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Sample records for purified envelope glycoprotein

  1. Immunization of Rabbits with Highly Purified, Soluble, Trimeric Human Immunodeficiency Virus Type 1 Envelope Glycoprotein Induces a Vigorous B Cell Response and Broadly Cross-Reactive Neutralization

    PubMed Central

    Quinnan, Gerald V.; Onabajo, Olusegun; Zhang, Pengfei; Yan, Lianying; Mattapallil, Joseph J.; Zhang, Zhiqiang; Dong, Ming; Lu, Min; Montefiori, David; LaBranche, Celia; Broder, Christopher C.

    2014-01-01

    Previously we described induction of cross-reactive HIV-1 neutralizing antibody responses in rabbits using a soluble HIV-1 gp140 envelope glycoprotein (Env) in an adjuvant containing monophosphoryl lipid A (MPL) and QS21 (AS02A). Here, we compared different forms of the same HIV-1 strain R2 Env for antigenic and biophysical characteristics, and in rabbits characterized the extent of B cell induction for specific antibody expression and secretion and neutralizing responses. The forms of this Env that were produced in and purified from stably transformed 293T cells included a primarily dimeric gp140, a trimeric gp140 appended to a GCN4 trimerization domain (gp140-GCN4), gp140-GCN4 with a 15 amino acid flexible linker between the gp120 and gp41 ectodomain (gp140-GCN4-L), also trimeric, and a gp140 with the flexible linker purified from cell culture supernatants as either dimer (gp140-L(D)) or monomer (gp140-L(M)). Multimeric states of the Env proteins were assessed by native gel electrophoresis and analytical ultracentrifugation. The different forms of gp140 bound broadly cross-reactive neutralizing (BCN) human monoclonal antibodies (mAbs) similarly in ELISA and immunoprecipitation assays. All Envs bound CD4i mAbs in the presence and absence of sCD4, as reported for the R2 Env. Weak neutralization of some strains of HIV-1 was seen after two additional doses in AS02A. Rabbits that were given a seventh dose of gp140-GCN4-L developed BCN responses that were weak to moderate, similar to our previous report. The specificity of these responses did not appear similar to that of any of the known BCN human mAbs. Induction of spleen B cell and plasma cells producing immunoglobulins that bound trimeric gp140-GCN4-L was vigorous, based on ELISpot and flow cytometry analyses. The results demonstrate that highly purified gp140-GCN4-L trimer in adjuvant elicits BCN responses in rabbits accompanied by vigorous B cell induction. PMID:24846288

  2. 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.

  3. 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.

  4. 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.

  5. The HTLV-I envelope glycoproteins: structure and functions.

    PubMed

    Delamarre, L; Rosenberg, A R; Pique, C; Pham, D; Callebaut, I; Dokhélar, M C

    1996-01-01

    The human T-cell lymphotropic virus type I (HTLV-I) envelope has a structural organization shared by all retroviral envelopes, which contain two mature viral glycoproteins deriving from a common precursor: an external surface protein (SU), associated with a transmembrane protein (TM) responsible for anchoring the SU-TM complex at the cell surface or in the viral envelope. Our understanding of the tertiary structure of these proteins is extremely poor. The intracellular maturation follows the normal cellular secretory pathway, resulting in expression of the mature glycoproteins at the cell surface. The five potential N-glycosylation sites are glycosylated. Most mutations artificially introduced into the glycoproteins result in loss of function, mostly due to abnormal intracellular maturation. This probably indicates a very compact structure of these proteins, where the entire structure is involved in correct conformation. Studies using neutralizing antibodies or mutagenesis have defined functional domains in the SU protein, which is responsible for receptor binding. These domains occur throughout the SU glycoprotein. Sequence analysis of the HTLV-I TM predicts a structure, and probably functions, similar to other retrovirus TMs: involvement of this glycoprotein in the different oligomerization steps leading to a fusogenic SU-TM complex and in the fusion process itself. These features remain to be proven, and it is not yet understood why the free HTLV-I viral particle is not infectious. PMID:8797709

  6. Envelope Glycoprotein Trimers as HIV-1 Vaccine Immunogens

    PubMed Central

    Sattentau, Quentin J.

    2013-01-01

    The HIV-1 envelope glycoprotein spike is the target of neutralizing antibody attack, and hence represents the only relevant viral antigen for antibody-based vaccine design. Various approaches have been attempted to recapitulate Env in membrane-anchored and soluble forms, and these will be discussed here in the context of recent successes and challenges still to be overcome. PMID:26344344

  7. Synthesis of cell envelope glycoproteins of Cryptococcus laurentii.

    PubMed

    Schutzbach, John; Ankel, Helmut; Brockhausen, Inka

    2007-05-21

    Fungi of the genus Cryptococcus are encapsulated basidiomycetes that are ubiquitously found in the environment. These organisms infect both lower and higher animals. Human infections that are common in immune-compromised individuals have proven difficult to cure or even control with currently available antimycotics that are quite often toxic to the host. The virulence of Cryptococcus has been linked primarily to its polysaccharide capsule, but also to cell-bound glycoproteins. In this review, we show that Cryptococcus laurentii is an excellent model for studies of polysaccharide and glycoprotein synthesis in the more pathogenic relative C. neoformans. In particular, we will discuss the structure and biosynthesis of O-linked carbohydrates on cell envelope glycoproteins of C. laurentii. These O-linked structures are synthesized by at least four mannosyltransferases, two galactosyltransferases, and at least one xylosyltransferase that have been characterized. These glycosyltransferases have no known homologues in human tissues. Therefore, enzymes involved in the synthesis of cryptococcal glycoproteins, as well as related enzymes involved in capsule synthesis, are potential targets for the development of specific inhibitors for treatment of cryptococcal disease. PMID:17316583

  8. 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

  9. 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.

  10. 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

  11. 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. PMID:26939061

  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. 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.

  14. 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

  15. Interactome analysis of herpes simplex virus 1 envelope glycoprotein H.

    PubMed

    Hirohata, Yoshitaka; Kato, Akihisa; Oyama, Masaaki; Kozuka-Hata, Hiroko; Koyanagi, Naoto; Arii, Jun; Kawaguchi, Yasushi

    2015-06-01

    Herpes simplex virus 1 (HSV-1) envelope glycoprotein H (gH) is important for viral entry into cells and nuclear egress of nucleocapsids. To clarify additional novel roles of gH during HSV-1 replication, host cell proteins that interact with gH were screened for by tandem affinity purification coupled with mass spectrometry-based proteomics in 293T cells transiently expressing gH. This screen identified 123 host cell proteins as potential gH interactors. Of these proteins, general control nonderepressive-1 (GCN1), a trans-acting positive effector of GCN2 kinase that regulates phosphorylation of the α subunit of translation initiation factor 2 (eIF2α), was subsequently confirmed to interact with gH in HSV-1-infected cells. eIF2α phosphorylation is known to downregulate protein synthesis, and various viruses have evolved mechanisms to prevent the accumulation of phosphorylated eIF2α in infected cells. Here, it was shown that GCN1 knockdown reduces phosphorylation of eIF2α in HSV-1-infected cells and that the gH-null mutation increases eIF2α in HSV-1-infected cells, whereas gH overexpression in the absence of other HSV-1 proteins reduces eIF2α phosphorylation. These findings suggest that GCN1 can regulate eIF2α phosphorylation in HSV-1-infected cells and that the GCN1-binding viral partner gH is necessary and sufficient to prevent the accumulation of phosphorylated eIF2α. Our database of 123 host cell proteins potentially interacting with gH will be useful for future studies aimed at unveiling further novel functions of gH and the roles of cellular proteins in HSV-1-infected cells. PMID:25808324

  16. Autoantibodies induced by chimeric cytokine - HIV envelope glycoprotein immunogens

    PubMed Central

    Isik, Gözde; van Montfort, Thijs; Chung, Nancy P.Y.; Moore, John P.; Sanders, Rogier W.

    2014-01-01

    Cytokines are often used as adjuvants to increase the immunogenicity of vaccines as they can improve the immune response and/or direct it into a desired direction. As an alternative to co-delivering antigens and cytokines separately they can be fused into a composite protein, with the advantage that both moieties act on the same immune cells. The HIV-1 envelope glycoprotein (Env) spike, located on the outside of virus particles and the only relevant protein for the induction of neutralizing antibodies (NAbs), is poorly immunogenic. The induction of anti-Env Abs can be improved by coupling Env proteins to co-stimulatory molecules such as a proliferation inducing ligand (APRIL). Here, we evaluated the immunogenicity of chimeric molecules containing uncleaved Env gp140 fused to the species-matched cytokines IL-21 or GM-CSF in rabbits and mice. Each cytokine was either fused to the C-terminus of Env or embedded within Env at the position of the variable loops 1 and 2 (V1V2). The cytokine components of the chimeric Env-GM-CSF and Env-IL-21 molecules were functional in vitro, but none of the Env-cytokine fusion proteins resulted in improved Ab responses in vivo. Both the Env-GM-CSF and the Env-IL-21 molecules induced strong anti-cytokine Ab responses, in both test species. These autoimmune responses were independent of the location of the cytokine in the chimeric Env molecules; in that they were induced by cytokines inserted within the V1V2 of Env or fused to its Ct. The induction of undesired autoimmune responses should be considered when using cytokines as co-stimulatory molecules in fusion proteins. PMID:24729614

  17. Identification and antigenicity of the major envelope glycoprotein of lymphadenopathy-associated virus

    SciTech Connect

    Montagnier, L.; Clavel, F.; Krust, B.; Chamaret, S.; Rey, F.; Barre-Sinoussi, F.; Chermann, J.C.

    1985-07-15

    The major envelope glycoprotein of the causative agent of Acquired Immune Deficiency Syndrome (AIDS) lymphadenopathy-associated virus (LAV) has been identified and characterized. The glycoprotein has an apparent molecular weight of 110,000-120,000 under denaturing conditions in polyacrylamide gel electrophoresis. Upon deglycosylation by a specific endoglycosydase, its size is reduced to 80,000. Cellular precursors of this glycoprotein have been detected with apparent molecular weight of 150,000 and 135,000. Nearly all AIDS and pre-AIDS patients have detectable antibodies against this viral glycoprotein.

  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. Native oligomeric human immunodeficiency virus type 1 envelope glycoprotein elicits diverse monoclonal antibody reactivities.

    PubMed Central

    Earl, P L; Broder, C C; Long, D; Lee, S A; Peterson, J; Chakrabarti, S; Doms, R W; Moss, B

    1994-01-01

    We synthesized and purified a recombinant human immunodeficiency virus type 1 (HIV-1) envelope (Env) glycoprotein, lacking the gp120/gp41 cleavage site as well as the transmembrane domain, that is secreted principally as a stable oligomer. Mice were immunized with separated monomeric and oligomeric HIV-1 Env glycoproteins to analyze the repertoire of antibody responses to the tertiary and quaternary structure of the protein. Hybridomas were generated and assayed for reactivity by immunoprecipitation of nondenatured Env protein. A total of 138 monoclonal antibodies (MAbs) were generated and cloned, 123 of which were derived from seven animals immunized with oligomeric Env. Within this group, a significant response was obtained against the gp41 ectodomain; 49 MAbs recognized epitopes in gp41, 82% of which were conformational. The influence of conformation on gp120 antigenicity was less pronounced, with 40% of the anti-gp120 MAbs binding to conformational epitopes, many of which blocked CD4 binding. Surprisingly, less than 7% of the MAbs derived from mice immunized with oligomeric Env recognized the V3 loop. In addition, MAbs to linear epitopes in the C-terminal domain of gp120 were not obtained, suggesting that this region of the protein may be partially masked in the oligomeric molecule. A total of 15 MAbs were obtained from two mice immunized with monomeric Env. Nearly half of these recognized the V3 loop, suggesting that this region may be a less predominant epitope in the context of oligomeric Env than in monomeric protein. Thus, immunization with oligomeric Env generates a large proportion of antibodies to conformational epitopes in both gp120 and gp41, many of which may be absent from monomeric Env. Images PMID:7512157

  20. B epitopes and selection pressures in feline immunodeficiency virus envelope glycoproteins.

    PubMed Central

    Pancino, G; Chappey, C; Saurin, W; Sonigo, P

    1993-01-01

    In order to map linear B epitopes in feline immunodeficiency virus (FIV) envelope glycoproteins (Env), a random library of FIV Env polypeptides fused to beta-galactosidase and expressed in Escherichia coli was screened by using sera from experimentally FIV-infected cats. We mapped five antibody-binding domains in the surface envelope glycoprotein (SU1 to SU5) and four in the transmembrane envelope glycoprotein (TM1 to TM4). Immunological analysis with 48 serum samples from naturally or experimentally infected cats of diverse origins revealed a broad group reactivity for epitopes SU2, TM2, and TM3, whereas SU3 appeared as strictly type specific. To study selection pressures acting on the identified immunogenic domains, we analyzed structural constraints and distribution of synonymous and nonsynonymous mutations (amino acids unchanged or changed). Two linear B epitopes (SU3 and TM4) appeared to be submitted to positive selection for change, a pattern of evolution predicting their possible involvement in antiviral protection. These experiments provide a pertinent choice of oligopeptides for further analysis of the protective response against FIV envelope glycoproteins, as a model to understand the role of antibody escape in lentiviral persistence and to design feline AIDS vaccines. Images PMID:7678301

  1. Expression and characterization of glycophospholipid-anchored human immunodeficiency virus type 1 envelope glycoproteins.

    PubMed Central

    Salzwedel, K; Johnston, P B; Roberts, S J; Dubay, J W; Hunter, E

    1993-01-01

    Four chimeric human immunodeficiency virus type 1 (HIV-1) env genes were constructed which encoded the extracellular domain of either the wild-type or a cleavage-defective HIV-1 envelope glycoprotein (gp160) fused at one of two different positions in env to a C-terminal glycosyl-phosphatidylinositol (GPI) attachment signal from the mouse Thy-1.1 glycoprotein. All four of the constructs encoded glycoproteins that were efficiently expressed when Rev was supplied in trans, and the two cleavable forms were processed normally to gp120 and a chimeric "gp41." The chimeric glycoproteins, in contrast to the wild-type glycoprotein, could be cleaved from the surface of transfected cells by treatment with phosphatidylinositol-specific phospholipase C, indicating that they were anchored in the plasma membrane by a GPI moiety. These GPI-anchored glycoproteins were transported intracellularly at a rate only slightly lower than that of the full-length HIV-1 glycoprotein and were present on the cell surface in equivalent amounts. Nevertheless, all four glycoproteins were defective in mediating both cell-cell and virus-cell fusion as determined by syncytium formation in COS-1-HeLa-T4 cell mixtures and trans complementation of an env-defective HIV-1 genome. Images PMID:8102410

  2. Enhancement of feline immunodeficiency virus infection after immunization with envelope glycoprotein subunit vaccines.

    PubMed Central

    Siebelink, K H; Tijhaar, E; Huisman, R C; Huisman, W; de Ronde, A; Darby, I H; Francis, M J; Rimmelzwaan, G F; Osterhaus, A D

    1995-01-01

    Cats were immunized three times with different recombinant feline immunodeficiency virus (FIV) candidate vaccines. Recombinant vaccinia virus (rVV)-expressed envelope glycoprotein with (vGR657) or without (vGR657 x 15) the cleavage site and an FIV envelope bacterial fusion protein (beta-Galactosidase-Env) were incorporated into immune-stimulating complexes or adjuvanted with Quil A. Although all immunized cats developed antibodies against the envelope protein, only the cats vaccinated with the rVV-expressed envelope glycoproteins developed antibodies which neutralized FIV infection of Crandell feline kidney cells. These antibodies failed to neutralize infection of thymocytes with a molecularly cloned homologous FIV. After the third immunization the cats were challenged with homologous FIV. Two weeks after challenge the cell-associated viral load proved to be significantly higher in the cats immunized with vGR657 and vGR657 x 15 than in the other cats. The cats immunized with vGR657 and vGR657 x 15 also developed antibodies against the Gag proteins more rapidly than the cats immunized with beta-Galactosidase-Env or the control cats. This suggested that immunization with rVV-expressed glycoprotein of FIV results in enhanced infectivity of FIV. It was shown that the observed enhancement could be transferred to naive cats with plasma collected at the day of challenge. PMID:7745719

  3. The Human Cytomegalovirus-Specific UL1 Gene Encodes a Late-Phase Glycoprotein Incorporated in the Virion Envelope

    PubMed Central

    Shikhagaie, Medya; Mercé-Maldonado, Eva; Isern, Elena; Muntasell, Aura; Albà, M. Mar; López-Botet, Miguel; Hengel, Hartmut

    2012-01-01

    We have investigated the previously uncharacterized human cytomegalovirus (HCMV) UL1 open reading frame (ORF), a member of the rapidly evolving HCMV RL11 family. UL1 is HCMV specific; the absence of UL1 in chimpanzee cytomegalovirus (CCMV) and sequence analysis studies suggest that UL1 may have originated by the duplication of an ancestor gene from the RL11-TRL cluster (TRL11, TRL12, and TRL13). Sequence similarity searches against human immunoglobulin (Ig)-containing proteins revealed that HCMV pUL1 shows significant similarity to the cellular carcinoembryonic antigen-related (CEA) protein family N-terminal Ig domain, which is responsible for CEA ligand recognition. Northern blot analysis revealed that UL1 is transcribed during the late phase of the viral replication cycle in both fibroblast-adapted and endotheliotropic strains of HCMV. We characterized the protein encoded by hemagglutinin (HA)-tagged UL1 in the AD169-derived HB5 background. UL1 is expressed as a 224-amino-acid type I transmembrane glycoprotein which becomes detectable at 48 h postinfection. In infected human fibroblasts, pUL1 colocalized at the cytoplasmic site of virion assembly and secondary envelopment together with TGN-46, a marker for the trans-Golgi network, and viral structural proteins, including the envelope glycoprotein gB and the tegument phosphoprotein pp28. Furthermore, analyses of highly purified AD169 UL1-HA epitope-tagged virions revealed that pUL1 is a novel constituent of the HCMV envelope. Importantly, the deletion of UL1 in HCMV TB40/E resulted in reduced growth in a cell type-specific manner, suggesting that pUL1 may be implicated in regulating HCMV cell tropism. PMID:22345456

  4. Evaluation of glycoproteins purified from adult and larval camel ticks (Hyalomma dromedarii) as a candidate vaccine

    PubMed Central

    El Hakim, Amr E.; Abdel-Shafy, Sobhy; Abouelella, Amira M. K.; Hamed, Ragaa R.

