharinate

    DOEpatents

    Rai, Dhanpat; Moore, Robert C.; Tucker, Mark D.

    2010-09-07

    A method of purifying isosaccharinate by mixing sodium carbonate, potassium carbonate, sodium hydroxide or potassium hydroxide with calcium isosaccharinate, removing the precipitated calcium carbonate and adjusting the pH to between approximately 4.5 to 5.0 thereby removing excess carbonate and hydroxide to provide an acidic solution containing isosaccharinate.

  19. Identification of peptides that bind hepatitis C virus envelope protein E2 and inhibit viral cellular entry from a phage-display peptide library.

    PubMed

    Lü, Xin; Yao, Min; Zhang, Jian-Min; Yang, Jing; Lei, Ying-Feng; Huang, Xiao-Jun; Jia, Zhan-Sheng; Ma, Li; Lan, Hai-Yun; Xu, Zhi-Kai; Yin, Wen

    2014-05-01

    Hepatitis C virus (HCV) envelope protein E2 is required for the entry of HCV into cells. Viral envelope proteins interact with cell receptors in a multistep process, which may be a promising target for the development of novel antiviral agents. In this study, a heptapeptide M13 phage-display library was screened for peptides that bind specifically to prokaryotically expressed, purified truncated HCV envelope protein E2. ELISA assay was used to quantify the binding of the peptides to HCV E2 protein. Flow cytometry, quantitative reverse-transcription PCR and western blotting were used to investigate the inhibition effect of one peptide on HCV infection in hepatoma cells (Huh7.5) in vitro. Four peptides capable of binding specifically to HCV E2 protein were obtained after three rounds of biopanning. Peptide C18 (WPWHNHR), with the highest affinity for binding HCV E2 protein, was synthesized. The results showed that peptide C18 inhibited the viral infectivity of both HCV pseudotype particles (HCVpp) harboring HCV envelope glycoproteins and cell-culture produced HCV (HCVcc). Thus, this study demonstrated that peptide C18 is a potential candidate for anti-HCV therapy as a novel viral entry inhibitor.

  20. Purified water quality study

    SciTech Connect

    Spinka, H.; Jackowski, P.

    2000-04-03

    Argonne National Laboratory (HEP) is examining the use of purified water for the detection medium in cosmic ray sensors. These sensors are to be deployed in a remote location in Argentina. The purpose of this study is to provide information and preliminary analysis of available water treatment options and associated costs. This information, along with the technical requirements of the sensors, will allow the project team to determine the required water quality to meet the overall project goals.

  1. PROCESS OF PURIFYING URANIUM

    DOEpatents

    Seaborg, G.T.; Orlemann, E.F.; Jensen, L.H.

    1958-12-23

    A method of obtaining substantially pure uranium from a uranium composition contaminated with light element impurities such as sodium, magnesium, beryllium, and the like is described. An acidic aqueous solution containing tetravalent uranium is treated with a soluble molybdate to form insoluble uranous molybdate which is removed. This material after washing is dissolved in concentrated nitric acid to obtaln a uranyl nitrate solution from which highly purified uranium is obtained by extraction with ether.

  2. Multitasking: Making the Most out of the Retroviral Envelope.

    PubMed

    Varela, Mariana; Palmarini, Massimo

    2010-08-01

    Evasion of the host's immune system is a required step for the establishment of viral infection. In this article, we discuss the recent findings of Heidmann and colleagues demonstrating that some retroviruses possess an immune suppressive (IS) domain "encrypted" within their envelope glycoprotein that is required to establish a successful infection in immunocompetent hosts [1].

  3. The effect of ginger extract on glycoproteins of Raji cells.

    PubMed

    Zamani, Zahra; Nassir-Ud-Din; Kohan, Haleemeh Kabini; Kadivar, Mehdi; Kalyee, Zahra; Rad, Behzad Laame; Iravani, Ayda; Rahimi, Nourooz Ali; Wahabi, Farideh; Sadeghi, Sedigheh; Pourfallah, Fatemeh; Arjmand, Mohammad

    2014-01-15

    Protein glycosylation is associated with the development and progression of specific diseases, including cancers. The ginger rhizome is known to have anti-cancer and anti-fungal properties. This investigation was carried out to study the effect of ginger on glycoproteins of Raji cells. A 10% yield of ginger extract was mixed with 0.01% DMSO and added to 6 x 10(4) Raji cells at different concentrations for 24, 48 and 72 h at 37 degrees C. Their half maximal inhibitory concentration (IC50) was determined and analyzed statistically using Graphpad prism software. Cell extracts were prepared and their glycoproteins purified using lectin-affinity chromatography (Q proteome total glycoprotein and O glycoprotein kits) and SDS PAGE was carried out. IC50 of ginger extract on Raji cells was 20 microg mL(-1) at 72 h with < 0.01 significance. Silver staining of purified glycoprotiens in Raji cells indicated the presence of O-glycans and N-glycans. N-linked mannose and N-linked sialic acids were detected with the total glycoprotein kit. O-linked galactose and O-linked sialic acids were identified with the O-glycoprotein. Ginger reduced the expression of O-linked sialic acid and also N-linked mannose on Raji cells but had no effect on other glycoproteins. Sialic acid is now well known as a cancer marker and investigations are on to use it as a drug-target in cancerous tissues.

  4. Polymorphism in purified guanylate cyclase from vertebrate rod photoreceptors.

    PubMed Central

    Hayashi, F; Yamazaki, A

    1991-01-01

    Guanylate cyclase from rod photoreceptors of amphibian (toad, Bufo marinus, and frog, Rana catesbeiana) and bovine retinas was solubilized and purified by a single chromatography step on a GTP-agarose column. Silver staining of purified amphibian enzymes in SDS/polyacrylamide gels disclosed a doublet band (110 and 115 kDa), while the bovine enzyme appeared as a singlet band (110 kDa). The identification of these guanylate cyclases was confirmed using three chromatography systems with the purified enzymes. Specific binding to Con A-Sepharose suggested that rod guanylate cyclase is a glycoprotein. Two-dimensional gel electrophoresis of purified toad, frog, and bovine enzymes resolved two, three, and five variants, respectively, that differed in isoelectric point. Two variants of toad guanylate cyclase showed differences in various characterizations. These data suggest multiple mechanisms for regulation of guanylate cyclase activity in vertebrate rod photoreceptors. Images PMID:1675787

  5. Exhaust gas purifying device

    SciTech Connect

    Sakurai, S.; Hamada, S.

    1985-04-23

    An exhaust gas purifying device for use with a diesel engine comprising a filter block disposed in an engine exhaust passage for collecting exhaust gas particulates, and a heater for incinerating the collected exhaust gas particulates. The filter block has parallel channels defined therein and separated from one another by porous partition walls, some of the channels being closed at their inlet ends with blind plugs while the other channels are closed at their outlet ends with blind plugs. The heater is supported by the blind plugs.

  6. Expression and characterization of a soluble rubella virus E1 envelope protein.

    PubMed

    Seto, N O; Gillam, S

    1994-10-01

    Individual specific antigenic rubella virus (RV) structural proteins are required for accurate serological diagnosis of acute and congenital rubella infections as well as rubella immune status. The RV envelope glycoprotein E1 is the major target antigen and plays an important role in viral-specific immune responses. The native virion is difficult to produce in large quantities and the protein subunits are also difficult to isolate without loss of antigenicity. The production of a soluble RV E1 (designated E1 delta Tm) using the baculovirus-insect cell expression system is described. In contrast to wild-type RV E1, the genetically engineered E1 delta Tm protein lacks a transmembrane anchor. It behaved as a secretory protein and was secreted abundantly from insect cells. Pulse-chase studies were used to examine the synthesis, glycosylation, and secretion of E1 delta Tm by the insect cells. The secreted E1 delta Tm protein was purified from serum-free medium by one-step immunochromatography. The purified E1 delta Tm protein retained full antigenicity and may be a convenient source of E1 protein for use in diagnostic assay and rubella vaccine development. PMID:7852960

  7. Selective interactions of the human immunodeficiency virus-inactivating protein cyanovirin-N with high-mannose oligosaccharides on gp120 and other glycoproteins.

    PubMed

    Shenoy, S R; O'Keefe, B R; Bolmstedt, A J; Cartner, L K; Boyd, M R

    2001-05-01

    The virucidal protein cyanovirin-N (CV-N) mediates its highly potent anti-human immunodeficiency virus activity, at least in part, through interactions with the viral envelope glycoprotein gp120. Here we dissect in further detail the mechanism of CV-N's glycosylation-dependent binding to gp120. Isothermal titration calorimetry (ITC) binding studies of CV-N with endoglycosidase H-treated gp120 showed that binding was completely abrogated by removal of high-mannose oligosaccharides from the glycoprotein. Additional ITC and circular dichroism spectral studies with CV-N and other glycoproteins as well showed that CV-N discriminately bound only glycoproteins that contain high-mannose oligosaccharides. Binding experiments with RNase B indicated that the single high-mannose oligosaccharide on that enzyme mediated all of its binding with CV-N (K(d) = 0.602 microM). A finer level of oligosaccharide selectivity of CV-N was revealed in affinity chromatography-liquid chromatography-mass spectrometry experiments, which showed that CV-N preferentially bound only oligomannose-8 (Man-8) and oligomannose-9 isoforms of RNase B. Finally, we biophysically characterized the interaction of CV-N with a purified, single oligosaccharide, Man-8. The binding affinity of Man-8 for CV-N is unusually strong (K(d) = 0.488 microM), several hundredfold greater than observed for oligosaccharides and their protein lectins (K(d) = 1 microM--1 mM), further establishing a critical role of high-mannose oligosaccharides in CV-N binding to glycoproteins.

  8. GENERAL VIEW OF PURIFIERS ON SECOND FLOOR. (THE PURIFIERS DATE ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    GENERAL VIEW OF PURIFIERS ON SECOND FLOOR. (THE PURIFIERS DATE FROM CA. 1910 AND WERE MANUFACTURED BY THE ALLIS CHALMERS COMPANY OF MILWAUKEE, WISCONSIN.) - Patterson Milling Company, Feed Mill, Water & Point Streets, Saltsburg, Indiana County, PA

  9. KDN-containing glycoprotein from loach skin mucus.

    PubMed

    Nakagawa, H; Hama, Y; Sumi, T; Li, S C; Li, Y T

    2001-01-01

    It has been widely recognized that the mucus coat of fish plays a variety of important physical, chemical, and physiological functions. One of the major constituents of the mucus coat is mucus glycoprotein. We found that sialic acids in the skin mucus of the loach, Misgurnus anguillicaudatus, consisted predominantly of KDN. Subsequently, we isolated KDN-containing glycoprotein from loach skin mucus and characterized its chemical nature and structure. Loach mucus glycoprotein was purified from the Tris-HCl buffer extract of loach skin mucus by DEAE-cellulose chromatography, Nuclease P1 treatment, and Sepharose CL-6B gel filtration. The purified mucus glycoprotein was found to contain 38.5 KDN, 0.5% NeuAc, 25.0% GalNAc, 3.5% Gal, 0.5% GlcNAc and 28% amino acids. Exhaustive Actinase digestion of the glycoprotein yielded a glycopeptide with a higher sugar content and higher Thr and Ser contents. The molecular size of this glycopeptide was approximately 1/12 of the intact glycoprotein. These results suggest that approximately 11 highly glycosylated polypeptide units are linked in tandem through nonglycosylated peptides to form the glycoporotein molecule. The oligosaccharide alditols liberated from the loach mucus glycoprotein by alkaline borohydride treatment were separated by Sephadex G-25 gel filtration and HPLC. The purified sugar chains were analyzed b --> 6GalNAc-ol, KDNalpha2 --> 3(GalNAcbeta1 --> 14)GalNAc-ol, KDNalpha2 --> 6(GalNAcalpha1 --> 3)GalNAc-ol, KDNalpha2 --> 6(Gal3alpha1--> 3)GalNAc-ol, and NeuAcalpha2 --> 6Gal NAc-ol. It is estimated that one loach mucus glycoprotein molecule contains more than 500 KDN-containing sugar chains that are linked to Thr and Ser residues of the protein core through GalNAc. PMID:14533798

  10. KDN-containing glycoprotein from loach skin mucus.

    PubMed

    Nakagawa, H; Hama, Y; Sumi, T; Li, S C; Li, Y T

    2001-01-01

    It has been widely recognized that the mucus coat of fish plays a variety of important physical, chemical, and physiological functions. One of the major constituents of the mucus coat is mucus glycoprotein. We found that sialic acids in the skin mucus of the loach, Misgurnus anguillicaudatus, consisted predominantly of KDN. Subsequently, we isolated KDN-containing glycoprotein from loach skin mucus and characterized its chemical nature and structure. Loach mucus glycoprotein was purified from the Tris-HCl buffer extract of loach skin mucus by DEAE-cellulose chromatography, Nuclease P1 treatment, and Sepharose CL-6B gel filtration. The purified mucus glycoprotein was found to contain 38.5 KDN, 0.5% NeuAc, 25.0% GalNAc, 3.5% Gal, 0.5% GlcNAc and 28% amino acids. Exhaustive Actinase digestion of the glycoprotein yielded a glycopeptide with a higher sugar content and higher Thr and Ser contents. The molecular size of this glycopeptide was approximately 1/12 of the intact glycoprotein. These results suggest that approximately 11 highly glycosylated polypeptide units are linked in tandem through nonglycosylated peptides to form the glycoporotein molecule. The oligosaccharide alditols liberated from the loach mucus glycoprotein by alkaline borohydride treatment were separated by Sephadex G-25 gel filtration and HPLC. The purified sugar chains were analyzed b --> 6GalNAc-ol, KDNalpha2 --> 3(GalNAcbeta1 --> 14)GalNAc-ol, KDNalpha2 --> 6(GalNAcalpha1 --> 3)GalNAc-ol, KDNalpha2 --> 6(Gal3alpha1--> 3)GalNAc-ol, and NeuAcalpha2 --> 6Gal NAc-ol. It is estimated that one loach mucus glycoprotein molecule contains more than 500 KDN-containing sugar chains that are linked to Thr and Ser residues of the protein core through GalNAc.

  11. Natural Air Purifier

    NASA Technical Reports Server (NTRS)

    1993-01-01

    NASA environmental research has led to a plant-based air filtering system. Dr. B.C. Wolverton, a former NASA engineer who developed a biological filtering system for space life support, served as a consultant to Terra Firma Environmental. The company is marketing the BioFilter, a natural air purifier that combines activated carbon and other filter media with living plants and microorganisms. The filter material traps and holds indoor pollutants; plant roots and microorganisms then convert the pollutants into food for the plant. Most non-flowering house plants will work. After pollutants have been removed, the cleansed air is returned to the room through slits in the planter. Terra Firma is currently developing a filter that will also disinfect the air.

  12. Glycoproteins: Occurrence and Significance

    NASA Astrophysics Data System (ADS)

    Wittmann, Valentin

    Protein glycosylation is regarded as the most complex form of post-translational modification leading to a heterogeneous expression of glycoproteins as mixtures of glycoforms. This chapter describes the structure and occurrence of glycoproteins with respect to their glycan chains. Discussed are different carbohydrate-peptide linkages including GPI anchors, common structures of N- and O-glycans, and the structure of glycosaminoglycans contained in proteoglycans. Also covered are the bacterial cell wall polymer peptidoglycan and the glycopeptide antibiotics of the vancomycin group. Properties and functions of the glycans contained in glycoproteins are dealt with in the next chapter of this book.

  13. Effect of cytochalasin B on the maturation of enveloped viruses

    PubMed Central

    1979-01-01

    The maturation of two enveloped viruses, influenza and vesicular stomatitis, occurs in cells treated with cytochalasin B. Virions produced in the presence of 50 microgram/ml cytochalasin B (CB) appear to be as infectious as those from control cells, indicating that polymerized actin is not required for the assembly of functional viral components. CB inhibits the release of influenza virus from treated cells, a phenomenon which appears to be a result of the synthesis of an aberrant neuraminidase (NA) glycoprotein; virions grown in CB-treated cells had a 90% reduction in specific enzymatic activity. We found that both influenza viral glycoproteins (NA and Hemagglutinin glycoprotein) had faster electrophoretic mobilities and were more heterogeneous in CB- treated cells as compared with controls. We also observed complete inhibition of incorporation of labeled glucosamine into viral glycoproteins in the presence of the drug. It was of interest that CB- induced inhibition of glycosylation appeared to cause loss of neuraminidase function, whereas hemagglutinating activity was not noticeably impaired. The presence of altered glycoproteins did not significantly diminish the infectivity of either influenza virus or vesicular stomatitis virus. Our results indicate that no step in the maturation of enveloped viruses is dependent upon an intact cytoskeletal network. PMID:222875

  14. Tegument Assembly and Secondary Envelopment of Alphaherpesviruses.

    PubMed

    Owen, Danielle J; Crump, Colin M; Graham, Stephen C

    2015-09-01

    Alphaherpesviruses like herpes simplex virus are large DNA viruses characterized by their ability to establish lifelong latent infection in neurons. As for all herpesviruses, alphaherpesvirus virions contain a protein-rich layer called "tegument" that links the DNA-containing capsid to the glycoprotein-studded membrane envelope. Tegument proteins mediate a diverse range of functions during the virus lifecycle, including modulation of the host-cell environment immediately after entry, transport of virus capsids to the nucleus during infection, and wrapping of cytoplasmic capsids with membranes (secondary envelopment) during virion assembly. Eleven tegument proteins that are conserved across alphaherpesviruses have been implicated in the formation of the tegument layer or in secondary envelopment. Tegument is assembled via a dense network of interactions between tegument proteins, with the redundancy of these interactions making it challenging to determine the precise function of any specific tegument protein. However, recent studies have made great headway in defining the interactions between tegument proteins, conserved across alphaherpesviruses, which facilitate tegument assembly and secondary envelopment. We summarize these recent advances and review what remains to be learned about the molecular interactions required to assemble mature alphaherpesvirus virions following the release of capsids from infected cell nuclei. PMID:26393641

  15. Tegument Assembly and Secondary Envelopment of Alphaherpesviruses

    PubMed Central

    Owen, Danielle J.; Crump, Colin M.; Graham, Stephen C.

    2015-01-01

    Alphaherpesviruses like herpes simplex virus are large DNA viruses characterized by their ability to establish lifelong latent infection in neurons. As for all herpesviruses, alphaherpesvirus virions contain a protein-rich layer called “tegument” that links the DNA-containing capsid to the glycoprotein-studded membrane envelope. Tegument proteins mediate a diverse range of functions during the virus lifecycle, including modulation of the host-cell environment immediately after entry, transport of virus capsids to the nucleus during infection, and wrapping of cytoplasmic capsids with membranes (secondary envelopment) during virion assembly. Eleven tegument proteins that are conserved across alphaherpesviruses have been implicated in the formation of the tegument layer or in secondary envelopment. Tegument is assembled via a dense network of interactions between tegument proteins, with the redundancy of these interactions making it challenging to determine the precise function of any specific tegument protein. However, recent studies have made great headway in defining the interactions between tegument proteins, conserved across alphaherpesviruses, which facilitate tegument assembly and secondary envelopment. We summarize these recent advances and review what remains to be learned about the molecular interactions required to assemble mature alphaherpesvirus virions following the release of capsids from infected cell nuclei. PMID:26393641

  16. Demethoxycurcumin modulates human P-glycoprotein function via uncompetitive inhibition of ATPase hydrolysis activity.

    PubMed

    Teng, Yu-Ning; Hsieh, Yow-Wen; Hung, Chin-Chuan; Lin, Hui-Yi

    2015-01-28

    Curcuminoids are major components of Curcuma longa L., which is widely used as spice in food. This study aimed at identifying whether curcumin, demethoxycurcumin, and bisdemethoxycurcumin could modulate efflux function of human P-glycoprotein and be used as chemosensitizers in cancer treatments. Without altering P-glycoprotein expression levels and conformation, the purified curcuminoids significantly inhibited P-glycoprotein efflux function. In rhodamine 123 efflux and calcein-AM accumulation assays, demethoxycurcumin demonstrated the highest inhibition potency (inhibitory IC50 = 1.56 ± 0.13 μM) among the purified curcuminoids, as well as in the fold of reversal assays. Demethoxycurcumin inhibited P-glycoprotein-mediated ATP hydrolysis under concentrations of <1 μM and efficiently inhibited 200 μM verapamil-stimulated ATPase activity, indicating a high affinity of demethoxycurcumin for P-glycoprotein. These results suggested that demethoxycurcumin may be a potential additive natural product in combination with chemotherapeutic agents in drug-resistant cancers.

  17. [Prokaryotic expression and immunogenicity analysis of glycoprotein from infectious hematopoietic necrosis virus].

    PubMed

    Xu, Li-ming; Liu, Hong-bai; Yin, Jia-sheng; Lu, Tong-yan

    2013-09-01

    In order to detect Infectious hematopoietic necrosis virus with immunological methods, the surface glycoprotein of a recent IHNV-Sn isolated from farmed rainbow trout ( Oncorhynchus mykiss ) in China was amplified and cloned into pET27b(+) vector (designated as pET27b-G ). The expression of recombinant plasmid pET27b-G in E. coli BL21(DE3) was induced and determined by SDS-PAGE analysis. The predicted molecular weight of glycoprotein protein was approximately 55 kD and was confirmed in this study. The inclusion body of glycoprotein was treated with urea at different urea concentrations, and dialyzed into PBS buffer. Purified glycoprotein with high concentration was obtained after dialyzed in the PBS buffer. Antisera against glycoprotein were produced from immunized rabbits. The prepared antisera could react specifically with both the recombinant glycoprotein and natural glycoprotein of the IHNV-Sn isolated in the test of indirect ELISA, and the titer against the recombinant glycoprotein was 1:20,000. IFA showed that the antisera can recognize the glycoprotein located on the surface of IHNV-Sn and IHNV reference strain. These results indicated that the expressed glycoprotein was immunogenical and antigenical and could be functional as the natural IHNV glycoprotein. These results established a foundation for further study on vaccine and rapid diagnosis of IHNV.

  18. Variability of mammalian liver nuclear-envelope preparations.

    PubMed Central

    Agutter, P S; Gleed, C D

    1980-01-01

    The composition, density and enzymic activities of sheep liver nuclear-envelope preparations were found to vary markedly according to the concentrations of nuclei during the lysis stage. The effect of nuclear concentration on the properties of the purified envelopes could not be attributed to bound Mg2+ or to other ions, and appeared to result from some component of the nucleus which was not eluted during lysis. The implications of these findings for studies on the nuclear envelope are discussed. Images Fig. 3. PMID:7305915

  19. Substitution of specific cysteine residues in E1 glycoprotein of classical swine fever virus strain Brescia affects formation of E1-E2 heterodimers and alters virulence in swine

    Technology Transfer Automated Retrieval System (TEKTRAN)

    E1, along with E^rns and E2, is one of the three envelope glycoproteins of Classical Swine Fever Virus (CSFV). E1 and E2 are anchored to the virus envelope at their carboxyl termini and E^rns loosely associates with the viral envelope. In infected cells, E2 forms homodimers and heterodimers with E1,...

  20. Secondary cell-wall-specific glycoprotein(s) from French bean hypocotyls.

    PubMed Central

    Wojtaszek, P; Bolwell, G P

    1995-01-01

    Specific labeling of secondary cell walls of tracheary elements and of xylary and phloem fibers has been observed when wheat germ agglutinin (WGA) and anti-WGA antibodies were used during ultrastructural studies of French bean (Phaseolus vulgaris L.) hypocotyls. In this report we demonstrate that at least part of this labeling is due to the presence of secondary cell-wall-specific glycoproteins. Three major novel glycoproteins with relative molecular weights of 55,000, 86,000, and 90,000, purified by means of WGA-Sepharose affinity chromatography, have been characterized. Their amino acid composition indicates that they are not the members of known classes of structural cell-wall proteins, since they contain no hydroxyproline, a lower level of glycine than seen in glycine-rich proteins, and very little proline. N-terminal sequences of all three proteins show no significant homology with other proteins. Antibodies were raised against electrophoretically pure 90-kD glycoprotein. These were used to localize this protein in secondary cell walls of xylem tracheary elements and in xylary and phloem fibers, i.e. in the same compartments where labeling with WGA has been observed. To our knowledge this is one of the first biochemical and ultrastructural demonstrations of secondary cell-wall-specific glycoproteins. PMID:7630932

  1. Immunogenicity of Escherichia coli expressed envelope 2 protein of Chikungunya virus.

    PubMed

    Tripathi, Nagesh K; Priya, Raj; Shrivastava, Ambuj

    2014-01-01

    Chikungunya fever, a re-emerging infection, is an arthropod-borne viral disease prevalent in different parts of the world, particularly Africa and South East Asia. Chikungunya virus envelope 2 protein is involved in binding to host receptors and it contains specific epitopes that elicit virus neutralizing antibodies. A highly immunogenic, recombinant Chikungunya virus envelope 2 protein was produced by bioreactor in Escherichia coli for development of a suitable diagnostic and vaccine candidate. This protein was refolded and further purified to achieve biologically active protein. The biological function of refolded and purified recombinant envelope 2 protein of Chikungunya virus was confirmed by its ability to generate envelope 2 specific antibodies with high titers in animal models. These findings suggest that recombinant envelope 2 protein of Chikungunya virus in combination with compatible adjuvant is highly immunogenic. Thus, recombinant envelope 2 protein can be a potential diagnostic reagent and vaccine candidate against Chikungunya virus infection.

  2. Characterization of the purified Chlamydomonas minus agglutinin

    PubMed Central

    1985-01-01

    Chlamydomonas flagellar sexual agglutinins are responsible for the adhesion of opposite mating-type (plus and minus) gametes during the first stages of mating. Purification and partial characterization of the plus agglutinin was previously reported (Adair, W. S., C. J. Hwang, and U. W. Goodenough, 1983, Cell, 33:183-193). Here we characterize the purified minus molecule. We show it to be a high molecular weight, hydroxyproline-rich glycoprotein that migrates in the 3% stacking region of an SDS-polyacrylamide gel and is absent from two nonagglutinating minus mutants. Plus and minus agglutinins are remarkably similar, although nonidentical, in amino acid composition, molecular morphology, and reactivity in vivo and in vitro with monoclonal antibodies raised against the plus agglutinin. Moreover, the adhesiveness of both plus and minus agglutinins, when coupled to agarose beads, is abolished by thermolysin, trypsin, periodate, alkaline borohydride, reducing agents, or heat, but unaffected by exo- or endoglycosidases. The minus agglutinin, however, migrates just ahead of the plus molecule on SDS PAGE, is excluded from an anion-exchange (Mono Q) column, elutes earlier during hydrophobic interaction (Bio-gel TSK Phenyl 5PW) chromatography, and is sensitive to chymotrypsin digestion (unlike the plus agglutinin); therefore, it differs from the plus agglutinin in apparent molecular weight, net charge, relative hydrophobicity and proteolytic susceptibility. Nevertheless, our results generally demonstrate a high degree of homology between these complementary cell-cell recognition/adhesion molecules, which suggests that they are specified by genes that have a common evolutionary origin. PMID:2411736

  3. The Bacterial Cell Envelope

    PubMed Central

    Silhavy, Thomas J.; Kahne, Daniel; Walker, Suzanne

    2010-01-01

    The bacteria cell envelope is a complex multilayered structure that serves to protect these organisms from their unpredictable and often hostile environment. The cell envelopes of most bacteria fall into one of two major groups. Gram-negative bacteria are surrounded by a thin peptidoglycan cell wall, which itself is surrounded by an outer membrane containing lipopolysaccharide. Gram-positive bacteria lack an outer membrane but are surrounded by layers of peptidoglycan many times thicker than is found in the Gram-negatives. Threading through these layers of peptidoglycan are long anionic polymers, called teichoic acids. The composition and organization of these envelope layers and recent insights into the mechanisms of cell envelope assembly are discussed. PMID:20452953

  4. Arms Race between Enveloped Viruses and the Host ERAD Machinery

    PubMed Central

    Frabutt, Dylan A.; Zheng, Yong-Hui

    2016-01-01

    Enveloped viruses represent a significant category of pathogens that cause serious diseases in animals. These viruses express envelope glycoproteins that are singularly important during the infection of host cells by mediating fusion between the viral envelope and host cell membranes. Despite low homology at protein levels, three classes of viral fusion proteins have, as of yet, been identified based on structural similarities. Their incorporation into viral particles is dependent upon their proper sub-cellular localization after being expressed and folded properly in the endoplasmic reticulum (ER). However, viral protein expression can cause stress in the ER, and host cells respond to alleviate the ER stress in the form of the unfolded protein response (UPR); the effects of which have been observed to potentiate or inhibit viral infection. One important arm of UPR is to elevate the capacity of the ER-associated protein degradation (ERAD) pathway, which is comprised of host quality control machinery that ensures proper protein folding. In this review, we provide relevant details regarding viral envelope glycoproteins, UPR, ERAD, and their interactions in host cells. PMID:27657106

  5. [Proteomic Analyses of Purified Particles of the Rabies Virus].

    PubMed

    Tu, Zhongzhong; Gong, Wenjie; Zhang, Yan; Feng, Ye; Li, Nan; Tu, Changchun

    2015-05-01

    The rabies virus (RABV) is an enveloped RNA virus. It mainly damages the central nervous system and causes anencephaly in mammals and humans. There is now compelling evidence that enveloped virions released from infected cells can carry many host proteins, some of which may play an important part in viral replication. Several host proteins have been reported to be incorporated into RABV particles. However, a systematic study to reveal the proteomics of RABV particles has not been conducted. In the present study, after virus culture and purification by sucrose density gradient ultracentrifugation, a proteomics approach was used to analyze the protein composition of purified RABV particles to understand the molecular mechanisms of virus-cell interactions. Fifty host proteins, along with five virus-encoded structural proteins, were identified in purified RABV particles. These proteins could be classified into ten categories according to function: intracellular trafficking (14%), molecular chaperone (12%), cytoskeletal (24%), signal transduction (8%), transcription regulation (12%), calcium ion-binding (6%), enzyme binding (6%), metabolic process (2%), ubiquitin (2%) and other (14%). Of these, four proteins (beta-actin, p-tubulin, Cofilin, Hsc70) were validated by western blotting to be present in purified RABV particles. This novel study of the composition of host proteins in RABV particles may aid investigation of the mechanism of RABV replication. PMID:26470524

  6. [Proteomic Analyses of Purified Particles of the Rabies Virus].

    PubMed

    Tu, Zhongzhong; Gong, Wenjie; Zhang, Yan; Feng, Ye; Li, Nan; Tu, Changchun

    2015-05-01

    The rabies virus (RABV) is an enveloped RNA virus. It mainly damages the central nervous system and causes anencephaly in mammals and humans. There is now compelling evidence that enveloped virions released from infected cells can carry many host proteins, some of which may play an important part in viral replication. Several host proteins have been reported to be incorporated into RABV particles. However, a systematic study to reveal the proteomics of RABV particles has not been conducted. In the present study, after virus culture and purification by sucrose density gradient ultracentrifugation, a proteomics approach was used to analyze the protein composition of purified RABV particles to understand the molecular mechanisms of virus-cell interactions. Fifty host proteins, along with five virus-encoded structural proteins, were identified in purified RABV particles. These proteins could be classified into ten categories according to function: intracellular trafficking (14%), molecular chaperone (12%), cytoskeletal (24%), signal transduction (8%), transcription regulation (12%), calcium ion-binding (6%), enzyme binding (6%), metabolic process (2%), ubiquitin (2%) and other (14%). Of these, four proteins (beta-actin, p-tubulin, Cofilin, Hsc70) were validated by western blotting to be present in purified RABV particles. This novel study of the composition of host proteins in RABV particles may aid investigation of the mechanism of RABV replication.

  7. Reduction of Influenza Virus Envelope's Fusogenicity by Viral Fusion Inhibitors.

    PubMed

    Rowse, Michael; Qiu, Shihong; Tsao, Jun; Yamauchi, Yohei; Wang, Guoxin; Luo, Ming

    2016-01-01

    During cell entry of an enveloped virus, the viral membrane must be fused with the cellular membrane. The virus envelope has a unique structure consisting of viral proteins and a virus-specific lipid composition, whereas the host membrane has its own structure with host membrane proteins. Compound 136 was previously found to bind in close proximity to the viral envelope and inhibit influenza virus entry. We showed here that the 136-treated influenza virus still caused hemolysis. When liposomes were used as the target membrane for 136-treated viruses, aberrant fusion occurred; few liposomes fused per virion, and glycoproteins were not distributed evenly across fusion complexes. Additionally, large fusion aggregates did not form, and in some instances, neck-like structures were found. Based on previous results and hemolysis, fusion inhibition by 136 occurs post-scission but prior to lipid mixing. PMID:27622947

  8. Enveloped particles in the serum of chronic hepatitis C patients

    SciTech Connect

    Petit, Marie-Anne . E-mail: petit@lyon.inserm.fr; Lievre, Marjory . E-mail: marjory.lievre@free.fr; Peyrol, Simone . E-mail: peyrol@laennec.univ-lyon1.fr; De Sequeira, Sylvie . E-mail: desequeira@lyon.inserm.fr; Berthillon, Pascale . E-mail: berthillon@lyon.inserm.fr; Ruigrok, Rob W.H. . E-mail: ruigrok@embl-grenoble.fr; Trepo, Christian . E-mail: trepo@lyon.inserm.fr

    2005-06-05

    HCV particles were isolated from the plasma of chronically infected patients. The virus was analysed by sucrose density gradient centrifugation. The fractions were tested for viral RNA, core antigen and envelope proteins by using a monoclonal antibody directed against the natural E1E2 complex (D32.10). Two populations of particles containing RNA plus core antigen were separated: the first with a density of 1.06-1.08 g/ml did not contain the envelope proteins; the second with a density between 1.17 and 1.21 g/ml expressed both E1 and E2 glycoproteins. Electron microscopy of the enveloped population after immunoprecipitation with D32.10 showed spherical particles with a rather featureless surface and with a diameter around 40 nm. Immuno-gold staining gave evidence that the E1E2 complex was indeed positioned at the surface of these particles.

  9. EBV glycoproteins: where are we now?

    PubMed Central

    Hutt-Fletcher, Lindsey M

    2015-01-01

    Glycoproteins are critical to virus entry, to spread within and between hosts and can modify the behavior of cells. Many viruses carry only a few, most found in the virion envelope. EBV makes more than 12, providing flexibility in how it colonizes its human host. Some are dedicated to getting the virus through the cell membrane and on toward the nucleus of the cell, some help guide the virus back out and on to the next cell in the same or a new host. Yet others undermine host defenses helping the virus persist for a lifetime, maintaining a presence that is mostly tolerated and serves to perpetuate EBV as one of the most common infections of man. PMID:26843889

  10. Methods for purifying carbon materials

    DOEpatents

    Dailly, Anne; Ahn, Channing; Yazami, Rachid; Fultz, Brent T.

    2009-05-26

    Methods of purifying samples are provided that are capable of removing carbonaceous and noncarbonaceous impurities from a sample containing a carbon material having a selected structure. Purification methods are provided for removing residual metal catalyst particles enclosed in multilayer carbonaceous impurities in samples generate by catalytic synthesis methods. Purification methods are provided wherein carbonaceous impurities in a sample are at least partially exfoliated, thereby facilitating subsequent removal of carbonaceous and noncarbonaceous impurities from the sample. Methods of purifying carbon nanotube-containing samples are provided wherein an intercalant is added to the sample and subsequently reacted with an exfoliation initiator to achieve exfoliation of carbonaceous impurities.

  11. Targeted entry via somatostatin receptors using a novel modified retrovirus glycoprotein that delivers genes at levels comparable to those of wild-type viral glycoproteins.

    PubMed

    Li, Fang; Ryu, Byoung Y; Krueger, Robin L; Heldt, Scott A; Albritton, Lorraine M

    2012-01-01

    Here we report a novel viral glycoprotein created by replacing a natural receptor-binding sequence of the ecotropic Moloney murine leukemia virus envelope glycoprotein with the peptide ligand somatostatin. This new chimeric glycoprotein, which has been named the Sst receptor binding site (Sst-RBS), gives targeted transduction based on three criteria: (i) a gain of the use of a new entry receptor not used by any known virus; (ii) targeted entry at levels comparable to gene delivery by wild-type ecotropic Moloney murine leukemia virus and vesicular stomatitis virus (VSV) G glycoproteins; and (iii) a loss of the use of the natural ecotropic virus receptor. Retroviral vectors coated with Sst-RBS gained the ability to bind and transduce human 293 cells expressing somatostatin receptors. Their infection was specific to target somatostatin receptors, since a synthetic somatostatin peptide inhibited infection in a dose-dependent manner and the ability to transduce mouse cells bearing the natural ecotropic receptor was effectively lost. Importantly, vectors coated with the Sst-RBS glycoprotein gave targeted entry of up to 1 × 10(6) transducing U/ml, a level comparable to that seen with infection of vectors coated with the parental wild-type ecotropic Moloney murine leukemia virus glycoprotein through the ecotropic receptor and approaching that of infection of VSV G-coated vectors through the VSV receptor. To our knowledge, this is the first example of a glycoprotein that gives targeted entry of retroviral vectors at levels comparable to the natural capacity of viral envelope glycoproteins.

  12. Characterization of I/F1 glycoprotein as a receptor for Mycoplasma pneumoniae.

    PubMed Central

    Hengge, U R; Kirschfink, M; König, A L; Nicklas, W; Roelcke, D

    1992-01-01

    Serologic evidence of anti-I and anti-Fl cold agglutinins occurring in mycoplasma infections led to the isolation of I/Fl glycoprotein from human erythrocyte membranes. Mycoplasma pneumoniae bound to purified I/Fl glycoprotein in a dose-dependent fashion depending on sialylated carbohydrate determinants. This was shown by the decreased binding of mycoplasmas to either sialidase-treated I/Fl glycoprotein (dot blot analysis) or sialidase-treated erythrocytes (hemagglutination test). Structural properties of the receptor for optimal binding could be explored by hemagglutination inhibition assays. Glycophorins were excluded as receptors. These results indicate that Fl (and I) antigens are receptors for M. pneumoniae. Images PMID:1370278

  13. FRACTIONAL CRYSTALLIZATION FEED ENVELOPE

    SciTech Connect

    HERTING DL

    2008-03-19

    Laboratory work was completed on a set of evaporation tests designed to establish a feed envelope for the fractional crystallization process. The feed envelope defines chemical concentration limits within which the process can be operated successfully. All 38 runs in the half-factorial design matrix were completed successfully, based on the qualitative definition of success. There is no feed composition likely to be derived from saltcake dissolution that would cause the fractional crystallization process to not meet acceptable performance requirements. However, some compositions clearly would provide more successful operation than other compositions.

  14. Effects of glycosylation on antigenicity and immunogenicity of classical swine fever virus envelope proteins

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Classical swine fever virus (CSFV) harbors three envelope glycoproteins (E(rns), E1 and E2). Previous studies have demonstrated that removal of specific glycosylation sites within these proteins yielded attenuated and immunogenic CSFV mutants. Here we analyzed the effects of lack of glycosylation of...

  15. Purifying Aluminum by Vacuum Distillation

    NASA Technical Reports Server (NTRS)

    Du Fresne, E. R.

    1985-01-01

    Proposed method for purifying aluminum employs one-step vacuum distillation. Raw material for process impure aluminum produced in electrolysis of aluminum ore. Impure metal melted in vacuum. Since aluminum has much higher vapor pressure than other constituents, boils off and condenses on nearby cold surfaces in proportions much greater than those of other constituents.

  16. Furin cleavage of the SARS coronavirus spike glycoprotein enhances cell-cell fusion but does not affect virion entry

    SciTech Connect

    Follis, Kathryn E.; York, Joanne; Nunberg, Jack H. . E-mail: jack.nunberg@umontana.edu

    2006-07-05

    The fusogenic potential of Class I viral envelope glycoproteins is activated by proteloytic cleavage of the precursor glycoprotein to generate the mature receptor-binding and transmembrane fusion subunits. Although the coronavirus (CoV) S glycoproteins share membership in this class of envelope glycoproteins, cleavage to generate the respective S1 and S2 subunits appears absent in a subset of CoV species, including that responsible for the severe acute respiratory syndrome (SARS). To determine whether proteolytic cleavage of the S glycoprotein might be important for the newly emerged SARS-CoV, we introduced a furin recognition site at single basic residues within the putative S1-S2 junctional region. We show that furin cleavage at the modified R667 position generates discrete S1 and S2 subunits and potentiates membrane fusion activity. This effect on the cell-cell fusion activity by the S glycoprotein is not, however, reflected in the infectivity of pseudotyped lentiviruses bearing the cleaved glycoprotein. The lack of effect of furin cleavage on virion infectivity mirrors that observed in the normally cleaved S glycoprotein of the murine coronavirus and highlights an additional level of complexity in coronavirus entry.

  17. Cloning and characterization of functional subtype A HIV-1 envelope variants transmitted through breastfeeding.

    PubMed

    Rainwater, Stephanie M J; Wu, Xueling; Nduati, Ruth; Nedellec, Rebecca; Mosier, Donald; John-Stewart, Grace; Mbori-Ngacha, Dorothy; Overbaugh, Julie

    2007-03-01

    Previous studies of HIV-1 variants transmitted from mother-to-infant have focused primarily on computational analyses of partial envelope gene sequences, rather than analyses of functional envelope variants. There are very few examples of well-characterized functional envelope clones from mother-infant pairs, especially from envelope variants representing the most prevalent subtypes worldwide. To address this, we amplified the envelope variants present in 4 mother-infant transmission pairs, all of whom were infected with subtype A and three of whom presumably transmitted HIV-1 during the breastfeeding period. Functional envelope clones were constructed, either encoding full-length envelope sequences from the mother and baby or by making chimeric envelope clones in a common backbone sequence. The infant envelope sequences were genetically homogeneous compared to the maternal viruses, and pseudoviruses bearing these envelopes all used CCR5 as a coreceptor. The infant viruses were generally resistant to neutralization by maternal antibodies present near the time of transmission. There were no notable differences in sensitivity of the mother and infant envelope variants to neutralization by heterologous plasma or monoclonal antibodies 2G12 and b12, or to inhibition by sCD4, PSC-RANTES or TAK779. This collection of viral envelopes, which can be used for making pseudotyped viruses, may be useful for examining the efficacy of interventions to block mother-infant transmission, including sera from vaccine candidates, purified antibodies under consideration for passive immunization and viral entry inhibitors.

  18. Preparation of envelope membrane fractions from Arabidopsis chloroplasts for proteomic analysis and other studies.

    PubMed

    Salvi, Daniel; Moyet, Lucas; Seigneurin-Berny, Daphné; Ferro, Myriam; Joyard, Jacques; Rolland, Norbert

    2011-01-01

    Plastids are semiautonomous organelles restricted to plants and protists. These plastids are surrounded by a double membrane system, or envelope. These envelope membranes contain machineries to import nuclear-encoded proteins, and transporters for ions or metabolites, but are also essential for a range of plastid-specific metabolisms. Targeted semiquantitative proteomic investigations have revealed specific cross-contaminations by other cell or plastid compartments that may occur during chloroplast envelope purification. This article describes procedures developed to recover highly purified envelope fractions starting from Percoll-purified Arabidopsis chloroplasts, gives an overview of possible cross-contaminations, provides some tricks to limit these cross-contaminations, and lists immunological markers and methods that can be used to assess the purity of the envelope fractions.

  19. Cellufine sulfate column chromatography as a simple, rapid, and effective method to purify dengue virus.

    PubMed

    Kanlaya, Rattiyaporn; Thongboonkerd, Visith

    2016-08-01

    Conventional method to purify/concentrate dengue virus (DENV) is time-consuming with low virus recovery yield. Herein, we applied cellufine sulfate column chromatography to purify/concentrate DENV based on the mimicry between heparan sulfate and DENV envelope protein. Comparative analysis demonstrated that this new method offered higher purity (as determined by less contamination of bovine serum albumin) and recovery yield (as determined by greater infectivity). Moreover, overall duration used for cellufine sulfate column chromatography to purify/concentrate DENV was approximately 1/20 of that of conventional method. Therefore, cellufine sulfate column chromatography serves as a simple, rapid, and effective alternative method for DENV purification/concentration.

  20. Role of zona pellucida glycoproteins during fertilization in humans.

    PubMed

    Gupta, Satish Kumar

    2015-04-01

    In the last decade, scientific investigations pertaining to the role of zona pellucida (ZP) glycoproteins during fertilization in humans have led to new insights. This has been achieved using purified native/recombinant human zona proteins and transgenic mice expressing human ZP glycoproteins. The proposed model in mice of ZP glycoprotein-3 (ZP3) acting as primary sperm receptor and ZP glycoprotein-2 (ZP2) as secondary sperm receptor has been modified for sperm-egg binding in humans. ZP glycoprotein-1 (ZP1), ZP3, and ZP glycoprotein-4 (ZP4) have been shown to bind to the capacitated human sperm. ZP2 binds to the acrosome-reacted human spermatozoa. Further, the eggs obtained from transgenic mice expressing human ZP2 alone or in conjunction with other human instead of mouse zona proteins showed binding of human sperm, suggesting that ZP2 might also play a role in sperm-egg binding. This function has been mapped to a domain corresponding to amino acid residues 51-144 of ZP2. In contrast to mice, where ZP3 is the primary agonist for inducing the acrosome reaction, in humans, the acrosome reaction can be mediated by ZP1, ZP3, and ZP4. The effect of mutations in the genes encoding zona proteins on the ZP morphology and infertility has not been established. Further, the role of autoantibodies against ZP in women with 'unexplained infertility' leading to poor outcome of in vitro fertilization is currently controversial and needs further investigations. Understanding the role of ZP glycoproteins during human fertilization facilitates the development of new contraceptives and strategies to overcome the problem of infertility.

  1. Jacketed lamp bulb envelope

    DOEpatents

    MacLennan, Donald A.; Turner, Brian P.; Gitsevich, Aleksandr; Bass, Gary K.; Dolan, James T.; Kipling, Kent; Kirkpatrick, Douglas A.; Leng, Yongzhang; Levin, Izrail; Roy, Robert J.; Shanks, Bruce; Smith, Malcolm; Trimble, William C.; Tsai, Peter

    2001-01-01

    A jacketed lamp bulb envelope includes a ceramic cup having an open end and a partially closed end, the partially closed end defining an aperture, a lamp bulb positioned inside the ceramic cup abutting the aperture, and a reflective ceramic material at least partially covering a portion of the bulb not abutting the aperture. The reflective ceramic material may substantially fill an interior volume of the ceramic cup not occupied by the bulb. The ceramic cup may include a structural feature for aiding in alignment of the jacketed lamp bulb envelope in a lamp. The ceramic cup may include an external flange about a periphery thereof. One example of a jacketed lamp bulb envelope includes a ceramic cup having an open end and a closed end, a ceramic washer covering the open end of the ceramic cup, the washer defining an aperture therethrough, a lamp bulb positioned inside the ceramic cup abutting the aperture, and a reflective ceramic material filling an interior volume of the ceramic cup not occupied by the bulb. A method of packing a jacketed lamp bulb envelope of the type comprising a ceramic cup with a lamp bulb disposed therein includes the steps of filling the ceramic cup with a flowable slurry of reflective material, and applying centrifugal force to the cup to pack the reflective material therein.

  2. Targeting Nuclear Envelope Repair.

    PubMed

    2016-06-01

    Migrating cancer cells undergo repeated rupture of the protective nuclear envelope as they squeeze through small spaces in the surrounding tissue, compromising genomic integrity. Inhibiting both general DNA repair and the mechanism that seals these tears may enhance cell death and curb metastasis. PMID:27130435

  3. COMMON ENVELOPE: ENTHALPY CONSIDERATION

    SciTech Connect

    Ivanova, N.; Chaichenets, S.

    2011-04-20

    In this Letter, we discuss a modification to the criterion for the common envelope (CE) event to result in envelope dispersion. We emphasize that the current energy criterion for the CE phase is not sufficient for an instability of the CE, nor for an ejection. However, in some cases, stellar envelopes undergo stationary mass outflows, which are likely to occur during the slow spiral-in stage of the CE event. We propose the condition for such outflows, in a manner similar to the currently standard {alpha}{sub CE}{lambda}-prescription but with an addition of P/{rho} term in the energy balance equation, accounting therefore for the enthalpy of the envelope rather than merely the gas internal energy. This produces a significant correction, which might help to dispense with an unphysically high value of energy efficiency parameter during the CE phase, currently required in the binary population synthesis studies to make the production of low-mass X-ray binaries with a black hole companion to match the observations.

  4. Process for purifying geothermal steam

    DOEpatents

    Li, Charles T.

    1980-01-01

    Steam containing hydrogen sulfide is purified and sulfur recovered by passing the steam through a reactor packed with activated carbon in the presence of a stoichiometric amount of oxygen which oxidizes the hydrogen sulfide to elemental sulfur which is adsorbed on the bed. The carbon can be recycled after the sulfur has been recovered by vacuum distillation, inert gas entrainment or solvent extraction. The process is suitable for the purification of steam from geothermal sources which may also contain other noncondensable gases.

  5. Process for purifying geothermal steam

    DOEpatents

    Li, C.T.

    Steam containing hydrogen sulfide is purified and sulfur recovered by passing the steam through a reactor packed with activated carbon in the presence of a stoichiometric amount of oxygen which oxidizes the hydrogen sulfide to elemental sulfur which is adsorbed on the bed. The carbon can be recycled after the sulfur has been recovered by vacuum distillation, inert gas entrainment or solvent extraction. The process is suitable for the purification of steam from geothermal sources which may also contain other noncondensable gases.

  6. Enhanced detection of glycoproteins in polyacrylamide gels.

    PubMed

    Muñoz, G; Marshall, S; Cabrera, M; Horvat, A

    1988-05-01

    A highly sensitive and simple method to enhance detection of glycoproteins resolved by either one- or two-dimensional polyacrylamide gel electrophoresis is described. The method is a modification of the procedure described by D. Fargeaud et al. (D. Fargeaud, J. C. Benoit, F. Kato, and G. Chappuis (1984) Arch. Virol. 80, 69-82) that uses concanavalin A conjugated with fluorescein isothyocyanate to detect the carbohydrate moiety of glycoproteins. Briefly, the electrophoresed gel is exposed to the fluorescent lectin, thoroughly washed, and sequentially transferred to 50% methanol in deionized water and to absolute methanol. The result is an abrupt dehydration of the gel which turns evenly white and stiff. At least a twofold enhancement of fluorescence is obtained as detected by exposing the treated gel to an appropriate uv source. The sensitivity of the procedure allows us to detect purified immunoglobulin molecules by their carbohydrate content in the range of 0.2 microgram of total protein. The specificity of the detection is demonstrated by a comparison with the corresponding polypeptide profile obtained by silver nitrate staining of the gel. PMID:3394948

  7. Enhanced detection of glycoproteins in polyacrylamide gels.

    PubMed

    Muñoz, G; Marshall, S; Cabrera, M; Horvat, A

    1988-05-01

    A highly sensitive and simple method to enhance detection of glycoproteins resolved by either one- or two-dimensional polyacrylamide gel electrophoresis is described. The method is a modification of the procedure described by D. Fargeaud et al. (D. Fargeaud, J. C. Benoit, F. Kato, and G. Chappuis (1984) Arch. Virol. 80, 69-82) that uses concanavalin A conjugated with fluorescein isothyocyanate to detect the carbohydrate moiety of glycoproteins. Briefly, the electrophoresed gel is exposed to the fluorescent lectin, thoroughly washed, and sequentially transferred to 50% methanol in deionized water and to absolute methanol. The result is an abrupt dehydration of the gel which turns evenly white and stiff. At least a twofold enhancement of fluorescence is obtained as detected by exposing the treated gel to an appropriate uv source. The sensitivity of the procedure allows us to detect purified immunoglobulin molecules by their carbohydrate content in the range of 0.2 microgram of total protein. The specificity of the detection is demonstrated by a comparison with the corresponding polypeptide profile obtained by silver nitrate staining of the gel.

  8. Glycoprotein import: a common feature of complex plastids?

    PubMed

    Peschke, Madeleine; Hempel, Franziska

    2013-10-01

    Complex plastids evolved by secondary endosymbiosis and are, in contrast to primary plastids, surrounded by 3 or 4 envelope membranes. Recently, we provided evidence that in diatoms proteins exist that get N-glycosylated during transport across the outermost membrane of the complex plastid. This gives rise to unique questions on the transport mechanisms of these bulky proteins, which get transported across up to 3 further membranes into the plastid stroma. Here we discuss our results in an evolutionary context and speculate about the existence of plastidal glycoproteins in other organisms with complex plastids.

  9. Solar purifier of drinking water

    SciTech Connect

    Fawzy, I.O.

    1987-01-01

    Around 1920, ultraviolet radiation was used in Switzerland and France for water purification. Now, it is in use in more than 2000 European water works. In the United States, between 1916 and 1928, four municipal water installations of ultraviolet apparatus were in operation. By 1939, they were all abandoned in favor of chlorination primarily because of economy and the inadequacy of technology available at that time. In recent years, ultraviolet purification has had a comeback, partly because of the realization of what chlorination is doing to the environment and partly due to the vast advances in UV technology. Although solar ultraviolet radiation has a marginal biocidal effect, a property designed solar purifier could be a viable option in certain application. Among possible uses are: (1) rural single-family dwellings; (2) underdeveloped countries; and (3) small usage rates where electric power is not available. A solar purifier model is presented in this study. The data it provided illustrates that it can be effective in treating partially contaminated water.

  10. Process for purifying zirconium sponge

    SciTech Connect

    Abodishish, H.A.M.; Kimball, L.S.

    1992-03-31

    This patent describes a Kroll reduction process wherein a zirconium sponge contaminated with unreacted magnesium and by-product magnesium chloride is produced as a regulus, a process for purifying the zirconium sponge. It comprises: distilling magnesium and magnesium chloride from: a regulus containing a zirconium sponge and magnesium and magnesium chloride at a temperature above about 800{degrees} C and at an absolute pressure less than about 10 mmHg in a distillation vessel to purify the zirconium sponge; condensing the magnesium and the magnesium chloride distilled from the zirconium sponge in a condenser; and then backfilling the vessel containing the zirconium sponge and the condenser containing the magnesium and the magnesium chloride with a gas; recirculating the gas between the vessel and the condenser to cool the zirconium sponge from above about 800{degrees} C to below about 300{degrees} C; and cooling the recirculating gas in the condenser containing the condensed magnesium and the condensed magnesium chloride as the gas cools the zirconium sponge to below about 300{degrees} C.

  11. Purified discord and multipartite entanglement

    SciTech Connect

    Brown, Eric G.; Webster, Eric J.; Martín-Martínez, Eduardo; Kempf, Achim

    2013-10-15

    We study bipartite quantum discord as a manifestation of a multipartite entanglement structure in the tripartite purified system. In particular, we find that bipartite quantum discord requires the presence of both bipartite and tripartite entanglement in the purification. This allows one to understand the asymmetry of quantum discord, D(A,B)≠D(B,A) in terms of entanglement monogamy. As instructive special cases, we study discord for qubits and Gaussian states in detail. As a result of this we shed new light on a counterintuitive property of Gaussian states: the presence of classical correlations necessarily requires the presence of quantum correlations. Finally, our results also shed new light on a protocol for remote activation of entanglement by a third party. -- Highlights: •Bipartite quantum discord as a manifestation of multipartite entanglement. •Relevance of quantum discord as a utilizable resource for quantum info. tasks. •Quantum discord manifests itself in entanglement in the purified state. •Relation between asymmetry of discord and entanglement monogamy. •Protocol for remote activation of entanglement by a third party.

  12. Isolation of the envelope of vesicular stomatitis virus.

    PubMed Central

    Taube, S E; Rothfield, L I

    1978-01-01

    Vesicular stomatitis virus was disrupted by a combination of freezing and thawing, osmotic shock, and sonic treatment. Subviral components were separated by isopycnic centrifugation. The low-density, lipid-rich fractions were pooled and shown to contain primarily viral glycoprotein. Further purification of this material resulted in the isolation of a preparation of vesicles which contained only the G protein and the same phospholipids as in the intact virions and exhibited spikelike structures similar to those on intact vesicular stomatitis virions. We conclude that we have isolated fragments of native vesicular stomatitis virus envelopes. Images PMID:209217

  13. HSV-1 Glycoproteins Are Delivered to Virus Assembly Sites Through Dynamin-Dependent Endocytosis.

    PubMed

    Albecka, Anna; Laine, Romain F; Janssen, Anne F J; Kaminski, Clemens F; Crump, Colin M

    2016-01-01

    Herpes simplex virus-1 (HSV-1) is a large enveloped DNA virus that belongs to the family of Herpesviridae. It has been recently shown that the cytoplasmic membranes that wrap the newly assembled capsids are endocytic compartments derived from the plasma membrane. Here, we show that dynamin-dependent endocytosis plays a major role in this process. Dominant-negative dynamin and clathrin adaptor AP180 significantly decrease virus production. Moreover, inhibitors targeting dynamin and clathrin lead to a decreased transport of glycoproteins to cytoplasmic capsids, confirming that glycoproteins are delivered to assembly sites via endocytosis. We also show that certain combinations of glycoproteins colocalize with each other and with the components of clathrin-dependent and -independent endocytosis pathways. Importantly, we demonstrate that the uptake of neutralizing antibodies that bind to glycoproteins when they become exposed on the cell surface during virus particle assembly leads to the production of non-infectious HSV-1. Our results demonstrate that transport of viral glycoproteins to the plasma membrane prior to endocytosis is the major route by which these proteins are localized to the cytoplasmic virus assembly compartments. This highlights the importance of endocytosis as a major protein-sorting event during HSV-1 envelopment.

  14. Palmitoylation Strengthens Cholesterol-dependent Multimerization and Fusion Activity of Human Cytomegalovirus Glycoprotein B (gB).

    PubMed

    Patrone, Marco; Coroadinha, Ana Sofia; Teixeira, Ana P; Alves, Paula M

    2016-02-26

    Herpesviruses are a large order of animal enveloped viruses displaying a virion fusion mechanism of unusual complexity. Their multipartite machinery has a conserved core made of the gH/gL ancillary complexes and the homo-trimeric fusion protein glycoprotein B (gB). Despite its essential role in starting the viral infection, gB interaction with membrane lipids is still poorly understood. Here, evidence is provided demonstrating that human cytomegalovirus (HCMV) gB depends on the S-palmitoylation of its endodomain for an efficient interaction with cholesterol-rich membrane patches. We found that, unique among herpesviral gB proteins, the HCMV fusion factor has a Cys residue in the C-terminal region that is palmitoylated and mediates methyl-β-cyclodextrin-sensitive self-association of purified gB. A cholesterol-dependent virus-like particle trap assay, based on co-expression of the HIV Gag protein, confirmed that this post-translational modification is functional in the context of cellular membranes. Mutation of the palmitoylated Cys residue to Ala or inhibition of protein palmitoylation decreased HCMV gB export via Gag particles. Moreover, purified gBC777A showed an increased kinetic sensitivity in a cholesterol depletion test, demonstrating that palmitoyl-gB limits outward cholesterol diffusion. Finally, gB palmitoylation was required for full fusogenic activity in human epithelial cells. Altogether, these results uncover the palmitoylation of HCMV gB and its role in gB multimerization and activity.

  15. Methods for Purifying Enzymes for Mycoremediation

    NASA Technical Reports Server (NTRS)

    Cullings, Kenneth W. (Inventor); DeSimone, Julia C. (Inventor); Paavola, Chad D. (Inventor)

    2014-01-01

    A process for purifying laccase from an ectomycorrhizal fruiting body is disclosed. The process includes steps of homogenization, sonication, centrifugation, filtration, affinity chromatography, ion exchange chromatography, and gel filtration. Purified laccase can also be separated into isomers.

  16. Refrigerated cryogenic envelope

    DOEpatents

    Loudon, John D.

    1976-11-16

    An elongated cryogenic envelope including an outer tube and an inner tube coaxially spaced within said inner tube so that the space therebetween forms a vacuum chamber for holding a vacuum. The inner and outer tubes are provided with means for expanding or contracting during thermal changes. A shield is located in the vacuum chamber intermediate the inner and outer tubes; and, a refrigeration tube for directing refrigeration to the shield is coiled about at least a portion of the inner tube within the vacuum chamber to permit the refrigeration tube to expand or contract along its length during thermal changes within said vacuum chamber.

  17. A new Ebola virus nonstructural glycoprotein expressed through RNA editing.

    PubMed

    Mehedi, Masfique; Falzarano, Darryl; Seebach, Jochen; Hu, Xiaojie; Carpenter, Michael S; Schnittler, Hans-Joachim; Feldmann, Heinz

    2011-06-01

    Ebola virus (EBOV), an enveloped, single-stranded, negative-sense RNA virus, causes severe hemorrhagic fever in humans and nonhuman primates. The EBOV glycoprotein (GP) gene encodes the nonstructural soluble glycoprotein (sGP) but also produces the transmembrane glycoprotein (GP₁,₂) through transcriptional editing. A third GP gene product, a small soluble glycoprotein (ssGP), has long been postulated to be produced also as a result of transcriptional editing. To identify and characterize the expression of this new EBOV protein, we first analyzed the relative ratio of GP gene-derived transcripts produced during infection in vitro (in Vero E6 cells or Huh7 cells) and in vivo (in mice). The average percentages of transcripts encoding sGP, GP₁,₂, and ssGP were approximately 70, 25, and 5%, respectively, indicating that ssGP transcripts are indeed produced via transcriptional editing. N-terminal sequence similarity with sGP, the absence of distinguishing antibodies, and the abundance of sGP made it difficult to identify ssGP through conventional methodology. Optimized 2-dimensional (2D) gel electrophoresis analyses finally verified the expression and secretion of ssGP in tissue culture during EBOV infection. Biochemical analysis of recombinant ssGP characterized this protein as a disulfide-linked homodimer that was exclusively N glycosylated. In conclusion, we have identified and characterized a new EBOV nonstructural glycoprotein, which is expressed as a result of transcriptional editing of the GP gene. While ssGP appears to share similar structural properties with sGP, it does not appear to have the same anti-inflammatory function on endothelial cells as sGP.

  18. Epitope mapping of the infectious hematopoietic necrosis virus glycoprotein by flow cytometry.

    PubMed

    Xu, Li-Ming; Liu, Miao; Zhao, Jing-Zhuang; Cao, Yong-Sheng; Yin, Jia-Sheng; Liu, Hong-Bai; Lu, Tongyan

    2014-10-01

    The glycoprotein of infectious hematopoietic necrosis virus was truncated to ten overlapping fragments. All fragments were displayed on the inner membrane of the Escherichia coli periplasm. After disruption of the outer membrane, spheroplasts that had anchored with the glycoprotein fragment were incubated with an anti-glycoprotein polyclonal antibody. Prey pairs were detected and quantitated by flow cytometry with all fragments but one, G2, reacting with the polyclonal antibody. The antigenicity of all ten fragments was analyzed using conventional methods, and epitopes were localized in all fragments, except for G2 and were consistent with FCM analysis. Antigenicity of purified glycoprotein fusion proteins was confirmed by western blotting and ELISA. This method provides a rapid, quantitative and simple strategy for identifying linear B cell epitopes of a given protein.

  19. [Purification and analysis of Cimicifuga foetida glycoprotein ( CF- I )].

    PubMed

    Sun, Yu-jun; Chen, Yan; Song, Zhi-yin; Zhou, Dong-wen

    2007-02-01

    A kind of glycoprotein ( CF- I ) was extracted from Cimicifuga foetida and purified by DEAE-Cellulose ( DEAE-52) and Sepharose CL-4B. It was identified to be homogeneous glycoprotein complex by electrophoresis and fast protein liquid chromatography (FPLC). alpha-glucosidic bond was detrermined by IR. Typical absorption of polysaccharide was shown in its IR spectrum. It had no typical absorption of nucleic acid or pigment by UV scanning. Glucose, galactose, mannose and arabinose were identified in CF- I with the molar ratio of 11. 94: 2. 18: 1. 38: 1 by GC. Its average MW was estimated to be 5. 8 x 10' by gel filtration. The content of total saccharide, protein and acid polysaccharide was 78% , 14. 4% and 23% respectively. It might be composed of 1-->4 and 1-->6 linked glucopyranose by periodate oxidation and Smith degration.

  20. Identification of a Neutralizing Epitope within Antigenic Domain 5 of Glycoprotein B of Human Cytomegalovirus

    PubMed Central

    Wiegers, Anna-Katharina; Sticht, Heinrich; Winkler, Thomas H.; Britt, William J.

    2014-01-01

    ABSTRACT Human cytomegalovirus (HCMV) is an important, ubiquitous pathogen that causes severe clinical disease in immunocompromised individuals, such as organ transplant recipients and infants infected in utero. The envelope glycoprotein B (gB) of HCMV is a major antigen for the induction of virus-neutralizing antibodies. We have begun to define target structures within gB that are recognized by virus-neutralizing antibodies. Antigenic domain 5 (AD-5) of gB has been identified as an important target for neutralizing antibodies in studies using human monoclonal antibodies (MAbs). Anti-AD-5 MAbs share a target site on gB, despite originating from different, healthy, HCMV-infected donors. Mutational analysis of AD-5 identified tyrosine 280 in combination with other surface-exposed residues (the YNND epitope) as critical for antibody binding. The YNND epitope is strictly conserved among different HCMV strains. Recombinant viruses carrying YNND mutations in AD-5 were resistant to virus-neutralizing MAbs. Competition enzyme-linked immunosorbent assays (ELISAs) with human HCMV-convalescent-phase sera from unselected donors confirmed the conserved antibody response for the YNND epitope in HCMV-infected individuals and, because a significant fraction of the gB AD-5 response was directed against the YNND epitope, further argued that this epitope is a major target of anti-AD-5 antibody responses. In addition, affinity-purified polyclonal anti-AD-5 antibodies prepared from individual sera showed reactivity to AD-5 and neutralization activity toward gB mutant viruses that were similar to those of AD-5-specific MAbs. Taken together, our data indicate that the YNND epitope represents an important target for anti-gB antibody responses as well as for anti-AD-5 virus-neutralizing antibodies. IMPORTANCE HCMV is a major global health concern, and a vaccine to prevent HCMV disease is a widely recognized medical need. Glycoprotein B of HCMV is an important target for neutralizing

  1. Analysis of Glycoproteins for Biomarker Discovery

    PubMed Central

    He, Jintang; Liu, Yashu; Wu, Jing; Lubman, David M.

    2012-01-01

    Summary Glycoproteins play an important role in cell signaling and cell-cell interaction. The alterations of glycoproteins are often relevant to progression of diseases and these changed glycoproteins can be important biomarkers. The lectin-based glycoproteomic technology has extensively been used for high-throughput screening of potential glycoprotein biomarkers. Here we describe a multi-lectin affinity chromatography and label-free quantitative glycoproteomic approach for discovery of glycoprotein biomarkers relevant to differentiation of glioblastoma stem cells. PMID:23625399

  2. Mucus glycoprotein secretion by tracheal explants: effects of pollutants

    SciTech Connect

    Last, J.A.; Kaizu, T.

    1980-04-01

    Tracheal slices incubated with radioactive precursors in tissue culture medium secrete labeled mucus glycoproteins into the culture medium. We have used an in vivtro approach, a combined method utilizing exposure to pneumotoxins in vivo coupled with quantitation of mucus secretion rates in vitro, to study the effects of inhaled pollutants on mucus biosynthesis by rat airways. In addition, we have purified the mucus glycoproteins secreted by rat tracheal explants in order to determine putative structural changes that might by the basis for the observed augmented secretion rates after exposure of rats to H2SO4 aerosols in combination with high ambient levels of ozone. After digestion with papain, mucus glycoproteins secreted by tracheal explants may be separated into five fractions by ion-exchange chromatography, with recovery in high yield, on columns of DEAE-cellulose. Each of these five fractions, one neutral and four acidic, migrates as a single unique spot upon cellulose acetate electrophoresis at pH values of 8.6 and 1.2. The neutral fraction, which is labeled with (3H) glucosamine, does not contain radioactivity when Na2 35SO4 is used as the precursor. Acidic fractions I to IV are all labeled with either 3H-glucosamine or Na2 35SO4 as precursor. Acidic fraction II contains sialic acid as the terminal sugar on its oligosaccharide side chains, based upon its chromatographic behavior on columns of wheat-germ agglutinin-Agarose. Treatment of this fraction with neuraminidase shifts its elution position in the gradient to a lower salt concentration, coincident with acidic fraction I. After removal of terminal sialic acid residues with either neuraminidase or low pH treatment, the resultant terminal sugar on the oligosaccharide side chains is fucose. These results are identical with those observed with mucus glycoproteins secreted by cultured human tracheal explants and purified by these same techniques.

  3. Opioid binding properties of the purified kappa receptor from human placenta

    SciTech Connect

    Ahmed, M.S.; Zhou, D.; Cavinato, A.G.; Maulik, D.

    1989-01-01

    A glycoprotein with a molecular weight of 63,000 has been purified, in an active form, from human placental villus tissue membranes. The binding properties of this glycoprotein to opioid alkaloids and peptides indicates that it is the kappa opiate receptor of human placenta. The receptor binds the tritiated ligands etorphine, bremazocine, ethylketocyclazocine and naloxone specifically and reversibly with Kd values of 3.3, 4.4, 5.1 and 7.0nM, respectively. The binding of /sup 3/H-Bremazocine to the purified receptor is inhibited by the following compounds with the corresponding Ki values EKC, 1.3 x 10/sup -8/M; Dynorphin 1-8, 3.03 x 10/sup -7/; U50,488H, 4.48 x 10/sup -9/; U69-593,2.28 x 10/sup -8/, morphine, 4.05 x 10/sup -6/ DADLE, 6.47 x 10/sup -6/ and naloxone, 2.64 x 10/sup -8/. The purified receptor binds 8 nmole of /sup 3/H-Etorphine and 1.7 nmole /sup 3/H-BZC per mg protein. The theoretical binding capacity of a protein of this molecular weight is 15.8. Although the iodinated purified receptor appears by autoradiography as one band on SDS-PAGE, yet homogeneity of the preparation is not claimed.

  4. Lubrication by glycoprotein brushes.

    NASA Astrophysics Data System (ADS)

    Zappone, Bruno; Ruths, Marina; Greene, George W.; Israelachvili, Jacob

    2006-03-01

    Grafted polyelectrolyte brushes show excellent lubricating properties under water and have been proposed as a model to study boundary lubrication in biological system. Lubricin, a glycoprotein of the synovial fluid, is considered the major boundary lubricant of articular joints. Using the Surface Force Apparatus, we have measured normal and friction forces between model surfaces (negatively charged mica, positively charged poly-lysine and aminothiol, hydrophobic alkanethiol) bearing adsorbed layers of lubricin. Lubricin layers acts like a versatile anti-adhesive, adsorbing on all the surfaces considered and creating a repulsion similar to the force between end-grafted polymer brushes. Analogies with polymer brushes also appear from bridging experiment, where proteins molecules are end-adsorbed on two opposing surfaces at the same time. Lubricin `brushes' show good lubricating ability at low applied pressures (P<0.5MPa), especially on negatively charged surfaces like mica. At higher load, the adsorbed layers wears and fails lubricating the surfaces, while still protecting the underlying substrate from wearing. Lubricin might thus be a first example of biological polyelectrolytes providing `brush-like' lubrication and wear-protection.

  5. Structural changes of envelope proteins during alphavirus fusion

    SciTech Connect

    Li, Long; Jose, Joyce; Xiang, Ye; Kuhn, Richard J.; Rossmann, Michael G.

    2010-12-08

    Alphaviruses are enveloped RNA viruses that have a diameter of about 700 {angstrom} and can be lethal human pathogens. Entry of virus into host cells by endocytosis is controlled by two envelope glycoproteins, E1 and E2. The E2-E1 heterodimers form 80 trimeric spikes on the icosahedral virus surface, 60 with quasi-three-fold symmetry and 20 coincident with the icosahedral three-fold axes arranged with T = 4 quasi-symmetry. The E1 glycoprotein has a hydrophobic fusion loop at one end and is responsible for membrane fusion. The E2 protein is responsible for receptor binding and protects the fusion loop at neutral pH. The lower pH in the endosome induces the virions to undergo an irreversible conformational change in which E2 and E1 dissociate and E1 forms homotrimers, triggering fusion of the viral membrane with the endosomal membrane and then releasing the viral genome into the cytoplasm. Here we report the structure of an alphavirus spike, crystallized at low pH, representing an intermediate in the fusion process and clarifying the maturation process. The trimer of E2-E1 in the crystal structure is similar to the spikes in the neutral pH virus except that the E2 middle region is disordered, exposing the fusion loop. The amino- and carboxy-terminal domains of E2 each form immunoglobulin-like folds, consistent with the receptor attachment properties of E2.

  6. Hantavirus Gc glycoprotein: evidence for a class II fusion protein.

    PubMed

    Tischler, Nicole D; Gonzalez, Angel; Perez-Acle, Tomas; Rosemblatt, Mario; Valenzuela, Pablo D T

    2005-11-01

    Hantavirus cell entry is promoted by its envelope glycoproteins, Gn and Gc, through cell attachment and by fusion between viral and endosomal membranes at low pH. However, the role of Gn and Gc in receptor binding and cell fusion has not yet been defined. In this work, a sequence presenting characteristics similar to those of class II fusion peptides (FPs) of alphavirus E1 and flavivirus E proteins is identified within the hantavirus Gc glycoprotein. A three-dimensional comparative molecular model based on crystallographic data of tick-borne encephalitis virus E protein is proposed for the Andes virus (ANDV) Gc ectodomain, which supports a feasible class II fusion-protein fold. In vitro experimental evidence is provided for the binding activity of the ANDV FP candidate to artificial membranes, as demonstrated by fluorescence anisotropy assays. Taken together, these results support the hypothesis that the Gc glycoprotein of hantaviruses and of other members of the family Bunyaviridae directs the viral fusion activity and that it may be classified as a class II viral fusion protein.

  7. Folding of synthetic homogeneous glycoproteins in the presence of a glycoprotein folding sensor enzyme.

    PubMed

    Dedola, Simone; Izumi, Masayuki; Makimura, Yutaka; Seko, Akira; Kanamori, Akiko; Sakono, Masafumi; Ito, Yukishige; Kajihara, Yasuhiro

    2014-03-10

    UDP-glucose:glycoprotein glucosyltransferase (UGGT) plays a key role in recognizing folded and misfolded glycoproteins in the glycoprotein quality control system of the endoplasmic reticulum. UGGT detects misfolded glycoproteins and re-glucosylates them as a tag for misfolded glycoproteins. A flexible model to reproduce in vitro folding of a glycoprotein in the presence of UGGT in a mixture containing correctly folded, folding intermediates, and misfolded glycoproteins is described. The data demonstrates that UGGT can re-glucosylate all intermediates in the in vitro folding experiments, thus indicating that UGGT inspects not only final folded products, but also the glycoprotein folding intermediates.

  8. Anisotropic charged core envelope star

    NASA Astrophysics Data System (ADS)

    Mafa Takisa, P.; Maharaj, S. D.

    2016-08-01

    We study a charged compact object with anisotropic pressures in a core envelope setting. The equation of state is quadratic in the core and linear in the envelope. There is smooth matching between the three regions: the core, envelope and the Reissner-Nordström exterior. We show that the presence of the electric field affects the masses, radii and compactification factors of stellar objects with values which are in agreement with previous studies. We investigate in particular the effect of electric field on the physical features of the pulsar PSR J1614-2230 in the core envelope model. The gravitational potentials and the matter variables are well behaved within the stellar object. We demonstrate that the radius of the core and the envelope can vary by changing the parameters in the speed of sound.

  9. Salivary Mucin 19 Glycoproteins

    PubMed Central

    Culp, David J.; Robinson, Bently; Cash, Melanie N.; Bhattacharyya, Indraneel; Stewart, Carol; Cuadra-Saenz, Giancarlo

    2015-01-01

    Saliva functions in innate immunity of the oral cavity, protecting against demineralization of teeth (i.e. dental caries), a highly prevalent infectious disease associated with Streptococcus mutans, a pathogen also linked to endocarditis and atheromatous plaques. Gel-forming mucins are a major constituent of saliva. Because Muc19 is the dominant salivary gel-forming mucin in mice, we studied Muc19−/− mice for changes in innate immune functions of saliva in interactions with S. mutans. When challenged with S. mutans and a cariogenic diet, total smooth and sulcal surface lesions are more than 2- and 1.6-fold higher in Muc19−/− mice compared with wild type, whereas the severity of lesions are up to 6- and 10-fold higher, respectively. Furthermore, the oral microbiota of Muc19−/− mice display higher levels of indigenous streptococci. Results emphasize the importance of a single salivary constituent in the innate immune functions of saliva. In vitro studies of S. mutans and Muc19 interactions (i.e. adherence, aggregation, and biofilm formation) demonstrate Muc19 poorly aggregates S. mutans. Nonetheless, aggregation is enhanced upon adding Muc19 to saliva from Muc19−/− mice, indicating Muc19 assists in bacterial clearance through formation of heterotypic complexes with salivary constituents that bind S. mutans, thus representing a novel innate immune function for salivary gel-forming mucins. In humans, expression of salivary MUC19 is unclear. We find MUC19 transcripts in salivary glands of seven subjects and demonstrate MUC19 glycoproteins in glandular mucous cells and saliva. Similarities and differences between mice and humans in the expression and functions of salivary gel-forming mucins are discussed. PMID:25512380

  10. Crystallization and preliminary X-ray analysis of Chandipura virus glycoprotein G

    PubMed Central

    Baquero, Eduard; Buonocore, Linda; Rose, John K.; Bressanelli, Stéphane; Gaudin, Yves; Albertini, Aurélie A.

    2012-01-01

    Fusion in members of the Rhabdoviridae virus family is mediated by the G glycoprotein. At low pH, the G glycoprotein catalyzes fusion between viral and endosomal membranes by undergoing a major conformational change from a pre-fusion trimer to a post-fusion trimer. The structure of the G glycoprotein from vesicular stomatitis virus (VSV G), the prototype of Vesiculovirus, has recently been solved in its trimeric pre-fusion and post-fusion conformations; however, little is known about the structural details of the transition. In this work, a soluble form of the ectodomain of Chandipura virus G glycoprotein (CHAV Gth) was purified using limited proteolysis of purified virus; this soluble ectodomain was also crystallized. This protein shares 41% amino-acid identity with VSV G and thus its structure could provide further clues about the structural transition of rhabdoviral glycoproteins induced by low pH. Crystals of CHAV Gth obtained at pH 7.5 diffracted X-rays to 3.1 Å resolution. These crystals belonged to the orthorhombic space group P21212, with unit-cell parameters a = 150.3, b = 228.2, c = 78.8 Å. Preliminary analysis of the data based on the space group and the self-rotation function indicated that there was no trimeric association of the protomers. This unusual oligomeric status could result from the presence of fusion intermediates in the crystal. PMID:22949203

  11. A chimeric measles virus with a lentiviral envelope replicates exclusively in CD4+/CCR5+ cells

    SciTech Connect

    Mourez, Thomas; Mesel-Lemoine, Mariana; Combredet, Chantal; Najburg, Valerie; Cayet, Nadege; Tangy, Frederic

    2011-10-25

    We generated a replicating chimeric measles virus in which the hemagglutinin and fusion surface glycoproteins were replaced with the gp160 envelope glycoprotein of simian immunodeficiency virus (SIVmac239). Based on a previously cloned live-attenuated Schwarz vaccine strain of measles virus (MV), this chimera was rescued at high titers using reverse genetics in CD4+ target cells. Cytopathic effect consisted in the presence of large cell aggregates evolving to form syncytia, as observed during SIV infection. The morphology of the chimeric virus was identical to that of the parent MV particles. The presence of SIV gp160 as the only envelope protein on chimeric particles surface altered the cell tropism of the new virus from CD46+ to CD4+ cells. Used as an HIV candidate vaccine, this MV/SIVenv chimeric virus would mimic transient HIV-like infection, benefiting both from HIV-like tropism and the capacity of MV to replicate in dendritic cells, macrophages and lymphocytes.

  12. Hantavirus Gn and Gc Glycoproteins Self-Assemble into Virus-Like Particles

    PubMed Central

    Acuña, Rodrigo; Cifuentes-Muñoz, Nicolás; Márquez, Chantal L.; Bulling, Manuela; Klingström, Jonas; Mancini, Roberta; Lozach, Pierre-Yves

    2014-01-01

    How hantaviruses assemble and exit infected cells remains largely unknown. Here, we show that the expression of Andes (ANDV) and Puumala (PUUV) hantavirus Gn and Gc envelope glycoproteins lead to their self-assembly into virus-like particles (VLPs) which were released to cell supernatants. The viral nucleoprotein was not required for particle formation. Further, a Gc endodomain deletion mutant did not abrogate VLP formation. The VLPs were pleomorphic, exposed protrusions and reacted with patient sera. PMID:24335294

  13. Chimeric Bovine Respiratory Syncytial Virus with Attachment and Fusion Glycoproteins Replaced by Bovine Parainfluenza Virus Type 3 Hemagglutinin-Neuraminidase and Fusion Proteins

    PubMed Central

    Stope, Matthias B.; Karger, Axel; Schmidt, Ulrike; Buchholz, Ursula J.

    2001-01-01

    Chimeric bovine respiratory syncytial viruses (BRSV) expressing glycoproteins of bovine parainfluenza virus type 3 (BPIV-3) instead of BRSV glycoproteins were generated from cDNA. In the BRSV antigenome cDNA, the open reading frames of the major BRSV glycoproteins, attachment protein G and fusion protein F, were replaced individually or together by those of the BPIV-3 hemagglutinin-neuraminidase (HN) and/or fusion (F) glycoproteins. Recombinant virus could not be recovered from cDNA when the BRSV F open reading frame was replaced by the BPIV-3 F open reading frame. However, cDNA recovery of the chimeric virus rBRSV-HNF, with both glycoproteins replaced simultaneously, and of the chimeric virus rBRSV-HN, with the BRSV G protein replaced by BPIV-3 HN, was successful. The replication rates of both chimeras were similar to that of standard rBRSV. Moreover, rBRSV-HNF was neutralized by antibodies specific for BPIV-3, but not by antibodies specific to BRSV, demonstrating that the BRSV glycoproteins can be functionally replaced by BPIV-3 glycoproteins. In contrast, rBRSV-HN was neutralized by BRSV-specific antisera, but not by BPIV-3 specific sera, showing that infection of rBRSV-HN is mediated by BRSV F. Hemadsorption of cells infected with rBRSV-HNF and rBRSV-HN proved that BPIV-3 HN protein expressed by rBRSV is functional. Colocalization of the BPIV-3 glycoproteins with BRSV M protein was demonstrated by confocal laser scan microscopy. Moreover, protein analysis revealed that the BPIV-3 glycoproteins were present in chimeric virions. Taken together, these data indicate that the heterologous glycoproteins were not only expressed but were incorporated into the envelope of recombinant BRSV. Thus, the envelope glycoproteins derived from a member of the Respirovirus genus can together functionally replace their homologs in a Pneumovirus background. PMID:11533200

  14. Mason-Pfizer monkey virus: analysis and localization of virion proteins and glycoproteins.

    PubMed Central

    Schochetman, G; Kortright, K; Schlom, J

    1975-01-01

    The polypeptide composition of Mason-Pfizer monkey virus was determined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Six major polypeptides of molecular weights 68,000, 27,000, 20,000, 14,000, 12,000, and 10,000 were resolved regardless of the cell type (i.e., two human and two rhesus) in which the virus was grown. Protein gp68 (68,000) represented the major virus glycoprotein and protein gp20 (20,000) represented a minor glycoprotein of the virion, again regardless of the cell type of origin of the virus. Protein gp68 appears to be located on the outer surface of the viral envelope, as demonstrated by lactoperoxidase catalyzed iodination of intact virions. Additional glycoproteins were shown to be virion associated; their presence depended, however, on the cell type in which the virus was propagated. PMID:810603

  15. Structure of a trimeric variant of the Epstein-Barr virus glycoprotein B

    SciTech Connect

    Backovic, Marija; Longnecker, Richard; Jardetzky, Theodore S

    2009-03-16

    Epstein-Barr virus (EBV) is a herpesvirus that is associated with development of malignancies of lymphoid tissue. EBV infections are life-long and occur in >90% of the population. Herpesviruses enter host cells in a process that involves fusion of viral and cellular membranes. The fusion apparatus is comprised of envelope glycoprotein B (gB) and a heterodimeric complex made of glycoproteins H and L. Glycoprotein B is the most conserved envelope glycoprotein in human herpesviruses, and the structure of gB from Herpes simplex virus 1 (HSV-1) is available. Here, we report the crystal structure of the secreted EBV gB ectodomain, which forms 16-nm long spike-like trimers, structurally homologous to the postfusion trimers of the fusion protein G of vesicular stomatitis virus (VSV). Comparative structural analyses of EBV gB and VSV G, which has been solved in its pre and postfusion states, shed light on gB residues that may be involved in conformational changes and membrane fusion. Also, the EBV gB structure reveals that, despite the high sequence conservation of gB in herpesviruses, the relative orientations of individual domains, the surface charge distributions, and the structural details of EBV gB differ from the HSV-1 protein, indicating regions and residues that may have important roles in virus-specific entry.