    2011-01-01

    In order to identify antigens that can help prevent camel tick infestations, three major glycoproteins (GLPs) about 97, 66 and 40 kDa in size were purified from adult and larval Egyptian ticks, Hyalomma (H.) dromedarii, using a single-step purification method with Con-A sepharose. The purified GLPs were evaluated as vaccines against camel tick infestation in rabbits. The rabbits received three intramuscular inoculations of GLPs (20 µg/animal) on days 0, 14, and 28. In the immunoblot analysis, Sera from the immunized rabbits recognized the native GLPs and other proteins from larval and adult H. dromedarii ticks along with those from other tick species such as Rhipicephalus sanguineus but not Ornithodoros moubata. The effects of immunity induced by these GLPs were determined by exposing rabbits to adult H. dromedarii ticks. These results demonstrated that GLP immunization led to a slightly decreased reproductive index and significantly reduced rates of egg hatchability. These results demonstrated that immunization with the purified GLPs can provide protection against infestation by H. dromedarii and some other tick species. Further studies are needed to confirm the effectiveness of immunization with GLPs against other tick species. PMID:21897098

  5. 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.

  6. 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. PMID:26611586

  7. 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

  8. Identification of glycosaminoglycan-binding sites within hepatitis C virus envelope glycoprotein E2*.

    PubMed

    Olenina, L V; Kuzmina, T I; Sobolev, B N; Kuraeva, T E; Kolesanova, E F; Archakov, A I

    2005-11-01

    Heparan sulphate is one of the candidate receptors for hepatitis C virus (HCV). Envelope glycoproteins of HCV have been proposed to be responsible for recognition and binding with cell receptors. They are characterized by great genetic polymorphism. In this study the mapping of regions with glycosaminoglycan-binding properties within HCV envelope proteins has been undertaken. We prepared a set of overlapping peptides corresponding to conserved regions of these envelope proteins and analysed them by solid phase heparin-binding assay. The search for established glycosaminoglycan-binding motifs in the HCV envelope proteins showed the absence of the sites corresponding to the glycosaminoglycan-binding patterns in consensus sequence. We identified one highly conserved and two less conserved heparin-binding sequences within the envelope protein E2 based on solid phase assay results. We did not find any differences in binding efficiency of these peptides with heparin, heparan sulphate or dextran sulphate. Our data supported the specific association between HCV envelope protein E2 and cell surface glycosaminoglycans. We hypothesize that identified regions from E2 can contribute to HCV binding to cell surface glycosaminoglycans. PMID:16255759

  9. 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

  10. Virion envelope glycoproteins as epidermiological markers of Venezuelan encephalitis virus isolates.

    PubMed Central

    Wiebe, M E; Scherer, W F

    1980-01-01

    Virion polypeptide compositions of 26 isolates of Venezuelan encephalitis virus were analyzed by a reproducible and comparative technique of discontinuous sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis. Although the molecular weight of the core polypeptide for each isolate was 36,000, numbers and molecular weights of envelope glycoproteins were heterogeneous. Isolates associated with human, but not equine, disease usually had two glycoproteins of 50,000 to 51,000 and 51,000 to 55,000 molecular weight, whereas isolates associated with both human and equine disease usually had an additional, third polypeptide band of either 45,000 to 46,000 or 56,000 to 58,000 molecular weight. The former isolates were in hemagglutination inhibition subtypes I-D, I-E, III, or IV, and the latter were in subtypes I-A, I-B, I-C, or II. Thus virion envelope glycoproteins should be useful markers of Venezuelan encephalitis virus isolates in epidemiological investigations. PMID:7372798

  11. 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.

  12. 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.

  13. Pseudotyping of lentiviral vector with novel vesiculovirus envelope glycoproteins derived from Chandipura and Piry viruses.

    PubMed

    Hu, Shuang; Mohan Kumar, Dipu; Sax, Chelsea; Schuler, Clayton; Akkina, Ramesh

    2016-01-15

    While the envelope glycoprotein of vesicular stomatitis virus (VSV-G) is widely used for pseudotyping of lentiviral vectors, sub-optimal gene transfer into certain cell types and its sensitivity to inactivation by human complement hinders its broader applications. To find alternative candidates, here we evaluated two serologically distinct novel viral envelopes derived from Chandipura (CNV-G) and Piry (PRV-G) vesiculoviruses. Both permitted generation of high titer psuedotyped lentiviral vectors with a capacity for high efficiency gene transfer into various cell types from different species. In human lymphoid and hematopoietic stem cells, their transduction efficiency was significantly lower than that of VSV-G. However, both novel envelopes were found to be more resistant to inactivation by human serum complement compared to VSV-G. Thus CNV-G and PRV-G envelopes can be harnessed for multiple uses in the future based on the cell type that needs to be gene transduced and possibly for in vivo gene transfer. PMID:26650691

  14. A Directed Molecular Evolution Approach to Improved Immunogenicity of the HIV-1 Envelope Glycoprotein

    PubMed Central

    Du, Sean X.; Xu, Li; Zhang, Wenge; Tang, Susan; Boenig, Rebecca I.; Chen, Helen; Mariano, Ellaine B.; Zwick, Michael B.; Parren, Paul W. H. I.; Burton, Dennis R.; Wrin, Terri; Petropoulos, Christos J.; Ballantyne, John A.; Chambers, Michael; Whalen, Robert G.

    2011-01-01

    A prophylactic vaccine is needed to slow the spread of HIV-1 infection. Optimization of the wild-type envelope glycoproteins to create immunogens that can elicit effective neutralizing antibodies is a high priority. Starting with ten genes encoding subtype B HIV-1 gp120 envelope glycoproteins and using in vitro homologous DNA recombination, we created chimeric gp120 variants that were screened for their ability to bind neutralizing monoclonal antibodies. Hundreds of variants were identified with novel antigenic phenotypes that exhibit considerable sequence diversity. Immunization of rabbits with these gp120 variants demonstrated that the majority can induce neutralizing antibodies to HIV-1. One novel variant, called ST-008, induced significantly improved neutralizing antibody responses when assayed against a large panel of primary HIV-1 isolates. Further study of various deletion constructs of ST-008 showed that the enhanced immunogenicity results from a combination of effective DNA priming, an enhanced V3-based response, and an improved response to the constant backbone sequences. PMID:21738594

  15. 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.

  16. Receptor choice determinants in the envelope glycoproteins of amphotropic, xenotropic, and polytropic murine leukemia viruses.

    PubMed Central

    Battini, J L; Heard, J M; Danos, O

    1992-01-01

    The envelope glycoproteins (SU) of mammalian type C retroviruses possess an amino-terminal domain of about 200 residues, which is involved in binding a cell surface receptor. In this domain, highly conserved amino acid sequences are interrupted by two segments of variable length and sequence, VRA and VRB. We have studied the role of these variable regions in receptor recognition and binding by constructing chimeric molecules in which portions of the amino-terminal domains from amphotropic (4070A), xenotropic (NZB), and polytropic (MCF 247) murine leukemia virus SU proteins were permuted. These chimeras, which exchanged either one or two variable regions, were expressed at the surface of replication-defective viral particles by a pseudotyping assay. Wild-type or recombinant env genes were transfected into a cell line producing Moloney murine leukemia virus particles devoid of envelope glycoproteins in which a retrovirus vector genome carrying an Escherichia coli lacZ gene was packaged. The host range and sensitivity to interference of pseudotyped virions were assayed, and we observed which permutations resulted in receptor switch or loss of function. Our results indicate that the determinants of receptor choice are found within the just 120 amino acids of SU proteins. Downstream sequences contribute to the stabilization of the receptor-specific structure. PMID:1310758

  17. Phage-displayed peptides mimicking the discontinuous neutralization sites of puumala Hantavirus envelope glycoproteins.

    PubMed

    Heiskanen, T; Lundkvist, A; Soliymani, R; Koivunen, E; Vaheri, A; Lankinen, H

    1999-09-30

    We selected peptide ligands mimicking the surface structure of discontinuous binding sites of Puumala hantavirus-neutralizing monoclonal antibodies from a random 18-amino acid peptide library containing a disulfide bridge in a fixed position and displayed on a filamentous phage. The varying of selection conditions, either by shortening of the association time or by competitive elution with antigen, was crucial for the selection of peptide inserts that could be aligned with the primary sequences of the envelope glycoproteins G1 and G2. Correspondingly, when the envelope glycoprotein sequences were synthesized as overlapping peptides as spots on membrane, the same site in primary structure was found as with phage display, which corroborates the use of the two methods in mapping of conformational epitopes. Also, epitopes reactive with early-phase sera from Puumala virus infection were defined with the pepspot assay in the amino-terminal region of G1. Similarities of the selected phage clones to a monoclonal antibody-escape mutant site and to a linear early-phase epitope were found. PMID:10502511

  18. Co-assembly of Viral Envelope Glycoproteins Regulates Their Polarized Sorting in Neurons

    PubMed Central

    Mardones, Gonzalo A.; Bonifacino, Juan S.

    2014-01-01

    Newly synthesized envelope glycoproteins of neuroinvasive viruses can be sorted in a polarized manner to the somatodendritic and/or axonal domains of neurons. Although critical for transneuronal spread of viruses, the molecular determinants and interregulation of this process are largely unknown. We studied the polarized sorting of the attachment (NiV-G) and fusion (NiV-F) glycoproteins of Nipah virus (NiV), a paramyxovirus that causes fatal human encephalitis, in rat hippocampal neurons. When expressed individually, NiV-G exhibited a non-polarized distribution, whereas NiV-F was specifically sorted to the somatodendritic domain. Polarized sorting of NiV-F was dependent on interaction of tyrosine-based signals in its cytosolic tail with the clathrin adaptor complex AP-1. Co-expression of NiV-G with NiV-F abolished somatodendritic sorting of NiV-F due to incorporation of NiV-G•NiV-F complexes into axonal transport carriers. We propose that faster biosynthetic transport of unassembled NiV-F allows for its proteolytic activation in the somatodendritic domain prior to association with NiV-G and axonal delivery of NiV-G•NiV-F complexes. Our study reveals how interactions of viral glycoproteins with the host's transport machinery and between themselves regulate their polarized sorting in neurons. PMID:24831812

  19. Utilization of C-C chemokine receptor 5 by the envelope glycoproteins of a pathogenic simian immunodeficiency virus, SIVmac239.

    PubMed Central

    Marcon, L; Choe, H; Martin, K A; Farzan, M; Ponath, P D; Wu, L; Newman, W; Gerard, N; Gerard, C; Sodroski, J

    1997-01-01

    We examined chemokine receptors for the ability to facilitate the infection of CD4-expressing cells by viruses containing the envelope glycoproteins of a pathogenic simian immunodeficiency virus, SIVmac239. Expression of either human or simian C-C chemokine receptor CCR5 allowed the SIVmac239 envelope glycoproteins to mediate virus entry and cell-to-cell fusion. Thus, distantly related immunodeficiency viruses such as SIV and the primary human immunodeficiency virus type 1 isolates can utilize CCR5 as an entry cofactor. PMID:9032394

  20. Human immunodeficiency virus type 1 and 2 envelope glycoproteins oligomerize through conserved sequences.

    PubMed Central

    Center, R J; Kemp, B E; Poumbourios, P

    1997-01-01

    Hetero-oligomerization between human immunodeficiency virus type 2 (HIV-2) envelope glycoprotein (Env) truncation mutants and epitope-tagged gp160 is dependent on the presence of gp41 transmembrane protein (TM) amino acids 552 to 589, a putative amphipathic alpha-helical sequence. HIV-2 Env truncation mutants containing this sequence were also able to form cross-type hetero-oligomers with HIV-1 Env. HIV-2/HIV-1 hetero-oligomerization was, however, more sensitive to disruption by mutagenesis or increased temperature. The conservation of the Env oligomerization function of the HIV-1 and HIV-2 alpha-helical sequences suggests that retroviral TM alpha-helical motifs may have a universal role in oligomerization. PMID:9188654

  1. Virion incorporation of envelope glycoproteins with long but not short cytoplasmic tails is blocked by specific, single amino acid substitutions in the human immunodeficiency virus type 1 matrix.

    PubMed Central

    Freed, E O; Martin, M A

    1995-01-01

    Incorporation of envelope glycoproteins into a budding retrovirus is an essential step in the formation of an infectious virus particle. By using site-directed mutagenesis, we identified specific amino acid residues in the matrix domain of the human immunodeficiency virus type 1 (HIV-1) Gag protein that are critical to the incorporation of HIV-1 envelope glycoproteins into virus particles. Pseudotyping analyses were used to demonstrate that two heterologous envelope glycoproteins with short cytoplasmic tails (the envelope of the amphotropic murine leukemia virus and a naturally truncated HIV-2 envelope) are efficiently incorporated into HIV-1 particles bearing the matrix mutations. Furthermore, deletion of the cytoplasmic tail of HIV-1 transmembrane envelope glycoprotein gp41 from 150 to 7 or 47 residues reversed the incorporation block imposed by the matrix mutations. These results suggest the existence of a specific functional interaction between the HIV-1 matrix and the gp41 cytoplasmic tail. PMID:7853546

  2. 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

  3. 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

  4. 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

  5. [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

  6. Membrane anchoring domain of herpes simplex virus glycoprotein gB is sufficient for nuclear envelope localization.

    PubMed Central

    Gilbert, R; Ghosh, K; Rasile, L; Ghosh, H P

    1994-01-01

    We have used the glycoprotein gB of herpes simplex virus type 1 (gB-1), which buds from the inner nuclear membrane, as a model protein to study localization of membrane proteins in the nuclear envelope. To determine whether specific domains of gB-1 glycoprotein are involved in localization in the nuclear envelope, we have used deletion mutants of gB-1 protein as well as chimeric proteins constructed by replacing the domains of the cell surface glycoprotein G of vesicular stomatitis virus with the corresponding domains of gB. Mutant and chimeric proteins expressed in COS cells were localized by immunoelectron microscopy. A chimeric protein (gB-G) containing the ectodomain of gB and the transmembrane and cytoplasmic domains of G did not localize in the nuclear envelope. When the ectodomain of G was fused to the transmembrane and cytoplasmic domains of gB, however, the resulting chimeric protein (G-gB) was localized in the nuclear envelope. Substitution of the transmembrane domain of G with the 69 hydrophobic amino acids containing the membrane anchoring domain of gB allowed the hybrid protein (G-tmgB) to be localized in the nuclear envelope, suggesting that residues 721 to 795 of gB can promote retention of proteins in the nuclear envelope. Deletion mutations in the hydrophobic region further showed that a transmembrane segment of 21 hydrophobic amino acids, residues 774 to 795 of gB, was sufficient for localization in the nuclear envelope. Since wild-type gB and the mutant and chimeric proteins that were localized in the nuclear envelope were also retained in the endoplasmic reticulum, the membrane spanning segment of gB could also influence retention in the endoplasmic reticulum. Images PMID:8139012

  7. 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. PMID:11482987

  8. Characterization of hepatitis C virus envelope glycoprotein complexes expressed by recombinant vaccinia viruses.

    PubMed

    Ralston, R; Thudium, K; Berger, K; Kuo, C; Gervase, B; Hall, J; Selby, M; Kuo, G; Houghton, M; Choo, Q L

    1993-11-01

    We constructed recombinant vaccinia virus vectors for expression of the structural region of hepatitis C virus (HCV). Infection of mammalian cells with a vector (vv/HCV1-906) encoding C-E1-E2-NS2 generated major protein species of 22 kDa (C), 33 to 35 kDa (E1), and 70 to 72 kDa (E2), as observed previously with other mammalian expression systems. The bulk of the E1 and E2 expressed by vv/HCV1-906 was found integrated into endoplasmic reticulum membranes as core-glycosylated species, suggesting that these E1 and E2 species represent intracellular forms of the HCV envelope proteins. HCV E1 and E2 formed E1-E2 complexes which were precipitated by either anti-E1 or anti-E2 serum and which sedimented at approximately 15 S on glycerol density gradients. No evidence of intermolecular disulfide bonding between E1 and E2 was detected. E1 and E2 were copurified to approximately 90% purity by mild detergent extraction followed by chromatography on Galanthus nivalus lectin-agarose and DEAE-Fractogel. Immunization of chimpanzees with purified E1-E2 generated high titers of anti-E1 and anti-E2 antibodies. Further studies, to be reported separately, demonstrated that purified E1-E2 complexes were recognized at high frequency by HCV+ human sera (D. Y. Chien, Q.-L. Choo, R. Ralston, R. Spaete, M. Tong, M. Houghton, and G. Kuo, Lancet, in press) and generated protective immunity in chimpanzees (Q.-L. Choo, G. Kuo, R. Ralston, A. Weiner, D. Chien, G. Van Nest, J. Han, K. Berger, K. Thudium, J. Kansopon, J. McFarland, A. Tabrizi, K. Ching, B. Mass, L. B. Cummins, E. Muchmore, and M. Houghton, submitted for publication), suggesting that these purified HCV envelope proteins display native HCV epitopes. PMID:8411378

  9. 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

  10. 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

  11. Robust Neutralizing Antibodies Elicited by HIV-1 JRFL Envelope Glycoprotein Trimers in Nonhuman Primates

    PubMed Central

    Feng, Yu; Sharma, Shailendra Kumar; McKee, Krisha; Karlsson Hedestam, Gunilla B.; LaBranche, Celia C.; Montefiori, David C.; Mascola, John R.

    2013-01-01

    Host cell-mediated proteolytic cleavage of the human immunodeficiency virus type 1 (HIV-1) gp160 precursor glycoprotein into gp120 and gp41 subunits is required to generate fusion-competent envelope glycoprotein (Env) spikes. The gp120-directed broadly neutralizing monoclonal antibodies (bNabs) isolated from HIV-infected individuals efficiently recognize fully cleaved JRFL Env spikes; however, nonneutralizing gp120-directed monoclonal antibodies isolated from infected or vaccinated subjects recognize only uncleaved JRFL spikes. Therefore, as an immunogen, cleaved spikes that selectively present desired neutralizing epitopes to B cells may elicit cross-reactive neutralizing antibodies. Accordingly, we inoculated nonhuman primates (NHPs) with plasmid DNA encoding transmembrane-anchored, cleaved JRFL Env or by electroporation (EP). Priming with DNA expressing soluble, uncleaved gp140 trimers was included as a comparative experimental group of NHPs. DNA inoculation was followed by boosts with soluble JRFL gp140 trimers, and control NHPs were inoculated with soluble JRFL protein trimers without DNA priming. In the TZM-bl assay, elicitation of neutralizing antibodies against HIV-1 tier 1 isolates was robust following the protein boost. Neutralization of tier 2 isolates was detected, but only in animals primed with plasmid DNA and boosted with trimeric protein. Using the more sensitive A3R5 assay, consistent neutralization of both clade B and C tier 2 isolates was detected from all regimens assessed in the current study, exceeding levels achieved by our previous vaccine regimens in primates. Together, these data suggest a potential advantage of B cell priming followed by a rest interval and protein boosting to present JRFL Env spikes to the immune system to better generate HIV-1 cross-clade neutralizing antibodies. PMID:24067980

  12. 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

  13. 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.

  14. 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.

  15. Production and characterization of mouse monoclonal antibodies reactive to Chikungunya envelope E2 glycoprotein.

    PubMed

    Bréhin, Anne-Claire; Rubrecht, Laetitia; Navarro-Sanchez, Martha Erika; Maréchal, Valérie; Frenkiel, Marie-Pascale; Lapalud, Priscilla; Laune, Daniel; Sall, Amadou Alpha; Desprès, Philippe

    2008-02-01

    Chikungunya fever is an arbovirosis of major impact in public health in Asia and Africa. Chikungunya (CHIK) virus is member of the genus Alphavirus and belongs to the Semliki Forest (SF) antigenic complex. We describe for the first time a panel of monoclonal antibodies (MAbs) reactive to CHIK envelope E2 glycoprotein. For the screening of E2-specific MAbs, we expressed a recombinant soluble CHIK E2 protein in Drosophila S2 cells. Analyzed by immunological methods, MAbs 3C3, 3E4, and 8A4 were selected on the basis of their reactivity. Their epitopes are located to the outer surface of CHIK virion. These MAbs have no cross reactivity with related members of SF antigenic complex with the notable exception of Igbo-Ora virus. Anti-CHIK E2 MAbs 3C3, 3E4, and 8A4 should be helpful for studying the biology of CHIK virus and pathogenesis of disease. The combination of 8A4 and 3E4 is suitable for developing a specific antigen-capture ELISA. PMID:17949772

  16. 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.

  17. Biochemical evidence of a role for matrix trimerization in HIV-1 envelope glycoprotein incorporation.

    PubMed

    Tedbury, Philip R; Novikova, Mariia; Ablan, Sherimay D; Freed, Eric O

    2016-01-12

    The matrix (MA) domain of HIV Gag has important functions in directing the trafficking of Gag to sites of assembly and mediating the incorporation of the envelope glycoprotein (Env) into assembling particles. HIV-1 MA has been shown to form trimers in vitro; however, neither the presence nor the role of MA trimers has been documented in HIV-1 virions. We developed a cross-linking strategy to reveal MA trimers in virions of replication-competent HIV-1. By mutagenesis of trimer interface residues, we demonstrated a correlation between loss of MA trimerization and loss of Env incorporation. Additionally, we found that truncating the long cytoplasmic tail of Env restores incorporation of Env into MA trimer-defective particles, thus rescuing infectivity. We therefore propose a model whereby MA trimerization is required to form a lattice capable of accommodating the long cytoplasmic tail of HIV-1 Env; in the absence of MA trimerization, Env is sterically excluded from the assembling particle. These findings establish MA trimerization as an obligatory step in the assembly of infectious HIV-1 virions. As such, the MA trimer interface may represent a novel drug target for the development of antiretrovirals. PMID:26711999

  18. Retromer Regulates HIV-1 Envelope Glycoprotein Trafficking and Incorporation into Virions

    PubMed Central

    Groppelli, Elisabetta; Jolly, Clare

    2014-01-01

    The envelope glycoprotein (Env) of the Human Immunodeficiency Virus Type-1 (HIV-1) is a critical determinant of viral infectivity, tropism and is the main target for humoral immunity; however, little is known about the cellular machinery that directs Env trafficking and its incorporation into nascent virions. Here we identify the mammalian retromer complex as a novel and important cellular factor regulating Env trafficking. Retromer mediates endosomal sorting and is most closely associated with endosome-to-Golgi transport. Consistent with this function, inactivating retromer using RNAi targeting the cargo selective trimer complex inhibited retrograde trafficking of endocytosed Env to the Golgi. Notably, in HIV-1 infected cells, inactivating retromer modulated plasma membrane expression of Env, along with Env incorporation into virions and particle infectivity. Mutagenesis studies coupled with coimmunoprecipitations revealed that retromer-mediated trafficking requires the Env cytoplasmic tail that we show binds directly to retromer components Vps35 and Vps26. Taken together these results provide novel insight into regulation of HIV-1 Env trafficking and infectious HIV-1 morphogenesis and show for the first time a role for retromer in the late-steps of viral replication and assembly of a virus. PMID:25393110

  19. A recombinant pseudotyped lentivirus expressing the envelope glycoprotein of Hantaan virus induced protective immunity in mice

    PubMed Central

    2013-01-01

    Background Hantaviruses cause acute hemorrhagic fever with renal syndrome (HFRS). Currently, several types of inactivated HFRS vaccines are widely used, however the limited ability of these immunogen to elicit neutralizing antibodies restricts vaccine efficacy. Development of an effective vaccine to overcome this weakness is must. Methods In the present study, a recombinant pseudotyped lentivirus bearing the hantaan virus (HTNV) envelope glycoproteins (GP), rLV-M, was constructed. C57BL/6 mice were immunized with the rLV-M and a series of immunological assays were conducted to determine the immunogenicity of the recombinant pseudotyped lentivirus. The humoral and cell-mediated immune responses induced by rLV-M were compared with those of the inactivated HFRS vaccine. Results Indirect immunofluorescence assay (IFA) showed the rLV-M expressed target proteins in HEK-293cells. In mice, the rLV-M efficiently induced GP-specific humoral responses and protection against HTNV infection. Furthermore, the rLV-M induced higher neutralizing antibody titers than the inactivated HFRS vaccine control. Conclusions The results indicated the potential of using a pseudotyped lentivirus as a delivery vector for a hantavirus vaccine immunogen. PMID:24093752

  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. Composition and Antigenic Effects of Individual Glycan Sites of a Trimeric HIV-1 Envelope Glycoprotein

    PubMed Central

    Behrens, Anna-Janina; Vasiljevic, Snezana; Pritchard, Laura K.; Harvey, David J.; Andev, Rajinder S.; Krumm, Stefanie A.; Struwe, Weston B.; Cupo, Albert; Kumar, Abhinav; Zitzmann, Nicole; Seabright, Gemma E.; Kramer, Holger B.; Spencer, Daniel I.R.; Royle, Louise; Lee, Jeong Hyun; Klasse, Per J.; Burton, Dennis R.; Wilson, Ian A.; Ward, Andrew B.; Sanders, Rogier W.; Moore, John P.; Doores, Katie J.; Crispin, Max

    2016-01-01

    Summary The HIV-1 envelope glycoprotein trimer is covered by an array of N-linked glycans that shield it from immune surveillance. The high density of glycans on the trimer surface imposes steric constraints limiting the actions of glycan-processing enzymes, so that multiple under-processed structures remain on specific areas. These oligomannose glycans are recognized by broadly neutralizing antibodies (bNAbs) that are not thwarted by the glycan shield but, paradoxically, target it. Our site-specific glycosylation analysis of a soluble, recombinant trimer (BG505 SOSIP.664) maps the extremes of simplicity and diversity of glycan processing at individual sites and reveals a mosaic of dense clusters of oligomannose glycans on the outer domain. Although individual sites usually minimally affect the global integrity of the glycan shield, we identify examples of how deleting some glycans can subtly influence neutralization by bNAbs that bind at distant sites. The network of bNAb-targeted glycans should be preserved on vaccine antigens. PMID:26972002

  2. 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.

  3. 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.

  4. 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

  5. Identification of multiple pregnancy-associated glycoproteins (PAGs) purified from the European bison (Eb; Bison bonasus L.) placentas.

    PubMed

    Kiewisz, J; Melo de Sousa, N; Beckers, J F; Panasiewicz, G; Gizejewski, Z; Szafranska, B

    2009-06-01

    This paper describes the first identified chorionic PAGs in the European bison (Eb), named EbPAGs, predominantly expressed during early and mid-pregnancy (45-120 day post-coitum; dpc). Many EbPAGs were extracted from various cotyledonary tissues, precipitated, chromatographed (DEAE and VVA: Vicia villosa agglutinin), electrophoresed (1D- and 2D-PAGE), analysed by heterologous (cross-species) Western blotting and then micro-sequenced by Edman degradation. Finally, twelve selected VVA-purified isoforms (Ip 3.7-7.4) were entirely characterised. Nine identified NH(2)-terminal micro-sequences were found to be PAGs. On 45 dpc, three identified forms were named: EbPAG(67AkDa) (RGSNLTHPLRNIGDLFYVGN), EbPAG(55BkDa) (RGSNLTHPL) and EbPAG(50CkDa) (SQISLRGSNLTI). On 60 dpc, the next three forms were named: EbPAG(71DkDa) (RGSNLTIHPLRNIIDLFYVG), EbPAG(55EkDa) (RGSNLTHPLRNI) and EbPAG(50FkDa) (SQISLRGS). On 120 dpc, three other forms were named: EbPAG(71GkDa) (RGSNLTHPLRNIRDLFYVG), EbPAG(60HkDa) (RGSNLTTHPLRNIKDLVVYM) and EbPAG(50IkDa) (SGSNLTTV). These EbPAG ((A-I)) sequences are unique, as they are not identical to any other PAGs purified previously in related species of the Bovidae family. However, the EbPAGs (A-I forms) have some sequence resemblance to internal sequences of various full-length polypeptide PAG precursors (in silico translated from cloned cDNAs) identified in domestic cattle. Three other novel native isoforms (J1, J2 and K): EbUPG(45kDa) J1 (SKDNYKNYIPLIVPFAT), EbUPG(45kDa) J2 (SKDNQKNYIPLIVPFAT) and EbUPG(76kDa) K (SPEFTV), were temporarily named 'unknown placental glycoproteins' (UPGs), due to their efficient VVA-purification (specific for glycoproteins only) and a lack of considerable consensus to previously sequenced placental glycoproteins in the Bovidae family. This is the first study identifying NH(2)-terminals of multiple/diverse EbPAGs and some EbUPGs purified from the synepitheliochorial cotyledonary placenta of the endangered Bison bonasus (Red List

  6. 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. PMID:25462351

  7. Human plasmacytoid dendritic cells efficiently capture HIV-1 envelope glycoproteins via CD4 for antigen presentation.

    PubMed

    Sandgren, Kerrie J; Smed-Sörensen, Anna; Forsell, Mattias N; Soldemo, Martina; Adams, William C; Liang, Frank; Perbeck, Leif; Koup, Richard A; Wyatt, Richard T; Karlsson Hedestam, Gunilla B; Loré, Karin

    2013-07-01

    Advances in HIV-1 vaccine clinical trials and preclinical research indicate that the virus envelope glycoproteins (Env) are likely to be an essential component of a prophylactic vaccine. Efficient Ag uptake and presentation by dendritic cells (DCs) is important for strong CD4(+) Th cell responses and the development of effective humoral immune responses. In this study, we examined the capacity of distinct primary human DC subsets to internalize and present recombinant Env to CD4(+) T cells. Consistent with their specific receptor expression, skin DCs bound and internalized Env via C-type lectin receptors, whereas blood DC subsets, including CD1c(+) myeloid DCs, CD123(+) plasmacytoid DCs (PDCs), and CD141(+) DCs exhibited a restricted repertoire of C-type lectin receptors and relied on CD4 for uptake of Env. Despite a generally poor capacity for Ag uptake compared with myeloid DCs, the high expression of CD4 on PDCs allowed them to bind and internalize Env very efficiently. CD4-mediated uptake delivered Env to EEA1(+) endosomes that progressed to Lamp1(+) and MHC class II(+) lysosomes where internalized Env was degraded rapidly. Finally, all three blood DC subsets were able to internalize an Env-CMV pp65 fusion protein via CD4 and stimulate pp65-specific CD4(+) T cells. Thus, in the in vitro systems described in this paper, CD4-mediated uptake of Env is a functional pathway leading to Ag presentation, and this may therefore be a mechanism used by blood DCs, including PDCs, for generating immune responses to Env-based vaccines. PMID:23729440

  8. EXPRESSION SYSTEM-DEPENDENT MODULATION OF HIV-1 ENVELOPE GLYCOPROTEIN ANTIGENICITY AND IMMUNOGENICITY

    PubMed Central

    Kong, Leopold; Sheppard, Neil C.; Stewart-Jones, Guillaume B.E.; Robson, Cynthia L.; Chen, Hongying; Xu, Xiaodong; Krashias, George; Bonomelli, Camille; Scanlan, Christopher N.; Kwong, Peter D.; Jeffs, Simon A.; Jones, Ian M.; Sattentau, Quentin J.