  16. Multifamily Envelope Leakage Model

    SciTech Connect

    Faakye, Omari; Griffiths, Dianne

    2015-05-08

    “The cost for blower testing is high, because it is labor intensive, and it may disrupt occupants in multiple units. This high cost and disruption deter program participants, and dissuade them from pursuing energy improvements that would trigger air leakage testing, such as improvements to the building envelope.” This statement found in a 2012 report by Heschong Mahone Group for several California interests emphasizes the importance of reducing the cost and complexity of blower testing in multifamily buildings. Energy efficiency opportunities are being bypassed. The cost of single blower testing is on the order of $300. The cost for guarded blower door testing—the more appropriate test for assessing energy savings opportunities—could easily be six times that, and that’s only if you have the equipment and simultaneous access to multiple apartments. Thus, the proper test is simply not performed. This research seeks to provide an algorithm for predicting the guarded blower door test result based upon a single, total blower door test.

  17. Antiviral activity of purified human breast milk mucin.

    PubMed

    Habte, Habtom H; Kotwal, Girish J; Lotz, Zoë E; Tyler, Marilyn G; Abrahams, Melissa; Rodriques, Jerry; Kahn, Delawir; Mall, Anwar S

    2007-01-01

    Human breast milk is known to contain numerous biologically active components which protect breast fed infants against microbes, viruses, and toxins. The purpose of this study was to purify and characterize the breast milk mucin and determine its anti-poxvirus activity. In this study human milk mucin, free of contaminant protein and of sufficient quantity for further analysis, was isolated and purified by Sepharose CL-4B gel filtration and cesiumchloride density-gradient centrifugation. Based on the criteria of size and appearance of the bands and their electrophoretic mobility on sodium dodecyl sulfate polyacrylamide-gel electrophoresis, Western blotting together with the amino acid analysis, it is very likely that the human breast milk mucin is MUC1. It was shown that this breast milk mucin inhibits poxvirus activity by 100% using an inhibition assay with a viral concentration of 2.4 million plaque-forming units/ml. As the milk mucin seems to aggregate poxviruses prior to their entry into host cells, it is possible that this mucin may also inhibit other enveloped viruses such as HIV from entry into host cells. PMID:17361093

  18. Development and validation of glycoprotein-based native-subunit vaccine for fish against Aeromonas hydrophila.

    PubMed

    Çiftci, A; Onuk, E E; Çiftci, G; Fındık, A; Söğüt, M Ü; Didinen, B I; Aksoy, A; Üstünakın, K; Gülhan, T; Balta, F; Altun, S

    2016-08-01

    Aeromonas hydrophila is known to be causative agent of an infection named as Bacterial haemorrhagic septicaemia or red pest in freshwater fish. The aim of this study was to develop and validate the glycoprotein-based fish vaccine against Aeromonas hydrophila. For this aim, after identification and characterization of A. hydrophila isolates from fish farms, one A. hydrophila isolate was selected as vaccine strain. Antigenic glycoproteins of this vaccine strain were determined by Western blotting and glycan detection kit. The connection types of these glycoproteins were examined by glycoprotein differentiation kit. Two glycoproteins, molecular weights of 19 and 38 kDa, with SNA connection type were selected for use in vaccination trials. After their purification by SNA-specific lectin and size-exclusion chromatography, protection studies with purified proteins were performed. For challenge trials, four experimental fish groups were designated: Group I (with montanide), Group II (with montanide and ginseng), Group III [with Al(OH)3 ] and Group IV [with Al(OH)3 and ginseng]. The survival ratings of fish were determined, and protection was calculated as 21.56%, 29.41%, 69.83% and 78.88% in groups I, II, III and IV, respectively. In conclusion, A. hydrophila glycoproteins with Al(OH)3 and ginseng could be used as a safe and effective vaccine for fish. PMID:27144782

  19. Bacterially expressed and refolded envelope protein (domain III) of dengue virus type-4 binds heparan sulfate.

    PubMed

    Pattnaik, Priyabrata; Babu, J Pradeep; Verma, Shailendra Kumar; Tak, Vijay; Rao, P V Lakshmana

    2007-02-01

    An arboviral infection like dengue fever/dengue hemorrhagic fever (DHF) with high morbidity and mortality rate are extensively prevalent in several parts of the world. Global efforts have been directed towards development of vaccine for prevention of dengue. However, lack of thorough understanding about biology and pathogenesis of dengue virus restricts us from development of an effective vaccine. Here we report molecular interaction of domain III of envelope protein of dengue virus type-4 with heparan sulfate. A codon optimized synthetic gene encoding domain III of dengue virus type-4 envelope protein was expressed in Escherichia coli and purified under denaturing conditions, refolded and purified to homogeneity. Refolded Den4-DIII was characterized using biochemical and biophysical methods and shown to be pure and homogeneous. The purified protein was recognized in Western analyses by monoclonal antibody specific for the 6x His tag as well as the H241 monoclonal antibody. The in vitro refolded recombinant protein preparation was biologically functional and found to bind cell free heparan sulfate. This is the first report providing molecular evidence on binding of dengue-4 envelope protein to heparan sulfate. We developed a homology model of dengue-4 envelope protein (domain III) and mapped the possible amino acid residues critical for binding to heparan sulfate. Domain III envelope protein of dengue virus is a lead vaccine candidate. Our findings further the understanding on biology of dengue virus and will help in development of bioassay for the proposed vaccine candidate.

  20. Hydrogen purifier module with membrane support

    DOEpatents

    A hydrogen purifier utilizing a hydrogen-permeable membrane to purify hydrogen from mixed gases containing hydrogen is disclosed. Improved mechanical support for the permeable membrane is described, enabling forward or reverse differential pressurization of the membrane, which further stabilizes the membrane from wrinkling upon hydrogen uptake.

    2012-07-24

    A hydrogen purifier utilizing a hydrogen-permeable membrane to purify hydrogen from mixed gases containing hydrogen is disclosed. Improved mechanical support for the permeable membrane is described, enabling forward or reverse differential pressurization of the membrane, which further stabilizes the membrane from wrinkling upon hydrogen uptake.

  1. Phosphatidylserine receptors: enhancers of enveloped virus entry and infection

    PubMed Central

    Moller-Tank, Sven; Maury, Wendy

    2014-01-01

    A variety of both RNA and DNA viruses envelop their capsids in a lipid bilayer. One of the more recently appreciated benefits this envelope is incorporation of phosphatidylserine (PtdSer). Surface exposure of PtdSer disguises viruses as apoptotic bodies; tricking cells into engulfing virions. This mechanism is termed apoptotic mimicry. Several PtdSer receptors have been identified to enhance virus entry and we have termed this group of proteins PtdSer-mediated virus entry enhancing receptors or PVEERs. These receptors enhance entry of a broad range of enveloped viruses. Internalization of virions by PVEERs provides a broad mechanism of entry with little investment by the virus itself and may allow some viruses to attach to cells, thereby making viral glycoprotein/cellular receptor interactions more probable. Alternatively, other viruses may rely entirely on PVEERs for internalization into endosomes. This review provides an overview of PtdSer receptors that serve as PVEERs and the biology behind virion/PVEER interaction. PMID:25277499

  2. Intra-nuclear localization of two envelope proteins, gB and gD, of herpes simplex virus.

    PubMed

    Stannard, L M; Himmelhoch, S; Wynchank, S

    1996-01-01

    The envelopes of herpes simplex virus (HSV) particles are acquired from the inner nuclear membrane (INM) of the infected cell and virus-coded glycoproteins are present in the envelope of mature virions. Our ultrastructural study examined the process of virus envelopment and the targeting of two major viral glycoproteins, gB and gD, to the INM in HSV-infected human embryonic fibroblasts. It was shown that envelopment and transport of virus particles from the nucleus is facilitated by the formation of a dynamic tubulo-reticulum arising from the INM. Capsids were assembled in the nucleus and collected within INM tubules which protruded into the perinuclear space and thence into the cisternae of the endoplasmic reticulum (ER). Envelopment occurred by constriction and fusion of the tubular channel walls, releasing enveloped virions into the ER. Transport to the cell surface took place in membrane-bound compartments and probably followed the normal secretory pathway through the Golgi apparatus. Immunogold probes, tagged with specific monoclonal antibodies, were used to localize gB and gD during the process of virus maturation. Cytoplasmic membranes were not labelled, but probes bound inside the nucleus, mainly at sites of virus assembly. Labelling occurred on the nucleoplasmic side of the INM which surrounded capsids in the process of envelopment, but not on the outside of that membrane, although characteristic gB glycoprotein spikes were labelled on the envelopes of extracellular virus particles and on virions in trans-Golgi transport vesicles just prior to their release from the infected cell. gB was not detected on the surface of enveloped virions in the perinuclear space, or the cisternae of the ER or cis-Golgi, which suggests that the specific epitope was masked during that stage of intracellular processing. gD probes bound to virion envelopes and also to the tegument region of some particles found in both perinuclear and extracellular sites. We postulate the

  3. Proteomic analysis of Singapore grouper iridovirus envelope proteins and characterization of a novel envelope protein VP088.

    PubMed

    Zhou, Sheng; Wan, Qingjiao; Huang, Youhua; Huang, Xiaohong; Cao, Jianhao; Ye, Lili; Lim, Teck-Kwang; Lin, Qingsong; Qin, Qiwei

    2011-06-01

    Singapore grouper iridovirus (SGIV) is an enveloped virus causing heavy economic losses to marine fish culture. The envelope fractions of SGIV were separated from the purified virions by Triton X-100 treatment, and subjected to 1-DE-MALDI-TOF/TOF-MS/MS and LC-MALDI-TOF/TOF-MS/MS analysis. A total of 19 virus-encoded envelope proteins were identified in this study and 73.7% (13/17) of them were predicted to be membrane proteins. Three viral envelope proteins were uniquely identified by 1-DE-MALDI, whereas another ten proteins were identified only by LC-MALDI, with six proteins identified by both workflows. VP088 was chosen as a representative of proteomic identification and characterized further. VP088 was predicted to be a viral transmembrane envelope protein which contains two RGD (Arg-Gly-Asp) motifs, three transmembrane domains, and five N-glycosylation sites. VP088 gene transcript was first detected at 12 h p.i. and reached the peak at 48 h p.i. Combined with the drug inhibition assay, VP088 gene was identified as a late (L) gene. Recombinant VP088 (rVP088) was expressed in Escherichia coli, and the specific antiserum against rVP088 was raised. VP088 was proved to be a viral envelope protein by Western blot and immunoelectron microscopy (IEM). Furthermore, rVP088 can bind to a 94 kDa host cell membrane protein, suggesting that VP088 might function as an attaching protein. Neutralization assay also suggested that VP088 is involved in SGIV infection. This study will lead to a better understanding of molecular mechanisms of the iridoviral pathogenesis and virus-host interactions.

  4. Insights into the structure and substrate interactions of the P-glycoprotein multidrug transporter from spectroscopic studies.

    PubMed

    Sharom, F J; Liu, R; Romsicki, Y; Lu, P

    1999-12-01

    The P-glycoprotein multidrug transporter is a 170-kDa efflux pump which exports a diverse group of natural products, chemotherapeutic drugs, and hydrophobic peptides across the plasma membrane, driven by ATP hydrolysis. The transporter has been proposed to interact with its drug substrates within the membrane environment; however, much remains to be learned about the nature and number of the drug binding site(s). The two nucleotide binding domains are responsible for ATP binding and hydrolysis, which is coupled to drug movement across the membrane. In recent years, P-glycoprotein has been purified and functionally reconstituted in amounts large enough to allow biophysical studies. The use of spectroscopic techniques has led to insights into both its secondary and tertiary structure, and its interaction with nucleotides and drugs. In this review, we will summarise what has been learned by application to purified P-glycoprotein of fluorescence spectroscopy, circular dichroism spectroscopy and infra-red spectroscopy.

  5. Effects of retroviral envelope-protein cleavage upon trafficking, incorporation, and membrane fusion

    SciTech Connect

    Apte, Swapna; Sanders, David Avram

    2010-09-15

    Retroviral envelope glycoproteins undergo proteolytic processing by cellular subtilisin-like proprotein convertases at a polybasic amino-acid site in order to produce the two functional subunits, SU and TM. Most previous studies have indicated that envelope-protein cleavage is required for rendering the protein competent for promoting membrane fusion and for virus infectivity. We have investigated the role of proteolytic processing of the Moloney murine leukemia virus envelope-protein through site-directed mutagenesis of the residues near the SU-TM cleavage site and have established that uncleaved glycoprotein is unable either to be incorporated into virus particles efficiently or to induce membrane fusion. Additionally, the results suggest that cleavage of the envelope protein plays an important role in intracellular trafficking of protein via the cellular secretory pathway. Based on our results it was concluded that a positively charged residue located at either P2 or P4 along with the arginine at P1 is essential for cleavage.

  6. Rapid intramolecular turnover of N-linked glycans in plasma membrane glycoproteins. Extension of intramolecular turnover to the core sugars in plasma membrane glycoproteins of hepatoma.

    PubMed

    Tauber, R; Park, C S; Becker, A; Geyer, R; Reutter, W

    1989-12-01

    Plasma membrane glycoproteins of rat hepatocytes undergo a rapid terminal deglycosylation in that the terminal sugars of the oligosaccharide side chains are rapidly removed from the otherwise intact glycoproteins [Tauber, R., Park, C.S. & Reutter, W. (1983) Proc. Natl Acad. Sci. USA 80, 4026-4029]. The present paper demonstrates that this rapid intramolecular turnover of plasma membrane glycoproteins is not restricted to peripheral sugars but, in contrast to liver, in hepatoma the core sugars of the oligosaccharide chains are also involved. Intramolecular turnover was measured in Morris hepatoma 7777 in five plasma membrane glycoproteins with Mr of 85,000 (hgp85), 105,000 (hgp105), 115,000 (hgp115), 125,000 (hgp125), 175,000 (hgp175) (hgp = hepatoma glycoprotein) that were isolated and purified to homogeneity by concanavalin-A--Sepharose affinity chromatography and semipreparative SDS gel electrophoresis. Analysis of the carbohydrates of hgp85, hgp105, hgp115 and hgp125 revealed the presence of N-linked oligosaccharides containing L-fucose, D-galactose, D-mannose and N-acetyl-D-glucosamine, but only of trace amounts of N-acetyl-D-galactosamine; hgp175 additionally contained significant amounts of N-acetyl-D-galactosamine, indicating the presence of both N- and O-linked oligosaccharides. As shown by digestion with endoglucosaminidase H, the N-linked oligosaccharides of hgp105, hgp115, hgp125 and hgp175 were of the complex type, whereas hgp85 also contained oligosaccharides of the high-mannose type. Half-lives of the turnover of the oligosacharide chains and of the protein backbone of the five glycoproteins were measured in the plasma membrane in pulse-chase experiments in vivo, using L-[3H]fucose as a marker of terminal sugars, D-[3H]mannose as marker of a core sugar and L-[3H]leucine for labelling the protein backbone. Protein backbones of the five glycoproteins were degraded with individual half-lives ranging over 41-90 h with a mean of 66 h. Compared to the

  7. A multigene family encodes the human cytomegalovirus glycoprotein complex gcII (gp47-52 complex)

    SciTech Connect

    Gretch, D.R.; Stinski, M.F. ); Kari, B.; Gehrz, R.C. )

    1988-06-01

    The HXLF (HindIII-X left reading frame) gene family is a group of five genes that share one or two regions of homology and are arranged in tandem within the short unique component of the human cytomegalovirus genome. These genes were cloned into an SP6 expression vector in both the sense and antisense orientations. An abundant 1.62-kilobase (kb) bicistronic mRNA, predicted to originate from HXLF1 and HXLF2, was detected in the cytoplasm of infected human fibroblast cells by Northern (RNA) blot analysis. Less abundant RNAs of 1.0 and 0.8 kb, predicted to originate from the HXLF5 and HXLF2 genes, respectively, were also detected. Monocistronic, bicistronic, and polycistronic RNAs synthesized in vitro by using SP6 polymerase were translated in rabbit reticulocyte lysates with or without canine pancreatic microsomal membranes. The HXLF1 or the HXLF1 and HXLF2 translation products were detected when the above mRNAs were used. The HXLF3, HXLF4, and HXLF5 gene products were not detected by in vitro translation of the SP6-derived polycistronic mRNA. The amino acid composition of gp47-52 purified from virion envelopes has the highest similarity to the predicted amino acid composition of the HXLF1 plus HXLF2 open reading frames, but it is more similar to HXLF2 than to HXLF1. The Northern blot results imply that gp47-52 is synthesized predominantly from the abundant 1.62-kb bicistronic mRNA encoded by the HXLF1 and HXLF2 genes. However, the glycoprotein could also be synthesized by the monocistronic 0.8-kb mRNA encoded by the HXLF2 gene as well as by the mRNAs predicted from the other HXLF genes.

  8. Method for purifying bidentate organophosphorus compounds

    DOEpatents

    Schulz, Wallace W.

    1977-01-01

    Bidentate organophosphorus compounds useful for extracting actinide elements from acidic nuclear waste solutions are purified of undesirable acidic impurities by contacting the compounds with ethylene glycol which preferentially extracts the impurities found in technical grade bidentate compounds.

  9. Transient transfection of purified Babesia bovis merozoites

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Transient transfection of intraerythrocytic Babesia bovis parasites has been previously reported. In this study, we describe the development and optimization of methods for transfection of purified B. bovis merozoites using either nucleofection (Amaxa) or conventional electroporation (Gene Pulser II...

  10. Characterization of the rabbit homolog of human MUC1 glycoprotein isolated from bladder by affinity chromatography on immobilized jacalin.

    PubMed

    Higuchi, T; Xin, P; Buckley, M S; Erickson, D R; Bhavanandan, V P

    2000-07-01

    The urinary bladder is lined by transitional epithelium, the glycocalyx on the luminal surface has interesting properties and is implicated in protective functions. Glycoconjugates are major components of the glycocalyx, but their biochemical nature is not well understood. Previous studies on rabbit bladder indicated the presence of significant levels of sialoglycoproteins compared to glycosaminoglycans in the epithelium. In this study, rabbit explant cultures were radiolabeled by precursor sugars or amino acids and a major lectin-reactive glycoprotein of rabbit bladder mucosa was isolated by affinity chromatography on jacalin-agarose. The radiolabeled glycoprotein was purified to homogeneity by a second cycle on the lectin column, followed by gel filtration and density gradient centrifugation. The average molecular mass of the glycoprotein was estimated to be 245 kDa and 210 kDa by gel filtration and SDS-PAGE, respectively. Its buoyant density was 1.40 g/ml, suggesting a carbohydrate content of approximately 50%. The percent distribution of glucosamine-derived tritium label in sialic acid, galactosamine, and glucosamine was 30, 52, and 18, respectively. The glycoprotein consisted entirely of small sialylated and neutral oligosaccharides O-glycosidically linked to serine and threonine residues. The same glycoprotein could be immunoprecipitated with an antibody against the carboxy terminal 17 amino acid peptide of human MUC1 mucin glycoprotein. This suggests that this mucin glycoprotein is the rabbit homolog of MUC1 glycoprotein, which has been previously established to be a component of human bladder urothelium and has been purified from human urine and biochemically characterized.

  11. Apoptosis induced by glycoprotein (150-kDa) isolated from Solanum nigrum L. is not related to intracellular reactive oxygen species (ROS) in HCT-116 cells.

    PubMed

    Lee, Sei-Jung; Lim, Kye-Taek

    2006-04-01

    This study was carried out to investigate the apoptotic effects of glycoprotein [Solanum nigrum L. (SNL) glycoprotein, 150-kDa] isolated from Solanum nigrum L., which has been used as an antipyretic and anticancer agent in folk medicine. With the purified SNL glycoprotein, we evaluated the cytotoxic and apoptotic effects of SNL glycoprotein on HCT-116 cells, DNA fragmentation and nuclear staining assays, respectively. SNL glycoprotein has an apparent cytotoxic and apoptotic effect at a concentration of 40 microg/ml after 4 h. To further verify the apoptotic effect, we investigated the changes in activity of the apoptotic-related proteins [Bid, cytochrome c, caspases and poly(ADP-ribose)polymerase (PARP)] triggered by SNL glycoprotein, using a western blot analysis. The results in this study indicated that SNL glycoprotein has a stimulatory effect on Bid activation, resulting in the release of cytochrome c, the stimulation of caspase-8, -9 and -3 activities, and the cleavage of PARP in HCT-116 cells. However, SNL glycoprotein did not significantly stimulate an increase in levels of intracellular reactive oxygen species (ROS). From the results in this experiment, it is suggested that SNL glycoprotein induces apoptosis through the mitochondrial apoptotic signal pathway in HCT-116 cells, rather than through intracellular ROS. PMID:16208518

  12. Alteration of a second putative fusion peptide of structural glycoprotein E2 of Classical Swine Fever Virus alters virus replication and virulence in swine

    Technology Transfer Automated Retrieval System (TEKTRAN)

    E2, the major envelope glycoprotein of Classical Swine Fever Virus (CSFV), is involved in several critical virus functions including cell attachment, host range susceptibility, and virulence in natural hosts. Functional structural analysis of E2 based on Wimley-White interfacial hydrophobicity dis...

  13. Effect of specific amino acid substitutions in the putative fusion peptide of structural glycoprotein E2 on Classical Swine Fever Virus replication

    Technology Transfer Automated Retrieval System (TEKTRAN)

    E2, along with E^rns and E1, is an envelope glycoprotein of Classical Swine Fever Virus (CSFV). E2 is involved in several virus functions including cell attachment, host range susceptibility and virulence in natural hosts. In infected cells, E2 forms homodimers as well as heterodimers with E1, media...

  14. HIV Neutralizing Antibodies Induced by Native-like Envelope Trimers

    PubMed Central

    Sanders, Rogier W.; van Gils, Marit J.; Derking, Ronald; Sok, Devin; Ketas, Thomas J.; Burger, Judith A.; Ozorowski, Gabriel; Cupo, Albert; Simonich, Cassandra; Goo, Leslie; Arendt, Heather; Kim, Helen J.; Lee, Jeong Hyun; Pugach, Pavel; Williams, Melissa; Debnath, Gargi; Moldt, Brian; van Breemen, Mariëlle J.; Isik, Gözde; Medina-Ramírez, Max; Back, Jaap Willem; Koff, Wayne; Julien, Jean-Philippe; Rakasz, Eva G.; Seaman, Michael S.; Guttman, Miklos; Lee, Kelly K.; Klasse, Per Johan; LaBranche, Celia; Schief, William R.; Wilson, Ian A.; Overbaugh, Julie; Burton, Dennis R.; Ward, Andrew B.; Montefiori, David C.; Dean, Hansi; Moore, John P.

    2015-01-01

    A challenge for HIV-1 immunogen design is inducing neutralizing antibodies (NAbs) against neutralization-resistant (Tier-2) viruses that dominate human transmissions. We show that a soluble recombinant HIV-1 envelope glycoprotein trimer that adopts a native conformation (BG505 SOSIP.664) induced NAbs potently against the sequence-matched Tier-2 virus in rabbits and similar but weaker responses in macaques. The trimer also consistently induced cross-reactive NAbs against more sensitive (Tier-1) viruses. Tier-2 NAbs recognized conformational epitopes that differed between animals and in some cases overlapped with those recognized by broadly neutralizing antibodies (bNAbs), whereas Tier-1 responses targeted linear V3 epitopes. A second trimer, B41 SOSIP.664, also induced a strong autologous Tier-2 NAb response in rabbits. Thus, native-like trimers represent a promising starting point for developing HIV-1 vaccines aimed at inducing bNAbs. PMID:26089353

  15. Personnel occupied woven envelope robot

    NASA Technical Reports Server (NTRS)

    Wessling, F. C.

    1986-01-01

    The use of nonmetallic or fabric structures for space application is considered. The following structures are suggested: (1) unpressurized space hangars; (2) extendable tunnels for soft docking; and (3) manned habitat for space stations, storage facilities, and work structures. The uses of the tunnel as a passageway: for personnel and equipment, eliminating extravehicular activity, for access to a control cabin on a space crane and between free flyers and the space station are outlined. The personnal occupied woven envelope robot (POWER) device is shown. The woven envelope (tunnel) acts as part of the boom of a crane. Potential applications of POWER are outlined. Several possible deflection mechanisms and design criteria are determined.

  16. Carbon chemistry of circumstellar envelopes

    NASA Technical Reports Server (NTRS)

    Bieging, John H.

    1990-01-01

    The chemical composition of envelopes surrounding cool evolved stars, as determined from microwave spectroscopic observations, is reviewed. Emphasis is placed on recent observations with the new large mm-wavelength telescopes and interferometer arrays, and on new theoretical work, especially concerning ion-molecule chemistry of carbon-bearing in these envelopes. Thermal (as opposed to maser) emission lines are discussed. Much progress has been made in the past few years in the theoretical understanding of these objects. It is already clear, however, that observations with the new generation of mm-telescopes will require substantial improvements in the theoretical models to achieve a thorough understanding of the data now becoming available.

  17. The Domains of Glycoprotein D Required To Block Apoptosis Depend on Whether Glycoprotein D Is Present in the Virions Carrying Herpes Simplex Virus 1 Genome Lacking the Gene Encoding the Glycoprotein

    PubMed Central

    Zhou, Guoying; Roizman, Bernard

    2001-01-01

    An earlier report showed that viruses lacking the open reading frames encoding glycoproteins J and D but containing the glycoprotein D in their envelopes (gD−/+ stocks) and viruses lacking both the open reading frames and the glycoproteins in their envelopes (gD−/− stocks) induce apoptosis (G. Zhou, V. Galvan, G. Campadelli-Fiume, and B. Roizman, J. Virol. 74:11782–11791, 2000). Furthermore, apoptosis was blocked by delivery in trans of genes expressing glycoprotein D or J. Whereas gD−/− stocks attach but cannot initiate productive infection, gD−/+ stocks infect cells and produce gD−/− progeny virus. The difference in the infectivity of these two stocks suggested the possibility that the requirements for blocking apoptosis may be different. To test this hypothesis, we cloned into baculoviruses the entire wild-type glycoprotein D (Bac-gD-WT), the ectodomain only (Bac-gD-A), the ectodomain and the transmembrane domain (Bac-gD-B), the ectodomain and the cytoplasmic domain without the transmembrane domain (Bac-gD-C), or the transmembrane domain and the carboxyl-terminal cytoplasmic domain (Bac-gD-D). We report the following. Apoptosis induced by gD−/+ stocks was blocked by delivery in trans of recombinant baculovirus Bac-gD-WT, Bac-gD-A, Bac-gD-B, or Bac-gD-C but not of Bac-gD. Apoptosis induced by gD−/− stocks was blocked by Bac-gD-WT or by a mixture of Bac-gD-B and Bac-gD-D but not by any baculoviruses expressing truncated glycoprotein D alone or by the mixture of Bac-gD-A and Bac-gD-D. We conclude that the requirements to block apoptosis induced by the two virus stocks are different. The gD ectodomain is sufficient to block apoptosis induced by gD, whereas both the ectodomain and the cytoplasmic domain are required to block apoptosis induced by gD−/− stocks. The results indicate that in the case of gD−/− stocks, the transmembrane domain is required either to deliver the ectodomain to the appropriate intracellular compartment or to

  18. Study on the extraction and purification of glycoprotein from the yellow seahorse, Hippocampus kuda Bleeker.

    PubMed

    Su, Yuting; Xu, Yongjian

    2015-07-01

    The optimum parameters of extraction for glycoprotein from seahorse were examined and determined by Box-Behnken combined with ultrasonic extraction technology. Column chromatography of glycoprotein was used for further purification. The optimal extraction conditions of seahorse glycoprotein were extracting time 4.3 h, salt concentration 0.08 mol/L, extracting temperature 73°C, raw material, and water ratio 1:6. At the optimal conditions, the yield of saccharide reached to 1.123%, and the yield of protein reached to 5.898%. For purifying the crude glycoprotein, the stage renounces of DEAE-52 column chromatography were done, respectively, with 0.05, 0.1, 0.5 mol/L NaHCO3 solution, and further purification was done with Sephadex G-100 column chromatography. Finally, two pieces of seahorse glycoprotein were obtained by the column chromatography, that is, HG-11 and HG-21. The saccharide content was 56.7975% and 39.479%, the protein content was 30.5475% and 51.747%, respectively.

  19. A role for the dystrophin-glycoprotein complex as a transmembrane linker between laminin and actin

    PubMed Central

    1993-01-01

    The dystrophin-glycoprotein complex was tested for interaction with several components of the extracellular matrix as well as actin. The 156-kD dystrophin-associated glycoprotein (156-kD dystroglycan) specifically bound laminin in a calcium-dependent manner and was inhibited by NaCl (IC50 = 250 mM) but was not affected by 1,000-fold (wt/wt) excesses of lactose, IKVAV, or YIGSR peptides. Laminin binding was inhibited by heparin (IC50 = 100 micrograms/ml), suggesting that one of the heparin-binding domains of laminin is involved in binding dystroglycan while negatively charged oligosaccharide moieties on dystroglycan were found to be necessary for its laminin-binding activity. No interaction between any component of the dystrophin- glycoprotein complex and fibronectin, collagen I, collagen IV, entactin, or heparan sulfate proteoglycan was detected by 125I-protein overlay and/or extracellular matrix protein-Sepharose precipitation. In addition, laminin-Sepharose quantitatively precipitated purified dystrophin-glycoprotein complex, demonstrating that the laminin-binding site is accessible when dystroglycan is associated with the complex. Dystroglycan of nonmuscle tissues also bound laminin. However, the other proteins of the striated muscle dystrophin-glycoprotein complex appear to be absent, antigenically dissimilar or less tightly associated with dystroglycan in nonmuscle tissues. Finally, we show that the dystrophin-glycoprotein complex cosediments with F-actin but does not bind calcium or calmodulin. Our results support a role for the striated muscle dystrophin-glycoprotein complex in linking the actin- based cytoskeleton with the extracellular matrix. Furthermore, our results suggest that dystrophin and dystroglycan may play substantially different functional roles in nonmuscle tissues. PMID:8349731

  20. Structure and Function of RSV Surface Glycoproteins

    PubMed Central

    McLellan, Jason S.; Ray, William C.; Peeples, Mark E.

    2014-01-01

    The two major glycoproteins on the surface of the RSV virion, the attachment glycoprotein (G) and the fusion (F) glycoprotein, control the initial phases of infection. G targets the ciliated cells of the airways, and F causes the virion membrane to fuse with a target cell membrane. The F protein is the major target for antiviral drug development, and both G and F glycoproteins are the antigens targeted by neutralizing antibodies induced by infection. In this chapter we review the structure and function of the RSV surface glycoproteins, including recent X-ray crystallographic data of the F glycoprotein in its pre- and postfusion conformations, and discuss how this information informs antigen selection and vaccine development. PMID:24362685

  1. Enveloped virus-like particles as vaccines against pathogenic arboviruses.

    PubMed

    Pijlman, Gorben P

    2015-05-01

    Arthropod-borne arboviruses form a continuous threat to human and animal health, but few arboviral vaccines are currently available. Advances in expression technology for complex, enveloped virus-like particles (eVLPs) create new opportunities to develop potent vaccines against pathogenic arboviruses. In this short review, I highlight the successes and challenges in eVLP production for members of the three major arbovirus families: Flaviviridae (e.g., dengue, West Nile, Japanese encephalitis); Bunyaviridae (e.g., Rift Valley fever); and Togaviridae (e.g., chikungunya). The results from pre-clinical testing will be discussed as well as specific constraints to the large-scale manufacture and purification of eVLPs, which are complex assemblies of membranes and viral glycoproteins. Insect cells emerge as ideal substrates for correct arboviral glycoprotein folding and posttranslational modification to yield high quality eVLPs. Furthermore, baculovirus expression in insect cell culture is scalable and has a proven safety record in industrial human and veterinary vaccine manufacturing. In conclusion, eVLPs produced in insect cells using modern biotechnology have a realistic potential to be used in novel vaccines against arboviral diseases.

  2. Enveloped virus-like particles as vaccines against pathogenic arboviruses.

    PubMed

    Pijlman, Gorben P

    2015-05-01

    Arthropod-borne arboviruses form a continuous threat to human and animal health, but few arboviral vaccines are currently available. Advances in expression technology for complex, enveloped virus-like particles (eVLPs) create new opportunities to develop potent vaccines against pathogenic arboviruses. In this short review, I highlight the successes and challenges in eVLP production for members of the three major arbovirus families: Flaviviridae (e.g., dengue, West Nile, Japanese encephalitis); Bunyaviridae (e.g., Rift Valley fever); and Togaviridae (e.g., chikungunya). The results from pre-clinical testing will be discussed as well as specific constraints to the large-scale manufacture and purification of eVLPs, which are complex assemblies of membranes and viral glycoproteins. Insect cells emerge as ideal substrates for correct arboviral glycoprotein folding and posttranslational modification to yield high quality eVLPs. Furthermore, baculovirus expression in insect cell culture is scalable and has a proven safety record in industrial human and veterinary vaccine manufacturing. In conclusion, eVLPs produced in insect cells using modern biotechnology have a realistic potential to be used in novel vaccines against arboviral diseases. PMID:25692281

  3. Disulfide Bonds in Hepatitis C Virus Glycoprotein E1 Control the Assembly and Entry Functions of E2 Glycoprotein

    PubMed Central

    Wahid, Ahmed; Helle, François; Descamps, Véronique; Duverlie, Gilles; Penin, François

    2013-01-01

    Class II membrane fusion proteins have been described in viruses in which the envelope proteins are derived from a precursor polyprotein containing two transmembrane glycoproteins arranged in tandem. Although the second protein, which carries the membrane fusion function, is in general well characterized, the companion protein, which is a protein chaperone for the folding of the fusion protein, is less well characterized for some viruses, like hepatitis C virus (HCV). To investigate the role of the class II companion glycoprotein E1 of HCV, we chose to target conserved cysteine residues in the protein, and we systematically mutated them in a full-length infectious HCV clone by reverse genetics. All the mutants were infectious, albeit with lower titers than the wild-type virus. The reduced infectivity was in part due to a decrease in viral assembly, as revealed by measurement of intracellular infectivity and by quantification of core protein released from cells transfected with mutant genomes. Analyses of mutated proteins did not show any major defect in folding. However, the mutations reduced virus stability, and they could also affect the density of infectious viral particles. Mutant viruses also showed a defect in cell-to-cell transmission. Finally, our data indicate that HCV glycoprotein E1 can also affect the fusion protein E2 by modulating its recognition by the cellular coreceptor CD81. Therefore, in the context of HCV, our data identify an additional function of a class II companion protein as a molecule that can control the binding capacity of the fusion protein. PMID:23175356

  4. Effect of glycoprotein-processing inhibitors on fucosylation of glycoproteins

    SciTech Connect

    Schwarz, P.M.; Elbein, A.D.

    1985-11-25

    Influenza viral hemagglutinin contains L-fucose linked alpha 1,6 to some of the innermost GlcNAc residues of the complex oligosaccharides. To determine what structural features of the oligosaccharide were required for fucosylation influenza virus-infected MDCK cells were incubated in the presence of various inhibitors of glycoprotein processing to stop trimming at different points. After several hours of incubation with the inhibitors, (5,6-TH)fucose and (1- UC)mannose were added to label the glycoproteins, and cells were incubated in inhibitor and isotope for about 40 h to produce mature virus. Glycopeptides were prepared from the viral and the cellular glycoproteins, and these glycopeptides were isolated by gel filtration on Bio-Gel P-4. The glycopeptides were then digested with endo-beta-N-acetylglucosaminidase H and rechromatographed on the Bio-Gel column. In the presence of castanospermine or 2,5-dihydroxymethyl-3,4-dihydroxypyrrolidine, both inhibitors of glucosidase I, most of the radioactive mannose was found in Glc3Man7-9GlcNAc structures, and these did not contain radioactive fucose. In the presence of deoxymannojirimycin, an inhibitor of mannosidase I, most of the ( UC)mannose was in a Man9GlcNAc structure which was also not fucosylated. However, in the presence of swainsonine, an inhibitor of mannosidase II, the ( UC)mannose was mostly in hybrid types of oligosaccharides, and these structures also contained radioactive fucose. Treatment of the hybrid structures with endoglucosaminidase H released the (TH)fucose as a small peptide (Fuc-GlcNAc-peptide), whereas the ( UC)mannose remained with the oligosaccharide. The data support the conclusion that the addition of fucose linked alpha 1,6 to the asparagine-linked GlcNAc is dependent upon the presence of a beta 1,2-GlcNAc residue on the alpha 1,3-mannose branch of the core structure.

  5. Reconceptualization of the Budget Envelope.

    ERIC Educational Resources Information Center

    Jefferson, Anne L.

    This paper reconceptualizes the purposes of education's budget envelope. Citing numerous examples of how policymakers consider resource allocations apart from the main concerns of individual programs, the people reallocations affect, and education's most important programs, it suggests that policymakers and finance officers reemphasize program and…

  6. Rabies virus glycoprotein as a carrier for anthrax protective antigen

    SciTech Connect

    Smith, Mary Ellen; Koser, Martin; Xiao Sa; Siler, Catherine; McGettigan, James P.; Calkins, Catherine; Pomerantz, Roger J.; Dietzschold, Bernhard; Schnell, Matthias J. . E-mail: matthias.schnell@jefferson.edu

    2006-09-30

    Live viral vectors expressing foreign antigens have shown great promise as vaccines against viral diseases. However, safety concerns remain a major problem regarding the use of even highly attenuated viral vectors. Using the rabies virus (RV) envelope protein as a carrier molecule, we show here that inactivated RV particles can be utilized to present Bacillus anthracis protective antigen (PA) domain-4 in the viral membrane. In addition to the RV glycoprotein (G) transmembrane and cytoplasmic domains, a portion of the RV G ectodomain was required to express the chimeric RV G anthrax PA on the cell surface. The novel antigen was also efficiently incorporated into RV virions. Mice immunized with the inactivated recombinant RV virions exhibited seroconversion against both RV G and anthrax PA, and a second inoculation greatly increased these responses. These data demonstrate that a viral envelope protein can carry a bacterial protein and that a viral carrier can display whole polypeptides compared to the limited epitope presentation of previous viral systems.

  7. Identification of the origin and localization of chorion (egg envelope) proteins in an ancient fish, the white sturgeon, Acipenser transmontanus.

    PubMed

    Murata, Kenji; Conte, Fred S; McInnis, Elizabeth; Fong, Tak Hou; Cherr, Gary N

    2014-06-01

    In many modern teleost fish, chorion (egg envelope) glycoproteins are synthesized in the liver of females, and the expression of those genes is controlled by endogenous estrogen released from the ovary during maturation. However, among the classical teleosts, such as salmonid, carp, and zebrafish, the chorion glycoproteins are synthesized in the oocyte, as in higher vertebrates. Sturgeon, which are members of the subclass Chondrostei, represent an ancient lineage of ray-finned fishes that differ from other teleosts in that their sperm possess acrosomes, their eggs have numerous micropyles, and early embryo development is similar to that of amphibians. In order to understand the molecular mechanisms of chorion formation and the phylogenetic relationship between sturgeon and other teleosts, we used specific antibodies directed against the primary components of sturgeon chorion glycoproteins, using immunoblotting and immunocytochemistry approaches. The origin of each chorion glycoprotein was determined through analyses of both liver and ovary, and their localization during ovarian development was investigated. Our data indicate that the origin of the major chorion glycoproteins of sturgeon, ChG1, ChG2, and ChG4, derive not only from the oocyte itself but also from follicle cells in the ovary, as well as from hepatocytes. In the follicle cell layer, granulosa cells were found to be the primary source of ChGs during oogenesis in white sturgeon. The unique origins of chorion glycoproteins in sturgeon suggest that sturgeons are an intermediate form in the evolution of the teleost lineage.