    2010-01-01

    Recombinant expression systems differ in the type of glycosylation they impart on expressed antigens such as the Human Immunodeficiency Virus Type-1 (HIV-1) envelope glycoproteins, potentially affecting their biological properties. We performed head-to-head antigenic, immunogenic and molecular profiling of two distantly-related Env surface (gp120) antigens produced in different systems: a) mammalian (293F) cells in the presence of kifunensine which impart only high mannose glycans; b) insect (Spodoptera frugiperda, Sf9) cells, which confer mainly paucimannosidic glycans; c) Sf9 cells recombinant for mammalian glycosylation enzymes (Sf9 Mimic™), which impart high mannose, hybrid and complex glycans without sialic acid; d) 293F cells, which impart high mannose, hybrid and complex glycans with sialic acid. Molecular models revealed a significant difference in gp120 glycan coverage between the Sf9- and wild-type mammalian cell-derived material that is predicted to impact upon ligand binding sites proximal to glycans. Modelling of solvent-exposed surface electrostatic potentials showed that sialic acid imparts a significant negative surface charge that may influence gp120 antigenicity and immunogenicity. Gp120 expressed in systems that do not incorporate sialic acid displayed increased ligand binding to the CD4-binding and CD4–induced sites compared to those expressed in the system that does, and imparted other more subtle differences in antigenicity in a gp120 subtype-specific manner. Non-sialic acid-containing gp120 was significantly more immunogenic than the sialyated version when administered in two different adjuvants, and induced higher titres of antibodies competing for CD4 binding site ligand-gp120 interaction. These findings suggest that non-sialic acid imparting systems yield gp120 immunogens with modified antigenic and immunogenic properties, considerations which should be considered when selecting expression systems for glycosylated antigens to be used

  9. Comparative Immunogenicity of Evolved V1V2-Deleted HIV-1 Envelope Glycoprotein Trimers

    PubMed Central

    Tong, Tommy; van Montfort, Thijs; Eggink, Dirk; Montefiori, David; Olson, William C.; Moore, John P.; Binley, James M.; Berkhout, Ben; Sanders, Rogier W.

    2013-01-01

    Despite almost 30 years of research, no effective vaccine has yet been developed against HIV-1. Probably such a vaccine would need to induce both an effective T cell and antibody response. Any vaccine component focused on inducing humoral immunity requires the HIV-1 envelope (Env) glycoprotein complex as it is the only viral protein exposed on the virion surface. HIV-1 has evolved several mechanisms to evade broadly reactive neutralizing antibodies. One such a mechanism involves variable loop domains, which are highly flexible structures that shield the underlying conserved epitopes. We hypothesized that removal of such loops would increase the exposure and immunogenicity of these conserved regions. Env variable loop deletion however often leads to protein misfolding and aggregation because hydrophobic patches becoming solvent accessible. We have therefore previously used virus evolution to acquire functional Env proteins lacking the V1V2 loop. We then expressed them in soluble (uncleaved) gp140 forms. Three mutants were found to perform optimally in terms of protein expression, stability, trimerization and folding. In this study, we characterized the immune responses to these antigens in rabbits. The V1V2 deletion mutant ΔV1V2.9.VK induced a prominent response directed to epitopes that are not fully available on the other Env proteins tested but that effectively bound and neutralized the ΔV1V2 Env virus. This Env variant also induced more efficient neutralization of the tier 1 virus SF162. The immune refocusing effect was lost after booster immunization with a full-length gp140 protein with intact V1V2 loops. Collectively, this result suggests that deletion of variable domains could alter the specificity of the humoral immune response, but did not result in broad neutralization of neutralization-resistant virus isolates. PMID:23840716

  10. 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

  11. 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

  12. 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.

  13. 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

  14. 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

  15. 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

  16. 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

  17. 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.

  18. Identification of Two Intracellular Mechanisms Leading to Reduced Expression of Oncoretrovirus Envelope Glycoproteins at the Cell Surface

    PubMed Central

    Grange, Marie-Pierre; Blot, Vincent; Delamarre, Lelia; Bouchaert, Isabelle; Rocca, Anna; Dautry-Varsat, Alice; Dokhélar, Marie-Christine

    2000-01-01

    All retrovirus glycoproteins have a cytoplasmic domain that plays several roles in virus replication. We have determined whether and how the cytoplasmic domains of oncoretrovirus glycoproteins modulate their intracellular trafficking, by using chimeric proteins that combined the α-chain of the interleukin-2 receptor with the glycoprotein cytoplasmic domains of five oncoretroviruses: human T-cell leukemia virus type 1 (HTLV-1), Rous sarcoma virus (RSV), bovine leukemia virus (BLV), murine leukemia virus (MuLV), and Mason-Pfizer monkey virus (MPMV). All of these proteins were synthesized and matured in the same way as a control protein with no retrovirus cytoplasmic domain. However, the amounts of all chimeric proteins at the cell surface were smaller than that of the control protein. The protein appearing at and leaving the cell surface and endocytosis were measured in stable transfectants expressing the chimera. We identified two groups of proteins which followed distinct intracellular pathways. Group 1 included chimeric proteins that reached the cell surface normally but were rapidly endocytosed afterwards. This group included the chimeric proteins with HTLV-1, RSV, and BLV cytoplasmic domains. Group 2 included chimeric proteins that were not detected at the cell surface, despite normal intracellular concentrations, and were accumulated in the Golgi complex. This group included the chimeric proteins with MuLV and MPMV cytoplasmic domains. Finally, we verified that the MuLV envelope glycoproteins behaved in the same way as the corresponding chimeras. These results indicate that retroviruses have evolved two distinct mechanisms to ensure a similar biological feature: low concentrations of their glycoproteins at the cell surface. PMID:11090173

  19. Mutational Analysis of the Candidate Internal Fusion Peptide of the Avian Leukosis and Sarcoma Virus Subgroup A Envelope Glycoprotein

    PubMed Central

    Hernandez, Lorraine D.; White, Judith M.

    1998-01-01

    The transmembrane subunit (TM) of the avian leukosis and sarcoma virus (ALSV) envelope glycoprotein (Env) contains a stretch of conserved hydrophobic amino acids internal to its amino terminus (residues 21 to 42). By analogy with similar sequences in other viral envelope glycoproteins, this region has been proposed to be a fusion peptide. We investigated the role of this region by changing each of three hydrophobic residues (Ile-21, Val-30, and Ile-39) to glutamatic acid and lysine in the ALSV subgroup A Env. Like wild-type (wt) Env, all six mutant Env proteins were proteolytically processed, oligomerized, and expressed at the cell surface in a form that bound Tva, the ALSV subgroup A receptor. Like wt Env, Ile21Glu, Ile21Lys, Val30Glu, and Val30Lys changed conformation upon binding Tva, as assayed by sensitivity to thermolysin. Ile39Glu and Ile39Lys were cleaved by thermolysin in both the absence and presence of Tva. Although incorporated into virus particles at approximately equal levels, all mutant Envs were compromised in their ability to support infection. The mutants at residues 21 and 30 showed levels of infection 2 to 3 orders of magnitude lower than that of wt Env. The mutants at residue 39 were noninfectious. Furthermore, none of the mutants displayed activity in a cell-cell fusion assay. Our results support the contention that residues 21 to 42 of ALSV subgroup A Env constitute its fusion peptide. PMID:9525653

  20. 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

  1. Click labeling of unnatural sugars metabolically incorporated into viral envelope glycoproteins enables visualization of single particle fusion.

    PubMed

    Oum, Yoon Hyeun; Desai, Tanay M; Marin, Mariana; Melikyan, Gregory B

    2016-07-01

    Enveloped viruses infect target cells by fusing their membrane with cellular membrane through a process that is mediated by specialized viral glycoproteins. The inefficient and highly asynchronous nature of viral fusion complicates studies of virus entry on a population level. Single virus imaging in living cells has become an important tool for delineating the entry pathways and for mechanistic studies of viral fusion. We have previously demonstrated that incorporation of fluorescent labels into the viral membrane and trapping fluorescent proteins in the virus interior enables the visualization of single virus fusion in living cells. Here, we implement a new approach to non-invasively label the viral membrane glycoproteins through metabolic incorporation of unnatural sugars followed by click-reaction with organic fluorescent dyes. This approach allows for efficient labeling of diverse viral fusion glycoproteins on the surface of HIV pseudoviruses. Incorporation of a content marker into surface-labeled viral particles enables sensitive detection of single virus fusion with live cells. PMID:27033181

  2. 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.

  3. 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.

  4. The Sheep Tetherin Paralog oBST2B Blocks Envelope Glycoprotein Incorporation into Nascent Retroviral Virions

    PubMed Central

    Murphy, Lita; Varela, Mariana; Desloire, Sophie; Ftaich, Najate; Murgia, Claudio; Golder, Matthew; Neil, Stuart; Spencer, Thomas E.; Wootton, Sarah K.; Lavillette, Dimitri; Terzian, Christophe; Palmarini, Massimo

    2014-01-01

    ABSTRACT Bone marrow stromal cell antigen 2 (BST2) is a cellular restriction factor with a broad antiviral activity. In sheep, the BST2 gene is duplicated into two paralogs termed oBST2A and oBST2B. oBST2A impedes viral exit of the Jaagsiekte sheep retroviruses (JSRV), most probably by retaining virions at the cell membrane, similar to the “tethering” mechanism exerted by human BST2. In this study, we provide evidence that unlike oBST2A, oBST2B is limited to the Golgi apparatus and disrupts JSRV envelope (Env) trafficking by sequestering it. In turn, oBST2B leads to a reduction in Env incorporation into viral particles, which ultimately results in the release of virions that are less infectious. Furthermore, the activity of oBST2B does not seem to be restricted to retroviruses, as it also acts on vesicular stomatitis virus glycoproteins. Therefore, we suggest that oBST2B exerts antiviral activity using a mechanism distinct from the classical tethering restriction observed for oBST2A. IMPORTANCE BST2 is a powerful cellular restriction factor against a wide range of enveloped viruses. Sheep possess two paralogs of the BST2 gene called oBST2A and oBST2B. JSRV, the causative agent of a transmissible lung cancer of sheep, is known to be restricted by oBST2A. In this study, we show that unlike oBST2A, oBST2B impairs the normal cellular trafficking of JSRV envelope glycoproteins by sequestering them within the Golgi apparatus. We also show that oBST2B decreases the incorporation of envelope glycoprotein into JSRV viral particles, which in turn reduces virion infectivity. In conclusion, oBST2B exerts a novel antiviral activity that is distinct from those of BST2 proteins of other species. PMID:25339764

  5. Hepatitis C Virus (HCV) Envelope Glycoproteins E1 and E2 Contain Reduced Cysteine Residues Essential for Virus Entry*

    PubMed Central

    Fraser, Johanna; Boo, Irene; Poumbourios, Pantelis; Drummer, Heidi E.

    2011-01-01

    The HCV envelope glycoproteins E1 and E2 contain eight and 18 highly conserved cysteine residues, respectively. Here, we examined the oxidation state of E1E2 heterodimers incorporated into retroviral pseudotyped particles (HCVpp) and investigated the significance of free sulfhydryl groups in cell culture-derived HCV (HCVcc) and HCVpp entry. Alkylation of free sulfhydryl groups on HCVcc/pp with a membrane-impermeable sulfhydryl-alkylating reagent 4-(N-maleimido)benzyl-α-trimethylammonium iodide (M135) prior to virus attachment to cells abolished infectivity in a dose-dependent manner. Labeling of HCVpp envelope proteins with EZ-Link maleimide-PEG2-biotin (maleimide-biotin) detected free thiol groups in both E1 and E2. Unlike retroviruses that employ disulfide reduction to facilitate virus entry, the infectivity of alkylated HCVcc could not be rescued by addition of exogenous reducing agents. Furthermore, the infectivity of HCVcc bound to target cells was not affected by addition of M135 indicative of a change in glycoprotein oxidation state from reduced to oxidized following virus attachment to cells. By contrast, HCVpp entry was reduced by 61% when treated with M135 immediately following attachment to cells, suggesting that the two model systems might demonstrate variations in oxidation kinetics. Glycoprotein oxidation was not altered following binding of HCVpp incorporated E1E2 to soluble heparin or recombinant CD81. These results suggest that HCV entry is dependent on the presence of free thiol groups in E1 and E2 prior to cellular attachment and reveals a new essential component of the HCV entry process. PMID:21768113

  6. Hepatitis C virus (HCV) envelope glycoproteins E1 and E2 contain reduced cysteine residues essential for virus entry.

    PubMed

    Fraser, Johanna; Boo, Irene; Poumbourios, Pantelis; Drummer, Heidi E

    2011-09-16

    The HCV envelope glycoproteins E1 and E2 contain eight and 18 highly conserved cysteine residues, respectively. Here, we examined the oxidation state of E1E2 heterodimers incorporated into retroviral pseudotyped particles (HCVpp) and investigated the significance of free sulfhydryl groups in cell culture-derived HCV (HCVcc) and HCVpp entry. Alkylation of free sulfhydryl groups on HCVcc/pp with a membrane-impermeable sulfhydryl-alkylating reagent 4-(N-maleimido)benzyl-α-trimethylammonium iodide (M135) prior to virus attachment to cells abolished infectivity in a dose-dependent manner. Labeling of HCVpp envelope proteins with EZ-Link maleimide-PEG2-biotin (maleimide-biotin) detected free thiol groups in both E1 and E2. Unlike retroviruses that employ disulfide reduction to facilitate virus entry, the infectivity of alkylated HCVcc could not be rescued by addition of exogenous reducing agents. Furthermore, the infectivity of HCVcc bound to target cells was not affected by addition of M135 indicative of a change in glycoprotein oxidation state from reduced to oxidized following virus attachment to cells. By contrast, HCVpp entry was reduced by 61% when treated with M135 immediately following attachment to cells, suggesting that the two model systems might demonstrate variations in oxidation kinetics. Glycoprotein oxidation was not altered following binding of HCVpp incorporated E1E2 to soluble heparin or recombinant CD81. These results suggest that HCV entry is dependent on the presence of free thiol groups in E1 and E2 prior to cellular attachment and reveals a new essential component of the HCV entry process. PMID:21768113

  7. 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

  8. 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.

  9. Production of Hepatitis C Virus Lacking the Envelope-Encoding Genes for Single-Cycle Infection by Providing Homologous Envelope Proteins or Vesicular Stomatitis Virus Glycoproteins in trans ▿ †

    PubMed Central

    Li, Rui; Qin, Yan; He, Ying; Tao, Wanyin; Zhang, Nan; Tsai, Cheguo; Zhou, Paul; Zhong, Jin

    2011-01-01

    Hepatitis C virus (HCV) infection is a major worldwide health problem. The envelope glycoproteins are the major components of viral particles. Here we developed a trans-complementation system that allows the production of infectious HCV particles in whose genome the regions encoding envelope proteins are deleted (HCVΔE). The lack of envelope proteins could be efficiently complemented by the expression of homologous envelope proteins in trans. HCVΔE production could be enhanced significantly by previously described adaptive mutations in NS3 and NS5A. Moreover, HCVΔE could be propagated and passaged in packaging cells stably expressing HCV envelope proteins, resulting in only single-round infection in wild-type cells. Interestingly, we found that vesicular stomatitis virus (VSV) glycoproteins could efficiently rescue the production of HCV lacking endogenous envelope proteins, which no longer required apolipoprotein E for virus production. VSV glycoprotein-mediated viral entry could allow for the bypass of the natural HCV entry process and the delivery of HCV replicon RNA into HCV receptor-deficient cells. Our development provides a new tool for the production of single-cycle infectious HCV particles, which should be useful for studying individual steps of the HCV life cycle and may also provide a new strategy for HCV vaccine development. PMID:21159872

  10. Determinants of the Bovine Leukemia Virus Envelope Glycoproteins Involved in Infectivity, Replication and Pathogenesis.

    PubMed

    de Brogniez, Alix; Mast, Jan; Willems, Luc

    2016-01-01

    Interaction of viral envelope proteins with host cell membranes has been extensively investigated in a number of systems. However, the biological relevance of these interactions in vivo has been hampered by the absence of adequate animal models. Reverse genetics using the bovine leukemia virus (BLV) genome highlighted important functional domains of the envelope protein involved in the viral life cycle. For example, immunoreceptor tyrosine-based activation motifs (ITAM) of the envelope transmembrane protein (TM) are essential determinants of infection. Although cell fusion directed by the aminoterminal end of TM is postulated to be essential, some proviruses expressing fusion-deficient envelope proteins unexpectedly replicate at wild-type levels. Surprisingly also, a conserved N-linked glycosylation site of the extracellular envelope protein (SU) inhibits cell-to-cell transmission suggesting that infectious potential has been limited during evolution. In this review, we summarize the knowledge pertaining to the BLV envelope protein in the context of viral infection, replication and pathogenesis. PMID:27023592

  11. Determinants of the Bovine Leukemia Virus Envelope Glycoproteins Involved in Infectivity, Replication and Pathogenesis

    PubMed Central

    de Brogniez, Alix; Mast, Jan; Willems, Luc

    2016-01-01

    Interaction of viral envelope proteins with host cell membranes has been extensively investigated in a number of systems. However, the biological relevance of these interactions in vivo has been hampered by the absence of adequate animal models. Reverse genetics using the bovine leukemia virus (BLV) genome highlighted important functional domains of the envelope protein involved in the viral life cycle. For example, immunoreceptor tyrosine-based activation motifs (ITAM) of the envelope transmembrane protein (TM) are essential determinants of infection. Although cell fusion directed by the aminoterminal end of TM is postulated to be essential, some proviruses expressing fusion-deficient envelope proteins unexpectedly replicate at wild-type levels. Surprisingly also, a conserved N-linked glycosylation site of the extracellular envelope protein (SU) inhibits cell-to-cell transmission suggesting that infectious potential has been limited during evolution. In this review, we summarize the knowledge pertaining to the BLV envelope protein in the context of viral infection, replication and pathogenesis. PMID:27023592

  12. 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

  13. 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

  14. Mutants of the Rous sarcoma virus envelope glycoprotein that lack the transmembrane anchor and cytoplasmic domains: analysis of intracellular transport and assembly into virions.

    PubMed Central

    Perez, L G; Davis, G L; Hunter, E

    1987-01-01

    The envelope glycoprotein complex of Rous sarcoma virus consists of a knoblike, receptor-binding gp85 polypeptide that is linked through disulfide bonds to a membrane-spanning gp37 spike. We used oligonucleotide-directed mutagenesis to assess the role of the hydrophobic transmembrane region and hydrophilic cytoplasmic domain of gp37 in intracellular transport and assembly into virions. Early termination codons were introduced on either side of the hydrophobic transmembrane region, and the mutated env genes were expressed from the late promoter of simian virus 40. This resulted in the synthesis of glycoprotein complexes composed of a normal gp85 and a truncated gp37 molecule that lacked the cytoplasmic domain alone or both the cytoplasmic and transmembrane domains. The biosynthesis and intracellular transport of the truncated proteins were not significantly different from those of the wild-type glycoproteins, suggesting that any protein signals for biosynthesis and intracellular transport of this viral glycoprotein complex must reside in its extracellular domain. The glycoprotein complex lacking the cytoplasmic domain of gp37 is stably expressed on the cell surface in a manner similar to that of the wild type. In contrast, the complex lacking both the transmembrane and cytoplasmic domains is secreted as a soluble molecule into the media. It can be concluded, therefore, that the transmembrane domain alone is essential for anchoring the RSV env complex in the cell membrane and that the cytoplasmic domain is not required for anchor function. Insertion of the mutated genes into an infectious proviral genome allowed us to assess the ability of the truncated gene products to be assembled into virions and to determine whether such virions were infectious. Viral genomes encoding the secreted glycoprotein were noninfectious, whereas those encoding a glycoprotein complex lacking only the cytoplasmic domain of gp37 were infectious. Virions produced from these mutant

  15. Enhanced Central Nervous System Transduction with Lentiviral Vectors Pseudotyped with RVG/HIV-1gp41 Chimeric Envelope Glycoproteins

    PubMed Central

    Trabalza, Antonio; Eleftheriadou, Ioanna; Sgourou, Argyro; Liao, Ting-Yi; Patsali, Petros; Lee, Heyne

    2014-01-01

    ABSTRACT To investigate the potential benefits which may arise from pseudotyping the HIV-1 lentiviral vector with its homologous gp41 envelope glycoprotein (GP) cytoplasmic tail (CT), we created chimeric RVG/HIV-1gp41 GPs composed of the extracellular and transmembrane sequences of RVG and either the full-length gp41 CT or C terminus gp41 truncations sequentially removing existing conserved motifs. Lentiviruses (LVs) pseudotyped with the chimeric GPs were evaluated in terms of particle release (physical titer), biological titers, infectivity, and in vivo central nervous system (CNS) transduction. We report here that LVs carrying shorter CTs expressed higher levels of envelope GP and showed a higher average infectivity than those bearing full-length GPs. Interestingly, complete removal of GP CT led to vectors with the highest transduction efficiency. Removal of all C-terminal gp41 CT conserved motifs, leaving just 17 amino acids (aa), appeared to preserve infectivity and resulted in a significantly increased physical titer. Furthermore, incorporation of these 17 aa in the RVG CT notably enhanced the physical titer. In vivo stereotaxic delivery of LV vectors exhibiting the best in vitro titers into rodent striatum facilitated efficient transduction of the CNS at the site of injection. A particular observation was the improved retrograde transduction of neurons in connected distal sites that resulted from the chimeric envelope R5 which included the “Kennedy” sequence (Ken) and lentivirus lytic peptide 2 (LLP2) conserved motifs in the CT, and although it did not exhibit a comparable high titer upon pseudotyping, it led to a significant increase in distal retrograde transduction of neurons. IMPORTANCE In this study, we have produced novel chimeric envelopes bearing the extracellular domain of rabies fused to the cytoplasmic tail (CT) of gp41 and pseudotyped lentiviral vectors with them. Here we report novel effects on the transduction efficiency and physical titer

  16. Comparative evaluation of purified Taenia solium glycoproteins and crude metacestode extracts by immunoblotting for the serodiagnosis of human T. solium cysticercosis.

    PubMed Central

    Rodriguez-Canul, R; Allan, J C; Fletes, C; Sutisna, I P; Kapti, I N; Craig, P S

    1997-01-01

    A lentil-lectin purified glycoprotein (LL-Gp) and a crude saline extract of Taenia solium metacestodes were compared for the immunodiagnosis of human cysticercosis by immunoblotting. The LL-Gp preparation was 95% sensitive for antibodies against a range of seven antigens with molecular masses of 50 to 13 kDa, whereas the sensitivity of the crude saline extract for the detection of antibodies against two major polypeptide molecules (26 and 8 kDa) was 91%. Specificity was 100% with both sets of diagnostic antigens. Affinity-purified antibodies against the 26-kDa molecule from the crude saline extract recognized the 24-kDa diagnostic region in the LL-Gp-purified extract and vice versa, suggesting that the antigens had common epitopes recognized by cysticercotic sera. In addition, in a preliminary community study of 115 randomly selected people from Bali (Indonesia), seroprevalence by immunoblot assay varied from 7.8% (with the crude saline antigen extract) to 9.6% (with the LL-Gp-purified extract). The results of this study demonstrate that both antigenic preparations are applicable for the immunodiagnosis of T. solium cysticercosis. The crude T. solium metacestode antigen extract was as specific as the purified LL-Gp T. solium metacestode extract and simpler to produce but slightly less sensitive. PMID:9302208

  17. Importance of the intracytoplasmic domain of the simian immunodeficiency virus (SIV) envelope glycoprotein for pathogenesis.

    PubMed

    Luciw, P A; Shaw, K E; Shacklett, B L; Marthas, M L

    1998-12-01

    SIVmac1A11 and SIVmac239 are nonpathogenic and pathogenic molecular clones in rhesus macaques, respectively. Although these viruses exhibit approximately 98% nucleotide and amino acid sequence homology, differences are found in the length of the translation frames for several genes. SIVmac239 has a premature stop codon in nef, whereas SIVmac1A11 has a premature stop codon in vpr and two premature stop codons in the intracytoplasmic domain of the env-transmembrane (TM) subunit. Recombinant viruses, constructed through reciprocal exchange of large DNA restriction enzyme fragments between SIVmac1A11 and SIVmac239, were evaluated in adult rhesus macaques. This in vivo analysis revealed that two or more regions of the SIVmac genome were essential for high virus load and disease progression (Marthas et al., 1993. J. Virol. 67, 6047-6055). An important gap in knowledge remaining from this study was whether the premature stop codons in env-TM of recombinant virus SIV1A11/239gag-env/1A11 (Full-length vpr and nef, two stop codons in env-TM) reverted to coding triplets in vivo. Here, we report that viral sequences in macaques, which succumbed to an AIDS-like disease after infection with SIV1A11/239gag-env/1A11, exhibited reversion of both env-TM stop codons. In addition, antibodies to the intracytoplasmic domain of env-TM were detected in macaques containing revertant virus and showing disease; this finding indicates that this domain of the env glycoprotein was expressed in vivo. Thus selection for viral variants with full-length env-TM demonstrated that the cytoplasmic domain of the SIVmac env glycoprotein plays a role in viral persistence and immunodeficiency in primates. PMID:9875311

  18. 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. PMID:24725935

  19. 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.

  20. 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

  1. 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

  2. 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

  3. Envelope Glycoprotein Internalization Protects Human and Simian Immunodeficiency Virus-Infected Cells from Antibody-Dependent Cell-Mediated Cytotoxicity

    PubMed Central

    von Bredow, Benjamin; Arias, Juan F.; Heyer, Lisa N.; Gardner, Matthew R.; Farzan, Michael; Rakasz, Eva G.

    2015-01-01

    ABSTRACT The cytoplasmic tails of human and simian immunodeficiency virus (HIV and SIV, respectively) envelope glycoproteins contain a highly conserved, membrane-proximal endocytosis motif that prevents the accumulation of Env on the surface of infected cells prior to virus assembly. Using an assay designed to measure the killing of virus-infected cells by antibody-dependent cell-mediated cytotoxicity (ADCC), we show that substitutions in this motif increase the susceptibility of HIV-1- and SIV-infected cells to ADCC in a manner that directly correlates with elevated Env levels on the surface of virus-infected cells. In the case of HIV-1, this effect is additive with a deletion in vpu recently shown to enhance the susceptibility of HIV-1-infected cells to ADCC as a result of tetherin-mediated retention of budding virions on the cell surface. These results reveal a previously unappreciated role for the membrane-proximal endocytosis motif of gp41 in protecting HIV-1- and SIV-infected cells from antibody responses by regulating the amount of Env present on the cell surface. IMPORTANCE This study reveals an unappreciated role for the membrane-proximal endocytosis motif of gp41 in protecting HIV-1- and SIV-infected cells from elimination by Env-specific antibodies. Thus, strategies designed to interfere with this mechanism of Env internalization may improve the efficacy of antibody-based vaccines and antiretroviral therapies designed to enhance the immunological control of HIV-1 replication in chronically infected individuals. PMID:26269175

  4. 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.

  5. 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

  6. 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

  7. Modulation of the effector function of human monocytes for Mycobacterium avium by human immunodeficiency virus-1 envelope glycoprotein gp120.

    PubMed Central

    Shiratsuchi, H; Johnson, J L; Toossi, Z; Ellner, J J

    1994-01-01

    Disseminated Mycobacterium avium infection in AIDS is associated with high tissue burdens (10(9)-10(10) mycobacteria/g tissue) of organism. The basis for the extraordinary susceptibility of AIDS to M. avium infection is unclear. HIV or its constituents may alter mononuclear phagocyte functions resulting in enhanced intracellular M. avium growth. The effects of an envelope glycoprotein (gp120), a transmembrane protein (p121), and core proteins of HIV-1 on M. avium infection of human monocytes were examined. Preculturing monocytes with gp120 inhibited M. avium phagocytosis and consistently enhanced intracellular growth of six M. avium strains. Pretreatment with p121, gag5, or p24 did not inhibit phagocytosis nor enhance intracellular growth of M. avium. Incubation of gp120 with soluble CD4 before addition to monocyte cultures or pretreatment of monocytes with OKT4A abrogated gp120 effects on M. avium phagocytosis and intracellular growth. gp120 also augmented cytokine production by infected monocytes. These results suggest that gp120, but not p121 or core proteins, modulate monocyte phagocytosis and enhance intracellular growth of M. avium at least in part through monocyte CD4 receptors. Direct effects of HIV-1 products may, therefore, contribute to the diathesis of AIDS to develop disseminated M. avium infection and to the extensive replication of the organisms within tissue macrophages. Images PMID:8113420

  8. RSV Capsid Polymorphism Correlates with Polymerization Efficiency and Envelope Glycoprotein Content: Implications that Nucleation Controls Morphogenesis

    PubMed Central

    Butan, Carmen; Winkler, Dennis C.; Heymann, J. Bernard; Craven, Rebecca C.; Steven, Alasdair C.

    2008-01-01

    We used cryo-electron tomography to visualize Rous Sarcoma Virus, the prototypic alpharetrovirus. Its polyprotein Gag assembles into spherical procapsids, concomitant with budding. In maturation, Gag is dissected into its matrix (MA), capsid (CA) and nucleocapsid (NC) moieties. CA reassembles into cores housing the viral RNA and replication enzymes. Evidence suggests that a correctly formed core is essential for infectivity. The virions in our data set range from ~ 105 to ~ 175 nm in diameter. Their cores are highly polymorphic. We observe angular cores, including some that are distinctively “coffin-shaped” for which we propose a novel fullerene geometry; cores with continuous curvature including, rarely, fullerene cones; and tubular cores. Angular cores are the most voluminous and densely packed; tubes and some curved cores contain less material, suggesting incomplete packaging. From the tomograms, we measured the surface areas of cores and hence their contents of CA subunits. From the virion diameters, we estimated their original complements of Gag. We find that RSV virions, like HIV, contain unassembled CA subunits and the fraction of CA that is assembled correlates with core type; angular cores incorporate ~ 80% of the available subunits and open-ended tubes, ~ 30%. The number of glycoprotein spikes is variable (~ 0 to 118) and also correlates with core type; virions with angular cores average 82 spikes, whereas those with tubular cores, 14 spikes. These observations imply that initiation of capsid assembly, in which interactions of spike endodomains with the Gag layer play a role, is a critical determinant of core morphology. PMID:18206161

  9. Feline Tetherin Is Characterized by a Short N-Terminal Region and Is Counteracted by the Feline Immunodeficiency Virus Envelope Glycoprotein

    PubMed Central

    Celestino, Michele; Calistri, Arianna; Del Vecchio, Claudia; Salata, Cristiano; Chiuppesi, Flavia; Pistello, Mauro; Borsetti, Alessandra; Palù, Giorgio

    2012-01-01

    Tetherin (BST2) is the host cell factor that blocks the particle release of some enveloped viruses. Two putative feline tetherin proteins differing at the level of the N-terminal coding region have recently been described and tested for their antiviral activity. By cloning and comparing the two reported feline tetherins (called here cBST2504 and cBST2*) and generating specific derivative mutants, this study provides evidence that feline tetherin has a shorter intracytoplasmic domain than those of other known homologues. The minimal tetherin promoter was identified and assayed for its ability to drive tetherin expression in an alpha interferon-inducible manner. We also demonstrated that cBST2504 is able to dimerize, is localized at the cellular membrane, and impairs human immunodeficiency virus type 1 (HIV-1) particle release, regardless of the presence of the Vpu antagonist accessory protein. While cBST2504 failed to restrict wild-type feline immunodeficiency virus (FIV) egress, FIV mutants, bearing a frameshift at the level of the envelope-encoding region, were potently blocked. The transient expression of the FIV envelope glycoprotein was able to rescue mutant particle release from feline tetherin-positive cells but did not antagonize human BST2 activity. Moreover, cBST2504 was capable of specifically immunoprecipitating the FIV envelope glycoprotein. Finally, cBST2504 also exerted its function on HIV-2 ROD10 and on the simian immunodeficiency virus SIVmac239. Taken together, these results show that feline tetherin does indeed have a short N-terminal region and that the FIV envelope glycoprotein is the predominant factor counteracting tetherin restriction. PMID:22514338

  10. 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

  11. 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.

  12. Variable regions A and B in the envelope glycoproteins of feline leukemia virus subgroup B and amphotropic murine leukemia virus interact with discrete receptor domains.

    PubMed Central

    Tailor, C S; Kabat, D

    1997-01-01

    The surface (SU) envelope glycoproteins of feline leukemia virus subgroup B (FeLV-B) and amphotropic murine leukemia virus (A-MLV) are highly related, even in the variable regions VRA and VRB that have been shown to be required for receptor recognition. However, FeLV-B and A-MLV use different sodium-dependent phosphate symporters, Pit1 and Pit2, respectively, as receptors for infection. Pit1 and Pit2 are predicted to have 10 membrane-spanning domains and five extracellular loops. The close relationship of the retroviral envelopes enabled us to generate pseudotype virions carrying chimeric FeLV-B/A-MLV envelope glycoproteins. We found that some of the pseudotype viruses could not use Pit1 or Pit2 proteins but could efficiently utilize specific chimeric Pit1/Pit2 proteins as receptors. By studying Mus dunni tail fibroblasts expressing chimeric Pit1/Pit2 proteins and pseudotype virions carrying chimeric FeLV-B/A-MLV envelopes, we show that FeLV-B and A-MLV VRA and VRB interact in a modular manner with specific receptor domains. Our results suggest that FeLV-B VRA interacts with Pit1 extracellular loops 4 and 5 and that residues Phe-60 and Pro-61 of FeLV-B VRA are essential for receptor choice. However, this interaction is insufficient for infection, and an additional interaction between FeLV-B VRB and Pit1 loop 2 is essential. Similarly, A-MLV infection requires interaction of A-MLV VRA with Pit2 loops 4 and 5 and VRB with Pit2 loop 2, with residues Tyr-60 and Val-61 of A-MLV VRA being critical for receptor recognition. Together, our results suggest that FeLV-B and A-MLV infections require two major discrete interactions between the viral SU envelope glycoproteins and their respective receptors. We propose a common two-step mechanism for interaction between retroviral envelope glycoproteins and cell surface receptors. PMID:9371598

  13. Monoclonal Antibodies Directed against Conserved Epitopes on the Nucleocapsid Protein and the Major Envelope Glycoprotein of Equine Arteritis Virus

    PubMed Central

    Weiland, Emilie; Bolz, Sylvia; Weiland, Frank; Herbst, Werner; Raamsman, Martin J. B.; Rottier, Peter J. M.; De Vries, Antoine A. F.