  8. Functional Reconstitution into Liposomes of Purified Human RhCG Ammonia Channel

    PubMed Central

    Mouro-Chanteloup, Isabelle; Cochet, Sylvie; Chami, Mohamed; Genetet, Sandrine; Zidi-Yahiaoui, Nedjma; Engel, Andreas; Colin, Yves; Bertrand, Olivier; Ripoche, Pierre

    2010-01-01

    Background Rh glycoproteins (RhAG, RhBG, RhCG) are members of the Amt/Mep/Rh family which facilitate movement of ammonium across plasma membranes. Changes in ammonium transport activity following expression of Rh glycoproteins have been described in different heterologous systems such as yeasts, oocytes and eukaryotic cell lines. However, in these complex systems, a potential contribution of endogenous proteins to this function cannot be excluded. To demonstrate that Rh glycoproteins by themselves transport NH3, human RhCG was purified to homogeneity and reconstituted into liposomes, giving new insights into its channel functional properties. Methodology/Principal Findings An HA-tag introduced in the second extracellular loop of RhCG was used to purify to homogeneity the HA-tagged RhCG glycoprotein from detergent-solubilized recombinant HEK293E cells. Electron microscopy analysis of negatively stained purified RhCG-HA revealed, after image processing, homogeneous particles of 9 nm diameter with a trimeric protein structure. Reconstitution was performed with sphingomyelin, phosphatidylcholine and phosphatidic acid lipids in the presence of the C12E8 detergent which was subsequently removed by Biobeads. Control of protein incorporation was carried out by freeze-fracture electron microscopy. Particle density in liposomes was a function of the Lipid/Protein ratio. When compared to empty liposomes, ammonium permeability was increased two and three fold in RhCG-proteoliposomes, depending on the Lipid/Protein ratio (1/300 and 1/150, respectively). This strong NH3 transport was reversibly inhibited by mercuric and copper salts and exhibited a low Arrhenius activation energy. Conclusions/Significance This study allowed the determination of ammonia permeability per RhCG monomer, showing that the apparent PunitNH3 (around 1×10−3 µm3.s−1) is close to the permeability measured in HEK293E cells expressing a recombinant human RhCG (1.60×10−3 µm3.s−1), and in human red

  9. Performance assessment of O-18 water purifier.

    PubMed

    Kitano, H; Magata, Y; Tanaka, A; Mukai, T; Kuge, Y; Nagatsu, K; Konishi, J; Saji, H

    2001-02-01

    In the synthesis of 18F-FDG by the nucleophilic substitution method, 18O-H2O is usually used as target water. The target water should be recovered after synthesis and reused, because it is expensive, but recovered water contains impurities such as organic substances, and it must be purified before reuse. For this reason Sumitomo Heavy Industries, Ltd. developed an O-18 water purifier for elimination of organic substances in recovered water. This instrument consists of a UV irradiation unit and low-temperature distillation unit. Our institution had an opportunity to test use this instrument and evaluated its performance. The concentrations of organic substances after UV irradiation was greatly reduced, and recovery efficiency after distillation by the low-temperature distillation unit was very satisfactory at 99.3 +/- 0.5%. Furthermore, the yield of 18F-FDG from 18O-H20 purified with this instrument was sufficient for the clinical use. PMID:11355788

  10. Disulfide bond structure of glycoprotein D of herpes simplex virus types 1 and 2.

    PubMed Central

    Long, D; Wilcox, W C; Abrams, W R; Cohen, G H; Eisenberg, R J

    1992-01-01

    Glycoprotein D (gD) is a structural component of the herpes simplex virus envelope which is essential for virus penetration. The function of this protein is highly dependent on its structure, and its structure is dependent on maintenance of three intact disulfide bonds. gD contains six cysteines in its ectodomain whose spacing is conserved among all its homologs in other alphaherpesviruses as well as Marek's disease virus. For other proteins, conservation of cysteine spacing correlates with conservation of disulfide bond structure. We have now solved the disulfide bond structure of gD-1 and gD-2 of herpes simplex virus types 1 and 2, respectively. Two approaches were used. First, we constructed 15 double-Cys mutants of gD-1, representing all possible disulfide pairs. In each case, codons for cysteines were changed to serine. We reasoned that if two cysteines normally form a disulfide bond, double mutations which eliminate one proper bond should be less harmful to gD structure than double mutations which eliminate two disulfide bonds. The mutated genes were cloned into a eucaryotic expression vector, and the proteins were expressed in transiently transfected cells. Three double mutations, Cys-1,5, Cys-2,6, and Cys-3,4 permitted gD-1 folding, processing, transport to the cell surface, and function in virus infection, whereas 12 other double mutations each produced a malfolded and nonfunctional protein. Thus, the three functional double-Cys mutants may represent the actual partners in disulfide bond linkages. The second approach was to define the actual disulfide bond structure of gD by biochemical means. Purified native gD-2 was cleaved by CNBr and proteases, and the peptides were separated by high-performance liquid chromatography. Disulfide-linked peptides were subjected to N-terminal amino acid sequencing. The results show that cysteine 1 (amino acid [aa] 66) is bonded to cysteine 5 (aa 189), cysteine 2 (aa 106) is bonded to cysteine 6 (aa 202), and cysteine 3 (aa

  11. A sweet code for glycoprotein folding.

    PubMed

    Caramelo, Julio J; Parodi, Armando J

    2015-11-14

    Glycoprotein synthesis is initiated in the endoplasmic reticulum (ER) lumen upon transfer of a glycan (Glc3Man9GlcNAc2) from a lipid derivative to Asn residues (N-glycosylation). N-Glycan-dependent quality control of glycoprotein folding in the ER prevents exit to Golgi of folding intermediates, irreparably misfolded glycoproteins and incompletely assembled multimeric complexes. It also enhances folding efficiency by preventing aggregation and facilitating formation of proper disulfide bonds. The control mechanism essentially involves four components, resident lectin-chaperones (calnexin and calreticulin) that recognize monoglucosylated polymannose protein-linked glycans, lectin-associated oxidoreductase acting on monoglucosylated glycoproteins (ERp57), a glucosyltransferase that creates monoglucosylated epitopes in protein-linked glycans (UGGT) and a glucosidase (GII) that removes the glucose units added by UGGT. This last enzyme is the only mechanism component sensing glycoprotein conformations as it creates monoglucosylated glycans exclusively in not properly folded glycoproteins or in not completely assembled multimeric glycoprotein complexes. Glycoproteins that fail to properly fold are eventually driven to proteasomal degradation in the cytosol following the ER-associated degradation pathway, in which the extent of N-glycan demannosylation by ER mannosidases play a relevant role in the identification of irreparably misfolded glycoproteins.

  12. Functional roles of membrane glycoprotein CD36.

    PubMed

    Daviet, L; McGregor, J L

    1996-01-01

    Cell-cell and cell-extracellular matrix interactions are mediated by a number of membrane glycoproteins. On the basis of structural homologies, several families of cell adhesion molecules (integrins, selectins, immunoglobulins, cadherins, leucine-rich glycoproteins) have been established. Since 1991, a new family of CD36-like proteins has been identified. CD36 is a cell surface glycoprotein that interacts with a large variety of ligands. CD36 has been implicated in thrombosis, vascular biology, lipid metabolism and atherogenesis. In this review, we aim to summarize our present knowledge on this important, multifunctional glycoprotein. PMID:21043590

  13. Method for purifying bidentate organophosphorous compounds

    DOEpatents

    McIsaac, Lyle D.; Krupa, Joseph F.; Schroeder, Norman C.

    1981-01-01

    Bidentate organophosphorous compounds are purified of undesirable impurities by contacting a solution of the compounds with a mercuric nitrate solution to form an insoluble mercuric bidentate compound which precipitates while the impurities remain in solution. The precipitate is washed and then contacted with a mixture of an aqueous solution of a strong mercuric ion complexing agent and an organic solvent to complex the mercuric ion away from the bidentate compound which then dissolves in the solvent. The purified bidentate compounds are useful for extracting the actinide elements from aqueous acidic nuclear waste solutions.

  14. Acidic pH-Induced Conformations and LAMP1 Binding of the Lassa Virus Glycoprotein Spike

    PubMed Central

    Li, Sai; Sun, Zhaoyang; Pryce, Rhys; Parsy, Marie-Laure; Fehling, Sarah K.; Schlie, Katrin; Siebert, C. Alistair; Garten, Wolfgang; Bowden, Thomas A.; Strecker, Thomas; Huiskonen, Juha T.

    2016-01-01

    Lassa virus is an enveloped, bi-segmented RNA virus and the most prevalent and fatal of all Old World arenaviruses. Virus entry into the host cell is mediated by a tripartite surface spike complex, which is composed of two viral glycoprotein subunits, GP1 and GP2, and the stable signal peptide. Of these, GP1 binds to cellular receptors and GP2 catalyzes fusion between the viral envelope and the host cell membrane during endocytosis. The molecular structure of the spike and conformational rearrangements induced by low pH, prior to fusion, remain poorly understood. Here, we analyzed the three-dimensional ultrastructure of Lassa virus using electron cryotomography. Sub-tomogram averaging yielded a structure of the glycoprotein spike at 14-Å resolution. The spikes are trimeric, cover the virion envelope, and connect to the underlying matrix. Structural changes to the spike, following acidification, support a viral entry mechanism dependent on binding to the lysosome-resident receptor LAMP1 and further dissociation of the membrane-distal GP1 subunits. PMID:26849049

  15. Acidic pH-Induced Conformations and LAMP1 Binding of the Lassa Virus Glycoprotein Spike.

    PubMed

    Li, Sai; Sun, Zhaoyang; Pryce, Rhys; Parsy, Marie-Laure; Fehling, Sarah K; Schlie, Katrin; Siebert, C Alistair; Garten, Wolfgang; Bowden, Thomas A; Strecker, Thomas; Huiskonen, Juha T

    2016-02-01

    Lassa virus is an enveloped, bi-segmented RNA virus and the most prevalent and fatal of all Old World arenaviruses. Virus entry into the host cell is mediated by a tripartite surface spike complex, which is composed of two viral glycoprotein subunits, GP1 and GP2, and the stable signal peptide. Of these, GP1 binds to cellular receptors and GP2 catalyzes fusion between the viral envelope and the host cell membrane during endocytosis. The molecular structure of the spike and conformational rearrangements induced by low pH, prior to fusion, remain poorly understood. Here, we analyzed the three-dimensional ultrastructure of Lassa virus using electron cryotomography. Sub-tomogram averaging yielded a structure of the glycoprotein spike at 14-Å resolution. The spikes are trimeric, cover the virion envelope, and connect to the underlying matrix. Structural changes to the spike, following acidification, support a viral entry mechanism dependent on binding to the lysosome-resident receptor LAMP1 and further dissociation of the membrane-distal GP1 subunits.

  16. Interaction of a 60-kilodalton D-mannose-containing salivary glycoprotein with type 1 fimbriae of Escherichia coli.

    PubMed

    Babu, J P; Abraham, S N; Dabbous, M K; Beachey, E H

    1986-10-01

    A 60-kilodalton glycoprotein previously isolated and purified from human saliva (J. B. Babu, E. H. Beachey, D. L. Hasty, and W. A. Simpson, Infect. Immun. 51: 405-413, 1986) was found to interact with type 1 fimbriae and prevent adhesion of type 1 fimbriated Escherichia coli to animal cells in a D-mannose-sensitive manner. Purified salivary glycoprotein agglutinated type 1 fimbriated E. coli and, at subagglutinating concentrations, blocked the ability of type 1 fimbriated E. coli to attach to human buccal epithelial cells or agglutinate guinea pig erythrocytes. Both interactions were inhibited by alpha-methyl-D-mannoside but not by alpha-methyl-D-glucoside. Complexing of the glycoprotein to type 1 fimbriae was demonstrated by molecular sieve chromatography and modified Western blots. When mixed with type 1 fimbriae, the radiolabeled salivary glycoprotein coeluted with type 1 fimbriae from a column of Sepharose 4B. When blotted from a sodium dodecyl sulfate gel to nitrocellulose sheets, the glycoprotein interacted directly with type 1 fimbriae applied to the blots. Both of the latter interactions also were blocked by alpha-methyl-D-mannoside but not by alpha-methyl-D-glucoside. Chemical modification of the glycoprotein with sodium metaperiodate abolished its ability to interact with isolated type 1 fimbriae or type 1 fimbriated E. coli. These results suggest that the carbohydrate moiety of the 60-kilodalton glycoprotein serves as a receptor for type 1 fimbriae in the oral cavity, and we postulate that the interaction may cause agglutination and early removal of E. coli, thereby preventing colonization by these organisms of oropharyngeal mucosae and dental tissues. PMID:2875948

  17. Studies on the antitumor activity and isolation of the glycoprotein from the gonad of Chlamys (Azumapecten) farreri

    NASA Astrophysics Data System (ADS)

    Gu, Qian-Qun; Fang, Yu-Chun; Wang, Chang-Yun; Xiao, Hang; Qin, Yu-Qing

    1998-12-01

    After being degreased with acetone, the female gonad of Chlamys (Azumapecten) farreri was extracted with water, and then subjected to DEAE—cellulose column chromatography and purified by Sephadex G-100 chromatography to obtain a pure female gonad glycoprotein (FGGP). The IR spectra, electrophoresis and chemical analysis of FGGP indicated that it was a kind of acid glycoprotein and that the content of total protein and sugar were 54.8% and 40%, respectively. Pharmacological tests showed that FGGP could inhibit (inhibition rate of 40.87%) the growth of Sarcoma 180 of mice.

  18. Carbohydrate Structure of Sindbis Virus Glycoprotein E2 from Virus Grown in Hamster and Chicken Cells

    PubMed Central

    Burke, David; Keegstra, Kenneth

    1979-01-01

    Sindbis virus was used as a probe to examine glycosylation processes in two different species of cultured cells. Parallel studies were carried out analyzing the carbohydrate added to Sindbis glycoprotein E2 when the virus was grown in chicken embryo cells and BHK cells. The Pronase glycopeptides of Sindbis glycoprotein E2 were purified by a combination of ion-exchange and gel filtration chromatography. Four glycopeptides were resolved, ranging in molecular weight from 1,800 to 2,700. Structures are proposed for each of the four glycopeptides, based on data obtained by quantitative composition analyses, methylation analyses, and degradation of the glycopeptides using purified exo- and endoglycosidases. The largest three glycopeptides (S1, S2, and S3) have similar structures but differ in the extent of sialylation. All three contain N-acetylglucosamine, mannose, galactose, and fucose, in a structure similar to oligosaccharides found on other glycoproteins. Glycopeptide S1 has two residues of sialic acid, whereas glycopeptides S2 and S3 contain 1 and 0 residues of sialic acid, respectively. The smallest glycopeptide, S4, contains only N-acetyglucosamine and mannose, and is also similar to mannose-rich oligosaccharides found on other glycoproteins. Each of the complex glycopeptides (S1, S2, or S3) from virus grown in BHK cells is indistinguishable from the corresponding glycopeptides derived from virus grown in chicken cells. Glycopeptide S4 is also very similar in size, composition, and sugar linkages from virus derived from the two hosts. These results suggest that chicken cells and BHK cells have similar glycosylation mechanisms and glycosylate Sindbis glycoprotein E2 in nearly identical ways. PMID:430605

  19. Electrophoretic separator for purifying biologicals, part 1

    NASA Technical Reports Server (NTRS)

    Mccreight, L. R.

    1978-01-01

    A program to develop an engineering model of an electrophoretic separator for purifying biologicals is summarized. An extensive mathematical modeling study and numerous ground based tests were included. Focus was placed on developing an actual electrophoretic separator of the continuous flow type, configured and suitable for flight testing as a space processing applications rocket payload.

  20. Two systems developed for purifying inert atmospheres

    NASA Technical Reports Server (NTRS)

    Foster, M. S.; Johnson, C. E.; Kyle, M. L.

    1969-01-01

    Two systems, one for helium and one for argon, are used for purifying inert atmospheres. The helium system uses an activated charcoal bed at liquid nitrogen temperature to remove oxygen and nitrogen. The argon system uses heated titanium sponge to remove nitrogen and copper wool beds to remove oxygen. Both use molecular sieves to remove water vapor.

  1. Home Air Purifiers Eradicate Harmful Pathogens

    NASA Technical Reports Server (NTRS)

    2014-01-01

    Marshall Space Flight Center funded the University of Madison-Wisconsin to develop ethylene scrubbers to keep produce fresh in space. Akida Holdings of Jacksonville, Florida, licensed the technology and developed Airocide, an air purifier that can kill airborne pathogens. Previously designed for industrial spaces, there is now a specially designed unit for home use.

  2. Improvement of Linde Kryotechnik's internal purifier

    NASA Astrophysics Data System (ADS)

    Decker, Lutz; Meier, Albert; Wilhelm, Hanspeter

    2014-01-01

    With the recent shortage in supply of helium, recovery solutions have experienced a new focus with a tendency to recover streams with higher impurity content. This development calls for purifier systems operating efficiently and with low impact on liquefaction capacity for helium streams with impurity levels in the percentage range. Linde Kryotechnik has answered this demand by improving the performance of its purifier technology. Since 1983, its standardized helium liquefiers of the L- and former TCF-series type contain an internal purifier which already allows efficient impurity removal with minimized space demand. Along with a line dryer to absorb humidity, it is designed to remove air impurities up to 5 mol%. However, with increasing impurity level, liquefaction capacity reduced significantly being furthermore restricted to an upper level of approx. 180 l/h and continuous purification became limited in time. With the current redesign of this purifier, the impact on liquefaction capacity is now minimized without any limitation within the capacity range of the L-series plants. Continuous purification is hence ensured beyond previous maximum impurity content. This paper provides the key design changes and the achievable performance, which has been verified in the recent L-series plants delivered to customers.

  3. Method of purifying neutral organophosphorus extractants

    DOEpatents

    Horwitz, E. Philip; Gatrone, Ralph C.; Chiarizia, Renato

    1988-01-01

    A method for removing acidic contaminants from neutral mono and bifunctional organophosphorous extractants by contacting the extractant with a macroporous cation exchange resin in the H.sup.+ state followed by contact with a macroporous anion exchange resin in the OH.sup.- state, whereupon the resins take up the acidic contaminants from the extractant, purifying the extractant and improving its extraction capability.

  4. Paramyxovirus Glycoprotein Incorporation, Assembly and Budding: A Three Way Dance for Infectious Particle Production

    PubMed Central

    El Najjar, Farah; Schmitt, Anthony P.; Dutch, Rebecca Ellis

    2014-01-01

    Paramyxoviruses are a family of negative sense RNA viruses whose members cause serious diseases in humans, such as measles virus, mumps virus and respiratory syncytial virus; and in animals, such as Newcastle disease virus and rinderpest virus. Paramyxovirus particles form by assembly of the viral matrix protein, the ribonucleoprotein complex and the surface glycoproteins at the plasma membrane of infected cells and subsequent viral budding. Two major glycoproteins expressed on the viral envelope, the attachment protein and the fusion protein, promote attachment of the virus to host cells and subsequent virus-cell membrane fusion. Incorporation of the surface glycoproteins into infectious progeny particles requires coordinated interplay between the three viral structural components, driven primarily by the matrix protein. In this review, we discuss recent progress in understanding the contributions of the matrix protein and glycoproteins in driving paramyxovirus assembly and budding while focusing on the viral protein interactions underlying this process and the intracellular trafficking pathways for targeting viral components to assembly sites. Differences in the mechanisms of particle production among the different family members will be highlighted throughout. PMID:25105277

  5. Dynamic Viral Glycoprotein Machines: Approaches for Probing Transient States That Drive Membrane Fusion

    PubMed Central

    Garcia, Natalie K.; Lee, Kelly K.

    2016-01-01

    The fusion glycoproteins that decorate the surface of enveloped viruses undergo dramatic conformational changes in the course of engaging with target cells through receptor interactions and during cell entry. These refolding events ultimately drive the fusion of viral and cellular membranes leading to delivery of the genetic cargo. While well-established methods for structure determination such as X-ray crystallography have provided detailed structures of fusion proteins in the pre- and post-fusion fusion states, to understand mechanistically how these fusion glycoproteins perform their structural calisthenics and drive membrane fusion requires new analytical approaches that enable dynamic intermediate states to be probed. Methods including structural mass spectrometry, small-angle X-ray scattering, and electron microscopy have begun to provide new insight into pathways of conformational change and fusion protein function. In combination, the approaches provide a significantly richer portrait of viral fusion glycoprotein structural variation and fusion activation as well as inhibition by neutralizing agents. Here recent studies that highlight the utility of these complementary approaches will be reviewed with a focus on the well-characterized influenza virus hemagglutinin fusion glycoprotein system. PMID:26761026

  6. Dynamic Viral Glycoprotein Machines: Approaches for Probing Transient States That Drive Membrane Fusion.

    PubMed

    Garcia, Natalie K; Lee, Kelly K

    2016-01-11

    The fusion glycoproteins that decorate the surface of enveloped viruses undergo dramatic conformational changes in the course of engaging with target cells through receptor interactions and during cell entry. These refolding events ultimately drive the fusion of viral and cellular membranes leading to delivery of the genetic cargo. While well-established methods for structure determination such as X-ray crystallography have provided detailed structures of fusion proteins in the pre- and post-fusion fusion states, to understand mechanistically how these fusion glycoproteins perform their structural calisthenics and drive membrane fusion requires new analytical approaches that enable dynamic intermediate states to be probed. Methods including structural mass spectrometry, small-angle X-ray scattering, and electron microscopy have begun to provide new insight into pathways of conformational change and fusion protein function. In combination, the approaches provide a significantly richer portrait of viral fusion glycoprotein structural variation and fusion activation as well as inhibition by neutralizing agents. Here recent studies that highlight the utility of these complementary approaches will be reviewed with a focus on the well-characterized influenza virus hemagglutinin fusion glycoprotein system.

  7. Virion Glycoprotein-Mediated Immune Evasion by Human Cytomegalovirus: a Sticky Virus Makes a Slick Getaway.

    PubMed

    Gardner, Thomas J; Tortorella, Domenico

    2016-09-01

    The prototypic herpesvirus human cytomegalovirus (CMV) exhibits the extraordinary ability to establish latency and maintain a chronic infection throughout the life of its human host. This is even more remarkable considering the robust adaptive immune response elicited by infection and reactivation from latency. In addition to the ability of CMV to exist in a quiescent latent state, its persistence is enabled by a large repertoire of viral proteins that subvert immune defense mechanisms, such as NK cell activation and major histocompatibility complex antigen presentation, within the cell. However, dissemination outside the cell presents a unique existential challenge to the CMV virion, which is studded with antigenic glycoprotein complexes targeted by a potent neutralizing antibody response. The CMV virion envelope proteins, which are critical mediators of cell attachment and entry, possess various characteristics that can mitigate the humoral immune response and prevent viral clearance. Here we review the CMV glycoprotein complexes crucial for cell attachment and entry and propose inherent properties of these proteins involved in evading the CMV humoral immune response. These include viral glycoprotein polymorphism, epitope competition, Fc receptor-mediated endocytosis, glycan shielding, and cell-to-cell spread. The consequences of CMV virion glycoprotein-mediated immune evasion have a major impact on persistence of the virus in the population, and a comprehensive understanding of these evasion strategies will assist in designing effective CMV biologics and vaccines to limit CMV-associated disease. PMID:27307580

  8. The variable surface glycoproteins of Trypanosoma equiperdum are phosphorylated.

    PubMed Central

    Baltz, T; Giroud, C; Baltz, D; Duvillier, G; Degand, P; Demaille, J; Pautrizel, R

    1982-01-01

    The phosphoproteins from three Trypanosoma equiperdum variants were studied by labelling the parasites in vivo with 32P. Phosphoprotein analysis reveals the presence of a 58 000 mol. wt. phosphoprotein ( pp58 ) which is absent when live trypanosomes are pre-treated with proteinase K under conditions where only the surface coat containing the variable surface glycoprotein (VSG) is removed. Immunological and fingerprint analysis on labelled pp58 , purified from these variants by affinity chromatography on Concanavalin A-Sepharose, clearly identify this component as the VSG. Furthermore, the VSGs seem to be phosphorylated to the extent of 1 mol phosphate per mol glycoprotein. The phosphorylated region is located in the extreme C-terminal region representing approximately 10% of the total molecule. The phosphorylated residue is not an aliphatic or aromatic ester of serine, threonine, or tyrosine, nor an acyl phosphate involving an aspartyl or glutamyl residue, nor phosphohistidine. The evidence that VSGs are phosphorylated could have considerable implications for the transfer and function of these structures. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. PMID:6821334

  9. Envelope-receptor interactions in Nipah virus pathobiology.

    PubMed

    Lee, Benhur

    2007-04-01

    Nipah (NiV) and Hendra (HeV) viruses are members of the newly defined Henipavirus genus of the Paramyxoviridae. Nipah virus (NiV) is an emergent paramyxovirus that causes fatal encephalitis in up to 70% of infected patients, and there is increasing evidence of human-to-human transmission. NiV is designated a priority pathogen in the NIAID Biodefense Research Agenda, and could be a devastating agent of agrobioterrorism if used against the pig farming industry. Endothelial syncytium is a pathognomonic feature of NiV infections, and is mediated by the fusion (F) and attachment (G) envelope glycoproteins. This review summarizes what is known about the pathophysiology of NiV infections, and documents the identification of the NiV receptor. EphrinB2, the NiV and HeV receptor, is expressed on endothelial cells and neurons, consistent with the known cellular tropism for NiV. We discuss how the identification of the henipahvirus receptor sheds light on the pathobiology of NiV infection, and how it will spur the rational development of effective therapeutics. In addition, ephrinB3, a related protein, can serve as an alternative receptor, and we suggest that differential usage of ephrinB2 versus B3 may explain the variant pathogenic profiles observed between NiV and HeV. Thus, identifying the NiV receptors opens the door for a more comprehensive analysis of the envelope-receptor interactions in NiV pathobiology. Finally, we also describe how galectin-1 (an innate immune defense lectin) can interact with specific N-glycans on the Nipah envelope fusion protein, underscoring the potential role that innate immune defense mechanisms may play against emerging pathogens. PMID:17470911

  10. A Lectin Purified from Blood Red Bracket Mushroom, Pycnoporus sanguineus (Agaricomycetidae), Mycelium Displayed Affinity Toward Bovine Transferrin.

    PubMed

    Albores, Silvana; Moros, Maria; Cerdeiras, Maria Pia; de la Fuente, Jesus Martinez; Grazu, Valeria; Fraguas, Laura Franco

    2016-01-01

    Fungal lectins constitute excellent ligands for development of affinity adsorbents useful in affinity chromatography. In this work, a lectin was purified from Pycnoporus sanguineus (PSL) mycelium using 3 procedures: by affinity chromatography, using magnetic galactosyl-nanoparticles or galactose coupled to Sepharose, and by ionic exchange chromatography (IEC). The highest lectin yield was achieved by IEC (55%); SDS-PAGE of PSL showed 2 bands with molecular mass of 68.7 and 55.2 kDa and IEC displayed 2 bands at pi 5.5 and 5.2. The lectin agglutinates rat erythrocytes, exhibiting broad specificity toward several monosaccharides, including galactose. The agglutination was also inhibited by the glycoproteins fetal calf fetuin, bovine lactoferrin, bovine transferrin, and horseradish peroxidase. The lectin was then used to synthesize an affinity adsorbent (PSL-Sepharose) and the interaction with glycoproteins was evaluated by analyzing their chromatographic behaviors. The strongest interaction with the PSL-derivative was observed with transferrin, although lower interactions were also displayed toward fetuin and lactoferrin. These results indicate that the purified PSL constitutes an interesting ligand for the design of affinity adsorbents to be used (i.e., in glycoprotein purification). PMID:27279446

  11. Safeguards Envelope Progress FY08

    SciTech Connect

    Robert Bean; Richard Metcalf; Aaron Bevill

    2008-09-01

    The Safeguards Envelope Project met its milestones by creating a rudimentary safeguards envelope, proving the value of the approach on a small scale, and determining the most appropriate path forward. The Idaho Chemical Processing Plant’s large cache of reprocessing process monitoring data, dubbed UBER Data, was recovered and used in the analysis. A probabilistic Z test was used on a Markov Monte Carlo simulation of expected diversion data when compared with normal operating data. The data regarding a fully transient event in a tank was used to create a simple requirement, representative of a safeguards envelope, whose impact was a decrease in operating efficiency by 1.3% but an increase in material balance period of 26%. This approach is operator, state, and international safeguards friendly and should be applied to future reprocessing plants. Future requirements include tank-to-tank correlations in reprocessing facilities, detailed operations impact studies, simulation inclusion, automated optimization, advanced statistics analysis, and multi-attribute utility analysis.

  12. Transparent ceramic lamp envelope materials

    NASA Astrophysics Data System (ADS)

    Wei, G. C.

    2005-09-01

    Transparent ceramic materials with optical qualities comparable to single crystals of similar compositions have been developed in recent years, as a result of the improved understanding of powder-processing-fabrication- sintering-property inter-relationships. These high-temperature materials with a range of thermal and mechanical properties are candidate envelopes for focused-beam, short-arc lamps containing various fills operating at temperatures higher than quartz. This paper reviews the composition, structure and properties of transparent ceramic lamp envelope materials including sapphire, small-grained polycrystalline alumina, aluminium oxynitride, yttrium aluminate garnet, magnesium aluminate spinel and yttria-lanthana. A satisfactory thermal shock resistance is required for the ceramic tube to withstand the rapid heating and cooling cycles encountered in lamps. Thermophysical properties, along with the geometry, size and thickness of a transparent ceramic tube, are important parameters in the assessment of its resistance to fracture arising from thermal stresses in lamps during service. The corrosive nature of lamp-fill liquid and vapour at high temperatures requires that all lamp components be carefully chosen to meet the target life. The wide range of new transparent ceramics represents flexibility in pushing the limit of envelope materials for improved beamer lamps.

  13. Inhibition of the Hantavirus Fusion Process by Predicted Domain III and Stem Peptides from Glycoprotein Gc

    PubMed Central

    Barriga, Gonzalo P.; Villalón-Letelier, Fernando; Márquez, Chantal L.; Bignon, Eduardo A.; Acuña, Rodrigo; Ross, Breyan H.; Monasterio, Octavio; Mardones, Gonzalo A.; Vidal, Simon E.; Tischler, Nicole D.

    2016-01-01

    Hantaviruses can cause hantavirus pulmonary syndrome or hemorrhagic fever with renal syndrome in humans. To enter cells, hantaviruses fuse their envelope membrane with host cell membranes. Previously, we have shown that the Gc envelope glycoprotein is the viral fusion protein sharing characteristics with class II fusion proteins. The ectodomain of class II fusion proteins is composed of three domains connected by a stem region to a transmembrane anchor in the viral envelope. These fusion proteins can be inhibited through exogenous fusion protein fragments spanning domain III (DIII) and the stem region. Such fragments are thought to interact with the core of the fusion protein trimer during the transition from its pre-fusion to its post-fusion conformation. Based on our previous homology model structure for Gc from Andes hantavirus (ANDV), here we predicted and generated recombinant DIII and stem peptides to test whether these fragments inhibit hantavirus membrane fusion and cell entry. Recombinant ANDV DIII was soluble, presented disulfide bridges and beta-sheet secondary structure, supporting the in silico model. Using DIII and the C-terminal part of the stem region, the infection of cells by ANDV was blocked up to 60% when fusion of ANDV occurred within the endosomal route, and up to 95% when fusion occurred with the plasma membrane. Furthermore, the fragments impaired ANDV glycoprotein-mediated cell-cell fusion, and cross-inhibited the fusion mediated by the glycoproteins from Puumala virus (PUUV). The Gc fragments interfered in ANDV cell entry by preventing membrane hemifusion and pore formation, retaining Gc in a non-resistant homotrimer stage, as described for DIII and stem peptide inhibitors of class II fusion proteins. Collectively, our results demonstrate that hantavirus Gc shares not only structural, but also mechanistic similarity with class II viral fusion proteins, and will hopefully help in developing novel therapeutic strategies against hantaviruses

  14. A viral regulator of glycoprotein complexes contributes to human cytomegalovirus cell tropism.

    PubMed

    Li, Gang; Nguyen, Christopher C; Ryckman, Brent J; Britt, William J; Kamil, Jeremy P

    2015-04-01

    Viral glycoproteins mediate entry of enveloped viruses into cells and thus play crucial roles in infection. In herpesviruses, a complex of two viral glycoproteins, gH and gL (gH/gL), regulates membrane fusion events and influences virion cell tropism. Human cytomegalovirus (HCMV) gH/gL can be incorporated into two different protein complexes: a glycoprotein O (gO)-containing complex known as gH/gL/gO, and a complex containing UL128, UL130, and UL131 known as gH/gL/UL128-131. Variability in the relative abundance of the complexes in the virion envelope correlates with differences in cell tropism exhibited between strains of HCMV. Nonetheless, the mechanisms underlying such variability have remained unclear. We have identified a viral protein encoded by the UL148 ORF (UL148) that influences the ratio of gH/gL/gO to gH/gL/UL128-131 and the cell tropism of HCMV virions. A mutant disrupted for UL148 showed defects in gH/gL/gO maturation and enhanced infectivity for epithelial cells. Accordingly, reintroduction of UL148 into an HCMV strain that lacked the gene resulted in decreased levels of gH/gL/UL128-131 on virions and, correspondingly, decreased infectivity for epithelial cells. UL148 localized to the endoplasmic reticulum, but not to the cytoplasmic sites of virion envelopment. Coimmunoprecipitation results indicated that gH, gL, UL130, and UL131 associate with UL148, but that gO and UL128 do not. Taken together, the findings suggest that UL148 modulates HCMV tropism by regulating the composition of alternative gH/gL complexes.

  15. A viral regulator of glycoprotein complexes contributes to human cytomegalovirus cell tropism

    PubMed Central

    Li, Gang; Nguyen, Christopher C.; Ryckman, Brent J.; Britt, William J.; Kamil, Jeremy P.

    2015-01-01

    Viral glycoproteins mediate entry of enveloped viruses into cells and thus play crucial roles in infection. In herpesviruses, a complex of two viral glycoproteins, gH and gL (gH/gL), regulates membrane fusion events and influences virion cell tropism. Human cytomegalovirus (HCMV) gH/gL can be incorporated into two different protein complexes: a glycoprotein O (gO)-containing complex known as gH/gL/gO, and a complex containing UL128, UL130, and UL131 known as gH/gL/UL128-131. Variability in the relative abundance of the complexes in the virion envelope correlates with differences in cell tropism exhibited between strains of HCMV. Nonetheless, the mechanisms underlying such variability have remained unclear. We have identified a viral protein encoded by the UL148 ORF (UL148) that influences the ratio of gH/gL/gO to gH/gL/UL128-131 and the cell tropism of HCMV virions. A mutant disrupted for UL148 showed defects in gH/gL/gO maturation and enhanced infectivity for epithelial cells. Accordingly, reintroduction of UL148 into an HCMV strain that lacked the gene resulted in decreased levels of gH/gL/UL128-131 on virions and, correspondingly, decreased infectivity for epithelial cells. UL148 localized to the endoplasmic reticulum, but not to the cytoplasmic sites of virion envelopment. Coimmunoprecipitation results indicated that gH, gL, UL130, and UL131 associate with UL148, but that gO and UL128 do not. Taken together, the findings suggest that UL148 modulates HCMV tropism by regulating the composition of alternative gH/gL complexes. PMID:25831500

  16. Inhibition of the Hantavirus Fusion Process by Predicted Domain III and Stem Peptides from Glycoprotein Gc.

    PubMed

    Barriga, Gonzalo P; Villalón-Letelier, Fernando; Márquez, Chantal L; Bignon, Eduardo A; Acuña, Rodrigo; Ross, Breyan H; Monasterio, Octavio; Mardones, Gonzalo A; Vidal, Simon E; Tischler, Nicole D

    2016-07-01

    Hantaviruses can cause hantavirus pulmonary syndrome or hemorrhagic fever with renal syndrome in humans. To enter cells, hantaviruses fuse their envelope membrane with host cell membranes. Previously, we have shown that the Gc envelope glycoprotein is the viral fusion protein sharing characteristics with class II fusion proteins. The ectodomain of class II fusion proteins is composed of three domains connected by a stem region to a transmembrane anchor in the viral envelope. These fusion proteins can be inhibited through exogenous fusion protein fragments spanning domain III (DIII) and the stem region. Such fragments are thought to interact with the core of the fusion protein trimer during the transition from its pre-fusion to its post-fusion conformation. Based on our previous homology model structure for Gc from Andes hantavirus (ANDV), here we predicted and generated recombinant DIII and stem peptides to test whether these fragments inhibit hantavirus membrane fusion and cell entry. Recombinant ANDV DIII was soluble, presented disulfide bridges and beta-sheet secondary structure, supporting the in silico model. Using DIII and the C-terminal part of the stem region, the infection of cells by ANDV was blocked up to 60% when fusion of ANDV occurred within the endosomal route, and up to 95% when fusion occurred with the plasma membrane. Furthermore, the fragments impaired ANDV glycoprotein-mediated cell-cell fusion, and cross-inhibited the fusion mediated by the glycoproteins from Puumala virus (PUUV). The Gc fragments interfered in ANDV cell entry by preventing membrane hemifusion and pore formation, retaining Gc in a non-resistant homotrimer stage, as described for DIII and stem peptide inhibitors of class II fusion proteins. Collectively, our results demonstrate that hantavirus Gc shares not only structural, but also mechanistic similarity with class II viral fusion proteins, and will hopefully help in developing novel therapeutic strategies against hantaviruses

  17. Lipid rafts both in cellular membrane and viral envelope are critical for PRRSV efficient infection.

    PubMed

    Yang, Qian; Zhang, Qiong; Tang, Jun; Feng, Wen-Hai

    2015-10-01

    Porcine reproductive and respiratory syndrome virus (PRRSV) represents a significantly economical challenge to the swine industry worldwide. In this study, we investigated the importance of cellular and viral lipid rafts in PRRSV infection. First, we demonstrated that PRRSV glycoproteins, Gp3 and Gp4, were associated with lipid rafts during viral entry, and disruption of cellular lipid rafts inhibited PRRSV entry. We also showed the raft-location of CD163, which might contribute to the glycoproteins-raft association. Subsequently, raft disruption caused a significant reduction of viral RNA production. Moreover, Nsp9 was shown to be distributed in rafts, suggesting that rafts probably serve as a platform for PRRSV replication. Finally, we confirmed that disassembly of rafts on the virus envelope may affect the integrity of PRRSV particles and cause the leakage of viral proteins, which impaired PRRSV infectivity. These findings might provide insights on our understanding of the mechanism of PRRSV infection.

  18. Syncytial apoptosis signaling network induced by the HIV-1 envelope glycoprotein complex: an overview

    PubMed Central

    Nardacci, R; Perfettini, J-L; Grieco, L; Thieffry, D; Kroemer, G; Piacentini, M

    2015-01-01

    Infection by human immunodeficiency virus-1 (HIV-1) is associated with a progressive decrease in CD4 T-cell numbers and the consequent collapse of host immune defenses. The major pathogenic mechanism of AIDS is the massive apoptotic destruction of the immunocompetent cells, including uninfected cells. The latter process, also known as by-stander killing, operates by various mechanisms one of which involves the formation of syncytia which undergo cell death by following a complex pathway. We present here a detailed and curated map of the syncytial apoptosis signaling network, aimed at simplifying the whole mechanism that we have characterized at the molecular level in the last 15 years. The map was created using Systems Biology Graphical Notation language with the help of CellDesigner software and encompasses 36 components (proteins/genes) and 54 interactions. The simplification of this complex network paves the way for the development of novel therapeutic strategies to eradicate HIV-1 infection. Agents that induce the selective death of HIV-1-elicited syncytia might lead to the elimination of viral reservoirs and hence constitute an important complement to current antiretroviral therapies. PMID:26247731

  19. Supersite of immune vulnerability on the glycosylated face of HIV-1 envelope glycoprotein gp120

    PubMed Central

    Kong, Leopold; Lee, Jeong Hyun; Doores, Katie J.; Murin, Charles D.; Julien, Jean-Philippe; McBride, Ryan; Liu, Yan; Marozsan, Andre; Cupo, Albert; Klasse, Per-Johan; Hoffenberg, Simon; Caulfield, Michael; King, C. Richter; Hua, Yuanzi; Le, Khoa M.; Khayat, Reza; Deller, Marc C.; Clayton, Thomas; Tien, Henry; Feizi, Ten; Sanders, Rogier W.; Paulson, James C.; Moore, John P.; Stanfield, Robyn L.; Burton, Dennis R.; Ward, Andrew B.; Wilson, Ian A.