    2000-01-01

    We recently developed a highly effective immunization procedure for the generation of monoclonal antibodies (MAbs) directed against the porcine reproductive and respiratory syndrome virus (E. Weiland, M. Wieczorek-Krohmer, D. Kohl, K. K. Conzelmann, and F. Weiland, Vet. Microbiol. 66:171–186, 1999). The same method was used to produce a panel of 16 MAbs specific for the equine arteritis virus (EAV). Ten MAbs were directed against the EAV nucleocapsid (N) protein, and five MAbs recognized the major viral envelope glycoprotein (GL). Two of the EAV GL-specific MAbs and one antibody of unknown specificity neutralized virus infectivity. A comparison of the reactivities of the MAbs with 1 U.S. and 22 newly obtained European field isolates of EAV demonstrated that all N-specific MAbs, the three nonneutralizing anti-GL MAbs, and the weakest neutralizing MAb (MAb E7/d15-c9) recognized conserved epitopes. In contrast, the two MAbs with the highest neutralization titers bound to 17 of 23 (MAb E6/A3) and 10 of 23 (MAb E7/d15-c1) of the field isolates. Ten of the virus isolates reacted with only one of these two MAbs, indicating that they recognized different epitopes. The GL-specific MAbs and the strongly neutralizing MAb of unknown specificity (MAb E6/A3) were used for the selection of neutralization-resistant (NR) virus variants. The observation that the E6/A3-specific NR virus variants were neutralized by MAb E7/d15-c1 and that MAb E6/A3 blocked the infectivity of the E7/d15-c1-specific NR escape mutant confirmed that these antibodies reacted with distinct antigenic sites. Immunoelectron microscopy revealed for the first time that the antigenic determinants recognized by the anti-GL MAbs were localized on the virion surface. Surprisingly, although the immunofluorescence signal obtained with the neutralizing antibodies was relatively weak, they mediated binding of about three times as much gold granules to the viral envelope than the nonneutralizing anti-GL MAbs. PMID

  14. 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

  15. Conformational properties of the third variable loop of HIV-1AD8 envelope glycoprotein in the liganded conditions.

    PubMed

    Takeda, Satoshi; Takizawa, Mari; Miyauchi, Kosuke; Urano, Emiko; Fujino, Masayuki; Murakami, Toshio; Murakami, Tsutomu; Komano, Jun

    2016-06-17

    The conformational dynamics of the HIV-1 envelope glycoprotein gp120 and gp41 (Env) remains poorly understood. Here we examined how the V3 loop conformation is regulated in the liganded state using a panel of recombinant HIV-1NL4-3 clones bearing HIV-1AD8 Env by two experimental approaches, one adopting a monoclonal neutralizing antibody KD-247 (suvizumab) that recognizes the tip of the V3 loop, and the other assessing the function of the V3 loop. A significant positive correlation of the Env-KD-247 binding was detected between the liganded and unliganded conditions. Namely, the mutation D163G located in the V2 loop, which enhances viral susceptibility to KD-247 by 59.4-fold, had little effect on the sCD4-induced increment of the virus-KD-247 binding. By contrast, a virus with the S370N mutation in the C3 region increased the virus-KD-247 binding by 91.4-fold, although it did not influence the KD-247-mediated neutralization. Co-receptor usage and the susceptibility to CCR5 inhibitor Maraviroc were unaffected by D163G and S370N mutations. Collectively, these data suggest that the conformation of the liganded V3-loop of HIV-1AD8 Env is still under regulation of other Env domains aside from the V3 loop, including V2 and C3. Our results give an insight into the structural properties of HIV-1 Env and viral resistance to entry inhibitors by non-V3 loop mutations. PMID:27178216

  16. Preliminary mapping of non-conserved epitopes on envelope glycoprotein E2 of Bovine viral diarrhea virus type 1 and 2.

    PubMed

    Jelsma, H; Loeffen, W L A; van Beuningen, A; van Rijn, P A

    2013-09-27

    Bovine viral diarrhea virus (BVDV) belongs together with Classical swine fever virus (CSFV) and Border disease virus (BDV) to the genus Pestivirus in the Flaviviridae family. BVDV has been subdivided into two different species, BVDV1 and BVDV2 based on phylogenetic analysis. Subsequent characterization of both strains revealed major antigenic differences. Because the envelope glycoprotein E2 is the most immunodominant protein for all pestiviruses, the present study focused on epitope mapping by constructing chimeric BVDV type 1 and 2 E2 genes in expression plasmids. These plasmids with chimeric E2-genes were transfected in SK6 cells and transient expression was studied by immunostaining with a panel of MAbs specific for E2 of BVDV1 or BVDV2, resulting in the localization of type-specific antigenic domains at similar regions. These results indicate that E2 glycoproteins of both BVDV types exhibit a comparable antigenic structure, but with type specific epitopes. In addition, the antigenic resemblance with envelope glycoprotein E2 of Classical swine fever virus is discussed. PMID:23838147

  17. Intracellular distribution of the envelope glycoprotein of human immunodeficiency virus and its role in the production of cytopathic effect in CD4+ and CD4- human cell lines.

    PubMed Central

    Koga, Y; Sasaki, M; Nakamura, K; Kimura, G; Nomoto, K

    1990-01-01

    Human CD4+ and CD4- monocytoid cell lines were transfected with a constructed plasmid that has the envelope gene of human immunodeficiency virus under the transcriptional control of human metallothionein IIA promoter; the transfected cells were then cloned. These CD4+ and CD4- transfectant cell clones, both of which expressed almost the same amount of gp160 after induction with metal ions, were used for ultrastructural analysis of the distribution of the envelope glycoprotein in the cytoplasm. Immunofluorescence microscopy with an anti-envelope glycoprotein monoclonal antibody showed localized distribution of gp160 in the CD4+ cell clone and diffuse distribution of gp160 in the CD4- cell clone. These observations were substantiated by immunoelectron microscopy, in which the aggregated form of gp160 was observed in the cytoplasm of CD4+ cells but was scarce in that of CD4- cells. A notable finding was that the sites corresponding to the nuclear pores were occupied with aggregates of gp160 in CD4+ cells, exhibiting cytopathic effects. Both freeze-fracture and transmission electron microscopy also showed abnormal morphology around the nuclear pores and perinuclear space. These results support the possibility that such gp160 complexes accumulated around the nuclear pores primarily disturb the transportation of many molecules between the nucleus and the cytoplasm, resulting in a cytopathic effect in the CD4+ cell clone. Images PMID:2204721

  18. Antigenic Properties of the Human Immunodeficiency Virus Envelope Glycoprotein Gp120 on Virions Bound to Target Cells

    PubMed Central

    Mengistu, Meron; Ray, Krishanu; Lewis, George K.; DeVico, Anthony L.

    2015-01-01

    The HIV-1 envelope glycoprotein, gp120, undergoes multiple molecular interactions and structural rearrangements during the course of host cell attachment and viral entry, which are being increasingly defined at the atomic level using isolated proteins. In comparison, antigenic markers of these dynamic changes are essentially unknown for single HIV-1 particles bound to target cells. Such markers should indicate how neutralizing and/or non-neutralizing antibodies might interdict infection by either blocking infection or sensitizing host cells for elimination by Fc-mediated effector function. Here we address this deficit by imaging fluorescently labeled CCR5-tropic HIV-1 pseudoviruses using confocal and superresolution microscopy to track the exposure of neutralizing and non-neutralizing epitopes as they appear on single HIV-1 particles bound to target cells. Epitope exposure was followed under conditions permissive or non-permissive for viral entry to delimit changes associated with virion binding from those associated with post-attachment events. We find that a previously unexpected array of gp120 epitopes is exposed rapidly upon target cell binding. This array comprises both neutralizing and non-neutralizing epitopes, the latter being hidden on free virions yet capable of serving as potent targets for Fc-mediated effector function. Under non-permissive conditions for viral entry, both neutralizing and non-neutralizing epitope exposures were relatively static over time for the majority of bound virions. Under entry-permissive conditions, epitope exposure patterns changed over time on subsets of virions that exhibited concurrent variations in virion contents. These studies reveal that bound virions are distinguished by a broad array of both neutralizing and non-neutralizing gp120 epitopes that potentially sensitize a freshly engaged target cell for destruction by Fc-mediated effector function and/or for direct neutralization at a post-binding step. The elucidation of

  19. Optimized and enhanced DNA plasmid vector based in vivo construction of a neutralizing anti-HIV-1 envelope glycoprotein Fab.

    PubMed

    Muthumani, Kar; Flingai, Seleeke; Wise, Megan; Tingey, Colleen; Ugen, Kenneth E; Weiner, David B

    2013-10-01

    Monoclonal antibody preparations have demonstrated considerable clinical utility in the treatment of specific malignancies, as well as inflammatory and infectious diseases. Antibodies are conventionally delivered by passive administration, typically requiring costly large-scale laboratory development and production. Additional limitations include the necessity for repeat administrations, and the length of in vivo potency. Therefore, the development of methods to generate therapeutic antibodies and antibody like molecules in vivo, distinct from an active antigen-based immunization strategy, would have considerable clinical utility. In fact, adeno-associated viral (AAV) vector mediated delivery of immunoglobulin genes with subsequent generation of functional antibodies has recently been developed. As well, anon-viral vector mediated nucleic acid based delivery technology could permit the generation of therapeutic/prophylactic antibodies in vivo, obviating potential safety issues associated with viral vector based gene delivery. This delivery strategy has limitations as well, mainly due to very low in vivo production and expression of protein from the delivered gene. In the study reported here we have constructed an "enhanced and optimized" DNA plasmid technology to generate immunoglobulin heavy and light chains (i.e., Fab fragments) from an established neutralizing anti-HIV envelope glycoprotein monoclonal antibody (VRC01). This "enhanced" DNA (E-DNA) plasmid technology includes codon/RNA optimization, leader sequence utilization, as well as targeted potentiation of delivery and expression of the Fab immunoglobulin genes through use of "adaptive" in vivo electroporation. The results demonstrate that delivery by this method of a single administration of the optimized Fab expressing constructs resulted in generation of Fab molecules in mouse sera possessing high antigen specific binding and HIV neutralization activity for at least 7 d after injection, against diverse

  20. Optimized and enhanced DNA plasmid vector based in vivo construction of a neutralizing anti-HIV-1 envelope glycoprotein Fab

    PubMed Central

    Muthumani, Kar; Flingai, Seleeke; Wise, Megan; Tingey, Colleen; Ugen, Kenneth E; Weiner, David B

    2013-01-01

    Monoclonal antibody preparations have demonstrated considerable clinical utility in the treatment of specific malignancies, as well as inflammatory and infectious diseases. Antibodies are conventionally delivered by passive administration, typically requiring costly large-scale laboratory development and production. Additional limitations include the necessity for repeat administrations, and the length of in vivo potency. Therefore, the development of methods to generate therapeutic antibodies and antibody like molecules in vivo, distinct from an active antigen-based immunization strategy, would have considerable clinical utility. In fact, adeno-associated viral (AAV) vector mediated delivery of immunoglobulin genes with subsequent generation of functional antibodies has recently been developed. As well, anon-viral vector mediated nucleic acid based delivery technology could permit the generation of therapeutic/prophylactic antibodies in vivo, obviating potential safety issues associated with viral vector based gene delivery. This delivery strategy has limitations as well, mainly due to very low in vivo production and expression of protein from the delivered gene. In the study reported here we have constructed an “enhanced and optimized” DNA plasmid technology to generate immunoglobulin heavy and light chains (i.e., Fab fragments) from an established neutralizing anti-HIV envelope glycoprotein monoclonal antibody (VRC01). This “enhanced” DNA (E-DNA) plasmid technology includes codon/RNA optimization, leader sequence utilization, as well as targeted potentiation of delivery and expression of the Fab immunoglobulin genes through use of “adaptive” in vivo electroporation. The results demonstrate that delivery by this method of a single administration of the optimized Fab expressing constructs resulted in generation of Fab molecules in mouse sera possessing high antigen specific binding and HIV neutralization activity for at least 7 d after injection

  1. Enveloped Virus-Like Particle Expression of Human Cytomegalovirus Glycoprotein B Antigen Induces Antibodies with Potent and Broad Neutralizing Activity

    PubMed Central

    Kirchmeier, Marc; Fluckiger, Anne-Catherine; Soare, Catalina; Bozic, Jasminka; Ontsouka, Barthelemy; Ahmed, Tanvir; Diress, Abebaw; Pereira, Lenore; Schödel, Florian; Plotkin, Stanley; Dalba, Charlotte; Klatzmann, David

    2014-01-01

    A prophylactic vaccine to prevent the congenital transmission of human cytomegalovirus (HCMV) in newborns and to reduce life-threatening disease in immunosuppressed recipients of HCMV-infected solid organ transplants is highly desirable. Neutralizing antibodies against HCMV confer significant protection against infection, and glycoprotein B (gB) is a major target of such neutralizing antibodies. However, one shortcoming of past HCMV vaccines may have been their failure to induce high-titer persistent neutralizing antibody responses that prevent the infection of epithelial cells. We used enveloped virus-like particles (eVLPs), in which particles were produced in cells after the expression of murine leukemia virus (MLV) viral matrix protein Gag, to express either full-length CMV gB (gB eVLPs) or the full extracellular domain of CMV gB fused with the transmembrane and cytoplasmic domains from vesicular stomatitis virus (VSV)-G protein (gB-G eVLPs). gB-G-expressing eVLPs induced potent neutralizing antibodies in mice with a much greater propensity toward epithelial cell-neutralizing activity than that induced with soluble recombinant gB protein. An analysis of gB antibody binding titers and T-helper cell responses demonstrated that high neutralizing antibody titers were not simply due to enhanced immunogenicity of the gB-G eVLPs. The cells transiently transfected with gB-G but not gB plasmid formed syncytia, consistent with a prefusion gB conformation like those of infected cells and viral particles. Two of the five gB-G eVLP-induced monoclonal antibodies we examined in detail had neutralizing activities, one of which possessed particularly potent epithelial cell-neutralizing activity. These data differentiate gB-G eVLPs from gB antigens used in the past and support their use in a CMV vaccine candidate with improved neutralizing activity against epithelial cell infection. PMID:24334684

  2. Enveloped virus-like particle expression of human cytomegalovirus glycoprotein B antigen induces antibodies with potent and broad neutralizing activity.

    PubMed

    Kirchmeier, Marc; Fluckiger, Anne-Catherine; Soare, Catalina; Bozic, Jasminka; Ontsouka, Barthelemy; Ahmed, Tanvir; Diress, Abebaw; Pereira, Lenore; Schödel, Florian; Plotkin, Stanley; Dalba, Charlotte; Klatzmann, David; Anderson, David E

    2014-02-01

    A prophylactic vaccine to prevent the congenital transmission of human cytomegalovirus (HCMV) in newborns and to reduce life-threatening disease in immunosuppressed recipients of HCMV-infected solid organ transplants is highly desirable. Neutralizing antibodies against HCMV confer significant protection against infection, and glycoprotein B (gB) is a major target of such neutralizing antibodies. However, one shortcoming of past HCMV vaccines may have been their failure to induce high-titer persistent neutralizing antibody responses that prevent the infection of epithelial cells. We used enveloped virus-like particles (eVLPs), in which particles were produced in cells after the expression of murine leukemia virus (MLV) viral matrix protein Gag, to express either full-length CMV gB (gB eVLPs) or the full extracellular domain of CMV gB fused with the transmembrane and cytoplasmic domains from vesicular stomatitis virus (VSV)-G protein (gB-G eVLPs). gB-G-expressing eVLPs induced potent neutralizing antibodies in mice with a much greater propensity toward epithelial cell-neutralizing activity than that induced with soluble recombinant gB protein. An analysis of gB antibody binding titers and T-helper cell responses demonstrated that high neutralizing antibody titers were not simply due to enhanced immunogenicity of the gB-G eVLPs. The cells transiently transfected with gB-G but not gB plasmid formed syncytia, consistent with a prefusion gB conformation like those of infected cells and viral particles. Two of the five gB-G eVLP-induced monoclonal antibodies we examined in detail had neutralizing activities, one of which possessed particularly potent epithelial cell-neutralizing activity. These data differentiate gB-G eVLPs from gB antigens used in the past and support their use in a CMV vaccine candidate with improved neutralizing activity against epithelial cell infection. PMID:24334684

  3. Simple assay to screen for inhibitors of interaction between the human immunodeficiency virus envelope glycoprotein and its cellular receptor, CD4.