    2013-01-01

    A substantial fraction of broadly neutralizing antibodies (bnAbs) in certain HIV-infected donors recognizes glycan-dependent epitopes on HIV-1 gp120. Here, we elucidate how bnAb PGT 135 recognizes its Asn332 glycan-dependent epitope from its crystal structure with gp120, CD4 and Fab 17b at 3.1 Å resolution. PGT 135 interacts with glycans at Asn332, Asn392 and Asn386, using long CDR loops H1 and H3 to penetrate the glycan shield to access the gp120 protein surface. Electron microscopy reveals PGT 135 can accommodate the conformational and chemical diversity of gp120 glycans by altering its angle of engagement. The combined structural studies of PGT 135, PGT 128 and 2G12 show this Asn332-dependent epitope is highly accessible and much more extensive than initially appreciated, allowing for multiple binding modes and varied angles of approach, thereby representing a supersite of vulnerability for antibody neutralization. PMID:23708606

  20. Neutralizing activities of caprine antibodies towards conserved regions of the HCV envelope glycoprotein E2

    PubMed Central

    2011-01-01

    Anti HCV vaccine is not currently available and the present antiviral therapies fail to cure approximately half of the treated HCV patients. This study was designed to assess the immunogenic properties of genetically conserved peptides derived from the C-terminal region of HVR-1 and test their neutralizing activities in a step towards developing therapeutic and/or prophylactic immunogens against HCV infection. Antibodies were generated by vaccination of goats with synthetic peptides derived from HCV E2. Viral neutralizing capacity of the generated anti E2 antibodies was tested using in vitro assays. Goats immunized with E2 synthetic peptides termed p412 [a.a 412-419], p430 [a.a 430-447] and p517 [a.a 517-531] generated high titers of antibody responses 2 to 4.5 fold higher than comparable titers of antibodies to the same epitopes in chronic HCV patients. In post infection experiments of native HCV into cultured Huh7.5 cells anti p412 and anti p 517 were proven to be neutralizing to HCV genotype 4a from patients' sera (87.5% and 75% respectively). On the contrary anti p430 exhibited weak viral neutralization capacity on the same samples (31.25%). Furthermore Ab mixes containing anti p430 exhibited reduced viral neutralization properties. From these experiments one could predict that neutralization by Abs towards different E2-epitopes varies considerably and success in the enrichment of neutralization epitope-specific antibodies may be accompanied by favorable results in combating HCV infection. Also, E2 conserved peptides p517 and p412 represent potential components of a candidate peptide vaccine against HCV infection. PMID:21819575

  1. Nucleic acids encoding modified human immunodeficiency virus type 1 (HIV-1) group M consensus envelope glycoproteins

    DOEpatents

    Haynes, Barton F.; Gao, Feng; Korber, Bette T.; Hahn, Beatrice H.; Shaw, George M.; Kothe, Denise; Li, Ying Ying; Decker, Julie; Liao, Hua-Xin

    2011-12-06

    The present invention relates, in general, to an immunogen and, in particular, to an immunogen for inducing antibodies that neutralizes a wide spectrum of HIV primary isolates and/or to an immunogen that induces a T cell immune response. The invention also relates to a method of inducing anti-HIV antibodies, and/or to a method of inducing a T cell immune response, using such an immunogen. The invention further relates to nucleic acid sequences encoding the present immunogens.

  2. Excitation of gravity waves in common envelopes

    NASA Technical Reports Server (NTRS)

    Soker, Noam

    1992-01-01

    We study the excitation of gravity waves by a low-mass companion orbiting inside the envelope of a giant star, concentrating on brown dwarfs inside the envelope of asymptotic giant branch stars. Efficient g-wave excitations occur only after the brown dwarf has spiraled-in to the radiative zone, well inside the envelope, of the asymptotic giant branch star. The brown dwarf excites g-waves when its orbital radius is about 3-10 solar radii. At this stage of the evolution the envelope mass is below 0.1 solar mass. The g-waves propagate inward from the secondary orbit, carrying angular momentum and energy. We find that the angular momentum transport leads to an efficient spin-up of the inner envelopes. The differential rotation between the envelope and core and nonlinear wave effects, can cause a mixing of heavy elements from the core to the envelope.

  3. Incorporation of Hepatitis C Virus E1 and E2 Glycoproteins: The Keystones on a Peculiar Virion

    PubMed Central

    Vieyres, Gabrielle; Dubuisson, Jean; Pietschmann, Thomas

    2014-01-01

    Hepatitis C virus (HCV) encodes two envelope glycoproteins, E1 and E2. Their structure and mode of fusion remain unknown, and so does the virion architecture. The organization of the HCV envelope shell in particular is subject to discussion as it incorporates or associates with host-derived lipoproteins, to an extent that the biophysical properties of the virion resemble more very-low-density lipoproteins than of any virus known so far. The recent development of novel cell culture systems for HCV has provided new insights on the assembly of this atypical viral particle. Hence, the extensive E1E2 characterization accomplished for the last two decades in heterologous expression systems can now be brought into the context of a productive HCV infection. This review describes the biogenesis and maturation of HCV envelope glycoproteins, as well as the interplay between viral and host factors required for their incorporation in the viral envelope, in a way that allows efficient entry into target cells and evasion of the host immune response. PMID:24618856

  4. Microbiological testing of the Blairex Water Purifier.

    PubMed

    Meng, K E; Harris, M G

    1987-04-01

    The Blairex Water Purifier (previously called The Blairex Deionizer) is a filtration unit designed to purify tap water for uses that require distilled or deionized water. The unit is intended to offer soft contact lens wearers a more convenient and safe method of obtaining distilled water when using salt tablets or enzymatic cleaning tablets. In this study, the safety of these units was analyzed from a microbiological point of view. The microbial starting state of 18 factory sealed Blairex Water Purifiers was evaluated by filtering sterile water through each unit and enumerating the organisms in the effluent. Then a known number of specific microorganisms was filtered through each unit. For the next 30 days, subsequent sterile distilled water filtrations were done each day. The effluent was collected with each filtration and enumerated for microorganisms. The results indicated that the majority of Blairex units tested were not sterile from the onset. Several Blairex units evaluated did support bacterial growth, as the bacteria that were passed through the unit on day 1 of the study were found in the effluent in increasing numbers with use. The clinical implications of our findings are discussed. Each time Blairex units were obtained for evaluation, the units appeared different in either filter attachments, plastic composition, or shape. The results varied according to which type of Blairex unit was tested. PMID:3296767

  5. Steroidogenesis in amlodipine treated purified Leydig cells

    SciTech Connect

    Latif, Rabia; Lodhi, Ghulam Mustafa; Hameed, Waqas; Aslam, Muhammad

    2012-01-01

    Drugs have been shown to adversely affect male fertility and recently anti-hypertensive drugs were added to the list. The anti-fertility effects of amlodipine, a calcium channel blocker, are well-illustrated in in vivo experiments but lack an in vitro proof. The present study was designed to experimentally elucidate the effects of amlodipine on Leydig cell steroidogenesis and intracellular calcium in vitro. Leydig cells of Sprague–Dawley rats were isolated and purified by Percoll. Cells were incubated for 3 h with/without amlodipine in the presence/absence of LH, dbcAMP, Pregnenolone and 25-Hydroxycholesterol. Cytosolic calcium was measured in purified Leydig cells by fluorometric technique. The results showed significantly reduced (P < 0.05) steroidogenesis and intracellular calcium in amlodipine exposed rats. The site of amlodipine induced steroidogenic inhibition seems to be prior to the formation of Pregnenolone at the level of StAR protein. -- Highlights: ► Inhibition of steroidogenesis in isolated and purified Leydig cells by amlodipine. ► Site of inhibition was before Pregnenolone formation, at the level of StAR protein. ► Inhibition of LH stimulated rise in cytosolic calcium by amlodipine.

  6. Clinical applications of bacterial glycoproteins.

    PubMed

    Fulton, Kelly M; Smith, Jeffrey C; Twine, Susan M

    2016-01-01

    There is an ongoing race between bacterial evolution and medical advances. Pathogens have the advantages of short generation times and horizontal gene transfer that enable rapid adaptation to new host environments and therapeutics that currently outpaces clinical research. Antibiotic resistance, the growing impact of nosocomial infections, cancer-causing bacteria, the risk of zoonosis, and the possibility of biowarfare all emphasize the increasingly urgent need for medical research focussed on bacterial pathogens. Bacterial glycoproteins are promising targets for alternative therapeutic intervention since they are often surface exposed, involved in host-pathogen interactions, required for virulence, and contain distinctive glycan structures. The potential exists to exploit these unique structures to improve clinical prevention, diagnosis, and treatment strategies. Translation of the potential in this field to actual clinical impact is an exciting prospect for fighting infectious diseases. PMID:26971465

  7. A Strategy for O-Glycoproteomics of Enveloped Viruses—the O-Glycoproteome of Herpes Simplex Virus Type 1

    PubMed Central

    Bagdonaite, Ieva; Nordén, Rickard; Joshi, Hiren J.; Dabelsteen, Sally; Nyström, Kristina; Vakhrushev, Sergey Y.; Olofsson, Sigvard; Wandall, Hans H.

    2015-01-01

    Glycosylation of viral envelope proteins is important for infectivity and interaction with host immunity, however, our current knowledge of the functions of glycosylation is largely limited to N-glycosylation because it is difficult to predict and identify site-specific O-glycosylation. Here, we present a novel proteome-wide discovery strategy for O-glycosylation sites on viral envelope proteins using herpes simplex virus type 1 (HSV-1) as a model. We identified 74 O-linked glycosylation sites on 8 out of the 12 HSV-1 envelope proteins. Two of the identified glycosites found in glycoprotein B were previously implicated in virus attachment to immune cells. We show that HSV-1 infection distorts the secretory pathway and that infected cells accumulate glycoproteins with truncated O-glycans, nonetheless retaining the ability to elongate most of the surface glycans. With the use of precise gene editing, we further demonstrate that elongated O-glycans are essential for HSV-1 in human HaCaT keratinocytes, where HSV-1 produced markedly lower viral titers in HaCaT with abrogated O-glycans compared to the isogenic counterpart with normal O-glycans. The roles of O-linked glycosylation for viral entry, formation, secretion, and immune recognition are poorly understood, and the O-glycoproteomics strategy presented here now opens for unbiased discovery on all enveloped viruses. PMID:25830354

  8. Studies on a novel macrophage-specific calmodulin binding glycoprotein

    SciTech Connect

    Orlow, S.J.

    1986-01-01

    The murine macrophage-like cell line J774 and peritoneal exudate cells elicited with thioglycollate or starch contain a major calmodulin-binding protein which is absent in trifluoperazine-resistant variants of J774, resident peritoneal macrophages and these elicited with concanavalin A, lipopolysaccharide, proteose peptone or Bacillus Clamette Guerin. Resident murine peritoneal cells maintained in tissue culture for 3 days begin to accumulate this protein as do human peripheral blood monocytes after 7 days of culture. A specific competitive displacement radioimmunoassay was developed using a rabbit antiserum raised to the partially purified calmodulin binding protein and (/sup 125/I) calmodulin covalently crosslinked to the principal calmodulin binding protein in the preparation. The radioimmunoassay confirmed the unique cellular distribution of this protein suggesting that it may be a marker for certain stages of macrophage differentiation. Monoclonal antibodies were prepared and one of these was used to further purify the protein by immunoaffinity chromatography. A protein of molecular weight 50,000 to 60,000 was isolated. It could be selectively adsorbed to wheat germ agglutinin agarose and subsequently eluted with N-acetyl glucosamine. This property plus its sensitivity to endoglycosidase F led to the conclusion that it is a glycoprotein. The cellular distribution, subcellular localization and evidence of glycosylation suggest that this protein may be a macrophage-specific receptor with a high affinity for calcium-calmodulin.

  9. Targeting host-derived glycans on enveloped viruses for antibody-based vaccine design

    PubMed Central

    Crispin, Max; Doores, Katie J.

    2016-01-01

    The surface of enveloped viruses can be extensively glycosylated. Unlike the glycans coating pathogens such as bacteria and fungi, glycans on viruses are added and processed by the host-cell during biosynthesis. Glycoproteins are typically subjected to α-mannosidase processing and Golgi-mediated glycosyltransferase extension to form complex-type glycans. In envelope viruses, exceptions to this default pathway are common and lead to the presence of oligomannose-type glycan structures on the virion surface. In one extreme example, HIV-1 utilizes a high density of glycans to limit host antibody recognition of protein. However, the high density limits glycan processing and the resulting oligomannose structures can be recognised by broadly neutralising antibodies isolated form HIV-1 infected patients. Here we discuss how divergence from host-cell glycosylation can be targeted for vaccine design. PMID:25747313

  10. Crystal Structure of the Pre-fusion Nipah Virus Fusion Glycoprotein Reveals a Novel Hexamer-of-Trimers Assembly

    PubMed Central

    Dutta, Somnath; Yan, Lianying; Feng, YanRu; Wang, Lin-Fa; Skiniotis, Georgios; Lee, Benhur; Zhou, Z. Hong; Broder, Christopher C.; Aguilar, Hector C.; Nikolov, Dimitar B.

    2015-01-01

    Nipah virus (NiV) is a paramyxovirus that infects host cells through the coordinated efforts of two envelope glycoproteins. The G glycoprotein attaches to cell receptors, triggering the fusion (F) glycoprotein to execute membrane fusion. Here we report the first crystal structure of the pre-fusion form of the NiV-F glycoprotein ectodomain. Interestingly this structure also revealed a hexamer-of-trimers encircling a central axis. Electron tomography of Nipah virus-like particles supported the hexameric pre-fusion model, and biochemical analyses supported the hexamer-of-trimers F assembly in solution. Importantly, structure-assisted site-directed mutagenesis of the interfaces between F trimers highlighted the functional relevance of the hexameric assembly. Shown here, in both cell-cell fusion and virus-cell fusion systems, our results suggested that this hexamer-of-trimers assembly was important during fusion pore formation. We propose that this assembly would stabilize the pre-fusion F conformation prior to cell attachment and facilitate the coordinated transition to a post-fusion conformation of all six F trimers upon triggering of a single trimer. Together, our data reveal a novel and functional pre-fusion architecture of a paramyxoviral fusion glycoprotein. PMID:26646856

  11. Flexible Envelope Request Notation (FERN)

    NASA Technical Reports Server (NTRS)

    Zoch, David R.; Lavallee, David; Weinstein, Stuart

    1991-01-01

    The following topics are presented in view graph form and include the following: scheduling application; the motivation for the Flexible Envelope Request Notation (FERN); characteristics of FERN; types of information needed in requests; where information is stored in requests; FERN structures; generic requests; resource availability for pooled resources; expressive notation; temporal constraints; time formats; changes to FERN; sample FERN requests; the temporal relationship between two steps; maximum activity length to limit step delays; alternative requests; the temporal relationship between two activities; and idle resource usage between steps.

  12. Techniques and tactics used in determining the structure of the trimeric ebolavirus glycoprotein

    SciTech Connect

    Lee, Jeffrey E.; Fusco, Marnie L.; Abelson, Dafna M.; Hessell, Ann J.; Burton, Dennis R.; Saphire, Erica Ollmann

    2009-11-01

    Here, the techniques, tactics and strategies used to overcome a series of technical roadblocks in crystallization and phasing of the trimeric ebolavirus glycoprotein are described. The trimeric membrane-anchored ebolavirus envelope glycoprotein (GP) is responsible for viral attachment, fusion and entry. Knowledge of its structure is important both for understanding ebolavirus entry and for the development of medical interventions. Crystal structures of viral glycoproteins, especially those in their metastable prefusion oligomeric states, can be difficult to achieve given the challenges in production, purification, crystallization and diffraction that are inherent in the heavily glycosylated flexible nature of these types of proteins. The crystal structure of ebolavirus GP in its trimeric prefusion conformation in complex with a human antibody derived from a survivor of the 1995 Kikwit outbreak has now been determined [Lee et al. (2008 ▶), Nature (London), 454, 177–182]. Here, the techniques, tactics and strategies used to overcome a series of technical roadblocks in crystallization and phasing are described. Glycoproteins were produced in human embryonic kidney 293T cells, which allowed rapid screening of constructs and expression of protein in milligram quantities. Complexes of GP with an antibody fragment (Fab) promoted crystallization and a series of deglycosylation strategies, including sugar mutants, enzymatic deglycosylation, insect-cell expression and glycan anabolic pathway inhibitors, were attempted to improve the weakly diffracting glycoprotein crystals. The signal-to-noise ratio of the search model for molecular replacement was improved by determining the structure of the uncomplexed Fab. Phase combination with Fab model phases and a selenium anomalous signal, followed by NCS-averaged density modification, resulted in a clear interpretable electron-density map. Model building was assisted by the use of B-value-sharpened electron-density maps and the

  13. Analysis of a novel linkage unit of O-linked carbohydrates from the crystalline surface layer glycoprotein of Clostridium thermohydrosulfuricum S102-70.

    PubMed Central

    Messner, P; Christian, R; Kolbe, J; Schulz, G; Sleytr, U B

    1992-01-01

    The surface layer glycoprotein of Clostridium thermohydrosulfuricum S102-70 was shown to contain a new type of glycan chain. Different from all known eubacterial glycoproteins, the saccharide moiety consists only of six sugar residues without any repeat sequences. Proteolytic digestion of purified S-layer glycoprotein resulted in isolation of several glycopeptide fractions. These are composed of the same hexasaccharide portion but are linked to oligopeptides of different length. One of them contains only a single amino acid. As concluded from chemical analyses and proton and carbon nuclear magnetic resonance spectroscopy of this preparation, the hexasaccharide moiety is linked via a novel O-glycosidic linkage. This is a beta-D-glucose residue linked to the phenolic hydroxyl group of tyrosine in intact S-layer glycoprotein. Images PMID:1551844

  14. Crystal Structure of the Human Cytomegalovirus Glycoprotein B

    PubMed Central

    Burke, Heidi G.; Heldwein, Ekaterina E.

    2015-01-01

    Human cytomegalovirus (HCMV), a dsDNA, enveloped virus, is a ubiquitous pathogen that establishes lifelong latent infections and caused disease in persons with compromised immune systems, e.g., organ transplant recipients or AIDS patients. HCMV is also a leading cause of congenital viral infections in newborns. Entry of HCMV into cells requires the conserved glycoprotein B (gB), thought to function as a fusogen and reported to bind signaling receptors. gB also elicits a strong immune response in humans and induces the production of neutralizing antibodies although most anti-gB Abs are non-neutralizing. Here, we report the crystal structure of the HCMV gB ectodomain determined to 3.6-Å resolution, which is the first atomic-level structure of any betaherpesvirus glycoprotein. The structure of HCMV gB resembles the postfusion structures of HSV-1 and EBV homologs, establishing it as a new member of the class III viral fusogens. Despite structural similarities, each gB has a unique domain arrangement, demonstrating structural plasticity of gB that may accommodate virus-specific functional requirements. The structure illustrates how extensive glycosylation of the gB ectodomain influences antibody recognition. Antigenic sites that elicit neutralizing antibodies are more heavily glycosylated than those that elicit non-neutralizing antibodies, which suggest that HCMV gB uses glycans to shield neutralizing epitopes while exposing non-neutralizing epitopes. This glycosylation pattern may have evolved to direct the immune response towards generation of non-neutralizing antibodies thus helping HCMV to avoid clearance. HCMV gB structure provides a starting point for elucidation of its antigenic and immunogenic properties and aid in the design of recombinant vaccines and monoclonal antibody therapies. PMID:26484870

  15. Microvillar iron-binding glycoproteins isolated from the rabbit small intestine.

    PubMed Central

    O'Donnell, M W; Cox, T M

    1982-01-01

    Rabbit intestinal microvillus membranes possess high-affinity receptors for iron whose activity reflects homeostatic changes in mucosal iron transport. To isolate and characterize these membrane components, purified microvilli were radiolabelled with 59Fe(II) and solubilized in Triton X-100. 59Fe in 105000g supernatants co-eluted with a major broad protein peak (Mr approx. 100000) on gel-permeation chromatography and was rendered diffusible by Pronase digestion but not mild periodate degradation. Fluorescence studies with castor-bean lectin conjugates showed specific binding of this affinity probe exclusively to brush-border membranes in the intestinal epithelium. Affinity chromatography of solubilized membrane proteins showed binding to columns of immobilized lectin. Elution with D(+)-galactose released glycoprotein-bound 59Fe purified up to sevenfold over initial membrane extracts. The lectin bound up to 82% of protein-bound 59Fe. In contrast polyspecific antisera raised against rabbit microvilli in guinea-pigs precipitated less than 10% of solubilized radioactivity. Significantly more protein-bound 59Fe in detergent extracts of microvilli purified from bled animals interacted specifically with the lectin, suggesting that membrane glycoprotein receptors are involved in the homeostatic control of intestinal iron transport. Images PLATE 1 Fig. 5. Fig. 7. PMID:6282268

  16. Circumplanetary disc or circumplanetary envelope?

    NASA Astrophysics Data System (ADS)

    Szulágyi, J.; Masset, F.; Lega, E.; Crida, A.; Morbidelli, A.; Guillot, T.

    2016-08-01

    We present three-dimensional simulations with nested meshes of the dynamics of the gas around a Jupiter mass planet with the JUPITER and FARGOCA codes. We implemented a radiative transfer module into the JUPITER code to account for realistic heating and cooling of the gas. We focus on the circumplanetary gas flow, determining its characteristics at very high resolution (80 per cent of Jupiter's diameter). In our nominal simulation where the temperature evolves freely by the radiative module and reaches 13000 K at the planet, a circumplanetary envelope was formed filling the entire Roche lobe. Because of our equation of state is simplified and probably overestimates the temperature, we also performed simulations with limited maximal temperatures in the planet region (1000, 1500, and 2000 K). In these fixed temperature cases circumplanetary discs (CPDs) were formed. This suggests that the capability to form a CPD is not simply linked to the mass of the planet and its ability to open a gap. Instead, the gas temperature at the planet's location, which depends on its accretion history, plays also fundamental role. The CPDs in the simulations are hot and cooling very slowly, they have very steep temperature and density profiles, and are strongly sub-Keplerian. Moreover, the CPDs are fed by a strong vertical influx, which shocks on the CPD surfaces creating a hot and luminous shock-front. In contrast, the pressure supported circumplanetary envelope is characterized by internal convection and almost stalled rotation.

  17. Safeguards Envelope Progress FY10

    SciTech Connect

    Richard Metcalf

    2010-10-01

    The Safeguards Envelope is a strategy to determine a set of specific operating parameters within which nuclear facilities may operate to maximize safeguards effectiveness without sacrificing safety or plant efficiency. This paper details the additions to the advanced operating techniques that will be applied to real plant process monitoring (PM) data from the Idaho Chemical Processing Plant (ICPP). Research this year focused on combining disparate pieces of data together to maximize operating time with minimal downtime due to safeguards. A Chi-Square and Croiser's cumulative sum were both included as part of the new analysis. Because of a major issue with the original data, the implementation of the two new tests did not add to the existing set of tests, though limited one-variable optimization made a small increase in detection probability. Additional analysis was performed to determine if prior analysis would have caused a major security or safety operating envelope issue. It was determined that a safety issue would have resulted from the prior research, but that the security may have been increased under certain conditions.

  18. A glycoprotein from a folk medicinal plant, Withania somnifera, inhibits hyaluronidase activity of snake venoms.

    PubMed

    Machiah, Deepa K; Girish, K S; Gowda, T Veerabasappa

    2006-06-01

    Venom hyaluronidases help in rapid spreading of the toxins by destroying the integrity of the extra-cellular matrix of the tissues in the victims. A hyaluronidase inhibitor (WSG) is purified from a folk medicinal plant, Withania somnifera. The glycoprotein inhibited the hyaluronidase activity of cobra (Naja naja) and viper (Daboia russelii) venoms, which was demonstrated by zymogram assay and staining of the skin tissues for differential activity. WSG completely inhibited the activity of the enzyme at a concentration of 1:1 w/w of venom to WSG. Thus we are able to demonstrate that the glycoprotein inhibits hyaluronidase activity of the venoms. External application of the plant extract as an antidote in rural parts of India to snakebite victims appears to have a scientific basis.

  19. Air Purifiers Eliminate Pathogens, Preserve Food

    NASA Technical Reports Server (NTRS)

    2009-01-01

    NASA-funded researchers produced an ethylene reduction device for a plant growth unit. KES Science & Technology Inc., a Kennesaw, Georgia-based company specializing in sustaining perishable foods, licensed the ethylene scrubbing technology. KES partnered with Akida Holdings, of Jacksonville, Florida, which now markets the NASA-developed technology as AiroCide. According to the company, it is the only air purifier that completely destroys airborne bacteria, mold, fungi, mycotoxins, viruses, volatile organic compounds (like ethylene), and odors. What?s more, the devices have no filters that need changing and produce no harmful byproducts, such as the ozone created by some filtration systems.

  20. Immunocytochemical localization of water-soluble glycoproteins, including group 1 allergen, in pollen of ryegrass, Lolium perenne, using ferritin-labelled antibody.

    PubMed

    Vithanage, H I; Howlett, B J; Jobson, S; Knox, R B

    1982-11-01

    The cellular sites of the glycoproteins Group 1 allergen (glycoprotein 1) and Antigen A (glycoprotein 2) in mature ryegrass pollen have been investigated by immunoelectron microscopy. Radioimmunoassays confirm previous findings of cross-reactivity between the purified glycoprotein antigens at the high immunoglobulin G (IgG) concentrations used for localization. Freeze-drying of anthers followed by anhydrous processing has been employed because of the water solubility and mobility of the glycoproteins. A double-embedding technique has been developed. This involves, first, embedding anthers in the water-soluble plastic resin JB-4, sectioning and incubating in ferritin-labelled antisera by the indirect method. The sections are then embedded in Spurr's resin for ultra-thin sectioning. Both glycoproteins are found in the following sites: (1) exine and intine wall layers; (2) pollen cytoplasm; (3) the orbicules and anther loculus; and (4) the anther cuticle. In the exine arcades and surface and in the anther loculus, the ferritin label is bound to pollenkitt. The finding that the glycoproteins are in similar sites is predictable in view of the cross-specificity of the antisera. The extent of antibody penetration of the plastic sections has been examined; labelling is confined to cut grains and absent from intact grains.

  1. The nuclear envelope as a chromatin organizer

    PubMed Central

    Zuleger, Nikolaj; Robson, Michael I

    2011-01-01

    In the past 15 years our perception of nuclear envelope function has evolved perhaps nearly as much as the nuclear envelope itself evolved in the last 3 billion years. Historically viewed as little more than a diffusion barrier between the cytoplasm and the nucleoplasm, the nuclear envelope is now known to have roles in the cell cycle, cytoskeletal stability and cell migration, genome architecture, epigenetics, regulation of transcription, splicing and DNA replication. Here we will review both what is known and what is speculated about the role of the nuclear envelope in genome organization, particularly with respect to the positioning and repositioning of genes and chromosomes within the nucleus during differentiation. PMID:21970986

  2. Nonstationary envelope process and first excursion probability.

    NASA Technical Reports Server (NTRS)

    Yang, J.-N.

    1972-01-01

    The definition of stationary random envelope proposed by Cramer and Leadbetter, is extended to the envelope of nonstationary random process possessing evolutionary power spectral densities. The density function, the joint density function, the moment function, and the crossing rate of a level of the nonstationary envelope process are derived. Based on the envelope statistics, approximate solutions to the first excursion probability of nonstationary random processes are obtained. In particular, applications of the first excursion probability to the earthquake engineering problems are demonstrated in detail.

  3. Macaque Monoclonal Antibodies Targeting Novel Conserved Epitopes within Filovirus Glycoprotein

    PubMed Central

    Keck, Zhen-Yong; Enterlein, Sven G.; Howell, Katie A.; Vu, Hong; Shulenin, Sergey; Warfield, Kelly L.; Froude, Jeffrey W.; Araghi, Nazli; Douglas, Robin; Biggins, Julia; Lear-Rooney, Calli M.; Wirchnianski, Ariel S.; Lau, Patrick; Wang, Yong; Herbert, Andrew S.; Dye, John M.; Glass, Pamela J.; Holtsberg, Frederick W.; Foung, Steven K. H.

    2015-01-01

    . Since the nature of future outbreaks cannot be predicted, there is an urgent need for therapeutics with broad protective efficacy against multiple filoviruses. Here we describe a set of monoclonal antibodies cross-reactive with multiple filovirus species. These antibodies target novel conserved epitopes within the envelope glycoprotein and exhibit protective efficacy in mice. We further present novel concepts for combination of cross-reactive antibodies against multiple epitopes that show enhanced efficacy compared to monotherapy and provide complete protection in mice. These findings set the stage for further evaluation of these antibodies in nonhuman primates and development of effective pan-filovirus immunotherapeutics for use in future outbreaks. PMID:26468532

  4. [Biological role of heterogeneous glycoprotein structures].

    PubMed

    Jakab, Lajos

    2016-07-01

    Carbohydrate molecules connected mostly with covalent junctions to protein chains are called glycoproteins. These carbohydrate molecules are attached to the protein core in different qualities and order. When the protein core is connected with acidic components such as uronic acid or SO4 radicals, they are called proteoglycans. The currently used name "glycosaminoglycan" in this case is not entirely correct. In the living world polymannane structures occur, too. Glycoproteins do not only exceptionally hold acidic groups but they have neuraminic acid derivatives. Tissue, cellular and matrix structures, and mostly all serum "proteins" are mainly glycoproteins. In the everyday clinical practice glycoproteins are mentioned as proteins. Nevertheless, the inadequate use of the concept may cause errors in the attitudes, too. This paper aims to correct this notion, because the term of "glycobiology" has already been expanded to be an independent scientific field. The practical clinical consequences of recent knowledge in this field are also summarized including novel findings on glycoprotein structures and functions. The importance of the quantity of carbohydrates, and their structural arrangements are also presented. In short, significance of glycoprotein-carbohydrate structures, as well as their physiological and pathological roles are reviewed in order to introduce the field of "glycobiology". Orosomucoid and immunoglobulins are discussed separately. Orv. Hetil., 2016, 157(30), 1185-1192.

  5. Use of vesicular stomatitis virus pseudotypes bearing hantaan or seoul virus envelope proteins in a rapid and safe neutralization test.

    PubMed

    Ogino, Michiko; Ebihara, Hideki; Lee, Byoung-Hee; Araki, Koichi; Lundkvist, Ake; Kawaoka, Yoshihiro; Yoshimatsu, Kumiko; Arikawa, Jiro

    2003-01-01

    A vesicular stomatitis virus (VSV) pseudotype bearing hantavirus envelope glycoproteins was produced and used in a neutralization test as a substitute for native hantavirus. The recombinant VSV, in which the enveloped protein gene (G) was replaced by the green fluorescent protein gene and complemented with G protein expressed in trans (VSVDeltaG*G), was kindly provided by M. A. Whitt. 293T cells were transfected with plasmids for the expression of envelope glycoproteins (G1 and G2) of HTNV or SEOV and were then infected with VSVDeltaG*G. Pseudotype VSV with the Hantaan (VSVDeltaG*-HTN) or Seoul (VSVDeltaG*-SEO) envelope glycoproteins were harvested from the culture supernatant. The number of infectious units (IU) of the pseudotype VSVs ranged from 10(5) to 10(6)/ml. The infectivity of VSVDeltaG*-HTN and VSVDeltaG*-SEO was neutralized with monoclonal antibodies, immune rabbit sera, and sera from patients with hemorrhagic fever with renal syndrome, and the neutralizing titers were similar to those obtained with native hantaviruses. These results show that VSVDeltaG*-HTN and -SEO can be used as a rapid, specific, and safe neutralization test for detecting hantavirus-neutralizing antibodies as an effective substitute for the use of native hantaviruses. Furthermore, the IU of VSVDeltaG*-HTN and -SEO did not decrease by more than 10-fold when stored at 4 degrees C for up to 30 days. The stability of the pseudotype viruses allows distribution of the material to remote areas by using conventional cooling boxes for use as a diagnostic reagent.

  6. Effects of highly purified anthraquinoid compounds from Aloe vera on sensitive and multidrug resistant leukemia cells.

    PubMed

    Grimaudo, S; Tolomeo, M; Gancitano, R; Dalessandro, N; Aiello, E

    1997-01-01

    Folk medicine has attributed antitumor properties to preparations from Aloe vera. We have studied the effects of five purified compounds from the plant on human K562 leukemia and on its multidrug resistant (MDR) variant, K562/R. The glycosides aloin A and B, aloesin and aloeresin were devoid of antitumor activity up to 200 mu M concentrations. Only the aglycone aloe emodin produced reproducible antitumor effects, which, interestingly, were more pronounced in the MDR, P-glycoprotein overexpressing, cell line. Its IC50 was in fact 29 mu M in K562 and 10.5 mu M in K562/R. Aloe emodine caused mainly cytostasis and accumulation of the cells in the S and G(2)-M phases of the cell cycle during the first 48 h of treatment. Thereafter, massive cell death ensued. Research on the antitumor activity of compounds extracted from Aloe vera probably deserves continuation.

  7. Purification and characterization of novel whey glycoprotein WGP-88 which binds to a monoclonal antibody to PAS-4 glycoprotein in the bovine milk fat globule membrane.

    PubMed

    Hwangbo, S; Azuma, N; Kurisaki, J; Kanno, C

    1997-09-01

    A monoclonal antibody to the PAS-4 glycoprotein (78 kDa) of the bovine milk fat globule membrane (MFGM) specifically recognized PAS-4, and was named KAS4. A component recognized by KAS4 was found in whey protein, this being a glycoprotein of 88 kDa by SDS-PAGE and named WGP-88. WGP-88 was purified and characterized in comparison with PAS-4. WGP-88 had apparent pI values of 6.45 and 6.39, while those of PAS-4 were 7.39 and 7.35. Neuraminidase digestion shifted the pI values of WGP-88 to 6.57 and of PAS-4 to 7.52. WGP-88 was rich in polar amino residues (44.9 mol%), while PAS-4 was abundant in nonpolar amino acid residues (48.7 mol%). WGP-88 contained 17.1% of carbohydrate and PAS-4 had 7.2%. The results of reductive hydrolysis, N-glycanase digestion, and a lectin blot analysis suggested that N- and O-linked sugar chains were contained in both glycoproteins. WGP-88 and PAS-4 had a different N-terminal amino acid sequence. WGP-88 and PAS-4 respectively inhibited competitively the binding of KAS4 to PAS-4 and WGP-88. Our studies revealed WGP-88 recognized by KAS4 mAb to be a novel whey protein and to have different biochemical properties from those of PAS-4.

  8. Aberrant virion assembly and limited glycoprotein C production in varicella-zoster virus-infected neurons.

    PubMed

    Grose, Charles; Yu, Xiaoli; Cohrs, Randall J; Carpenter, John E; Bowlin, Jacqueline L; Gilden, Don

    2013-09-01

    Highly pure (>95%) terminally differentiated neurons derived from pluripotent stem cells appear healthy at 2 weeks after infection with varicella-zoster virus (VZV), and the cell culture medium contains no infectious virus. Analysis of the healthy-appearing neurons revealed VZV DNA, transcripts, and proteins corresponding to the VZV immediate early, early, and late kinetic phases of replication. Herein, we further characterized virus in these neuronal cells, focusing on (i) transcription and expression of late VZV glycoprotein C (gC) open reading frame 14 (ORF14) and (ii) ultrastructural features of virus particles in neurons. The analysis showed that gC was not expressed in most infected neurons and gC expression was markedly reduced in a minority of VZV-infected neurons. In contrast, expression of the early-late VZV gE glycoprotein (ORF68) was abundant. Transcript analysis also showed decreased gC transcription compared with gE. Examination of viral structure by high-resolution transmission electron microscopy revealed fewer viral particles than typically observed in cells productively infected with VZV. Furthermore, viral particles were more aberrant, in that most capsids in the nuclei lacked a dense core and most enveloped particles in the cytoplasm were light particles (envelopes without capsids). Together, these results suggest a considerable deficiency in late-phase replication and viral assembly during VZV infection of neurons in culture.

  9. The Role of Phlebovirus Glycoproteins in Viral Entry, Assembly and Release

    PubMed Central

    Spiegel, Martin; Plegge, Teresa; Pöhlmann, Stefan

    2016-01-01

    Bunyaviruses are enveloped viruses with a tripartite RNA genome that can pose a serious threat to animal and human health. Members of the Phlebovirus genus of the family Bunyaviridae are transmitted by mosquitos and ticks to humans and include highly pathogenic agents like Rift Valley fever virus (RVFV) and severe fever with thrombocytopenia syndrome virus (SFTSV) as well as viruses that do not cause disease in humans, like Uukuniemi virus (UUKV). Phleboviruses and other bunyaviruses use their envelope proteins, Gn and Gc, for entry into target cells and for assembly of progeny particles in infected cells. Thus, binding of Gn and Gc to cell surface factors promotes viral attachment and uptake into cells and exposure to endosomal low pH induces Gc-driven fusion of the viral and the vesicle membranes. Moreover, Gn and Gc facilitate virion incorporation of the viral genome via their intracellular domains and Gn and Gc interactions allow the formation of a highly ordered glycoprotein lattice on the virion surface. Studies conducted in the last decade provided important insights into the configuration of phlebovirus Gn and Gc proteins in the viral membrane, the cellular factors used by phleboviruses for entry and the mechanisms employed by phlebovirus Gc proteins for membrane fusion. Here, we will review our knowledge on the glycoprotein biogenesis and the role of Gn and Gc proteins in the phlebovirus replication cycle. PMID:27455305

  10. The Role of Phlebovirus Glycoproteins in Viral Entry, Assembly and Release.

    PubMed

    Spiegel, Martin; Plegge, Teresa; Pöhlmann, Stefan

    2016-01-01

    Bunyaviruses are enveloped viruses with a tripartite RNA genome that can pose a serious threat to animal and human health. Members of the Phlebovirus genus of the family Bunyaviridae are transmitted by mosquitos and ticks to humans and include highly pathogenic agents like Rift Valley fever virus (RVFV) and severe fever with thrombocytopenia syndrome virus (SFTSV) as well as viruses that do not cause disease in humans, like Uukuniemi virus (UUKV). Phleboviruses and other bunyaviruses use their envelope proteins, Gn and Gc, for entry into target cells and for assembly of progeny particles in infected cells. Thus, binding of Gn and Gc to cell surface factors promotes viral attachment and uptake into cells and exposure to endosomal low pH induces Gc-driven fusion of the viral and the vesicle membranes. Moreover, Gn and Gc facilitate virion incorporation of the viral genome via their intracellular domains and Gn and Gc interactions allow the formation of a highly ordered glycoprotein lattice on the virion surface. Studies conducted in the last decade provided important insights into the configuration of phlebovirus Gn and Gc proteins in the viral membrane, the cellular factors used by phleboviruses for entry and the mechanisms employed by phlebovirus Gc proteins for membrane fusion. Here, we will review our knowledge on the glycoprotein biogenesis and the role of Gn and Gc proteins in the phlebovirus replication cycle. PMID:27455305

  11. Cryo-electron microscopy of hepatitis B virions reveals variability in envelope capsid interactions.

    PubMed

    Seitz, Stefan; Urban, Stephan; Antoni, Christoph; Böttcher, Bettina

    2007-09-19

    Hepatitis B virus (HBV) is a major human pathogen causing about 750,000 deaths per year. The virion consists of a nucleocapsid and an envelope formed by lipids, and three integral membrane proteins. Although we have detailed structural insights into the organization of the HBV core, the arrangement of the envelope in virions and its interaction with the nucleocapsid is elusive. Here we show the ultrastructure of hepatitis B virions purified from patient serum. We identified two morphological phenotypes, which appear as compact and gapped particles with nucleocapsids in distinguishable conformations. The overall structures of these nucleocapsids resemble recombinant cores with two alpha-helical spikes per asymmetric unit. At the charged tips the spikes are contacted by defined protrusions of the envelope proteins, probably via electrostatic interactions. The HBV envelope in the two morphotypes is to some extent variable, but the surface proteins follow a general packing scheme with up to three surface protein dimers per asymmetric unit. The variability in the structure of the envelope indicates that the nucleocapsid does not firmly constrain the arrangement of the surface proteins, but provides a general template for the packing.

  12. Cryo-electron microscopy of hepatitis B virions reveals variability in envelope capsid interactions

    PubMed Central

    Seitz, Stefan; Urban, Stephan; Antoni, Christoph; Böttcher, Bettina

    2007-01-01

    Hepatitis B virus (HBV) is a major human pathogen causing about 750 000 deaths per year. The virion consists of a nucleocapsid and an envelope formed by lipids, and three integral membrane proteins. Although we have detailed structural insights into the organization of the HBV core, the arrangement of the envelope in virions and its interaction with the nucleocapsid is elusive. Here we show the ultrastructure of hepatitis B virions purified from patient serum. We identified two morphological phenotypes, which appear as compact and gapped particles with nucleocapsids in distinguishable conformations. The overall structures of these nucleocapsids resemble recombinant cores with two α-helical spikes per asymmetric unit. At the charged tips the spikes are contacted by defined protrusions of the envelope proteins, probably via electrostatic interactions. The HBV envelope in the two morphotypes is to some extent variable, but the surface proteins follow a general packing scheme with up to three surface protein dimers per asymmetric unit. The variability in the structure of the envelope indicates that the nucleocapsid does not firmly constrain the arrangement of the surface proteins, but provides a general template for the packing. PMID:17762862

  13. Chemical synthesis of intentionally misfolded homogeneous glycoprotein: a unique approach for the study of glycoprotein quality control.

    PubMed

    Izumi, Masayuki; Makimura, Yutaka; Dedola, Simone; Seko, Akira; Kanamori, Akiko; Sakono, Masafumi; Ito, Yukishige; Kajihara, Yasuhiro

    2012-05-01

    Biosynthesis of glycoproteins in the endoplasmic reticulum employs a quality control system, which discriminates and excludes misfolded malfunctional glycoproteins from a correctly folded one. As chemical tools to study the glycoprotein quality control system, we systematically synthesized misfolded homogeneous glycoproteins bearing a high-mannose type oligosaccharide via oxidative misfolding of a chemically synthesized homogeneous glycopeptide. The endoplasmic reticulum folding sensor enzyme, UDP-glucose:glycoprotein glucosyltransferase (UGGT), recognizes a specific folding intermediate, which exhibits a molten globule-like hydrophobic nature.

  14. Inhibition of Arenavirus Infection by a Glycoprotein-Derived Peptide with a Novel Mechanism

    PubMed Central

    Spence, Jennifer S.; Melnik, Lilia I.; Badani, Hussain; Wimley, William C.

    2014-01-01

    ABSTRACT The family Arenaviridae includes a number of viruses of public health importance, such as the category A hemorrhagic fever viruses Lassa virus, Junin virus, Machupo virus, Guanarito virus, and Sabia virus. Current chemotherapy for arenavirus infection is limited to the nucleoside analogue ribavirin, which is characterized by considerable toxicity and treatment failure. Using Pichinde virus as a model arenavirus, we attempted to design glycoprotein-derived fusion inhibitors similar to the FDA-approved anti-HIV peptide enfuvirtide. We have identified a GP2-derived peptide, AVP-p, with antiviral activity and no acute cytotoxicity. The 50% inhibitory dose (IC50) for the peptide is 7 μM, with complete inhibition of viral plaque formation at approximately 20 μM, and its antiviral activity is largely sequence dependent. AVP-p demonstrates activity against viruses with the Old and New World arenavirus viral glycoprotein complex but not against enveloped viruses of other families. Unexpectedly, fusion assays reveal that the peptide induces virus-liposome fusion at neutral pH and that the process is strictly glycoprotein mediated. As observed in cryo-electron micrographs, AVP-p treatment causes morphological changes consistent with fusion protein activation in virions, including the disappearance of prefusion glycoprotein spikes and increased particle diameters, and fluorescence microscopy shows reduced binding by peptide-treated virus. Steady-state fluorescence anisotropy measurements suggest that glycoproteins are destabilized by peptide-induced alterations in viral membrane order. We conclude that untimely deployment of fusion machinery by the peptide could render virions less able to engage in on-pathway receptor binding or endosomal fusion. AVP-p may represent a potent, highly specific, novel therapeutic strategy for arenavirus infection. IMPORTANCE Because the only drug available to combat infection by Lassa virus, a highly pathogenic arenavirus, is toxic

  15. Recombinant pestivirus E2 glycoproteins prevent viral attachment to permissive and non permissive cells with different efficiency.

    PubMed

    Asfor, A S; Wakeley, P R; Drew, T W; Paton, D J

    2014-08-30

    Bovine viral diarrhoea virus (BVDV) is an economically important animal pathogen, which like other pestiviruses has similar molecular biological features to hepaciviruses, including human Hepatitis C virus. The pestivirus E2 glycoproteins are the major target for virus-neutralising antibodies, as well as playing a role in receptor binding and host range restriction. In this study, recombinant E2 glycoproteins (rE2) derived from three different pestivirus species were examined for their inhibitory effects on pestivirus infectivity in cell culture. Histidine-tagged rE2 glycoproteins of BVDV type 2 strain 178003, BVDV type 1 strain Oregon C24V and CSFV strain Alfort 187 were produced in Spodoptera frugiperda insect cells and purified under native conditions. The ability of rE2 glycoprotein to inhibit the infection of permissive cells by both homologous and heterologous virus was compared, revealing that the inhibitory effects of rE2 glycoproteins correlated with the predicted similarity of the E2 structures in the recombinant protein and the test virus. This result suggests that the sequence and structure of E2 are likely to be involved in the host specificity of pestiviruses at their point of uptake into cells.

  16. Phosphorylation of varicella-zoster virus glycoprotein gpI by mammalian casein kinase II and casein kinase I

    SciTech Connect

    Grose, C.; Jackson, W. ); Traugh, J.A. )

    1989-09-01

    Varicella-zoster virus (VZV) glycoprotein gpI is the predominant viral glycoprotein within the plasma membranes of infected cells. This viral glycoprotein is phosphorylated on its polypeptide backbone during biosynthesis. In this report, the authors investigated the protein kinases which participate in the phosphorylation events. Under in vivo conditions, VZV gpI was phosphorylated on its serine and threonine residues by protein kinases present within lysates of either VZV-infected or uninfected cells. Because this activity was diminished by heparin, a known inhibitor of casein kinase II, isolated gpI was incubated with purified casein kinase II and shown to be phosphorylated in an in vitro assay containing ({gamma}-{sup 32}P)ATP. The same glycoprotein was phosphorylated when ({sup 32}P)GTP was substituted for ({sup 32}P)ATP in the protein kinase assay. They also tested whether VZV gpI was phosphorylated by two other ubiquitous mammalian protein kinases--casein kinase I and cyclic AMP-dependent kinase--and found that only casein kinase I modified gpI. When the predicted 623-amino-acid sequence of gpI was examined, two phosphorylation sites known to be optimal for casein kinase II were observed. In summary, this study showed that VZV gpI was phosphorylated by each of two mammalian protein kinases (casein kinase I and casein kinase II) and that potential serine-threonine phosphorylation sites for each of these two kinases were present in the viral glycoprotein.

  17. Kinetic Validation of the Models for P-Glycoprotein ATP Hydrolysis and Vanadate-Induced Trapping. Proposal for Additional Steps

    PubMed Central

    Lugo, Miguel Ramón; Sharom, Frances Jane

    2014-01-01

    P-Glycoprotein, a member of the ATP-binding cassette (ABC) superfamily, is a multidrug transporter responsible for cellular efflux of hundreds of structurally unrelated compounds, including natural products, many clinically used drugs and anti-cancer agents. Expression of P-glycoprotein has been linked to multidrug resistance in human cancers. ABC transporters are driven by ATP hydrolysis at their two cytoplasmic nucleotide-binding domains, which interact to form a closed ATP-bound sandwich dimer. Intimate knowledge of the catalytic cycle of these proteins is clearly essential for understanding their mechanism of action. P-Glycoprotein has been proposed to hydrolyse ATP by an alternating mechanism, for which there is substantial experimental evidence, including inhibition of catalytic activity by trapping of ortho-vanadate at one nucleotide-binding domain, and the observation of an asymmetric occluded state. Despite many studies of P-glycoprotein ATPase activity over the past 20 years, no comprehensive kinetic analysis has yet been carried out, and some puzzling features of its behaviour remain unexplained. In this work, we have built several progressively more complex kinetic models, and then carried out simulations and detailed analysis, to test the validity of the proposed reaction pathway employed by P-glycoprotein for ATP hydrolysis. To establish kinetic parameters for the catalytic cycle, we made use of the large amount of published data on ATP hydrolysis by hamster P-glycoprotein, both purified and in membrane vesicles. The proposed kinetic scheme(s) include a high affinity priming reaction for binding of the first ATP molecule, and an independent pathway for ADP binding outside the main catalytic cycle. They can reproduce to varying degrees the observed behavior of the protein's ATPase activity and its inhibition by ortho-vanadate. The results provide new insights into the mode of action of P-glycoprotein, and some hypotheses about the nature of the

  18. Characterization of the oligosaccharide units of the bovine erythrocyte membrane glycoprotein.

    PubMed

    Emerson, W A; Kornfeld, S

    1976-04-20

    The major glycoprotein of the bovine erythrocyte membrane was purified by extraction of the ghosts with lithium 3,5-diiodosalicylate followed by phenol-water extraction and acidification. The glycoprotein contains 20% protein and 80% carbohydrate by weight and gives a single band on sodium dodecyl sulfate-polyacrylamide gels with an estimated molecular weight of 230000 daltons. The carbohydrate composition of the glycoprotein was determined to be (in residues relative to sialic acid): sialic acid, 1.0; fucose, less than 0.01; mannose, 0.1; galactose, 3.3; N-acetylgalactosamine, 0.9; and N-acetylglucosamine, 2.4. Pronase digestion of the isolated glycoprotein followed by Sephadex G-75 gel filtration resulted in the separation of a small pool of glycopeptides (pool III), which included all of the mannose-containing glycopeptides, from the bulk of the glycopeptide material which was in the void fractions of the column (pool I). Alkaline borohydride treatment released over 95% of the oligosaccharide units in pool I and approximately 30% of the oligosaccharide units in pool III. These oligosaccharides were isolated by gel filtration and ion-exchange chromatography. The oligosaccharides released from pool I had molecular weights of 1100-1400 daltons and contained sialic acid, galactose, and N-acetylglucosamine in molar ratios of 0.5-1:3:2 as well as a partial residue of N-acetylgalactosaminitol. The oligosaccharides released from pool III by alkali had molecular weights of 1300-1600 daltons and contained sialic acid, galactose, N-acetylglucosamine, N-acetylgalactosamine and N-ACETYLgalactosaminitol in molar ratios of 1-2:2:1:1:1. These data indicate that the majority of the oligosaccharide units of the bovine erythrocyte glycoprotein are linked O-glycosidically to the peptide backbone of the molecule. PMID:1268191

  19. Glycosylation of dengue virus glycoproteins and their interactions with carbohydrate receptors: possible targets for antiviral therapy.

    PubMed

    Idris, Fakhriedzwan; Muharram, Siti Hanna; Diah, Suwarni

    2016-07-01

    Dengue virus, an RNA virus belonging to the genus Flavivirus, affects 50 million individuals annually, and approximately 500,000-1,000,000 of these infections lead to dengue hemorrhagic fever or dengue shock syndrome. With no licensed vaccine or specific antiviral treatments available to prevent dengue infection, dengue is considered a major public health problem in subtropical and tropical regions. The virus, like other enveloped viruses, uses the host's cellular enzymes to synthesize its structural (C, E, and prM/M) and nonstructural proteins (NS1-5) and, subsequently, to glycosylate these proteins to produce complete and functional glycoproteins. The structural glycoproteins, specifically the E protein, are known to interact with the host's carbohydrate receptors through the viral proteins' N-glycosylation sites and thus mediate the viral invasion of cells. This review focuses on the involvement of dengue glycoproteins in the course of infection and the virus' exploitation of the host's glycans, especially the interactions between host receptors and carbohydrate moieties. We also discuss the recent developments in antiviral therapies that target these processes and interactions, focusing specifically on the use of carbohydrate-binding agents derived from plants, commonly known as lectins, to inhibit the progression of infection. PMID:27068162

  20. Direct Involvement of HERV-W Env Glycoprotein in Human Trophoblast Cell Fusion and Differentiation

    PubMed Central

    Frendo, Jean-Louis; Olivier, Delphine; Cheynet, Valérie; Blond, Jean-Luc; Bouton, Olivier; Vidaud, Michel; Rabreau, Michèle; Evain-Brion, Danièle; Mallet, François

    2003-01-01

    We recently demonstrated that the product of the HERV-W env gene, a retroviral envelope protein also dubbed syncytin, is a highly fusogenic membrane glycoprotein inducing the formation of syncytia on interaction with the type D mammalian retrovirus receptor. In addition, the detection of HERV-W Env protein (Env-W) expression in placental tissue sections led us to propose a role for this fusogenic glycoprotein in placenta formation. To evaluate this hypothesis, we analyzed the involvement of Env-W in the differentiation of primary cultures of human villous cytotrophoblasts that spontaneously differentiate by cell fusion into syncytiotrophoblasts in vitro. First, we observed that HERV-W env mRNA and glycoprotein expression are colinear with primary cytotrophoblast differentiation and with expression of human chorionic gonadotropin (hCG), a marker of syncytiotrophoblast formation. Second, we observed that in vitro stimulation of trophoblast cell fusion and differentiation by cyclic AMP is also associated with a concomitant increase in HERV-W env and hCG mRNA and protein expression. Finally, by using specific antisense oligonucleotides, we demonstrated that inhibition of Env-W protein expression leads to a decrease of trophoblast fusion and differentiation, with the secretion of hCG in culture medium of antisense oligonucleotide-treated cells being decreased by fivefold. Taken together, these results strongly support a direct role for Env-W in human trophoblast cell fusion and differentiation. PMID:12724415

  1. Crystal Structure of Glycoprotein C from a Hantavirus in the Post-fusion Conformation

    PubMed Central

    Willensky, Shmuel; Bignon, Eduardo A.; Tischler, Nicole D.; Dessau, Moshe

    2016-01-01

    Hantaviruses are important emerging human pathogens and are the causative agents of serious diseases in humans with high mortality rates. Like other members in the Bunyaviridae family their M segment encodes two glycoproteins, GN and GC, which are responsible for the early events of infection. Hantaviruses deliver their tripartite genome into the cytoplasm by fusion of the viral and endosomal membranes in response to the reduced pH of the endosome. Unlike phleboviruses (e.g. Rift valley fever virus), that have an icosahedral glycoprotein envelope, hantaviruses display a pleomorphic virion morphology as GN and GC assemble into spikes with apparent four-fold symmetry organized in a grid-like pattern on the viral membrane. Here we present the crystal structure of glycoprotein C (GC) from Puumala virus (PUUV), a representative member of the Hantavirus genus. The crystal structure shows GC as the membrane fusion effector of PUUV and it presents a class II membrane fusion protein fold. Furthermore, GC was crystallized in its post-fusion trimeric conformation that until now had been observed only in Flavi- and Togaviridae family members. The PUUV GC structure together with our functional data provides intriguing evolutionary and mechanistic insights into class II membrane fusion proteins and reveals new targets for membrane fusion inhibitors against these important pathogens. PMID:27783673

  2. Forcible destruction of severely misfolded mammalian glycoproteins by the non-glycoprotein ERAD pathway.

    PubMed

    Ninagawa, Satoshi; Okada, Tetsuya; Sumitomo, Yoshiki; Horimoto, Satoshi; Sugimoto, Takehiro; Ishikawa, Tokiro; Takeda, Shunichi; Yamamoto, Takashi; Suzuki, Tadashi; Kamiya, Yukiko; Kato, Koichi; Mori, Kazutoshi

    2015-11-23

    Glycoproteins and non-glycoproteins possessing unfolded/misfolded parts in their luminal regions are cleared from the endoplasmic reticulum (ER) by ER-associated degradation (ERAD)-L with distinct mechanisms. Two-step mannose trimming from Man9GlcNAc2 is crucial in the ERAD-L of glycoproteins. We recently showed that this process is initiated by EDEM2 and completed by EDEM3/EDEM1. Here, we constructed chicken and human cells simultaneously deficient in EDEM1/2/3 and analyzed the fates of four ERAD-L substrates containing three potential N-glycosylation sites. We found that native but unstable or somewhat unfolded glycoproteins, such as ATF6α, ATF6α(C), CD3-δ-ΔTM, and EMC1, were stabilized in EDEM1/2/3 triple knockout cells. In marked contrast, degradation of severely misfolded glycoproteins, such as null Hong Kong (NHK) and deletion or insertion mutants of ATF6α(C), CD3-δ-ΔTM, and EMC1, was delayed only at early chase periods, but they were eventually degraded as in wild-type cells. Thus, higher eukaryotes are able to extract severely misfolded glycoproteins from glycoprotein ERAD and target them to the non-glycoprotein ERAD pathway to maintain the homeostasis of the ER.

  3. Forcible destruction of severely misfolded mammalian glycoproteins by the non-glycoprotein ERAD pathway

    PubMed Central

    Ninagawa, Satoshi; Okada, Tetsuya; Sumitomo, Yoshiki; Horimoto, Satoshi; Sugimoto, Takehiro; Ishikawa, Tokiro; Takeda, Shunichi; Yamamoto, Takashi; Suzuki, Tadashi; Kamiya, Yukiko

    2015-01-01

    Glycoproteins and non-glycoproteins possessing unfolded/misfolded parts in their luminal regions are cleared from the endoplasmic reticulum (ER) by ER-associated degradation (ERAD)-L with distinct mechanisms. Two-step mannose trimming from Man9GlcNAc2 is crucial in the ERAD-L of glycoproteins. We recently showed that this process is initiated by EDEM2 and completed by EDEM3/EDEM1. Here, we constructed chicken and human cells simultaneously deficient in EDEM1/2/3 and analyzed the fates of four ERAD-L substrates containing three potential N-glycosylation sites. We found that native but unstable or somewhat unfolded glycoproteins, such as ATF6α, ATF6α(C), CD3-δ–ΔTM, and EMC1, were stabilized in EDEM1/2/3 triple knockout cells. In marked contrast, degradation of severely misfolded glycoproteins, such as null Hong Kong (NHK) and deletion or insertion mutants of ATF6α(C), CD3-δ–ΔTM, and EMC1, was delayed only at early chase periods, but they were eventually degraded as in wild-type cells. Thus, higher eukaryotes are able to extract severely misfolded glycoproteins from glycoprotein ERAD and target them to the non-glycoprotein ERAD pathway to maintain the homeostasis of the ER. PMID:26572623

  4. African Swine Fever Virus Undergoes Outer Envelope Disruption, Capsid Disassembly and Inner Envelope Fusion before Core Release from Multivesicular Endosomes

    PubMed Central

    Hernáez, Bruno; Guerra, Milagros; Salas, María L.

    2016-01-01

    African swine fever virus (ASFV) is a nucleocytoplasmic large DNA virus (NCLDV) that causes a highly lethal disease in domestic pigs. As other NCLDVs, the extracellular form of ASFV possesses a multilayered structure consisting of a genome-containing nucleoid successively wrapped by a thick protein core shell, an inner lipid membrane, an icosahedral protein capsid and an outer lipid envelope. This structural complexity suggests an intricate mechanism of internalization in order to deliver the virus genome into the cytoplasm. By using flow cytometry in combination with pharmacological entry inhibitors, as well as fluorescence and electron microscopy approaches, we have dissected the entry and uncoating pathway used by ASFV to infect the macrophage, its natural host cell. We found that purified extracellular ASFV is internalized by both constitutive macropinocytosis and clathrin-mediated endocytosis. Once inside the cell, ASFV particles move from early endosomes or macropinosomes to late, multivesicular endosomes where they become uncoated. Virus uncoating requires acidic pH and involves the disruption of the outer membrane as well as of the protein capsid. As a consequence, the inner viral membrane becomes exposed and fuses with the limiting endosomal membrane to release the viral core into the cytosol. Interestingly, virus fusion is dependent on virus protein pE248R, a transmembrane polypeptide of the inner envelope that shares sequence similarity with some members of the poxviral entry/fusion complex. Collective evidence supports an entry model for ASFV that might also explain the uncoating of other multienveloped icosahedral NCLDVs. PMID:27110717

  5. Induction slag reduction process for purifying metals

    DOEpatents

    Traut, Davis E.; Fisher, II, George T.; Hansen, Dennis A.

    1991-01-01

    A continuous method is provided for purifying and recovering transition metals such as neodymium and zirconium that become reactive at temperatures above about 500.degree. C. that comprises the steps of contacting the metal ore with an appropriate fluorinating agent such as an alkaline earth metal fluosilicate to form a fluometallic compound, and reducing the fluometallic compound with a suitable alkaline earth or alkali metal compound under molten conditions, such as provided in an induction slag metal furnace. The method of the invention is advantageous in that it is simpler and less expensive than methods used previously to recover pure metals, and it may be employed with a wide range of transition metals that were reactive with enclosures used in the prior art methods and were hard to obtain in uncontaminated form.

  6. Subpopulations in purified platelets adhering on glass.

    PubMed

    Donati, Alessia; Gupta, Swati; Reviakine, Ilya

    2016-01-01

    Understanding how platelet activation is regulated is important in the context of cardiovascular disorders and their management with antiplatelet therapy. Recent evidence points to different platelet subpopulations performing different functions. In particular, procoagulant and aggregating subpopulations have been reported in the literature in platelets treated with the GPVI agonists. How the formation of platelet subpopulations upon activation is regulated remains unclear. Here, it is shown that procoagulant and aggregating platelet subpopulations arise spontaneously upon adhesion of purified platelets on clean glass surfaces. Calcium ionophore treatment of the adhering platelets resulted in one platelet population expressing both the procoagulant and the adherent population markers phosphatidylserine and the activated form of GPIIb/IIIa, while all of the platelets expressed CD62P independently of the ionophore treatment. Therefore, all platelets have the capacity to express all three activation markers. It is concluded that platelet subpopulations observed in various studies reflect the dynamics of the platelet activation process. PMID:27338300

  7. Apparatus and methods for purifying lead

    DOEpatents

    Tunison, Harmon M.

    2016-01-12

    Disclosed is an exemplary method of purifying lead which includes the steps of placing lead and a fluoride salt blend in a container; forming a first fluid of molten lead at a first temperature; forming a second fluid of the molten fluoride salt blend at a second temperature higher than the first temperature; mixing the first fluid and the second fluid together; separating the two fluids; solidifying the molten fluoride salt blend at a temperature above a melting point of the lead; and removing the molten lead from the container. In certain exemplary methods the molten lead is removed from the container by decanting. In still other exemplary methods the molten salt blend is a Lewis base fluoride eutectic salt blend, and in yet other exemplary methods the molten salt blend contains sodium fluoride, lithium fluoride, and potassium fluoride.

  8. Method of separating and purifying gadolinium-153

    DOEpatents

    Bray, Lane A [Richland, WA; Corneillie, Todd M [Davis, CA

    2001-01-01

    The present invention is an improvement to the method of separating and purifying gadolinium from a mixture of gadolinium and europium having the steps of (a) dissolving the mixture in an acid; (b) reducing europium+3 to europium+2; and (c) precipitating the europium+2 with a sulfate ion in a superstoichiometric amount; wherein the improvement is achieved by using one or more of the following: (i) the acid is an anoic acid; (ii) the reducing is with zinc metal in the absence of a second metal or with an amount of the second metal that is ineffective in the reducing; (iii) adding a group IIA element after step (c) for precipitating the excess sulfate prior to repeating step (c); (iv) the sulfate is a sulfate salt with a monovalent cation; (v) adding cold europium+3 prior to repeating step (c).

  9. Polycation-induced assembly of purified tubulin.

    PubMed Central

    Erickson, H P; Voter, W A

    1976-01-01

    Several different polycations have been found that can substitute for the microtubule-associated proteins, or tau factor, in facilitating assembly of tubulin that has been purified by ion exchange chromatography. In low concentrations of the polycation diethylaminoethyl-dextran, 7 mg of tubulin is pelleted per 1 mg of polycation added. Under conditions favorable to microtubule assembly the entire pellet is seen by electron microscopy to consist of "double wall microtubules", which are essentially identical to normal microtubules in subunit structure and arrangement. When assembly is inhibited approximately the same amount of tubulin is pelleted, but it is in the form of clusters of curved sheets or filaments apparently related to tubulin rings. When conditions are changed to favor assembly, the tubulin within these clusters appears to reassemble to form the double wall microtubules. Images PMID:1066692

  10. Isolating and Purifying Clostridium difficile Spores.

    PubMed

    Edwards, Adrianne N; McBride, Shonna M

    2016-01-01

    The ability for the obligate anaerobe, Clostridium difficile to form a metabolically dormant spore is critical for the survival of this organism outside of the host. This spore form is resistant to a myriad of environmental stresses, including heat, desiccation, and exposure to disinfectants and antimicrobials. These intrinsic properties of spores allow C. difficile to survive long-term in an oxygenated environment, to be easily transmitted from host-to-host, and to persist within the host following antibiotic treatment. Because of the importance of the spore form to the C. difficile life cycle and treatment and prevention of C. difficile infection (CDI), the isolation and purification of spores are necessary to study the mechanisms of sporulation and germination, investigate spore properties and resistances, and for use in animal models of CDI. Here we provide basic protocols, in vitro growth conditions, and additional considerations for purifying C. difficile spores for a variety of downstream applications. PMID:27507337

  11. Catalyst for purifying diesel engine exhaust gases

    SciTech Connect

    Saito, K.; Ueda, K.; Ikeda, Y.; Ono, T.

    1986-10-14

    This patent describes a catalyst for purifying a diesel engine exhaust gas. The catalyst comprises a refractory three-dimensional structure having a gas filter function, a porous inorganic carrier supported on it, and (a) vanadium oxide and (b) at least one metal selected from the group consisting of platinum, rhodium and palladium supported on the carrier. The amount of component (a) is in the range of 0.2 to 40.0 g as V/sub 2/O/sub 5/ per liter of the structure and the amount of component (b) is in the range of 0.1 to 4.0 g as metal liter of the structure, wherein the mole ratio of component (a) to component (b) deposited as 1-90:1.

  12. Fitter apparatus for purifying exhaust gases

    SciTech Connect

    Oyobe, K.; Fukutani, M.; Ito, K.; Matsui, K.; Miwa, N.; Nomura, E.

    1985-05-28

    Filter apparatus having a honeycomb structure and a heater for purifying exhaust gases consists of cells or passages defined by porous partition walls. Alternate passages are provided respectively with upstream plugs and downstream plugs. Particulates in exhaust gases are trapped in the honeycomb structure and ignited by the heater. The upstream plugs define spaces upstream thereof in which particulates are trapped in addition to those trapped in the passages having downstream plugs. The heat of burning of the particulates trapped in the spaces facilitates the burning of the particulates trapped in the passages having downstream plugs. Therefore, the exhaust gas particualtes trapped and collected in the downstream portion of the structure can also be easily burned to regenerate the structure over its entire length.

  13. Defining glycoprotein cancer biomarkers by MS in conjunction with glycoprotein enrichment.

    PubMed

    Song, Ehwang; Mechref, Yehia

    2015-01-01

    Protein glycosylation is an important and common post-translational modification. More than 50% of human proteins are believed to be glycosylated to modulate the functionality of proteins. Aberrant glycosylation has been correlated to several diseases, such as inflammatory skin diseases, diabetes mellitus, cardiovascular disorders, rheumatoid arthritis, Alzheimer's and prion diseases, and cancer. Many approved cancer biomarkers are glycoproteins which are not highly abundant proteins. Therefore, effective qualitative and quantitative assessment of glycoproteins entails enrichment methods. This chapter summarizes glycoprotein enrichment methods, including lectin affinity, immunoaffinity, hydrazide chemistry, hydrophilic interaction liquid chromatography, and click chemistry. The use of these enrichment approaches in assessing the qualitative and quantitative changes of glycoproteins in different types of cancers are presented and discussed. This chapter highlights the importance of glycoprotein enrichment techniques for the identification and characterization of new reliable cancer biomarkers.

  14. Defining glycoprotein cancer biomarkers by MS in conjunction with glycoprotein enrichment

    PubMed Central

    Song, Ehwang; Mechref, Yehia

    2016-01-01

    Protein glycosylation is an important and common post-translational modification. More than 50% of human proteins are believed to be glycosylated to modulate the functionality of proteins. Aberrant glycosylation has been correlated to several diseases, such as inflammatory skin diseases, diabetes mellitus, cardiovascular disorders, rheumatoid arthritis, Alzheimer’s and prion diseases, and cancer. Many approved cancer biomarkers are glycoproteins which are not highly abundant proteins. Therefore, effective qualitative and quantitative assessment of glycoproteins entails enrichment methods. This chapter summarizes glycoprotein enrichment methods, including lectin affinity, immunoaffinity, hydrazide chemistry, hydrophilic interaction liquid chromatography, and click chemistry. The use of these enrichment approaches in assessing the qualitative and quantitative changes of glycoproteins in different types of cancers are presented and discussed. This chapter highlights the importance of glycoprotein enrichment techniques for the identification and characterization of new reliable cancer biomarkers. PMID:26330015

  15. Defining glycoprotein cancer biomarkers by MS in conjunction with glycoprotein enrichment.

    PubMed

    Song, Ehwang; Mechref, Yehia

    2015-01-01

    Protein glycosylation is an important and common post-translational modification. More than 50% of human proteins are believed to be glycosylated to modulate the functionality of proteins. Aberrant glycosylation has been correlated to several diseases, such as inflammatory skin diseases, diabetes mellitus, cardiovascular disorders, rheumatoid arthritis, Alzheimer's and prion diseases, and cancer. Many approved cancer biomarkers are glycoproteins which are not highly abundant proteins. Therefore, effective qualitative and quantitative assessment of glycoproteins entails enrichment methods. This chapter summarizes glycoprotein enrichment methods, including lectin affinity, immunoaffinity, hydrazide chemistry, hydrophilic interaction liquid chromatography, and click chemistry. The use of these enrichment approaches in assessing the qualitative and quantitative changes of glycoproteins in different types of cancers are presented and discussed. This chapter highlights the importance of glycoprotein enrichment techniques for the identification and characterization of new reliable cancer biomarkers. PMID:26330015

  16. 21 CFR 880.6710 - Medical ultraviolet water purifier.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Medical ultraviolet water purifier. 880.6710... Miscellaneous Devices § 880.6710 Medical ultraviolet water purifier. (a) Identification. A medical ultraviolet water purifier is a device intended for medical purposes that is used to destroy bacteria in water...

  17. 21 CFR 880.6710 - Medical ultraviolet water purifier.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Medical ultraviolet water purifier. 880.6710... Miscellaneous Devices § 880.6710 Medical ultraviolet water purifier. (a) Identification. A medical ultraviolet water purifier is a device intended for medical purposes that is used to destroy bacteria in water...

  18. 21 CFR 880.6710 - Medical ultraviolet water purifier.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Medical ultraviolet water purifier. 880.6710... Miscellaneous Devices § 880.6710 Medical ultraviolet water purifier. (a) Identification. A medical ultraviolet water purifier is a device intended for medical purposes that is used to destroy bacteria in water...

  19. Purified oxygen scavenging cell membrane fragments and use of same

    SciTech Connect

    Jacobson, K.B.; Adler, H.I.

    1988-10-18

    A process for purifying oxygen scavenging cell membrane fragments (OSCMF) and the use of same are disclosed. The novel purifying process involves salt precipitation and molecular exclusion chromatography. The unique feature of purified OSCMF is its ability to remove oxygen from organic reaction media and organic preparations without contaminating them to any substantial degree. 1 ref., 2 figs.

  20. 21 CFR 880.6500 - Medical ultraviolet air purifier.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Medical ultraviolet air purifier. 880.6500 Section... Miscellaneous Devices § 880.6500 Medical ultraviolet air purifier. (a) Identification. A medical ultraviolet air purifier is a device intended for medical purposes that is used to destroy bacteria in the air by...

  1. 21 CFR 880.6710 - Medical ultraviolet water purifier.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Medical ultraviolet water purifier. 880.6710... Miscellaneous Devices § 880.6710 Medical ultraviolet water purifier. (a) Identification. A medical ultraviolet water purifier is a device intended for medical purposes that is used to destroy bacteria in water...

  2. Four glycoproteins are expressed in the cat zona pellucida.

    PubMed

    Stetson, I; Avilés, M; Moros, C; García-Vázquez, F A; Gimeno, L; Torrecillas, A; Aliaga, C; Bernardo-Pisa, M V; Ballesta, J; Izquierdo-Rico, M J

    2015-04-15

    The mammalian oocyte is surrounded by a matrix called the zona pellucida (ZP). This envelope participates in processes such as acrosome reaction induction, sperm binding and may be involved in speciation. In cat (Felis catus), this matrix is composed of at least three glycoproteins called ZP2, ZP3, and ZP4. However, recent studies have pointed to the presence of a fourth protein in several mammals (rat, human, hamster or rabbit), meaning that a reevaluation of cat ZP is needed. For this reason, the objective of this research was to analyze the protein composition of cat ZP by means of proteomic analysis. Using ZP from ovaries and oocytes, several peptides corresponding to four proteins were detected, yielding a coverage of 33.17%, 71.50%, 50.23%, and 49.64% for ZP1, ZP2, ZP3, and ZP4, respectively. Moreover, the expression of four genes was confirmed by molecular analysis. Using total RNA isolated from cat ovaries, the complementary deoxyribonucleic acids encoding cat ZP were partially amplified by reverse-transcribed polymerase chain reaction. Furthermore, ZP1 was totally amplified for the first time in this species. As far as we are aware, this is the first study that confirms the presence of four proteins in cat ZP.

  3. Use of lambdagt11 to isolate genes for two pseudorabies virus glycoproteins with homology to herpes simplex virus and varicella-zoster virus glycoproteins

    SciTech Connect

    Petrovskis, E.A.; Timmins, J.G.; Post, L.E.

    1986-10-01

    A library of pseudorabies virus (PRV) DNA fragments was constructed in the expression cloning vector lambdagt11. The library was screened with antisera which reacted with mixtures of PRV proteins to isolate recombinant bacteriophages expressing PRV proteins. By the nature of the lambdagt11 vector, the cloned proteins were expressed in Escherichia coli as ..beta..-galactosidase fusion proteins. The fusion proteins from 35 of these phages were purified and injected into mice to raise antisera. The antisera were screened by several different assays, including immunoprecipitation of (/sup 14/C)glucosamine-labeled PRV proteins. This method identified phages expressing three different PRV glycoproteins: the secreted glycoprotein, gX; gI; and a glycoprotein that had not been previously identified, which we designate gp63. The gp63 and gI genes map adjacent to each other in the small unique region of the PRV genome. The DNA sequence was determined for the region of the genome encoding gp63 and gI. It was found that gp63 has a region of homology with a herpes simplex virus type 1 (HSV-1) protein, encoded by US7, and also with varicella-zoster virus (VZV) gpIV. The gI protein sequence has a region of homology with HSV-1 gE and VZV gpI. It is concluded that PRV, HSV, and VZV all have a cluster of homologous glycoprotein genes in the small unique components of their genomes and that the organization of these genes is conserved.

  4. Sturgeon hatching enzyme and the mechanism of egg envelope digestion: Insight into changes in the mechanism of egg envelope digestion during the evolution of ray-finned fish.

    PubMed

    Nagasawa, Tatsuki; Kawaguchi, Mari; Sano, Kaori; Yasumasu, Shigeki

    2015-12-01

    We investigated the evolution of the hatching enzyme gene using bester sturgeon (hybrid of Acipencer ruthenus and Huso huso), a basal member of ray-finned fishes. We purified the bester hatching enzyme from hatching liquid, yielding a single band on SDS-PAGE, then isolated its cDNA from embryos by PCR. The sturgeon hatching enzyme consists of an astacin family protease domain and a CUB domain. The CUB domains are present in frog and bird hatching enzymes, but not in teleostei, suggesting that the domain structure of sturgeon hatching enzyme is the tetrapod type. The purified hatching enzyme swelled the egg envelope, and selectively cleaved one of five egg envelope proteins, ZPAX. Xenopus hatching enzyme preferentially digests ZPAX, thus, the egg envelope digestion process is conserved between amphibians and basal ray-finned fish. Teleostei hatching enzymes cleave the repeat sequences at the N-terminal region of ZPB and ZPC, suggesting that the targets of the teleostei hatching enzymes differ from those of amphibians and sturgeons. Such repeat sequences were not found in the N-terminal region of ZPB and ZPC of amphibians and sturgeons. Our results suggest that the change in substrates of the hatching enzymes was accompanied by the mutation of the amino acid sequence of N-terminal regions of ZPB and ZPC. We conclude that the changes in the mechanism of egg envelope digestion, including the change in the domain structure of the hatching enzymes and the switch in substrate, occurred during the evolution of teleostei, likely triggered by the teleost-specific third whole genome duplication. J. Exp. Zool. (Mol. Dev. Evol.) 324B: 720-732, 2015. © 2015 Wiley Periodicals, Inc.

  5. Enhanced antagonism of BST-2 by a neurovirulent SIV envelope

    PubMed Central

    Matsuda, Kenta; Chen, Chia-Yen; Whitted, Sonya; Chertova, Elena; Roser, David J.; Wu, Fan; Plishka, Ronald J.; Ourmanov, Ilnour; Buckler-White, Alicia; Lifson, Jeffrey D.; Strebel, Klaus; Hirsch, Vanessa M.

    2016-01-01

    Current antiretroviral therapy (ART) is not sufficient to completely suppress disease progression in the CNS, as indicated by the rising incidence of HIV-1–associated neurocognitive disorders (HAND) among infected individuals on ART. It is not clear why some HIV-1–infected patients develop HAND, despite effective repression of viral replication in the circulation. SIV-infected nonhuman primate models are widely used to dissect the mechanisms of viral pathogenesis in the CNS. Here, we identified 4 amino acid substitutions in the cytoplasmic tail of viral envelope glycoprotein gp41 of the neurovirulent virus SIVsm804E that enhance replication in macrophages and associate with enhanced antagonism of the host restriction factor BM stromal cell antigen 2 (BST-2). Rhesus macaques were inoculated with a variant of the parental virus SIVsmE543-3 that had been engineered to contain the 4 amino acid substitutions present in gp41 of SIVsm804E. Compared with WT virus–infected controls, animals infected with mutant virus exhibited higher viral load in cerebrospinal fluid. Together, these results are consistent with a potential role for BST-2 in the CNS microenvironment and suggest that BST-2 antagonists may serve as a possible target for countermeasures against HAND. PMID:27159392

  6. Structural constraints determine the glycosylation of HIV-1 envelope trimers

    PubMed Central

    Pritchard, Laura K.; Vasiljevic, Snezana; Ozorowski, Gabriel; Seabright, Gemma E.; Cupo, Albert; Ringe, Rajesh; Kim, Helen J.; Sanders, Rogier W.; Doores, Katie J.; Burton, Dennis R.; Wilson, Ian A.; Ward, Andrew B.; Moore, John P.; Crispin, Max

    2015-01-01

    A highly glycosylated, trimeric envelope glycoprotein (Env) mediates HIV-1 cell entry. The high density and heterogeneity of the glycans shield Env from recognition by the immune system but, paradoxically, many potent broadly neutralizing antibodies (bNAbs) recognize epitopes involving this glycan shield. To better understand Env glycosylation and its role in bNAb recognition, we characterized a soluble, cleaved recombinant trimer (BG505 SOSIP.664) that is a close structural and antigenic mimic of native Env. Large, unprocessed oligomannose-type structures (Man8-9GlcNAc2) are notably prevalent on the gp120 components of the trimer, irrespective of the mammalian cell expression system or the bNAb used for affinity-purification. In contrast, gp41 subunits carry more highly processed glycans. The glycans on uncleaved, non-native oligomeric gp140 proteins are also highly processed. A homogeneous, oligomannose-dominated glycan profile is therefore a hallmark of a native Env conformation and a potential Achilles’ heel that can be exploited for bNAb recognition and vaccine design. PMID:26051934

  7. Cryo electron tomography of herpes simplex virus during axonal transport and secondary envelopment in primary neurons.

    PubMed

    Ibiricu, Iosune; Huiskonen, Juha T; Döhner, Katinka; Bradke, Frank; Sodeik, Beate; Grünewald, Kay

    2011-12-01

    During herpes simplex virus 1 (HSV1) egress in neurons, viral particles travel from the neuronal cell body along the axon towards the synapse. Whether HSV1 particles are transported as enveloped virions as proposed by the 'married' model or as non-enveloped capsids suggested by the 'separate' model is controversial. Specific viral proteins may form a recruitment platform for microtubule motors that catalyze such transport. However, their subviral location has remained elusive. Here we established a system to analyze herpesvirus egress by cryo electron tomography. At 16 h post infection, we observed intra-axonal transport of progeny HSV1 viral particles in dissociated hippocampal neurons by live-cell fluorescence microscopy. Cryo electron tomography of frozen-hydrated neurons revealed that most egressing capsids were transported independently of the viral envelope. Unexpectedly, we found not only DNA-containing capsids (cytosolic C-capsids), but also capsids lacking DNA (cytosolic A-/B-capsids) in mid-axon regions. Subvolume averaging revealed lower amounts of tegument on cytosolic A-/B-capsids than on C-capsids. Nevertheless, all capsid types underwent active axonal transport. Therefore, even few tegument proteins on the capsid vertices seemed to suffice for transport. Secondary envelopment of capsids was observed at axon terminals. On their luminal face, the enveloping vesicles were studded with typical glycoprotein-like spikes. Furthermore, we noted an accretion of tegument density at the concave cytosolic face of the vesicle membrane in close proximity to the capsids. Three-dimensional analysis revealed that these assembly sites lacked cytoskeletal elements, but that filamentous actin surrounded them and formed an assembly compartment. Our data support the 'separate model' for HSV1 egress, i.e. progeny herpes viruses being transported along axons as subassemblies and not as complete virions within transport vesicles. PMID:22194682

  8. Autoantibodies interacting with purified native thyrotropin receptor.

    PubMed

    Atger, M; Misrahi, M; Young, J; Jolivet, A; Orgiazzi, J; Schaison, G; Milgrom, E

    1999-11-01

    Native thyrotropin receptor (TSHR) was purified by immunoaffinity chromatography from membrane extracts of stably transfected L cells. An ELISA test was devised to study anti-TSHR autoantibodies directly. Comparison of native TSHR with bacterially expressed, denatured TSHR showed that the latter was not recognized by the autoantibodies, suggesting that they bind to conformational epitopes only present on the native receptor. The use of deglycosylated TSHR and of purified receptor ectodomain (alpha-subunit) showed that the autoantibodies recognized only the protein backbone moiety of the receptor and that their epitopes were localized entirely in its ectodomain. Autoantibodies were detected in 45 of 48 subjects with untreated Graves' disease and in 26 of 47 healthy volunteers. The affinity for the receptor was similar in the two groups (Kd = 0.25-1 x 10-10 M) and the autoantibodies belonged to the IgG class in all cases. Although the concentration of autoantibodies was higher in Graves' disease patients (3.50 +/- 0.36 mg.L-1) than in control subjects (1.76 +/- 0.21) (mean +/- SEM), there was an overlap between the groups. Receptor-stimulating autoantibodies (TSAb) were studied by measuring cAMP synthesis in stably transfected HEK 293 cells. Their characteristics (recognition of alpha-subunit, of deglycosylated TSHR, nonrecognition of bacterially expressed denatured receptor) were similar to those of the antibodies detected by the ELISA test. TSAb were only found in individuals with Graves' disease. The ELISA test measures total anti-TSHR antibodies, whereas the test using adenylate cyclase stimulation measures antibodies that recognize specific epitopes involved in receptor activation. Our observations thus disprove the hypothesis according to which Graves' disease is related to the appearance of anti-TSHR antibodies not present in normal subjects. Actually, anti-TSHR antibodies exist in many euthyroid subjects, in some cases even at concentrations higher than those

  9. Glycosylation of purified enveloped nucleocapsids of the granulosis virus infecting Plodia interpunctella as determined by lectin binding.

    PubMed

    Russell, D L; Consigli, R A

    1985-12-01

    Using various iodinated plant lectins and a sensitive in vitro lectin binding procedure in which the radiolabeled lectins are applied directly to viral proteins on nitrocellulose sheets transferred from sodium dodecyl sulfate-polyacrylamide gels (Bartles and Hubbard, 1984; Glass et al., 1981), the proteins of the granulosis virus infecting Plodia interpunctella were probed for carbohydrate moieties. Six proteins (Mr 39 700, 31 000, 29 900, 26 300, 17 800 and 12 600) could be detected using a probe for alpha-D-N-acetylgalactosamine. Three proteins were also detectable with the probe specific for alpha-L-fucose (Mr 44 900, 31 000 and 17 800). One protein (Mr 17 800) was detected with the probe for sialic acid.