    PubMed Central

    Chams, V; Idziorek, T; Maddon, P J; Klatzmann, D

    1992-01-01

    The binding of the human immunodeficiency virus envelope glycoprotein gp120 to the CD4 molecule is the initial step in the viral replicative cycle. This interaction is therefore an important target for therapeutic intervention for the treatment of human immunodeficiency virus infection. We designed an enzyme-linked immunosorbent assay which detects the interaction between recombinant soluble forms of CD4 and gp160. This assay could be used as an initial screen of libraries of synthetic chemical compounds and natural products. PMID:1605591

  4. Association of purified skeletal-muscle AMP deaminase with a histidine-proline-rich-glycoprotein-like molecule.

    PubMed Central

    Ranieri-Raggi, M; Montali, U; Ronca, F; Sabbatini, A; Brown, P E; Moir, A J; Raggi, A

    1997-01-01

    Denaturation of rabbit skeletal-muscle AMP deaminase in acidic medium followed by chromatography on DEAE-cellulose in 8 M urea atpH 8.0 allows separation of two main peptide components of similar apparent molecular mass (75-80 kDa) that we tentatively assume correspond to two different enzyme subunits. Whereas the amino acid composition of one of the two peptides is in good agreement with that derived from the nucleotide sequence of the known rat and human AMPD1 cDNAs, the second component shows much higher contents of proline, glycine and histidine. N-Terminal sequence analysis of the fragments liberated by limited proteolysis with trypsin of the novel peptide reveals a striking similarity to the fragments produced by plasmin cleavage of the rabbit plasma protein called histidine-proline-rich glycoprotein (HPRG). However, some divergence is observed between the sequence of one of the fragments liberated from AMP deaminase by a more extensive trypsinization and rabbit plasma HPRG in the region containing residues 472-477. A fragment with a blocked N-terminus, which was found among those liberated by proteolysis with pepsin of either whole AMP deaminase or the novel component of the enzyme, shows an amino acid composition quite different from that of the N-terminus of the known subunit of AMP deaminase. By coupling this observation with the detection in freshly prepared AMP deaminase of a low yield of the sequence (LTPTDX) corresponding to that of HPRG N-terminus, it can be deduced that in comparison with HPRG, the putative HPRG-like component of AMP deaminase contains an additional fragment with a blocked N-terminus, which is liberated by a proteolytic process during purification of the enzyme. The implications of the association to rabbit skeletal-muscle AMP deaminase of a HPRG-like protein species are discussed. PMID:9307011

  5. 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

  6. 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.

  7. Influence of Disulfide-Stabilized Structure on the Specificity of Helper T-Cell and Antibody Responses to HIV Envelope Glycoprotein gp120▿ †

    PubMed Central

    Mirano-Bascos, Denise; Steede, N. Kalaya; Robinson, James E.; Landry, Samuel J.

    2010-01-01

    CD4+ helper T cells specific for human immunodeficiency virus type 1 (HIV-1) are associated with control of viremia. Nevertheless, vaccines have had limited effectiveness thus far, in part because sequence variability and other structural features of the HIV envelope glycoprotein deflect the immune response. Previous studies indicated that CD4+ T-cell epitope dominance is controlled by antigen three-dimensional structure through its influence on antigen processing and presentation. In this work, three disulfide bonds in the outer domain of gp120 were individually deleted in order to destabilize the local three-dimensional structure and enhance the presentation of nearby weakly immunogenic epitopes. However, upon immunization of groups of BALB/c mice, the CD4+ T-cell response was broadly reduced for all three variants, and distinct epitope profiles emerged. For one variant, antibody titers were sharply increased, and the antibody exhibited significant CD4-blocking activity. PMID:20089653

  8. Galactosyl ceramide or a derivative is an essential component of the neural receptor for human immunodeficiency virus type 1 envelope glycoprotein gp120.

    PubMed Central

    Bhat, S; Spitalnik, S L; Gonzalez-Scarano, F; Silberberg, D H

    1991-01-01

    This report demonstrates that galactosyl ceramide (GalCer) or a molecule derived from it may serve as an alternative receptor for human immunodeficiency virus in the nervous system. Recombinant gp120, an envelope glycoprotein of human immunodeficiency virus type 1, specifically binds to GalCer and its derivatives. This specificity was studied by inhibiting binding of radioiodinated gp120 to GalCer with antibodies to GalCer, antibodies to gp120, and an excess of unlabeled gp120. Binding activity was also removed by absorbing gp120 with liposomes containing GalCer. In addition, studies using natural and semisynthetic lipids indicate that the linkage between galactose and ceramide is essential for binding. The significance of an alternative receptor for human immunodeficiency virus in the nervous system is discussed. Images PMID:1871126

  9. Proteinase-resistant factors in human erythrocyte membranes mediate CD4-dependent fusion with cells expressing human immunodeficiency virus type 1 envelope glycoproteins.

    PubMed Central

    Dragic, T; Picard, L; Alizon, M

    1995-01-01

    Murine CD4+ cells are resistant to human immunodeficiency virus type 1 (HIV-1) entry and to fusion with cells expressing HIV-1 envelope glycoproteins (Env). The role of human-specific factors in Env/CD4-mediated fusion is shown by the ability of transient cell hybrids formed between CD4+ murine cells and human HeLa cells to fuse with Env+ cells. Fusion events were observed when other human cells, including erythrocytes, were substituted for HeLa cells in the hybrids. Experiments with erythrocyte ghosts showed that the factors allowing Env/CD4-mediated fusion are located in the plasma membrane. These factors were fully active after extensive digestion of erythrocytes with proteinase K or pronase. Nonprotein components of human plasma membranes, possibly glycolipids, could therefore be required for Env/CD4-mediated fusion and virus entry. PMID:7815477

  10. 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

  11. 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.

  12. Mutations at the C-terminus of the simian immunodeficiency virus envelope glycoprotein affect gp120-gp41 stability on virions

    SciTech Connect

    Affranchino, Jose L.; Gonzalez, Silvia A. . E-mail: sigonzal@ub.edu.ar

    2006-03-30

    The transmembrane (TM) subunit of the envelope (Env) glycoprotein of the simian immunodeficiency virus (SIV) contains an unusually long cytoplasmic domain of 164 amino acids. Previously, we identified domains in the SIV TM cytoplasmic tail that are necessary for Env incorporation into virions and viral infectivity. In this study, we investigated the relevance to Env function of the highly conserved sequence comprising the immediate C-terminal 19 residues of TM. To this end, small in-frame deletions as well as a premature stop codon mutation were introduced into the coding region for the SIV TM C-terminus. All the mutant Env glycoproteins were expressed, processed and transported to the cell surface in an essentially wild-type manner. Moreover, the ability of the mutant Env proteins to mediate cell-to-cell fusion was similar to or slightly lower than that of the wild-type Env. However, viruses expressing the mutant Env glycoproteins were found to be poorly infectious in single-cycle infectivity assays. Further characterization of the TM mutant viruses revealed that while exhibiting wild-type levels of the TM protein, they contained significantly lower levels of the Env surface (SU) subunit, which is consistent with increased SU shedding from virions after Env incorporation. This phenotype was independent of Gag processing, since genetic inactivation of the viral protease did not increase SU retention by the resulting immature particles. Our findings indicate that deletions at the C-terminus of the SIV Env promote the instability of the SU-TM association on the virion surface and point to an important role for the TM cytoplasmic domain in modulating Env structure.

  13. A Trimeric, V2-Deleted HIV-1 Envelope Glycoprotein Vaccine Elicits Potent Neutralizing Antibodies but Limited Breadth of Neutralization in Human Volunteers

    PubMed Central

    Lally, Michelle A.; Elizaga, Marnie; Montefiori, David; Tomaras, Georgia D.; McElrath, M. Juliana; Hural, John; De Rosa, Stephen C.; Sato, Alicia; Huang, Yunda; Frey, Sharon E.; Sato, Paul; Donnelly, John; Barnett, Susan; Corey, Lawrence J.

    2011-01-01

    Background. A key missing element in the development of a successful human immunodeficiency virus (HIV) vaccine is an immunogen that can generate broadly cross-neutralizing antibodies against primary isolates of the virus. Methods. This phase 1 clinical trial employed a DNA prime and subunit envelope protein boost in an attempt to generate cellular and humoral immune responses that might be desirable in a protective HIV vaccine. Priming was performed via intramuscular injection with gag and env DNA adsorbed to polylactide coglycolide microspheres, followed by boosting with a recombinant trimeric envelope (Env) glycoprotein delivered in MF59 adjuvant. Results. The DNA prime and protein boost were generally safe and well-tolerated. Env-specific CD4+ cellular responses were generated that were predominantly detected after Env protein boosting. Neutralizing antibody responses against the homologous SF162 viral isolate were remarkably strong and were present in the majority of vaccine recipients, including a strong response against CD4-induced epitopes on gp120. Despite the promising potency of this vaccine approach, neutralization breadth against heterologous tier 2 strains of HIV-1 was minimal. Conclusions. Potent neutralization against neutralization-sensitive strains of HIV is achievable in humans through a DNA prime, recombinant oligomeric Env protein boost regimen. Eliciting substantial breadth of neutralization remains an elusive goal.  Clinical Trials Registration. NCT00073216. PMID:21451004

  14. 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

  15. High-efficiency gene transfer into CD34+ cells with a human immunodeficiency virus type 1-based retroviral vector pseudotyped with vesicular stomatitis virus envelope glycoprotein G.

    PubMed Central

    Akkina, R K; Walton, R M; Chen, M L; Li, Q X; Planelles, V; Chen, I S

    1996-01-01

    Currently, amphotropic retroviral vectors are widely used for gene transfer into CD34+ hematopoietic progenitor cells. The relatively low levels of transduction efficiency associated with these vectors in human cells is due to low viral titers and limitations in concentrating the virus because of the inherent fragility of retroviral envelopes. Here we show that a human immunodeficiency virus type 1 (HIV-1)-based retroviral vector containing the firefly luciferase reporter gene can be pseudotyped with a broad-host-range vesicular stomatitis virus envelope glycoprotein G (VSV-G). Higher-efficiency gene transfer into CD34+ cells was achieved with a VSV-G-pseudotyped HIV-1 vector than with a vector packaged in an amphotropic envelope. Concentration of virus without loss of viral infectivity permitted a higher multiplicity of infection, with a consequent higher efficiency of gene transfer, reaching 2.8 copies per cell. These vectors also showed remarkable stability during storage at 4 degrees C for a week. In addition, there was no significant loss of titer after freezing and thawing of the stock virus. The ability of VSV-G-pseudotyped retroviral vectors to achieve a severalfold increase in levels of transduction into CD34+ cells will allow high-efficiency gene transfer into hematopoietic progenitor cells for gene therapy purposes. Furthermore, since it has now become possible to infect CD34+ cells with pseudotyped HIV-1 with a high level of efficiency in vitro, many important questions regarding the effect of HIV-1 on lineage-specific differentiation of hematopoietic progenitors can now be addressed. PMID:8642689

  16. Peptides presenting the binding site of human CD4 for the HIV-1 envelope glycoprotein gp120

    PubMed Central

    Meier, Julia; Kassler, Kristin; Sticht, Heinrich

    2012-01-01

    Summary Based on the structure of the HIV-1 glycoprotein gp120 in complex with its cellular receptor CD4, we have designed and synthesized peptides that mimic the binding site of CD4 for gp120. The ability of these peptides to bind to gp120 can be strongly enhanced by increasing their conformational stability through cyclization, as evidenced by binding assays, as well as through molecular-dynamics simulations of peptide–gp120 complexes. The specificity of the peptide–gp120 interaction was demonstrated by using peptide variants, in which key residues for the interaction with gp120 were replaced by alanine or D-amino acids. PMID:23209523

  17. 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

  18. 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. PMID:26072370

  19. Core structure of the envelope glycoprotein GP2 from Ebola virus at 1.9-Å resolution

    PubMed Central

    Malashkevich, Vladimir N.; Schneider, Brian J.; McNally, Margaret L.; Milhollen, Michael A.; Pang, James X.; Kim, Peter S.

    1999-01-01

    Ebola virions contain a surface transmembrane glycoprotein (GP) that is responsible for binding to target cells and subsequent fusion of the viral and host-cell membranes. GP is expressed as a single-chain precursor that is posttranslationally processed into the disulfide-linked fragments GP1 and GP2. The GP2 subunit is thought to mediate membrane fusion. A soluble fragment of the GP2 ectodomain, lacking the fusion-peptide region and the transmembrane helix, folds into a stable, highly helical structure in aqueous solution. Limited proteolysis studies identify a stable core of the GP2 ectodomain. This 74-residue core, denoted Ebo-74, was crystallized, and its x-ray structure was determined at 1.9-Å resolution. Ebo-74 forms a trimer in which a long, central three-stranded coiled coil is surrounded by shorter C-terminal helices that are packed in an antiparallel orientation into hydrophobic grooves on the surface of the coiled coil. Our results confirm the previously anticipated structural similarity between the Ebola GP2 ectodomain and the core of the transmembrane subunit from oncogenic retroviruses. The Ebo-74 structure likely represents the fusion-active conformation of the protein, and its overall architecture resembles several other viral membrane-fusion proteins, including those from HIV and influenza. PMID:10077567

  20. The Mature Avian Leukosis Virus Subgroup A Envelope Glycoprotein Is Metastable, and Refolding Induced by the Synergistic Effects of Receptor Binding and Low pH Is Coupled to Infection

    PubMed Central

    Smith, Jason G.; Mothes, Walther; Blacklow, Stephen C.; Cunningham, James M.

    2004-01-01

    The spring-loaded model stipulates that influenza virus infection is coupled to the transition of the virus hemagglutinin (HA) from a metastable conformation to a highly stable conformation at low pH. The properties of retrovirus envelope glycoproteins indicate that infection is coupled to an analogous conformational change. As a test of this hypothesis, the requirements for avian leukosis virus A (ALV-A) infection were examined. These studies indicate that, like HA, the conformation of the mature ALV-A envelope glycoprotein is metastable and that infection is linked to refolding at low pH. However, unlike HA, low-pH activation is only observed after priming by receptor. Therefore, ALV-A infection is dependent on the synergistic effects of receptor binding and low pH, suggesting that receptor binding superimposes an additional constraint on activation of ALV-A fusion that proceeds by a mechanism comparable to that of influenza virus. PMID:14722295

  1. 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.

  2. Effects of interleukin 2 and large envelope glycoprotein (gp 120) of human immunodeficiency virus (HIV) on lymphocyte proliferative responses to cytomegalovirus.

    PubMed Central

    Krowka, J; Stites, D; Mills, J; Hollander, H; McHugh, T; Busch, M; Wilhelm, L; Blackwood, L

    1988-01-01

    Lymphocytes from many HIV-infected asymptomatic individuals or patients with AIDS-related conditions (ARC) and from all AIDS patients were unable to proliferate in vitro in response to UV-inactivated cytomegalovirus (CMV). The addition of recombinant IL2 (rIL2) restored proliferative responses of lymphocytes from most HIV-infected asymptomatic individuals and ARC patients to levels similar to those of HIV-seronegative (HIV-) CMV-seropositive (CMV+) individuals. In contrast, rIL2 augmented CMV-specific lymphocyte proliferation of only 33% (6/18) of AIDS patients. Proliferative responses to CMV with or without rIL2 did not correlate well with the levels of CD4+ lymphocytes, HIV antigen levels or ratios of CD4+ and CD8+ lymphocytes. Proliferative responses to CMV were inhibited by relatively high concentrations (greater than or equal to 10 micrograms/ml) of recombinant HIV envelope glycoprotein (rgp120) and this immunosuppression was completely overcome by rIL2. These results indicate that defects in antigen-driven lymphocyte responses of HIV-infected individuals are not simply the result of reduced numbers of CD4+ lymphocytes but are influenced by defects in IL2 pathways and by immunosuppressive effects of HIV gp120. PMID:2842093

  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. A Chimeric HIV-1 Envelope Glycoprotein Trimer with an Embedded Granulocyte-Macrophage Colony-stimulating Factor (GM-CSF) Domain Induces Enhanced Antibody and T Cell Responses*

    PubMed Central

    van Montfort, Thijs; Melchers, Mark; Isik, Gözde; Menis, Sergey; Huang, Po-Ssu; Matthews, Katie; Michael, Elizabeth; Berkhout, Ben; Schief, William R.; Moore, John P.; Sanders, Rogier W.