  10. Use of a resin-bound synthetic peptide for identifying a neutralizing antigenic determinant associated with the human immunodeficiency virus envelope.

    PubMed

    Kennedy, R C; Dreesman, G R; Chanh, T C; Boswell, R N; Allan, J S; Lee, T H; Essex, M; Sparrow, J T; Ho, D D; Kanda, P

    1987-04-25

    A polyamide-based solid-phase support containing an acid-stable p-(oxymethyl)benzoic acid handle to anchor the COOH-terminal amino acid was utilized in the production of synthetic peptides analogous to amino acid sequences 503-532 from the human immunodeficiency virus (HIV) envelope glycoprotein. The resin-bound peptide was used to induce an antibody response to the native form of glycoprotein 120 in both rabbits and mice. This epitope was detected on the surface of HIV-infected cells and was capable of inducing an in vitro neutralizing HIV antibody response. In addition, sera from some individuals exposed to HIV react with this peptide bound to the resin in a solid-phase immunoassay. These data indicate that we have identified a neutralizing antigenic determinant present on the amino-terminal glycoprotein 120 subunits of HIV by utilizing resin-bound synthetic peptides.

  11. Safeguards Envelope Progress FY09

    SciTech Connect

    Richard Metcalf; Robert Bean

    2009-09-01

    The Safeguards Envelope is a strategy to determine a set of specific operating parameters which nuclear facilities may operate within to maximize safeguards effectiveness without sacrificing safety or plant efficiency. This paper details advanced statistical techniques will be applied to real plant process monitoring (PM) data from the Idaho Chemical Processing Plant (ICPP). As a result of the U.S. having no operating nuclear chemical reprocessing plants, there has been a strong interest in obtaining process monitoring data from the ICPP. The ICPP was shut down in 1996 and a recent effort has been made to retrieve the PM data from storage in a data mining effort. In a simulation based on this data, multi-tank and multi-attribute correlations were tested against synthetic diversion scenarios. Kernel regression smoothing was used to fit a curve to the historical data, and multivariable, residual analysis and cumulative sum techniques set parameters for operating conditions. Diversion scenarios were created and tested, showing improved results when compared with a previous study utilizing only one-variable Z- testing7.

  12. Personnel occupied woven envelope robot

    NASA Technical Reports Server (NTRS)

    Wessling, Francis; Teoh, William; Ziemke, M. Carl

    1988-01-01

    The Personnel Occupied Woven Envelope Robot (POWER) provides an alternative to extravehicular activity (EVA) of space suited astronauts and/or use of long slender manipulator arms such as are used in the Shuttle Remote Manipulator System. POWER provides the capability for a shirt sleeved astronaut to perform such work by entering a control pod through air locks at both ends of an inflated flexible bellows (access tunnel). The exoskeleton of the tunnel is a series of six degrees of freedom (Six-DOF) articulated links compressible to 1/6 of their fully extended length. The operator can maneuver the control pod to almost any location within about 50 m of the base attachment to the space station. POWER can be envisioned as a series of hollow Six-DOF manipulator segments or arms wherein each arm grasps the shoulder of the next arm. Inside the hollow arms ia a bellow-type access tunnel. The control pod is the fist of the series of linked hollow arms. The fingers of the fist are conventional manipulator arms under direct visual control of the nearby operator in the pod. The applications and progress to date of the POWER system is given.

  13. On the common envelope efficiency

    NASA Astrophysics Data System (ADS)

    Zuo, Zhao-Yu; Li, Xiang-Dong

    2014-08-01

    In this work, we try to use the apparent luminosity versus displacement (i.e. LX versus R) correlation of high-mass X-ray binaries (HMXBs) to constrain the common envelope (CE) efficiency αCE, which is a key parameter affecting the evolution of the binary orbit during the CE phase. The major updates that are crucial for the CE evolution include a variable λ parameter and a new CE criterion for Hertzsprung gap donor stars, both of which are recently developed. We find that, within the framework of the standard energy formula for CE and core definition at mass X = 10 per cent, a high value of αCE, i.e. around 0.8-1.0, is more preferable, while αCE < ˜0.4 likely can not reconstruct the observed LX versus R distribution. However, due to an ambiguous definition for the core boundary in the literature, the used λ here still carries almost two order of magnitude uncertainty, which may translate directly to the expected value of αCE. We present the detailed components of current HMXBs and their spatial offsets from star clusters, which may be further testified by future observations of HMXB populations in nearby star-forming galaxies.

  14. Traceless labeling of glycoproteins and its application to the study of glycoprotein-protein interactions.

    PubMed

    Yang, Yung-Lin; Lee, Yen-Pin; Yang, Yen-Ling; Lin, Po-Chiao

    2014-02-21

    A new chemical method for the traceless labeling of glycoproteins with synthetic boronic acid (BA)-tosyl probes was successfully developed. The BA moiety acts as an affinity head to direct the formation of a cyclic boronate diester with the diol groups of glycans. Following this step, the electrophilic tosyl group is displaced by an SN2 reaction with a nucleophilic residue of the boronated glycoprotein, and finally, a reporter group is tagged onto the glycoprotein via an ether linkage. In the presence of polyols, a competition reaction recovers the native glycan of the tagged glycoprotein, conserving its biological significance. The BA-tosyl probes were used successfully for the specific labeling of glycosylated fetuins in a mixed protein pool and from crude Escherichia coli (E. coli) lysate. Further, a BA-tosyl-functionalized glass slide was used to fabricate glycoprotein microarrays with highly conserved glycans. By interacting with various lectins (carbohydrate-binding proteins), such as Concanavalin A (Con A) and wheat germ agglutinin (WGA), the types of carbohydrates and specific linkages of glycoproteins (α or β) could be systematically monitored. It is believed that the newly developed method will greatly accelerate the understanding of glycoproteins.

  15. Mimicking respiratory phosphorylation using purified enzymes.

    PubMed

    von Ballmoos, Christoph; Biner, Olivier; Nilsson, Tobias; Brzezinski, Peter

    2016-04-01

    The enzymes of oxidative phosphorylation is a striking example of the functional association of multiple enzyme complexes, working together to form ATP from cellular reducing equivalents. These complexes, such as cytochrome c oxidase or the ATP synthase, are typically investigated individually and therefore, their functional interplay is not well understood. Here, we present methodology that allows the co-reconstitution of purified terminal oxidases and ATP synthases in synthetic liposomes. The enzymes are functionally coupled via proton translocation where upon addition of reducing equivalents the oxidase creates and maintains a transmembrane electrochemical proton gradient that energizes the synthesis of ATP by the F1F0 ATP synthase. The method has been tested with the ATP synthases from Escherichia coli and spinach chloroplasts, and with the quinol and cytochrome c oxidases from E. coli and Rhodobacter sphaeroides, respectively. Unlike in experiments with the ATP synthase reconstituted alone, the setup allows in vitro ATP synthesis under steady state conditions, with rates up to 90 ATP×s(-1)×enzyme(-1). We have also used the novel system to study the phenomenon of "mild uncoupling" as observed in mitochondria upon addition of low concentrations of ionophores (e.g. FCCP, SF6847) and the recoupling effect of 6-ketocholestanol. While we could reproduce the described effects, our data with the in vitro system does not support the idea of a direct interaction between a mitochondrial protein and the uncoupling agents as proposed earlier. PMID:26707617

  16. Aldosterone-induced glycoproteins: electrophysiological-biochemical correlation.

    PubMed

    Szerlip, H M; Weisberg, L; Geering, K; Rossier, B C; Cox, M

    1988-05-01

    Aldosterone induces the synthesis of a group of glycoproteins (GP65,70) in toad urinary bladders which are potential effectors of the natriferic action of this hormone. In the present study we have confirmed that aldosterone produces a two-phase electrophysiological response. During the early phase (less than 3 h) short-circuit current and transepithelial conductance increase in parallel, while during the late phase (greater than 3 h) short-circuit current continues to increase without any further change in conductance. By biosynthetically labeling aldosterone-treated toad bladders with [35S]methionine either during the early (h 0-2 or 1-3) or the late (h 4-6 or 7-9) phases of the natriferic response, we have demonstrated that GP65,70 is synthesized as a late effect of aldosterone. Since synthesis of GP65,70 occurs at a time when the electromotive force of the Na+ pump is increasing, and since GP65,70 biochemically resembles the beta subunit of Na+/K+-ATPase, studies were undertaken to examine whether GP65,70 is the beta subunit. Purified amphibian renal beta subunit was analyzed by two-dimensional polyacrylamide gel electrophoresis and was found to have an isoelectric point and Mr value similar to those of GP65,70. However, when nitrocellulose blots containing wheat germ agglutinin-purified proteins from aldosterone-treated bladders were stained with monospecific polyclonal antibodies developed against the beta subunit, GP65,70 was not recognized, whereas a group of slightly more acidic proteins of similar Mr were recognized. Thus, GP65,70 is not the beta subunit of Na+/Ka+-ATPase. Further studies are needed to determine the cellular function of GP65,70. PMID:2835098

  17. Purification and structural characterization of herpes simplex virus glycoprotein C

    SciTech Connect

    Kikuchi, G.E.; Baker, S.A.; Merajver, S.D.; Coligan, J.E.; Levine, M.; Glorioso, J.C.; Nairn, R.

    1987-01-27

    Purification of herpes simplex virus glycoprotein C (gC) in microgram amounts yielded sufficient material for an analysis of its secondary structure. Purification was facilitated by using the mutant virus gC-3, which bears a point mutation that interrupts the putative hydrophobic membrane anchor sequence, causing the secretion of gC-3 protein into the cell culture medium. gC-3 protein was purified by size fractionation of concentrated culture medium from infected cells on a gel filtration column of Sephacryl S-200, followed by immunoaffinity chromatography on a column constructed of gC-specific monoclonal antibodies cross-linked to a protein A-Sepharose CL-4B matrix. Purified gC-3 had a molecular weight of 130,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the size expected for gC, was reactive with gC-specific monoclonal antibodies in protein immunoblots, and contained amino acid sequences characteristic of gC as determined by radiochemical amino acid microsequence analyses. Polyclonal antisera obtained from a rabbit immunized with gC-3 reacted with wild-type gC in immunoprecipitation, enzyme immunoassay, and immunoelectroblot (western blot) assays. Deglycosylation by treatment with trifluoromethanesulfonic acid reduced the molecular weight of gC-3 by approximately 35%. Analyses of both native and deglycosylated gC-3 by Raman spectroscopy showed that the native molecule consists of about 17%..cap alpha..-helix, 24% ..beta..-sheet, and 60% disordered secondary structures, whereas deglycosylated gC-3 consists of about 8% ..cap alpha..-helix, 10% ..beta..-sheet, 81% disordered structures. These data were in good agreement with the 11% ..cap alpha..-helix, 18% ..beta..-sheet, 61% ..beta..-turn, and 9% disordered structures calculated from Chou-Fasman analysis of the primary sequence of gC-3.

  18. Resource envelope concepts for mission planning

    NASA Technical Reports Server (NTRS)

    Ibrahim, K. Y.; Weiler, J. D.; Tokaz, J. C.

    1991-01-01

    Seven proposed methods for creating resource envelopes for Space Station Freedom mission planning are detailed. Four reference science activity models are used to illustrate the effect of adding operational flexibility to mission timelines. For each method, a brief explanation is given along with graphs to illustrate the application of the envelopes to the power and crew resources. The benefits and costs of each method are analyzed in terms of resource utilization. In addition to the effect on individual activities, resource envelopes are analyzed at the experiment level.

  19. Purification of a soluble glycoprotein from the uncalcified ecdysial cuticle of the blue crab Callinectes sapidus and its possible role in initial mineralization.

    PubMed

    Tweedie, Elizabeth P; Coblentz, Francie E; Shafer, Thomas H

    2004-07-01

    A heavily glycosylated soluble protein was purified using a combination of lectin affinity and size exclusion chromatography from a soluble extract of uncalcified dorsal cuticle of blue crab Callinectes sapidus removed at ecdysis. Similarities in apparent molecular mass and carbohydrate composition suggest that this protein is the same species previously shown to disappear from soluble extracts coincidentally with the onset of mineral deposition in the newly exposed post-molt cuticle. The amino acid sequence of the N-terminal portion of the core polypeptide was determined and polyclonal antibodies were raised against both the purified glycoprotein and the peptide. Immunoblots of unfractionated soluble extracts taken at various times post-molt illustrated that the anti-peptide antibody recognized several polypeptides with electrophoretic mobilities that differ from the purified glycoprotein. These bands may be deglycosylation products which would not have been purified due to different lectin affinity or size. Immunohistochemical analysis indicated uniform protein distribution in the exocuticle at ecdysis, but decreased antibody binding at the interprismatic septa by 2 h post-molt. The location of the protein is therefore the negative image of the calcification pattern in the exocuticle and provides a spatial pattern to correlate with the previously reported temporal events. This strengthens the hypothesis that the glycoprotein under investigation is an inhibitor of calcite nucleation or of initial amorphous calcium carbonate accumulation.

  20. Characterization of disease-associated N-linked glycoproteins.

    PubMed

    Tian, Yuan; Zhang, Hui

    2013-02-01

    N-linked glycoproteins play important roles in biological processes, including cell-to-cell recognition, growth, differentiation, and programmed cell death. Specific N-linked glycoprotein changes are associated with disease progression and identification of these N-linked glycoproteins has potential for use in disease diagnosis, prognosis, and prediction of treatments. In this review, we summarize common strategies for N-linked glycoprotein characterization and applications of these strategies to identification of glycoprotein changes associated with disease states. We also review the N-linked glycoproteins altered in diseases such as breast cancer, lung cancer, and prostate cancer. Although assays for these glycoproteins have potential clinical utility, research is needed to translate these glycoproteins to clinical biomarkers.

  1. Characterization of disease-associated N-linked glycoproteins

    PubMed Central

    Tian, Yuan; Zhang, Hui

    2013-01-01

    N-linked glycoproteins play important roles in biological processes, including cell-to-cell recognition, growth, differentiation, and programmed cell death. Specific N-linked glycoprotein changes are associated with disease progression and identification of these N-linked glycoproteins has potential for use in disease diagnosis, prognosis, and prediction of treatments. In this review, we summarize common strategies for N-linked glycoprotein characterization and applications of these strategies to identification of glycoprotein changes associated with disease states. We also review the N-linked glycoproteins altered in diseases such as breast cancer, lung cancer, and prostate cancer. Although assays for these glycoproteins have potential clinical utility, research is needed to translate these glycoproteins to clinical biomarkers. PMID:23255236

  2. Characterization of co-purified acellular pertussis vaccines.

    PubMed

    Xu, Yinghua; Tan, Yajun; Asokanathan, Catpagavalli; Zhang, Shumin; Xing, Dorothy; Wang, Junzhi

    2015-01-01

    Whole-cell pertussis vaccines (WPVs) have been completely replaced by the co-purified acellular vaccines (APVs) in China. To date few laboratory studies were reported for co-purified APVs in terms of their antigenic composition and protective immune responses. To further understand the antigenic composition in co-purified APVs, in the present study 2-dimensional gel electrophoresis-based proteomic technology was used to analyze the composition of co-purified APVs. The results showed that besides the main antigens pertussis toxin (PT) and filamentous hemagglutinin (FHA), co-purified APVs also contained pertactin (PRN), fimbriae (FIM) 2and3 and other minor protein antigens. Of the 9 proteins identified, 3 were differentially presented in products from manufacturer 1 and manufacturer 2. Compared with WPVs and purified APVs, co-purified APVs induced a mixed Th1/Th2 immune response with more toward to a Th1 response than the purified APVs in this study. These results hint that different immune mechanisms might be involved in protection induced by co-purified and purified APVs.

  3. Characterization of co-purified acellular pertussis vaccines

    PubMed Central

    Xu, Yinghua; Tan, Yajun; Asokanathan, Catpagavalli; Zhang, Shumin; Xing, Dorothy; Wang, Junzhi

    2015-01-01

    Whole-cell pertussis vaccines (WPVs) have been completely replaced by the co-purified acellular vaccines (APVs) in China. To date few laboratory studies were reported for co-purified APVs in terms of their antigenic composition and protective immune responses. To further understand the antigenic composition in co-purified APVs, in the present study 2-dimensional gel electrophoresis-based proteomic technology was used to analyze the composition of co-purified APVs. The results showed that besides the main antigens pertussis toxin (PT) and filamentous hemagglutinin (FHA), co-purified APVs also contained pertactin (PRN), fimbriae (FIM) 2and3 and other minor protein antigens. Of the 9 proteins identified, 3 were differentially presented in products from manufacturer 1 and manufacturer 2. Compared with WPVs and purified APVs, co-purified APVs induced a mixed Th1/Th2 immune response with more toward to a Th1 response than the purified APVs in this study. These results hint that different immune mechanisms might be involved in protection induced by co-purified and purified APVs. PMID:25610957

  4. Use of the quartz crystal microbalance to monitor ligand-induced conformational rearrangements in HIV-1 envelope protein gp120

    PubMed Central

    Lee, Hyun-Su; Contarino, Mark; Umashankara, M.; Schön, Arne; Freire, Ernesto; Smith, Amos B.; Chaiken, Irwin M.

    2010-01-01

    We evaluated the potential of a quartz crystal microbalance with dissipation monitoring (QCM-D) to provide a sensitive, label-free method for detecting the conformational rearrangement of glycoprotein gp120 upon binding to different ligands. This glycoprotein is normally found on the envelope of the HIV-1 virus and is involved in viral entry into host cells. It was immobilized on the surface of the sensing element of the QCM-D and was exposed to individual solutions of several different small-molecule inhibitors as well as to a solution of soluble form of the host cell receptor to which gp120 binds. Instrument responses to ligand-triggered changes were in qualitative agreement with conformational changes suggested by other biophysical methods. PMID:20016882

  5. Poly(A) binding proteins located at the inner surface of resealed nuclear envelopes.

    PubMed

    Prochnow, D; Riedel, N; Agutter, P S; Fasold, H

    1990-04-25

    We have used a photoreactive cross-linking reagent, poly(A/8-N3-A) (a poly(A) of average molecular mass of 100 kDa in which 5-10% of the A residues are replaced by 8-N3-A), to label poly(A) binding proteins of rat liver nuclear envelopes. This reagent was prepared by polymerizing a mixture of ADP and 8-N3-ADP with polynucleotide phosphorylase. The purified poly(A) was labeled in the 5'-position with a 32P group. In nuclear envelopes prepared by a low salt DNase I procedure, the poly(A/8-N3-A) labeled a protein-nucleic acid complex of approximately 270 kDa, which on degradation with RNase U2 or NaOH at pH 10 yielded two polypeptides of approximately 50 and 30 kDa. These photoreaction products were markedly decreased when resealed nuclear envelopes or non-nuclear envelope proteins were irradiated in the presence of poly(A/8-N3-A). The affinity labeling was intensified when resealed vesicles were made leaky by freezing or ultrasonication, suggesting that the poly(A) binding proteins are accessible from the nucleoplasmic but not the cytoplasmic face of the envelope. Moreover binding was specific for poly(A). Alternative reagents, random poly(A/8-N3-A,C,G,U) of about 100 kDa and poly(dA) (molecular mass between 350 and 515 kDa), showed a very low affinity for poly(A) recognition proteins in the low salt DNase I-treated nuclear envelopes; the 270-kDa band was labeled only weakly. The binding site was not protected by poly(A,C,G,U), weakly by poly(dA), and distinctly by poly(A).

  6. Solar envelope concepts: moderate density building applications

    NASA Astrophysics Data System (ADS)

    Knowles, R. L.; Berry, R. D.

    1980-04-01

    The public policy mechanism for guaranteeing solar access is conceptualized as a solar zoning envelope that allows the largest possible building bulk on a land parcel without shadowing neighboring properties during specified times. Step-by-step methods for generating solar envelopes are described with extensive drawings, showing a variety of urban platting and lot configurations. Development and design possibilities are examined on a selected set of Los Angeles sites with typically diverse urban characteristics. Envelope attributes suitable for encouraging moderate-density commercial and residential building are examined in the context of two hypothetical but realistic development programs: one for speculative office buildings and one for condominium housing. Numerous illustrations of envelope forms and prototypical building designs are provided.

  7. Personnel occupied woven envelope robot power

    NASA Technical Reports Server (NTRS)

    Wessling, F. C.

    1988-01-01

    The Personnel Occupied Woven Envelope Robot (POWER) concept has evolved over the course of the study. The goal of the project was the development of methods and algorithms for solid modeling for the flexible robot arm.

  8. Survival of an Enveloped Virus on Toys.

    PubMed

    Bearden, Richard L; Casanova, Lisa M

    2016-08-01

    Children's toys may carry respiratory viruses. Inactivation of a lipid-enveloped bacteriophage, Φ6, was measured on a nonporous toy at indoor temperature and relative humidity (RH). Inactivation was approximately 2log10 after 24 hours at 60% RH and 6.8log10 at 10 hours at 40% RH. Enveloped viruses can potentially survive on toys long enough to result in exposures. PMID:27144972

  9. Control load envelope shaping by live twist

    NASA Technical Reports Server (NTRS)

    Tarzanin, F. J., Jr.; Mirick, P. H.

    1974-01-01

    Rotor control systems experience a rapid load growth resulting from retreating blade stall during flight conditions of high blade loading or airspeeds. An investigation was undertaken to determine the effect of changing blade torsional properties over the rotor flight envelope. The results of this study show that reducing the blade stiffness to introduce more blade live twist significantly reduces the large retreating blade control loads, while expanding the flight envelope and reducing retreating blade stall loads.

  10. Creating a Lunar EVA Work Envelope

    NASA Technical Reports Server (NTRS)

    Griffin, Brand N.; Howard, Robert; Rajulu, Sudhakar; Smitherman, David

    2009-01-01

    A work envelope has been defined for weightless Extravehicular Activity (EVA) based on the Space Shuttle Extravehicular Mobility Unit (EMU), but there is no equivalent for planetary operations. The weightless work envelope is essential for planning all EVA tasks because it determines the location of removable parts, making sure they are within reach and visibility of the suited crew member. In addition, using the envelope positions the structural hard points for foot restraints that allow placing both hands on the job and provides a load path for reacting forces. EVA operations are always constrained by time. Tasks are carefully planned to ensure the crew has enough breathing oxygen, cooling water, and battery power. Planning first involves computers using a virtual work envelope to model tasks, next suited crew members in a simulated environment refine the tasks. For weightless operations, this process is well developed, but planetary EVA is different and no work envelope has been defined. The primary difference between weightless and planetary work envelopes is gravity. It influences anthropometry, horizontal and vertical mobility, and reaction load paths and introduces effort into doing "overhead" work. Additionally, the use of spacesuits other than the EMU, and their impacts on range of motion, must be taken into account. This paper presents the analysis leading to a concept for a planetary EVA work envelope with emphasis on lunar operations. There is some urgency in creating this concept because NASA has begun building and testing development hardware for the lunar surface, including rovers, habitats and cargo off-loading equipment. Just as with microgravity operations, a lunar EVA work envelope is needed to guide designers in the formative stages of the program with the objective of avoiding difficult and costly rework.

  11. A simple, inexpensive, robust and sensitive dot-blot assay for equal detection of the nonstructural-1 glycoprotein of all dengue virus serotypes

    PubMed Central

    2013-01-01

    Background Detection of dengue virus (DENV) soluble/excreted (s/e) form of the nonstructural-1 (NS1) glycoprotein in patient acute-phase sera is ideal for diagnosis. The commercially-available detection assays are, however, too expensive for routine use and have low specificity, particularly for the s/e NS1 glycoprotein of DENV-2 and DENV-4, which are important causes of lethal human disease worldwide. Methods Mouse monoclonal antibodies (MAbs) were generated and screened against s/e NS1 glycoprotein purified from each DENV serotype to obtain those that reacted equally with each serotype, but not with yellow fever virus (YFV) s/e NS1 glycoprotein or human serum proteins. One MAb, MAb 2C4.6, was further tested against these DENV glycoproteins in human sera using simple, peroxidase-labelled secondary antibody/substrate-developed dot-blot assays. Results Optimal quenching of endogenous human serum peroxidases was attained using 3% H2O2 in H20 for 5 min. MAb 2C4.6 showed an acceptable detection sensitivity of < 32 ng/ml for the s/e NS1 glycoprotein of each DENV serotype but did not cross-react with the YFV s/e NS1 glycoprotein or human serum proteins. By contrast, the LX1 epitope-specific MAb, 3D1.4, showed similar detection sensitivity against only the DENV-1 NS1 glycoprotein, consistent with results from commercial DENV s/e NS1 glycoprotein detection assays. DENV s/e NS1 glycoproteins were stable in human sera after drying on the nitrocellulose membranes and storage for one month at ambient temperature (28°C) before being processed. The total assay time was reduced to 3 h without any loss of detection sensitivity. This dot-blot format was ideal for the circulating immune complex disruption step, which is required for increased DENV s/e NS1 glycoprotein detection. Conclusions This is the first study to determine the detection sensitivity of MAbs against known concentrations of s/e NS1 glycoprotein from each DENV serotype. The preparation of patient serum samples for

  12. Cooling of neutron stars with diffusive envelopes

    NASA Astrophysics Data System (ADS)

    Beznogov, M. V.; Fortin, M.; Haensel, P.; Yakovlev, D. G.; Zdunik, J. L.

    2016-08-01

    We study the effects of heat blanketing envelopes of neutron stars on their cooling. To this aim, we perform cooling simulations using newly constructed models of the envelopes composed of binary ion mixtures (H-He, He-C, C-Fe) varying the mass of lighter ions (H, He or C) in the envelope. The results are compared with those calculated using the standard models of the envelopes which contain the layers of lighter (accreted) elements (H, He and C) on top of the Fe layer, varying the mass of accreted elements. The main effect is that the chemical composition of the envelopes influences their thermal conductivity and, hence, thermal insulation of the star. For illustration, we apply these results to estimate the internal temperature of the Vela pulsar and to study the cooling of neutron stars of ages of 105 - 106 yr at the photon cooling stage. The uncertainties of the cooling models associated with our poor knowledge of chemical composition of the heat insulating envelopes strongly complicate theoretical reconstruction of the internal structure of cooling neutron stars from observations of their thermal surface emission.

  13. Genetic diversity of koala retroviral envelopes.

    PubMed

    Xu, Wenqin; Gorman, Kristen; Santiago, Jan Clement; Kluska, Kristen; Eiden, Maribeth V

    2015-03-01

    Genetic diversity, attributable to the low fidelity of reverse transcription, recombination and mutation, is an important feature of infectious retroviruses. Under selective pressure, such as that imposed by superinfection interference, gammaretroviruses commonly adapt their envelope proteins to use alternative receptors to overcome this entry block. The first characterized koala retroviruses KoRV subgroup A (KoRV-A) were remarkable in their absence of envelope genetic variability. Once it was determined that KoRV-A was present in all koalas in US zoos, regardless of their disease status, we sought to isolate a KoRV variant whose presence correlated with neoplastic malignancies. More than a decade after the identification of KoRV-A, we isolated a second subgroup of KoRV, KoRV-B from koalas with lymphomas. The envelope proteins of KoRV-A and KoRV-B are sufficiently divergent to confer the ability to bind and employ distinct receptors for infection. We have now obtained a number of additional KoRV envelope variants. In the present studies we report these variants, and show that they differ from KoRV-A and KoRV-B envelopes in their host range and superinfection interference properties. Thus, there appears to be considerable variation among KoRVs envelope genes suggesting genetic diversity is a factor following the KoRV-A infection process.

  14. Genetic diversity of koala retroviral envelopes.

    PubMed

    Xu, Wenqin; Gorman, Kristen; Santiago, Jan Clement; Kluska, Kristen; Eiden, Maribeth V

    2015-03-01

    Genetic diversity, attributable to the low fidelity of reverse transcription, recombination and mutation, is an important feature of infectious retroviruses. Under selective pressure, such as that imposed by superinfection interference, gammaretroviruses commonly adapt their envelope proteins to use alternative receptors to overcome this entry block. The first characterized koala retroviruses KoRV subgroup A (KoRV-A) were remarkable in their absence of envelope genetic variability. Once it was determined that KoRV-A was present in all koalas in US zoos, regardless of their disease status, we sought to isolate a KoRV variant whose presence correlated with neoplastic malignancies. More than a decade after the identification of KoRV-A, we isolated a second subgroup of KoRV, KoRV-B from koalas with lymphomas. The envelope proteins of KoRV-A and KoRV-B are sufficiently divergent to confer the ability to bind and employ distinct receptors for infection. We have now obtained a number of additional KoRV envelope variants. In the present studies we report these variants, and show that they differ from KoRV-A and KoRV-B envelopes in their host range and superinfection interference properties. Thus, there appears to be considerable variation among KoRVs envelope genes suggesting genetic diversity is a factor following the KoRV-A infection process. PMID:25789509

  15. Hydrogen purifier module and method for forming the same

    DOEpatents

    DeVries, Peter David

    2012-02-07

    A hydrogen purifier utilizing a hydrogen permeable membrane, and a gas-tight seal, where the seal is uses a low temperature melting point metal, which upon heating above the melting point subsequently forms a seal alloy with adjacent metals, where the alloy has a melting point above the operational temperature of the purifier. The purifier further is constructed such that a degree of isolation exists between the metal that melts to form the seal and the active area of the purifier membrane, so that the active area of the purifier membrane is not corrupted. A method of forming a hydrogen purifier utilizing a hydrogen permeable membrane with a seal of the same type is also disclosed.

  16. Purification and molecular properties of two cadmium-binding glycoproteins from the hepatopancreas of a whelk, Buccinum tenuissimum.

    PubMed

    Dohi, Y; Ohba, K; Yoneyama, Y

    1983-05-30

    Two cytoplasmic cadmium-binding glycoproteins from the hepatopancreas of a whelk, Buccinum tenuissimum, were purified to homogeneity by Sephadex G-75 gel filtration and two DEAE-Sephadex A-25 chromatographies in the presence of 2-mercaptoethanol. These two cadmium-binding glycoproteins, termed FIIA and FIIB, each showed a single band after electrophoresis on 10% polyacrylamide gel at pH 8.9. The molecular weight of FIIA was estimated as 8000 and that of FIIB as 13000 by SDS-polyacrylamide gel electrophoresis. Based on molecular weights of 6500 and 11900 for the protein moiety of FIIA and FIIB, respectively, the total number of amino acid residues was 52 in the former and 94 in the latter. Three and two cysteine residues in FIIA and FIIB, respectively, were titratable with p-chloromercuribenzoate. FIIB also contained two more half-cystine residues. The sugar contents of FIIA and FIIB were about 20.5% and 8.7% by weight, respectively, consisting of galactose, mannose, fucose and amino sugar. The purified glycoproteins FIIA and FIIB contained about 0.6% and 1.0% cadmium by weight, respectively, and both showed strong metal-binding capacity, especially for cadmium, copper and mercury. The apparent cadmium dissociation constants for FIIA and FIIB after treatment with 2-mercaptoethanol were 7.3 X 10(-6) and 9.1 X 10(-7) M, respectively. Cadmium contents at saturation were nearly 6 and 8 gatom per mole for FIIA and FIIB, respectively.

  17. Phytochrome regulates GTP-binding protein activity in the envelope of pea nuclei

    NASA Technical Reports Server (NTRS)

    Clark, G. B.; Memon, A. R.; Thompson, G. A. Jr; Roux, S. J.

    1993-01-01

    Three GTP-binding proteins with apparent molecular masses of 27, 28 and 30 kDa have been detected in isolated nuclei of etiolated pea plumules. After LDS-PAGE and transfer to nitrocellulose these proteins bind [32P]GTP in the presence of excess ATP, suggesting that they are monomeric G proteins. When nuclei are disrupted, three proteins co-purify with the nuclear envelope fraction and are highly enriched in this fraction. The level of [32P]GTP-binding for all three protein bands is significantly increased when harvested pea plumules are irradiated by red light, and this effect is reversed by far-red light. The results indicate that GTP-binding activity associated with the nuclear envelope of plant cells is photoreversibly regulated by the pigment phytochrome.

  18. Characterization and action patterns of two beta-1,4-glucanases purified from cellulomonas uda CS1-1.

    PubMed

    Yoon, Min-Ho; Choi, Woo-Young

    2007-08-01

    Two beta-1,4-glucanases (DI and DIII fractions) were purified to homogeneity from the culture filtrate of a cellulolytic bacteria, Cellulomonas sp. CS1-1, which was classified as a novel species belonging to Cellulomonas uda based on chemotaxanomic and phylogenetic analyses. The molecular mass was estimated as 50,000 Da and 52,000 Da for DI and DIII, respectively. Moreover, DIII was identified as a glycoprotein with a pI of 3.8, and DI was identified as a non-glycoprotein with a pI of 5.3. When comparing the ratio of the CMC-saccharifying activity and CMC-liquefying activity, DI exhibited a steep slope, characteristic of an endoglucanase, whereas DIII exhibited a low slope, characteristic of an exoglucanase. The substrate specificity of the purified enzymes revealed that DI efficiently hydrolyzed CMC as well as xylan, whereas DIII exhibited a high activity on microcrystalline celluloses, such as Sigmacells. A comparison of the hydrolysis patterns for pNP-glucosides (DP 2-5) using an HPLC analysis demonstrated that the halosidic bond 3 from the nonreducing end was the preferential cleavage site for DI, whereas bond 2, from which the cellobiose unit is split off, was the preferential cleavage site for DIII. The partial N-terminal amino acid sequences for the purified enzymes were 1Ala-Gly-Ser-Thr-Leu-Gln-Ala-Ala-Ala-Ser-Glu-Ser-Gly-Arg-Tyr15- for DI and 1Ala-Asp-Ser-Asp-Phe-Asn-Leu-Tyr-Val-Ala-Glu-Asn-Ala-Met-Lys15- for DIII. The apparent sequences exhibited high sequence similarities with other bacterial beta-1,4-glucanases as well as beta-1,4-xylanases.

  19. Using proximity biotinylation to detect herpesvirus entry glycoprotein interactions: Limitations for integral membrane glycoproteins.

    PubMed

    Lajko, Michelle; Haddad, Alexander F; Robinson, Carolyn A; Connolly, Sarah A

    2015-09-01

    Herpesvirus entry into cells requires coordinated interactions among several viral transmembrane glycoproteins. Viral glycoproteins bind to receptors and interact with other glycoproteins to trigger virus-cell membrane fusion. Details of these glycoprotein interactions are not well understood because they are likely transient and/or low affinity. Proximity biotinylation is a promising protein-protein interaction assay that can capture transient interactions in live cells. One protein is linked to a biotin ligase and a second protein is linked to a short specific acceptor peptide (AP). If the two proteins interact, the ligase will biotinylate the AP, without requiring a sustained interaction. To examine herpesvirus glycoprotein interactions, the ligase and AP were linked to herpes simplex virus 1 (HSV1) gD and Epstein Barr virus (EBV) gB. Interactions between monomers of these oligomeric proteins (homotypic interactions) served as positive controls to demonstrate assay sensitivity. Heterotypic combinations served as negative controls to determine assay specificity, since HSV1 gD and EBV gB do not interact functionally. Positive controls showed strong biotinylation, indicating that viral glycoprotein proximity can be detected. Unexpectedly, the negative controls also showed biotinylation. These results demonstrate the special circumstances that must be considered when examining interactions among glycosylated proteins that are constrained within a membrane.

  20. Comment on the invariant envelope solution in rf photoinjectors.

    SciTech Connect

    Wang, C.-x.; Accelerator Systems Division

    2006-02-01

    The beam envelope equation has been used to address the beam dynamics in rf photoinjectors. A special solution of the envelope equation, known as the invariant envelope, plays a critical role in the theory of emittance compensation. In this comment, I will present a different view of the invariant envelope solution that better delineates its properties and simplifies the picture of beam dynamics.

  1. The ectodomain of the human T-cell leukemia virus type 1 TM glycoprotein is involved in postfusion events.

    PubMed Central

    Rosenberg, A R; Delamarre, L; Pique, C; Pham, D; Dokhélar, M C

    1997-01-01

    To examine the contribution of the transmembrane envelope glycoprotein (TM) to the infectivity of the human T-cell leukemia virus type 1 (HTLV-1), single amino acid substitutions were introduced throughout its ectodomain. The mutated envelopes were tested for intracellular maturation and for functions, including ability to elicit syncytium formation and ability to mediate cell-to-cell transmission of the virus. Three major phenotypes, defining three functionally distinct regions, were identified. (i) Mutations causing defects in intracellular maturation of the envelope precursor are mostly distributed in the central portion of the TM ectodomain, containing the immunosuppressive peptide. This region, which includes vicinal cysteines thought to form an intramolecular disulfide bridge, is probably essential for correct folding of the protein. (ii) Mutations resulting in reduced syncytium-forming ability despite correct intracellular maturation are clustered in the amino-terminal part of the TM ectodomain, within the leucine zipper-like motif. Similar motifs with a propensity to form coiled-coil structures have been implicated in the fusion process driven by other viral envelope proteins, and HTLV-1 may thus conform to this general rule for viral fusion. (iii) Mutants with increased syncytium-forming ability define a region immediately amino-terminal to the membrane-spanning domain. Surprisingly, these mutants exhibited severe defects in infectivity, despite competence for fusion. Existence of this phenotype indicates that capacity for cell-to-cell fusion is not sufficient to ensure viral entry, even in cell-to-cell transmission. The ectodomain of the TM glycoprotein thus may be involved in postfusion events required for full infectivity of HTLV-1, which perhaps represents a unique feature of this poorly infectious retrovirus. PMID:9311790

  2. Simulating Convection in Stellar Envelopes

    NASA Astrophysics Data System (ADS)

    Tanner, Joel

    Understanding convection in stellar envelopes, and providing a mathematical description of it, would represent a substantial advance in stellar astrophysics. As one of the largest sources of uncertainty in stellar models, existing treatments of convection fail to account for many of the dynamical effects of convection, such as turbulent pressure and asymmetry in the velocity field. To better understand stellar convection, we must be able to study and examine it in detail, and one of the best tools for doing so is numerical simulation. Near the stellar surface, both convective and radiative process play a critical role in determining the structure and gas dynamics. By following these processes from first principles, convection can be simulated self-consistently and accurately, even in regions of inefficient energy transport where existing descriptions of convection fail. Our simulation code includes two radiative transfer solvers that are based on different assumptions and approximations. By comparing simulations that differ only in their respective radiative transfer methods, we are able to isolate the effect that radiative efficiency has on the structure of the superadiabatic layer. We find the simulations to be in good general agreement, but they show distinct differences in the thermal structure in the superadiabatic layer and atmosphere. Using the code to construct a grid of three-dimensional radiation hydrodynamic simulations, we investigate the link between convection and various chemical compositions. The stellar parameters correspond to main-sequence stars at several surface gravities, and span a range in effective temperatures (4500 < Teff < 6400). Different chemical compositions include four metallicities (Z = 0.040, 0.020, 0.010, 0.001), three helium abundances (Y = 0.1, 0.2, 0.3) and several levels of alpha-element enhancement. Our grid of simulations shows that various convective properties, such as velocity and the degree of superadiabaticity, are

  3. Structural proteins of ribonucleic acid tumor viruses. Purification of envelope, core, and internal components.

    PubMed

    Strand, M; August, J T

    1976-01-25

    Murine type C virus structural proteins, the envelope glycopeptides, 30,000 dalton major core protein, and 15,000 dalton internal protein have each been purified to near homogeneity and in high yield from the smae batch of virus by use of phosphocellulose column chromatography and gel filtration procedures. Evidence that these proteins are specified by the viral genome was obtained by competition radioimmunoassay analysis, comparing these polypeptides from Rauscher virus cultivated in a variety of mammalian cell lines; all of the reactive antigenic determinants of these proteins appeared to be virus-specific.

  4. Site occupancy and glycan compositional analysis of two soluble recombinant forms of the attachment glycoprotein of Hendra virus.

    PubMed

    Colgrave, Michelle L; Snelling, Hayley J; Shiell, Brian J; Feng, Yan-Ru; Chan, Yee-Peng; Bossart, Katharine N; Xu, Kai; Nikolov, Dimitar B; Broder, Christopher C; Michalski, Wojtek P

    2012-04-01

    Hendra virus (HeV) continues to cause morbidity and mortality in both humans and horses with a number of sporadic outbreaks. HeV has two structural membrane glycoproteins that mediate the infection of host cells: the attachment (G) and the fusion (F) glycoproteins that are essential for receptor binding and virion-host cell membrane fusion, respectively. N-linked glycosylation of viral envelope proteins are critical post-translation modifications that have been implicated in roles of structural integrity, virus replication and evasion of the host immune response. Deciphering the glycan composition and structure on these glycoproteins may assist in the development of glycan-targeted therapeutic intervention strategies. We examined the site occupancy and glycan composition of recombinant soluble G (sG) glycoproteins expressed in two different mammalian cell systems, transient human embryonic kidney 293 (HEK293) cells and vaccinia virus (VV)-HeLa cells, using a suite of biochemical and biophysical tools: electrophoresis, lectin binding and tandem mass spectrometry. The N-linked glycans of both VV and HEK293-derived sG glycoproteins carried predominantly mono- and disialylated complex-type N-glycans and a smaller population of high mannose-type glycans. All seven consensus sequences for N-linked glycosylation were definitively found to be occupied in the VV-derived protein, whereas only four sites were found and characterized in the HEK293-derived protein. We also report, for the first time, the existence of O-linked glycosylation sites in both proteins. The striking characteristic of both proteins was glycan heterogeneity in both N- and O-linked sites. The structural features of G protein glycosylation were also determined by X-ray crystallography and interactions with the ephrin-B2 receptor are discussed.

  5. Morphologically complex protostellar envelopes : structure and kinematics

    NASA Astrophysics Data System (ADS)

    Tobin, John J.

    I present an in-depth study of protostars and their surrounding envelopes of dense gas and dust, using a multitude of observational methods to reveal new details of the star formation process. I use mid-infrared imaging from the Spitzer Space Telescope, combined with photometry spanning the near-infrared to millimeter wavelengths, to construct a model of the L1527 protostellar system. I modeled both the spectral energy distribution and resolved scattered light images to determine physical properties of the protostellar system. The nature of the apparent central point source in the Spitzer images was uncertain until high-resolution L-band imaging from the Gemini observatory resolved the point source into a disk in scattered light, having a radius of 200 AU. Protostellar envelopes are also often found to cast shadows against the 8 micron Galactic background in Spitzer imaging, enabling direct probes of envelope structure. The shadow images show that the dense envelopes around twenty-two Class 0 protostars are generally morphologically complex from 0.1 pc scales down to ˜1000 AU; they are often filamentary, and frequently non-axisymmetric. The observed envelope structure indicates a likely origin in turbulent cloud structure rather than a quasi-static/equilibrium formation. The complex envelope structure also may indicate an increased likelihood of fragmentation during collapse, forming close binaries. To further characterize these envelopes, I have observed them in the dense molecular gas tracers nthp and nht, both of which closely follow the 8 micron extinction morphology. The magnitude of the velocity gradients and envelope complexity on ˜10000 AU scales indicates that the velocity structure may reflect large-scale infall in addition to the often assumed rotation. Comparisons with three-dimensional filamentary and symmetric rotating collapse models reinforce the interpretation of velocities reflecting large-scale infall, showing that the structure of the envelope

  6. Structure based design towards the identification of novel binding sites and inhibitors for the chikungunya virus envelope proteins.

    PubMed

    Rashad, Adel A; Keller, Paul A

    2013-07-01

    Chikungunya virus is an emerging arbovirus that is widespread in tropical regions and is spreading quickly to temperate climates with recent epidemics in Africa, Asia, Europe and the Americas. It is having an increasingly major impact on humans with potentially life-threatening and debilitating arthritis. Thus far, neither vaccines nor medications are available to treat or control the virus and therefore, the development of medicinal chemistry is a vital and immediate issue that needs to be addressed. The viral envelope proteins play a major role during infection through mediation of binding and fusion with the infected cell surfaces. The possible binding target sites of the chikungunya virus envelope proteins have not previously been investigated; we describe here for the first time the identification of novel sites for potential binding on the chikungunya glycoprotein complexes and the identification of possible antagonists for these sites through virtual screening using two successive docking scores; FRED docking for fast precise screening, with the top hits then subjected to a ranking scoring using the AUTODOCK algorithm. Both the immature and the mature forms of the chikungunya envelope proteins were included in the study to increase the probability of finding positive and reliable hits. Some small molecules have been identified as good in silico chikungunya virus envelope proteins inhibitors and these could be good templates for drug design targeting this virus.

  7. 21 CFR 880.6500 - Medical ultraviolet air purifier.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... to ultraviolet radiation. (b) Classification. Class II (performance standards). ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Medical ultraviolet air purifier. 880.6500 Section... Miscellaneous Devices § 880.6500 Medical ultraviolet air purifier. (a) Identification. A medical ultraviolet...

  8. 21 CFR 880.6500 - Medical ultraviolet air purifier.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... to ultraviolet radiation. (b) Classification. Class II (performance standards). ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Medical ultraviolet air purifier. 880.6500 Section... Miscellaneous Devices § 880.6500 Medical ultraviolet air purifier. (a) Identification. A medical ultraviolet...

  9. 21 CFR 880.6500 - Medical ultraviolet air purifier.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... to ultraviolet radiation. (b) Classification. Class II (performance standards). ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Medical ultraviolet air purifier. 880.6500 Section... Miscellaneous Devices § 880.6500 Medical ultraviolet air purifier. (a) Identification. A medical ultraviolet...

  10. 21 CFR 880.6500 - Medical ultraviolet air purifier.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... to ultraviolet radiation. (b) Classification. Class II (performance standards). ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Medical ultraviolet air purifier. 880.6500 Section... Miscellaneous Devices § 880.6500 Medical ultraviolet air purifier. (a) Identification. A medical ultraviolet...

  11. 21 CFR 880.6710 - Medical ultraviolet water purifier.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... exposure to ultraviolet radiation. (b) Classification. Class II (performance standards). ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Medical ultraviolet water purifier. 880.6710... Miscellaneous Devices § 880.6710 Medical ultraviolet water purifier. (a) Identification. A medical...

  12. Toremifene interacts with and destabilizes the Ebola virus glycoprotein.

    PubMed

    Zhao, Yuguang; Ren, Jingshan; Harlos, Karl; Jones, Daniel M; Zeltina, Antra; Bowden, Thomas A; Padilla-Parra, Sergi; Fry, Elizabeth E; Stuart, David I

    2016-07-01

    Ebola viruses (EBOVs) are responsible for repeated outbreaks of fatal infections, including the recent deadly epidemic in West Africa. There are currently no approved therapeutic drugs or vaccines for the disease. EBOV has a membrane envelope decorated by trimers of a glycoprotein (GP, cleaved by furin to form GP1 and GP2 subunits), which is solely responsible for host cell attachment, endosomal entry and membrane fusion. GP is thus a primary target for the development of antiviral drugs. Here we report the first, to our knowledge, unliganded structure of EBOV GP, and high-resolution complexes of GP with the anticancer drug toremifene and the painkiller ibuprofen. The high-resolution apo structure gives a more complete and accurate picture of the molecule, and allows conformational changes introduced by antibody and receptor binding to be deciphered. Unexpectedly, both toremifene and ibuprofen bind in a cavity between the attachment (GP1) and fusion (GP2) subunits at the entrance to a large tunnel that links with equivalent tunnels from the other monomers of the trimer at the three-fold axis. Protein–drug interactions with both GP1 and GP2 are predominately hydrophobic. Residues lining the binding site are highly conserved among filoviruses except Marburg virus (MARV), suggesting that MARV may not bind these drugs. Thermal shift assays show up to a 14 °C decrease in the protein melting temperature after toremifene binding, while ibuprofen has only a marginal effect and is a less potent inhibitor. These results suggest that inhibitor binding destabilizes GP and triggers premature release of GP2, thereby preventing fusion between the viral and endosome membranes. Thus, these complex structures reveal the mechanism of inhibition and may guide the development of more powerful anti-EBOV drugs. PMID:27362232

  13. Featured Image: Orbiting Stars Share an Envelope

    NASA Astrophysics Data System (ADS)

    Kohler, Susanna

    2016-03-01

    This beautiful series of snapshots from a simulation (click for a better look!) shows what happens when two stars in a binary system become enclosed in the same stellar envelope. In this binary system, one of the stars has exhausted its hydrogen fuel and become a red giant, complete with an expanding stellar envelope composed of hydrogen and helium. Eventually, the envelope expands so much that the companion star falls into it, where it releases gravitational potential energy into the common envelope. A team led by Sebastian Ohlmann (Heidelberg Institute for Theoretical Studies and University of Wrzburg) recently performed hydrodynamic simulations of this process. Ohlmann and collaborators discovered that the energy release eventually triggers large-scale flow instabilities, which leads to turbulence within the envelope. This process has important consequences for how these systems next evolve (for instance, determining whether or not a supernova occurs!). You can check out the authors video of their simulated stellar inspiral below, or see their paper for more images and results from their study.CitationSebastian T. Ohlmann et al 2016 ApJ 816 L9. doi:10.3847/2041-8205/816/1/L9

  14. The cell envelope proteome of Aggregatibacter actinomycetemcomitans

    PubMed Central

    Smith, Kenneth P.; Fields, Julia G.; Voogt, Richard D.; Deng, Bin; Lam, Ying-Wai; Mintz, Keith P.

    2014-01-01

    Summary The cell envelope of Gram-negative bacteria serves a critical role in maintenance of cellular homeostasis, resistance to external stress, and host-pathogen interactions. Envelope protein composition is influenced by the physiological and environmental demands placed on the bacterium. In this study, we report a comprehensive compilation of cell envelope proteins from the periodontal and systemic pathogen Aggregatibacter actinomycetemcomitans VT1169, an afimbriated serotype b strain. The urea-extracted membrane proteins were identified by mass spectrometry-based shotgun proteomics. The membrane proteome, isolated from actively growing bacteria under normal laboratory conditions, included 648 proteins representing 28% of the predicted ORFs in the genome. Bioinformatic analyses were used to annotate and predict the cellular location and function of the proteins. Surface adhesins, porins, lipoproteins, numerous influx and efflux pumps, multiple sugar, amino acid and iron transporters, and components of the type I, II and V secretion systems were identified. Periplasmic space and cytoplasmic proteins with chaperone function were also identified. 107 proteins with unknown function were associated with the cell envelope. Orthologs of a subset of these uncharacterized proteins are present in other bacterial genomes, while others are found exclusively in A. actinomycetemcomitans. This knowledge will contribute to elucidating the role of cell envelope proteins in bacterial growth and survival in the oral cavity. PMID:25055881

  15. Characterizing Functional Domains for TIM-Mediated Enveloped Virus Entry

    PubMed Central

    Moller-Tank, Sven; Albritton, Lorraine M.; Rennert, Paul D.

    2014-01-01

    ABSTRACT T-cell immunoglobulin and mucin domain 1 (TIM-1) and other TIM family members were recently identified as phosphatidylserine (PtdSer)-mediated virus entry-enhancing receptors (PVEERs). These proteins enhance entry of Ebola virus (EBOV) and other viruses by binding PtdSer on the viral envelope, concentrating virus on the cell surface, and promoting subsequent internalization. The PtdSer-binding activity of the immunoglobulin-like variable (IgV) domain is essential for both virus binding and internalization by TIM-1. However, TIM-3, whose IgV domain also binds PtdSer, does not effectively enhance virus entry, indicating that other domains of TIM proteins are functionally important. Here, we investigate the domains supporting enhancement of enveloped virus entry, thereby defining the features necessary for a functional PVEER. Using a variety of chimeras and deletion mutants, we found that in addition to a functional PtdSer-binding domain PVEERs require a stalk domain of sufficient length, containing sequences that promote an extended structure. Neither the cytoplasmic nor the transmembrane domain of TIM-1 is essential for enhancing virus entry, provided the protein is still plasma membrane bound. Based on these defined characteristics, we generated a mimic lacking TIM sequences and composed of annexin V, the mucin-like domain of α-dystroglycan, and a glycophosphatidylinositol anchor that functioned as a PVEER to enhance transduction of virions displaying Ebola, Chikungunya, Ross River, or Sindbis virus glycoproteins. This identification of the key features necessary for PtdSer-mediated enhancement of virus entry provides a basis for more effective recognition of unknown PVEERs. IMPORTANCE T-cell immunoglobulin and mucin domain 1 (TIM-1) and other TIM family members are recently identified phosphatidylserine (PtdSer)-mediated virus entry-enhancing receptors (PVEERs). These proteins enhance virus entry by binding the phospholipid, PtdSer, present on the viral

  16. BacMam System for High-Level Expression of Recombinant Soluble and Membrane Glycoproteins for Structural Studies

    PubMed Central

    Dukkipati, Abhiram; Park, Hyun Ho; Waghray, Deepa; Fischer, Suzanne; Garcia, K. Christopher

    2008-01-01

    Baculovirus mediated gene transduction of mammalian cells (BacMam) is an emerging technique for rapid recombinant protein expression in mammalian cells. Towards this, we constructed two baculovirus transfer vectors that incorporate several mammalian transcriptional regulatory elements necessary for high level protein expression in mammalian cells. Using these vectors, we show that the BacMam system in combination with the 293 GnTI− cell line can be used for production of milligram quantities of soluble glycoproteins. Moreover, for crystallization trials, the purified glycoproteins are sensitive to EndoH treatment resulting in a loss of the bulk of the attached N-linked glycosylation. In addition, we also show that a combination of the BacMam system and 293GnTI− cell line can be used for producing milligram quantities of a GPCR-protein ligand complex suitable for crystallization trials. PMID:18782620

  17. Localization and synthesis of an antigenic determinant of herpes simplex virus glycoprotein D that stimulates the production of neutralizing antibody

    SciTech Connect

    Cohen, G.H.; Dietzschold, B.; Ponce de Leon, M.; Long, D.; Golub, E.; Varrichio, A.; Pereira, L.; Eisenberg, R.J.

    1984-01-01

    An antigenic determinant capable of inducing type-common herpes simplex virus (HSV)-neutralizing antibodies has been located on glycoprotein D (gD) of HSV type 1 (HSV-1). A peptide of 16 amino acids corresponding to residues 8 to 23 of the mature glycoprotein (residues 33 to 48 of the predicted gD-1 sequence) was synthesized. This peptide reacted with an anti-gD monoclonal antibody (group VII) previously shown to neutralize the infectivity of HSV-1 and HSV-2. The peptide was also recognized by polyclonal antibodies prepared against purified gD-1 but was less reactive with anti-gD2 sera. Sera from animals immunized with the synthetic peptide reacted with native gD and neutralized both HSV-1 and HSV-2.

  18. Portable self-contained solar powered water purifier

    SciTech Connect

    Sherman, M.

    1991-10-22

    This patent describes a portable self-contained solar powered water purifier. It comprises housing means for buoyantly supporting the purifier; solar cell means supported by the housing means above water to be treated; purification means depending from the housing means so as to be positioned in water to be treated and including sacrificial anode means providing ionized metallic ions for purifying the water and cathode means providing abstraction of electrons to facilitate the release of oxygen into the water; means for electrically connecting the solar cell means to the electrolytic purification means to enable the electrolytic purification means to purify water when the purifier is placed therein; and diode means for preventing reverse current flow between the anode means and cathode means.

  19. Binding of a neutralizing antibody to dengue virus alters the arrangement of surface glycoproteins

    SciTech Connect

    Lok, Shee-Mei; Kostyuchenko, Victor; Nybakken, Grant E.; Holdaway, Heather A.; Battisti, Anthony J.; Sukupolvi-Petty, Soila; Sedlak, Dagmar; Fremont, Daved H.; Chipman, Paul R.; Roehrig, John T.; Diamond, Michael S.; Kuhn, Richard J.; Rossmann, Michael G.

    2008-04-02

    The monoclonal antibody 1A1D-2 has been shown to strongly neutralize dengue virus serotypes 1, 2 and 3, primarily by inhibiting attachment to host cells. A crystal structure of its antigen binding fragment (Fab) complexed with domain III of the viral envelope glycoprotein, E, showed that the epitope would be partially occluded in the known structure of the mature dengue virus. Nevertheless, antibody could bind to the virus at 37 degrees C, suggesting that the virus is in dynamic motion making hidden epitopes briefly available. A cryo-electron microscope image reconstruction of the virus:Fab complex showed large changes in the organization of the E protein that exposed the epitopes on two of the three E molecules in each of the 60 icosahedral asymmetric units of the virus. The changes in the structure of the viral surface are presumably responsible for inhibiting attachment to cells.

  20. Amino acid mutations in Ebola virus glycoprotein of the 2014 epidemic.

    PubMed

    Giovanetti, Marta; Grifoni, Alba; Lo Presti, Alessandra; Cella, Eleonora; Montesano, Carla; Zehender, Gianguglielmo; Colizzi, Vittorio; Amicosante, Massimo; Ciccozzi, Massimo

    2015-06-01

    Zaire Ebola virus (EBOV) is an enveloped non-segmented negative strand RNA virus of 19 kb in length belonging to the family Filoviridae. The virus was isolated and identified in 1976 during the epidemic of hemorrhagic fever in Zaire. The most recent outbreak of EBOV among humans, was that occurred in the forested areas of south eastern Guinea, that began in February 2014 and is still ongoing. The recent Ebola outbreak, is affecting other countries in West Africa, in addiction to Guinea: Liberia, Nigeria, and Sierra Leone. In this article, a selective pressure analysis and homology modeling based on the G Glycoprotein (GP) sequences retrieved from public databases were used to investigate the genetic diversity and modification of antibody response in the recent outbreak of Ebola Virus. Structural and the evolutionary analysis underline the 2014 epidemic virus being under negative selective pressure does not change with respect to the old epidemic in terms of genome adaptation.

  1. Characterization of soluble glycoprotein D-mediated herpes simplex virus type 1 infection

    SciTech Connect

    Tsvitov, Marianna; Frampton, Arthur R.; Shah, Waris A.; Wendell, Steven K.; Ozuer, Ali; Kapacee, Zoher; Goins, William F.; Cohen, Justus B.; Glorioso, Joseph C. . E-mail: glorioso@pitt.edu

    2007-04-10

    Herpes simplex virus type 1 (HSV-1) entry into permissive cells involves attachment to cell-surface glycosaminoglycans (GAGs) and fusion of the virus envelope with the cell membrane triggered by the binding of glycoprotein D (gD) to cognate receptors. In this study, we characterized the observation that soluble forms of the gD ectodomain (sgD) can mediate entry of gD-deficient HSV-1. We examined the efficiency and receptor specificity of this activity and used sequential incubation protocols to determine the order and stability of the initial interactions required for entry. Surprisingly, virus binding to GAGs did not increase the efficiency of sgD-mediated entry and gD-deficient virus was capable of attaching to GAG-deficient cells in the absence of sgD. These observations suggested a novel binding interaction that may play a role in normal HSV infection.

  2. Ruthenium red preserves glycoprotein peplomers of C-type retroviruses for transmission electron microscopy.

    PubMed

    Fassel, T A; Raisch, K P; Chetty, N; Grossberg, S E; Kushnaryov, V M

    1998-07-01

    Peplomers, the glycoprotein projections of the outer viral envelope, are distinctive for many viruses. Peplomers of retroviral C-type particles are fragile and are not preserved in standard preparations for transmission electron microscopy of thin sections, whereas the peplomers of B- and D- type retroviruses are usually preserved. Ruthenium red, extensively used in transmission electron microscopy to enhance the preservation of glycosylated proteins, was used in the preparation of three retrovirus-producing lymphoblastoid cell lines: murine SC-1 cells producing the C-type murine leukemia retrovirus LP-BM5 that causes immunodeficiency, human DG-75 cells producing a murine leukemia retrovirus, and human C5/MJ cells producing human T-cell lymphotropic virus type I (HTLV-I). Fixation of cells was carried out with ruthenium red present in the glutaraldehyde, osmium tetroxide, and the ethanol dehydration through the 70% ethanol step. The detailed structure of peplomers of these three different viruses was well preserved. PMID:9735881

  3. Envelope Solitons in Acoustically Dispersive Vitreous Silica

    NASA Technical Reports Server (NTRS)

    Cantrell, John H.; Yost, William T.

    2012-01-01

    Acoustic radiation-induced static strains, displacements, and stresses are manifested as rectified or dc waveforms linked to the energy density of an acoustic wave or vibrational mode via the mode nonlinearity parameter of the material. An analytical model is developed for acoustically dispersive media that predicts the evolution of the energy density of an initial waveform into a series of energy solitons that generates a corresponding series of radiation-induced static strains (envelope solitons). The evolutionary characteristics of the envelope solitons are confirmed experimentally in Suprasil W1 vitreous silica. The value (-11.9 plus or minus 1.43) for the nonlinearity parameter, determined from displacement measurements of the envelope solitons via a capacitive transducer, is in good agreement with the value (-11.6 plus or minus 1.16) obtained independently from acoustic harmonic generation measurements. The agreement provides strong, quantitative evidence for the validity of the model.

  4. Drug design from the cryptic inhibitor envelope

    PubMed Central

    Lee, Chul-Jin; Liang, Xiaofei; Wu, Qinglin; Najeeb, Javaria; Zhao, Jinshi; Gopalaswamy, Ramesh; Titecat, Marie; Sebbane, Florent; Lemaitre, Nadine; Toone, Eric J.; Zhou, Pei

    2016-01-01

    Conformational dynamics plays an important role in enzyme catalysis, allosteric regulation of protein functions and assembly of macromolecular complexes. Despite these well-established roles, such information has yet to be exploited for drug design. Here we show by nuclear magnetic resonance spectroscopy that inhibitors of LpxC—an essential enzyme of the lipid A biosynthetic pathway in Gram-negative bacteria and a validated novel antibiotic target—access alternative, minor population states in solution in addition to the ligand conformation observed in crystal structures. These conformations collectively delineate an inhibitor envelope that is invisible to crystallography, but is dynamically accessible by small molecules in solution. Drug design exploiting such a hidden inhibitor envelope has led to the development of potent antibiotics with inhibition constants in the single-digit picomolar range. The principle of the cryptic inhibitor envelope approach may be broadly applicable to other lead optimization campaigns to yield improved therapeutics. PMID:26912110

  5. Purification and characterization of recombinant envelope protein GP5 of porcine reproductive and respiratory syndrome virus from E. coli.

    PubMed

    Liu, Chong; Liu, Chun-Jiang; Yuan, Xi-Gang; Zhang, Chenming

    2013-08-23

    The major envelope protein, GP5, in porcine reproductive and respiratory syndrome virus (PRRSV) plays critical roles in the assembly, invasion and immune response of PRRSV particle, and is one of the mostly studied candidates in the development of recombinant vaccines. In this research, a purification process including immobilized metal affinity chromatography (IMAC) and hydrophobic interaction chromatography (HIC) was developed to prepare recombinant envelope protein GP5 with His-tag from an E. coli strain transformed with pGEM-ORF5. The result of cell culture indicated that His-tagged GP5 protein was expressed mainly in soluble form. After cell disruption, His-tagged GP5 protein was successfully purified by Ni(2+)-chelating Sepharose Fast Flow with a yield and purity of 80.5% and 48%, respectively. Recombinant GP5 protein was further purified by HIC to achieve a purity of 95%. Moreover, the purified rGP5 is shown in monomeric form contrasting the dimeric or tetrameric form when purified by a CEX-HIC process directly from PRRSV virions as reported in a previous study.

  6. Sweet new world: glycoproteins in bacterial pathogens.

    PubMed

    Schmidt, M Alexander; Riley, Lee W; Benz, Inga

    2003-12-01

    In eukaryotes, the combinatorial potential of carbohydrates is used for the modulation of protein function. However, despite the wealth of cell wall and surface-associated carbohydrates and glycoconjugates, the accepted dogma has been that prokaryotes are not able to glycosylate proteins. This has now changed and protein glycosylation in prokaryotes is an accepted fact. Intriguingly, in Gram-negative bacteria most glycoproteins are associated with virulence factors of medically significant pathogens. Also, important steps in pathogenesis have been linked to the glycan substitution of surface proteins, indicating that the glycosylation of bacterial proteins might serve specific functions in infection and pathogenesis and interfere with inflammatory immune responses. Therefore, the carbohydrate modifications and glycosylation pathways of bacterial proteins will become new targets for therapeutic and prophylactic measures. Here we discuss recent findings on the structure, genetics and function of glycoproteins of medically important bacteria and potential applications of bacterial glycosylation systems for the generation of novel glycoconjugates.

  7. Immunogenicity of stabilized HIV-1 envelope trimers with reduced exposure of non-neutralizing epitopes

    PubMed Central

    de Taeye, Steven W.; Ozorowski, Gabriel; de la Peña, Alba Torrents; Guttman, Miklos; Julien, Jean-Philippe; van den Kerkhof, Tom L.G.M.; Burger, Judith A.; Pritchard, Laura K.; Pugach, Pavel; Yasmeen, Anila; Crampton, Jordan; Hu, Joyce; Bontjer, Ilja; Torres, Jonathan L.; Arendt, Heather; DeStefano, Joanne; Koff, Wayne C.; Schuitemaker, Hanneke; Eggink, Dirk; Berkhout, Ben; Dean, Hansi; LaBranche, Celia; Crotty, Shane; Crispin, Max; Montefiori, David C.; Klasse, P. J.; Lee, Kelly K.; Moore, John P.; Wilson, Ian A.; Ward, Andrew B.; Sanders, Rogier W.

    2016-01-01

    Summary The envelope glycoprotein trimer mediates HIV-1 entry into cells. The trimer is flexible, fluctuating between closed and more open conformations and sometimes sampling the fully open, CD4-bound form. We hypothesized that conformational flexibility could hinder the induction of broadly neutralizing antibodies (bNAbs). We therefore modified soluble Env trimers to stabilize their closed, ground states. The trimer variants were indeed stabilized in the closed conformation, with a reduced ability to undergo receptor-induced conformational changes and a decreased exposure of non-neutralizing V3-directed antibody epitopes. In rabbits, the stabilized trimers induced similar autologous Tier-1B or Tier-2 NAb titers to those elicited by the corresponding wild-type trimers, but lower levels of V3-directed Tier-1A NAbs. Stabilized, closed trimers might therefore be useful components of vaccines aimed at inducing bNAbs. PMID:26687358

  8. HIV-1 VACCINES. HIV-1 neutralizing antibodies induced by native-like envelope trimers.

    PubMed

    Sanders, Rogier W; van Gils, Marit J; Derking, Ronald; Sok, Devin; Ketas, Thomas J; Burger, Judith A; Ozorowski, Gabriel; Cupo, Albert; Simonich, Cassandra; Goo, Leslie; Arendt, Heather; Kim, Helen J; Lee, Jeong Hyun; Pugach, Pavel; Williams, Melissa; Debnath, Gargi; Moldt, Brian; van Breemen, Mariëlle J; Isik, Gözde; Medina-Ramírez, Max; Back, Jaap Willem; Koff, Wayne C; Julien, Jean-Philippe; Rakasz, Eva G; Seaman, Michael S; Guttman, Miklos; Lee, Kelly K; Klasse, Per Johan; LaBranche, Celia; Schief, William R; Wilson, Ian A; Overbaugh, Julie; Burton, Dennis R; Ward, Andrew B; Montefiori, David C; Dean, Hansi; Moore, John P

    2015-07-10

    A challenge for HIV-1 immunogen design is the difficulty of inducing neutralizing antibodies (NAbs) against neutralization-resistant (tier 2) viruses that dominate human transmissions. We show that a soluble recombinant HIV-1 envelope glycoprotein trimer that adopts a native conformation, BG505 SOSIP.664, induced NAbs potently against the sequence-matched tier 2 virus in rabbits and similar but weaker responses in macaques. The trimer also consistently induced cross-reactive NAbs against more sensitive (tier 1) viruses. Tier 2 NAbs recognized conformational epitopes that differed between animals and in some cases overlapped with those recognized by broadly neutralizing antibodies (bNAbs), whereas tier 1 responses targeted linear V3 epitopes. A second trimer, B41 SOSIP.664, also induced a strong autologous tier 2 NAb response in rabbits. Thus, native-like trimers represent a promising starting point for the development of HIV-1 vaccines aimed at inducing bNAbs.

  9. The Flavivirus Precursor Membrane-Envelope Protein Complex: Structure and Maturation

    SciTech Connect

    Li, Long; Lok, Shee-Mei; Yu, I-Mei; Zhang, Ying; Kuhn, Richard J.; Chen, Jue; Rossmann, Michael G.

    2008-09-17

    Many viruses go through a maturation step in the final stages of assembly before being transmitted to another host. The maturation process of flaviviruses is directed by the proteolytic cleavage of the precursor membrane protein (prM), turning inert virus into infectious particles. We have determined the 2.2 angstrom resolution crystal structure of a recombinant protein in which the dengue virus prM is linked to the envelope glycoprotein E. The structure represents the prM-E heterodimer and fits well into the cryo-electron microscopy density of immature virus at neutral pH. The pr peptide {beta}-barrel structure covers the fusion loop in E, preventing fusion with host cell membranes. The structure provides a basis for identifying the stages of its pH-directed conformational metamorphosis during maturation, ending with release of pr when budding from the host.

  10. Fluctuation Dynamics Analysis of gp120 Envelope Protein Reveals a Topologically Based Communication Network

    PubMed Central

    Shrivastava, Indira; LaLonde, Judith M.

    2012-01-01

    HIV infection is initiated by binding of the viral glycoprotein gp120, to the cellular receptor CD4. Upon CD4 binding, gp120 undergoes conformational change, permitting binding to the chemokine receptor. Crystal structures of gp120 ternary complex reveal the CD4 bound conformation of gp120. We report here the application of Gaussian Network Model (GNM) to the crystal structures of gp120 bound to CD4 or CD4 mimic and 17b, to study the collective motions of the gp120 core and determine the communication propensities of the residue network. The GNM fluctuation profiles identify residues in the inner domain and outer domain that may facilitate conformational change or stability, respectively. Communication propensities delineate a residue network that is topologically suited for signal propagation from the Phe43 cavity throughout the gp120 outer domain. . These results provide a new context for interpreting gp120 core envelope structure-function relationships. PMID:20718047

  11. Uncleaved prefusion-optimized gp140 trimers derived from analysis of HIV-1 envelope metastability

    NASA Astrophysics Data System (ADS)

    Kong, Leopold; He, Linling; de Val, Natalia; Vora, Nemil; Morris, Charles D.; Azadnia, Parisa; Sok, Devin; Zhou, Bin; Burton, Dennis R.; Ward, Andrew B.; Wilson, Ian A.; Zhu, Jiang

    2016-06-01

    The trimeric HIV-1 envelope glycoprotein (Env) is critical for host immune recognition and neutralization. Despite advances in trimer design, the roots of Env trimer metastability remain elusive. Here we investigate the contribution of two Env regions to metastability. First, we computationally redesign a largely disordered bend in heptad region 1 (HR1) of SOSIP trimers that connects the long, central HR1 helix to the fusion peptide, substantially improving the yield of soluble, well-folded trimers. Structural and antigenic analyses of two distinct HR1 redesigns confirm that redesigned Env closely mimics the native, prefusion trimer with a more stable gp41. Next, we replace the cleavage site between gp120 and gp41 with various linkers in the context of an HR1 redesign. Electron microscopy reveals a potential fusion intermediate state for uncleaved trimers containing short but not long linkers. Together, these results outline a general approach for stabilization of Env trimers from diverse HIV-1 strains.

  12. Cryo-EM structure of a native, fully glycosylated, cleaved HIV-1 envelope trimer.

    PubMed

    Lee, Jeong Hyun; Ozorowski, Gabriel; Ward, Andrew B

    2016-03-01

    The envelope glycoprotein trimer (Env) on the surface of HIV-1 recognizes CD4(+) T cells and mediates viral entry. During this process, Env undergoes substantial conformational rearrangements, making it difficult to study in its native state. Soluble stabilized trimers have provided valuable insights into the Env structure, but they lack the hydrophobic membrane proximal external region (MPER, an important target of broadly neutralizing antibodies), the transmembrane domain, and the cytoplasmic tail. Here we present (i) a cryogenic electron microscopy (cryo-EM) structure of a clade B virus Env, which lacks only the cytoplasmic tail and is stabilized by the broadly neutralizing antibody PGT151, at a resolution of 4.2 angstroms and (ii) a reconstruction of this form of Env in complex with PGT151 and MPER-targeting antibody 10E8 at a resolution of 8.8 angstroms. These structures provide new insights into the wild-type Env structure. PMID:26941313

  13. Uncleaved prefusion-optimized gp140 trimers derived from analysis of HIV-1 envelope metastability

    PubMed Central

    Kong, Leopold; He, Linling; de Val, Natalia; Vora, Nemil; Morris, Charles D.; Azadnia, Parisa; Sok, Devin; Zhou, Bin; Burton, Dennis R.; Ward, Andrew B.; Wilson, Ian A.; Zhu, Jiang

    2016-01-01

    The trimeric HIV-1 envelope glycoprotein (Env) is critical for host immune recognition and neutralization. Despite advances in trimer design, the roots of Env trimer metastability remain elusive. Here we investigate the contribution of two Env regions to metastability. First, we computationally redesign a largely disordered bend in heptad region 1 (HR1) of SOSIP trimers that connects the long, central HR1 helix to the fusion peptide, substantially improving the yield of soluble, well-folded trimers. Structural and antigenic analyses of two distinct HR1 redesigns confirm that redesigned Env closely mimics the native, prefusion trimer with a more stable gp41. Next, we replace the cleavage site between gp120 and gp41 with various linkers in the context of an HR1 redesign. Electron microscopy reveals a potential fusion intermediate state for uncleaved trimers containing short but not long linkers. Together, these results outline a general approach for stabilization of Env trimers from diverse HIV-1 strains. PMID:27349805

  14. Immunogenicity of Stabilized HIV-1 Envelope Trimers with Reduced Exposure of Non-neutralizing Epitopes.

    PubMed

    de Taeye, Steven W; Ozorowski, Gabriel; Torrents de la Peña, Alba; Guttman, Miklos; Julien, Jean-Philippe; van den Kerkhof, Tom L G M; Burger, Judith A; Pritchard, Laura K; Pugach, Pavel; Yasmeen, Anila; Crampton, Jordan; Hu, Joyce; Bontjer, Ilja; Torres, Jonathan L; Arendt, Heather; DeStefano, Joanne; Koff, Wayne C; Schuitemaker, Hanneke; Eggink, Dirk; Berkhout, Ben; Dean, Hansi; LaBranche, Celia; Crotty, Shane; Crispin, Max; Montefiori, David C; Klasse, P J; Lee, Kelly K; Moore, John P; Wilson, Ian A; Ward, Andrew B; Sanders, Rogier W

    2015-12-17

    The envelope glycoprotein trimer mediates HIV-1 entry into cells. The trimer is flexible, fluctuating between closed and more open conformations and sometimes sampling the fully open, CD4-bound form. We hypothesized that conformational flexibility and transient exposure of non-neutralizing, immunodominant epitopes could hinder the induction of broadly neutralizing antibodies (bNAbs). We therefore modified soluble Env trimers to stabilize their closed, ground states. The trimer variants were indeed stabilized in the closed conformation, with a reduced ability to undergo receptor-induced conformational changes and a decreased exposure of non-neutralizing V3-directed antibody epitopes. In rabbits, the stabilized trimers induced similar autologous Tier-1B or Tier-2 NAb titers to those elicited by the corresponding wild-type trimers but lower levels of V3-directed Tier-1A NAbs. Stabilized, closed trimers might therefore be useful components of vaccines aimed at inducing bNAbs. PMID:26687358

  15. [NESPRINS--nuclear envelope proteins ensuring integrity].

    PubMed

    Pershina, E G; Morozova, K N; Kiseleva, E V

    2014-01-01

    This review describes the nesprins (nuclear envelope spectrin-repeat proteins), which are recently discovered family of nuclear envelope proteins. These proteins play an important role in maintaining the cellular architecture and establish the link between the nucleus and other sub-cellular compartments. Many tissue-specific diseases including lipodystrophies, hearing loss, cardiac and skeletal myopathies are associated with nesprins mutations. These proteins comprise of multiple tissue specific isoforms which contain spectrin repeats providing interaction of nesprins with other nuclear membrane proteins, cytoskeleton and intranuclear matrix. We summarize recent findings and suggestions about nesprins structural organization and function inside the cell. Human diseases caused by abnormal nesprins expression are also described.

  16. Two distinct trimeric conformations of natively membrane-anchored full-length herpes simplex virus 1 glycoprotein B.

    PubMed

    Zeev-Ben-Mordehai, Tzviya; Vasishtan, Daven; Hernández Durán, Anna; Vollmer, Benjamin; White, Paul; Prasad Pandurangan, Arun; Siebert, C Alistair; Topf, Maya; Grünewald, Kay

    2016-04-12

    Many viruses are enveloped by a lipid bilayer acquired during assembly, which is typically studded with one or two types of glycoproteins. These viral surface proteins act as the primary interface between the virus and the host. Entry of enveloped viruses relies on specialized fusogen proteins to help merge the virus membrane with the host membrane. In the multicomponent herpesvirus fusion machinery, glycoprotein B (gB) acts as this fusogen. Although the structure of the gB ectodomain postfusion conformation has been determined, any other conformations (e.g., prefusion, intermediate conformations) have so far remained elusive, thus restricting efforts to develop antiviral treatments and prophylactic vaccines. Here, we have characterized the full-length herpes simplex virus 1 gB in a native membrane by displaying it on cell-derived vesicles and using electron cryotomography. Alongside the known postfusion conformation, a novel one was identified. Its structure, in the context of the membrane, was determined by subvolume averaging and found to be trimeric like the postfusion conformation, but appeared more condensed. Hierarchical constrained density-fitting of domains unexpectedly revealed the fusion loops in this conformation to be apart and pointing away from the anchoring membrane. This vital observation is a substantial step forward in understanding the complex herpesvirus fusion mechanism, and opens up new opportunities for more targeted intervention of herpesvirus entry. PMID:27035968

  17. Functional characterization of the Sindbis virus E2 glycoprotein by transposon linker-insertion mutagenesis

    SciTech Connect

    Navaratnarajah, Chanakha K.; Kuhn, Richard J. . E-mail: kuhnr@purdue.edu

    2007-06-20

    The glycoprotein envelope of alphaviruses consists of two proteins, E1 and E2. E1 is responsible for fusion and E2 is responsible for receptor binding. An atomic structure is available for E1, but one for E2 has not been reported. In this study, transposon linker-insertion mutagenesis was used to probe the function of different domains of E2. A library of mutants, containing 19 amino acid insertions in the E2 glycoprotein sequence of the prototype alphavirus, Sindbis virus (SINV), was generated. Fifty-seven independent E2 insertions were characterized, of which more than half (67%) gave rise to viable virus. The wild-type-like mutants identify regions that accommodate insertions without perturbing virus production and can be used to insert targeting moieties to direct SINV to specific receptors. The defective and lethal mutants give insight into regions of E2 important for protein stability, transport to the cell membrane, E1-E2 contacts, and receptor binding.

  18. Dynamic Remodeling of the Plastid Envelope Membranes – A Tool for Chloroplast Envelope in vivo Localizations

    PubMed Central

    Breuers, Frederique K. H.; Bräutigam, Andrea; Geimer, Stefan; Welzel, Ulla Y.; Stefano, Giovanni; Renna, Luciana; Brandizzi, Federica; Weber, Andreas P. M.

    2012-01-01

    Two envelope membranes delimit plastids, the defining organelles of plant cells. The inner and outer envelope membranes are unique in their protein and lipid composition. Several studies have attempted to establish the proteome of these two membranes; however, differentiating between them is difficult due to their close proximity. Here, we describe a novel approach to distinguish the localization of proteins between the two membranes using a straightforward approach based on live cell imaging coupled with transient expression. We base our approach on analyses of the distribution of GFP-fusions, which were aimed to verify outer envelope membrane proteomics data. To distinguish between outer envelope and inner envelope protein localization, we used AtTOC64–GFP and AtTIC40–GFP, as respective controls. During our analyses, we observed membrane proliferations and loss of chloroplast shape in conditions of protein over-expression. The morphology of the proliferations varied in correlation with the suborganellar distribution of the over-expressed proteins. In particular, while layers of membranes built up in the inner envelope membrane, the outer envelope formed long extensions into the cytosol. Using electron microscopy, we showed that these extensions were stromules, a dynamic feature of plastids. Since the behavior of the membranes is different and is related to the protein localization, we propose that in vivo studies based on the analysis of morphological differences of the membranes can be used to distinguish between inner and outer envelope localizations of proteins. To demonstrate the applicability of this approach, we demonstrated the localization of AtLACS9 to the outer envelope membrane. We also discuss protein impact on membrane behavior and regulation of protein insertion into membranes, and provide new hypotheses on the formation of stromules. PMID:22645566

  19. Infectious enveloped RNA virus antigenic chimeras.

    PubMed

    London, S D; Schmaljohn, A L; Dalrymple, J M; Rice, C M

    1992-01-01

    Random insertion mutagenesis has been used to construct infectious Sindbis virus structural protein chimeras containing a neutralization epitope from a heterologous virus, Rift Valley fever virus. Insertion sites, permissive for recovery of chimeric viruses with growth properties similar to the parental virus, were found in the virion E2 glycoprotein and the secreted E3 glycoprotein. For the E2 chimeras, the epitope was expressed on the virion surface and stimulated a partially protective immune response to Rift Valley fever virus infection in vivo. Besides providing a possible approach for developing live attenuated vaccine viruses, insertion of peptide ligands into virion surface proteins may ultimately allow targeting of virus infection to specific cell types.

  20. Cell wall O-glycoproteins and N-glycoproteins: aspects of biosynthesis and function.

    PubMed

    Nguema-Ona, Eric; Vicré-Gibouin, Maïté; Gotté, Maxime; Plancot, Barbara; Lerouge, Patrice; Bardor, Muriel; Driouich, Azeddine

    2014-01-01

    Cell wall O-glycoproteins and N-glycoproteins are two types of glycomolecules whose glycans are structurally complex. They are both assembled and modified within the endomembrane system, i.e., the endoplasmic reticulum (ER) and the Golgi apparatus, before their transport to their final locations within or outside the cell. In contrast to extensins (EXTs), the O-glycan chains of arabinogalactan proteins (AGPs) are highly heterogeneous consisting mostly of (i) a short oligo-arabinoside chain of three to four residues, and (ii) a larger β-1,3-linked galactan backbone with β-1,6-linked side chains containing galactose, arabinose and, often, fucose, rhamnose, or glucuronic acid. The fine structure of arabinogalactan chains varies between, and within plant species, and is important for the functional activities of the glycoproteins. With regards to N-glycans, ER-synthesizing events are highly conserved in all eukaryotes studied so far since they are essential for efficient protein folding. In contrast, evolutionary adaptation of N-glycan processing in the Golgi apparatus has given rise to a variety of organism-specific complex structures. Therefore, plant complex-type N-glycans contain specific glyco-epitopes such as core β,2-xylose, core α1,3-fucose residues, and Lewis(a) substitutions on the terminal position of the antenna. Like O-glycans, N-glycans of proteins are essential for their stability and function. Mutants affected in the glycan metabolic pathways have provided valuable information on the role of N-/O-glycoproteins in the control of growth, morphogenesis and adaptation to biotic and abiotic stresses. With regards to O-glycoproteins, only EXTs and AGPs are considered herein. The biosynthesis of these glycoproteins and functional aspects are presented and discussed in this review. PMID:25324850