    2011-01-01

    An effective HIV-1 vaccine should ideally induce strong humoral and cellular immune responses that provide sterilizing immunity over a prolonged period. Current HIV-1 vaccines have failed in inducing such immunity. The viral envelope glycoprotein complex (Env) can be targeted by neutralizing antibodies to block infection, but several Env properties limit the ability to induce an antibody response of sufficient quantity and quality. We hypothesized that Env immunogenicity could be improved by embedding an immunostimulatory protein domain within its sequence. A stabilized Env trimer was therefore engineered with the granulocyte-macrophage colony-stimulating factor (GM-CSF) inserted into the V1V2 domain of gp120. Probing with neutralizing antibodies showed that both the Env and GM-CSF components of the chimeric protein were folded correctly. Furthermore, the embedded GM-CSF domain was functional as a cytokine in vitro. Mouse immunization studies demonstrated that chimeric EnvGM-CSF enhanced Env-specific antibody and T cell responses compared with wild-type Env. Collectively, these results show that targeting and activation of immune cells using engineered cytokine domains within the protein can improve the immunogenicity of Env subunit vaccines. PMID:21515681

  5. Bitopic Membrane Topology of the Stable Signal Peptide in the Tripartite Junín Virus GP-C Envelope Glycoprotein Complex▿

    PubMed Central

    Agnihothram, Sudhakar S.; York, Joanne; Trahey, Meg; Nunberg, Jack H.

    2007-01-01

    The stable signal peptide (SSP) of the GP-C envelope glycoprotein of the Junín arenavirus plays a critical role in trafficking of the GP-C complex to the cell surface and in its membrane fusion activity. SSP therefore may function on both sides of the lipid membrane. In this study, we have investigated the membrane topology of SSP by confocal microscopy of cells treated with the detergent digitonin to selectively permeabilize the plasma membrane. By using an affinity tag to mark the termini of SSP in the properly assembled GP-C complex, we find that both the N and C termini reside in the cytosol. Thus, SSP adopts a bitopic topology in which the C terminus is translocated from the lumen of the endoplasmic reticulum to the cytoplasm. This model is supported by (i) the presence of two conserved hydrophobic regions in SSP (hφ1 and hφ2) and (ii) our previous demonstration that lysine-33 in the ectodomain loop is essential for pH-dependent membrane fusion. Moreover, we demonstrate that the introduction of a charged side chain or single amino acid deletion in the membrane-spanning hφ2 region significantly diminishes SSP association in the GP-C complex and abolishes membrane fusion activity. Taken together, our results suggest that bitopic membrane insertion of SSP is centrally important in the assembly and function of the tripartite GP-C complex. PMID:17267481

  6. 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

  7. Discovery of cyanovirin-N, a novel human immunodeficiency virus-inactivating protein that binds viral surface envelope glycoprotein gp120: potential applications to microbicide development.

    PubMed Central

    Boyd, M R; Gustafson, K R; McMahon, J B; Shoemaker, R H; O'Keefe, B R; Mori, T; Gulakowski, R J; Wu, L; Rivera, M I; Laurencot, C M; Currens, M J; Cardellina, J H; Buckheit, R W; Nara, P L; Pannell, L K; Sowder, R C; Henderson, L E

    1997-01-01

    We have isolated and sequenced a novel 11-kDa virucidal protein, named cyanovirin-N (CV-N), from cultures of the cyanobacterium (blue-green alga) Nostoc ellipsosporum. We also have produced CV-N recombinantly by expression of a corresponding DNA sequence in Escherichia coli. Low nanomolar concentrations of either natural or recombinant CV-N irreversibly inactivate diverse laboratory strains and primary isolates of human immunodeficiency virus (HIV) type 1 as well as strains of HIV type 2 and simian immunodeficiency virus. In addition, CV-N aborts cell-to-cell fusion and transmission of HIV-1 infection. Continuous, 2-day exposures of uninfected CEM-SS cells or peripheral blood lymphocytes to high concentrations (e.g., 9,000 nM) of CV-N were not lethal to these representative host cell types. The antiviral activity of CV-N is due, at least in part, to unique, high-affinity interactions of CV-N with the viral surface envelope glycoprotein gp120. The biological activity of CV-N is highly resistant to physicochemical denaturation, further enhancing its potential as an anti-HIV microbicide. PMID:9210678

  8. 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

  9. Identification of the V1 region as a linear neutralizing epitope of the simian immunodeficiency virus SIVmac envelope glycoprotein.

    PubMed Central

    Jurkiewicz, E; Hunsmann, G; Schäffner, J; Nisslein, T; Lüke, W; Petry, H

    1997-01-01

    The sequence variability of viral structure polypeptides has been associated with immune escape mechanisms. The V1 region of simian immunodeficiency virus (SIV) is a highly variable region of the SIVmac env gene. Here, we describe the V1 region as a linear neutralizing epitope. V1 region-specific neutralizing antibodies (NAb) were first demonstrated in a rabbit infected with a recombinant vaccinia virus carrying the env gene of human immunodeficiency virus type 2 strain ben (HIV-2ben). Since we detected in this animal V1 region-specific NAb that were able to neutralize not only human immunodeficiency virus type 2 but also SIVmac32H, we investigated whether a similar immune response is evoked in macaques (Macaca mulatta) either infected with SIVmac or immunized with the external glycoprotein (gp130) of the same virus. Distinctly lower NAb titers were found in the SIVmac-infected animals than in the gp130-immunized macaques. Since the NAb titers in both groups were high enough for competition experiments, we used five overlapping peptides encompassing the whole V1 region for a detailed identification of the epitope. In each of the 12 macaques investigated, we detected a high level of NAb reacting with at least one peptide located in the central part of the V1 region. The relatively high degree of divergence, especially within the central part of the V1 region, which characterized the evolution of the retroviral sequences from the original inoculum in the infected macaques suggests the development of escape mutants. Furthermore, 3 of 12 animals developed NAb directed against the amino-terminal end of the V1 region epitope. Sequence analysis, however, revealed relatively low levels of genetic drift and genetic variability within this part of the V1 region. The induction of V1 env-specific NAb not only in gp130-immunized macaques but also in SIVmac-infected animals in combination with the increased genetic variability of this region in vivo indicates a marked biological

  10. 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.

  11. Effects of L- and D-REKR amino acid-containing peptides on HIV and SIV envelope glycoprotein precursor maturation and HIV and SIV replication.

    PubMed Central

    Bahbouhi, Bouchaib; Chazal, Nathalie; Seidah, Nabil Georges; Chiva, Cristina; Kogan, Marcelo; Albericio, Fernando; Giralt, Ernest; Bahraoui, Elmostafa

    2002-01-01

    The aim of the present study was to evaluate the capacity of synthetic l- and d-peptides encompassing the HIV-1(BRU) gp160 REKR cleavage site to interfere with HIV and simian immuno-deficiency virus (SIV) replication and maturation of the envelope glycoprotein (Env) precursors. To facilitate their penetration into cells, a decanoyl (dec) group was added at the N-terminus. The sequences synthesized included dec5d or dec5l (decREKRV), dec9d or dec9l (decRVVQREKRV) and dec14d or dec14l (TKAKRRVVQREKRV). The peptide dec14d was also prepared with a chloromethane (cmk) group as C-terminus. Because l-peptides exhibit significant cytotoxicity starting at 35 microM, further characterization was conducted mostly with d-peptides, which exhibited no cytotoxicity at concentrations higher than 70 microM. The data show that only dec14d and dec14dcmk could inhibit HIV-1(BRU), HIV-2(ROD) and SIV(mac251) replication and their syncytium-inducing capacities. Whereas peptides dec5d and dec9d were inactive, dec14dcmk was at least twice as active as peptide dec14d. At the molecular level, our data show a direct correlation between anti-viral activity and the ability of the peptides to interfere with maturation of the Env precursors. Furthermore, we show that when tested in vitro the dec14d peptide inhibited PC7 with an inhibition constant K(i)=4.6 microM, whereas the peptide dec14l preferentially inhibited furin with a K(i)=28 microM. The fact that PC7 and furin are the major prohormone convertases reported to be expressed in T4 lymphocytes, the principal cell targets of HIV, suggests that they are involved in the maturation of HIV and SIV Env precursors. PMID:12071862

  12. In vivo alteration of humoral responses to HIV-1 envelope glycoprotein gp120 by antibodies to the CD4-binding site of gp120

    PubMed Central

    Visciano, Maria Luisa; Tuen, Michael; Gorny, Miroslaw K.; Hioe, Catarina E.

    2008-01-01

    The binding of antibodies to the CD4-binding site (CD4bs) of the HIV-1 envelope glycoprotein gp120 has been shown to induce gp120 to undergo conformational changes that can expose and/or shield specific epitopes on gp120. Here, we study alterations in the antigenicity and immunogenicity of gp120 when complexed with human monoclonal antibodies (mAbs) specific for the CD4bs of gp120. The data showed that gp120 bound by anti-CD4bs mAbs had enhanced reactivity with mAbs to the V3 and N-terminal regions, but not with mAb to the C terminus. Moreover, mice immunized with the gp120/anti-CD4bs mAb complexes produced higher titers of gp120-specific serum IgG and IgA than mice immunized with uncomplexed gp120 or other gp120/mAb complexes. Notably, the enhanced antibody production was directed against V3 and correlated with better exposure of V3 on the gp120/anti-CD4bs mAb complexes. The higher antibody reactivity was evident against the homologous V3LAI peptide, but not against heterologous V3 peptides. Potent neutralization activity against HIV-1LAI was also observed in the sera from mice immunized with gp120/anti-CD4bs mAb complexes, although the sera exhibited poor neutralizing activities against other viruses tested. These results indicate that the anti-CD4bs antibodies alter the antigenicity and immunogenicity of gp120, leading to enhanced production of anti-gp120 antibodies directed particularly against the V3 region. PMID:18054978

  13. Conformation-specific antibodies targeting the trimer-of-hairpins motif of the human T-cell leukemia virus type 1 transmembrane glycoprotein recognize the viral envelope but fail to neutralize viral entry.

    PubMed

    Mirsaliotis, Antonis; Nurkiyanova, Kulpash; Lamb, Daniel; Woof, Jenny M; Brighty, David W

    2007-06-01

    Human T-cell leukemia virus type 1 (HTLV-1) entry into cells is dependent upon the viral envelope glycoprotein-catalyzed fusion of the viral and cellular membranes. Following receptor activation of the envelope, the transmembrane glycoprotein (TM) is thought to undergo a series of fusogenic conformational transitions through a rod-like prehairpin intermediate to a compact trimer-of-hairpins structure. Importantly, synthetic peptides that interfere with the conformational changes of TM are potent inhibitors of membrane fusion and HTLV-1 entry, suggesting that TM is a valid target for antiviral therapy. To assess the utility of TM as a vaccine target and to explore further the function of TM in HTLV-1 pathogenesis, we have begun to examine the immunological properties of TM. Here we demonstrate that a recombinant trimer-of-hairpins form of the TM ectodomain is strongly immunogenic. Monoclonal antibodies raised against the TM immunogen specifically bind to trimeric forms of TM, including structures thought to be important for membrane fusion. Importantly, these antibodies recognize the envelope on virally infected cells but, surprisingly, fail to neutralize envelope-mediated membrane fusion or infection by pseudotyped viral particles. Our data imply that, even in the absence of overt membrane fusion, there are multiple forms of TM on virally infected cells and that some of these display fusion-associated structures. Finally, we demonstrate that many of the antibodies possess the ability to recruit complement to TM, suggesting that envelope-derived immunogens capable of eliciting a combination of neutralizing and complement-fixing antibodies would be of value as subunit vaccines for intervention in HTLV infections. PMID:17376912

  14. 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

  15. Comprehensive Analysis of Contributions from Protein Conformational Stability and Major Histocompatibility Complex Class II-Peptide Binding Affinity to CD4+ Epitope Immunogenicity in HIV-1 Envelope Glycoprotein

    PubMed Central

    Li, Tingfeng; Steede, N. Kalaya; Nguyen, Hong-Nam P.; Freytag, Lucy C.; McLachlan, James B.; Mettu, Ramgopal R.; Robinson, James E.

    2014-01-01

    ABSTRACT Helper T-cell epitope dominance in human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp120 is not adequately explained by peptide binding to major histocompatibility complex (MHC) proteins. Antigen processing potentially influences epitope dominance, but few, if any, studies have attempted to reconcile the influences of antigen processing and MHC protein binding for all helper T-cell epitopes of an antigen. Epitopes of gp120 identified in both humans and mice occur on the C-terminal flanks of flexible segments that are likely to be proteolytic cleavage sites. In this study, the influence of gp120 conformation on the dominance pattern in gp120 from HIV strain 89.6 was examined in CBA mice, whose MHC class II protein has one of the most well defined peptide-binding preferences. Only one of six dominant epitopes contained the most conserved element of the I-Ak binding motif, an aspartic acid. Destabilization of the gp120 conformation by deletion of single disulfide bonds preferentially enhanced responses to the cryptic I-Ak motif-containing sequences, as reported by T-cell proliferation or cytokine secretion. Conversely, inclusion of CpG in the adjuvant with gp120 enhanced responses to the dominant CD4+ T-cell epitopes. The gp120 destabilization affected secretion of some cytokines more than others, suggesting that antigen conformation could modulate T-cell functions through mechanisms of antigen processing. IMPORTANCE CD4+ helper T cells play an essential role in protection against HIV and other pathogens. Thus, the sites of helper T-cell recognition, the dominant epitopes, are targets for vaccine design; and the corresponding T cells may provide markers for monitoring infection and immunity. However, T-cell epitopes are difficult to identify and predict. It is also unclear whether CD4+ T cells specific for one epitope are more protective than T cells specific for other epitopes. This work shows that the three-dimensional (3D) structure of an

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

    PubMed

    Hötzel, Isidro; 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. PMID

  17. 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.

  18. Envelope polypeptides of Friend leukemia virus: purification and structural analysis.

    PubMed Central

    Schneider, J; Falk, H; Hunsmann, G

    1980-01-01

    Roughly 10% of surface glycoproteins in the envelope of mature Friend murine leukemia virus are coupled to membrane polypeptides by disulfide bridges. The remaining 90% of these glycoproteins are associated noncovalently. However, they could also be linked to membrane polypeptides by the treatment of purified Friend murine leukemia virus with 2,2'dithiobis(m-nitropyridine). These amphiphilic heterodimer polypeptides, gp84/86, were recovered almost quantitatively in the form of aggregates, termed rosettes, when prepared by solubilization of the viral membrane with Triton X-100 and subsequent velocity sedimentation. gp69/71 and p12(E)/15(E) were purified from these protein micelles after reduction of the disulfide bonds by gel chromatography. Electron micrographs of rosettes, as well as of purified p12(E)/15(E), showed structures different from native viral knobs. Isolated gp84/86 could be reassociated and then displayed more similarity to these viral surface projections. As shown by peptide mapping, the primary structures of the glycoproteins gp69/71 are highly related as are those of the membrane polypeptides p12(E) and p15(E). Furthermore, it was shown by two-dimensional polyacrylamide gel electrophoresis and re-electrophoresis of purified gp84/86 that the larger component, gp86, was composed of gp71 associated with p15(E) and p12(E), whereas the smaller component, gp84, was formed by gp69 bound only to p12(E). Images PMID:7411688

  19. Human parainfluenza type 3 virus hemagglutinin-neuraminidase glycoprotein: nucleotide sequence of mRNA and limited amino acid sequence of the purified protein.

    PubMed Central

    Elango, N; Coligan, J E; Jambou, R C; Venkatesan, S

    1986-01-01

    The nucleotide sequence of mRNA for the hemagglutinin-neuraminidase (HN) protein of human parainfluenza type 3 virus obtained from the corresponding cDNA clone had a single long open reading frame encoding a putative protein of 64,254 daltons consisting of 572 amino acids. The deduced protein sequence was confirmed by limited N-terminal amino acid microsequencing of CNBr cleavage fragments of native HN that was purified by immunoprecipitation. The HN protein is moderately hydrophobic and has four potential sites (Asn-X-Ser/Thr) of N-glycosylation in the C-terminal half of the molecule. It is devoid of both the N-terminal signal sequence and the C-terminal membrane anchorage domain characteristic of the hemagglutinin of influenza virus and the fusion (F0) protein of the paramyxoviruses. Instead, it has a single prominent hydrophobic region capable of membrane insertion beginning at 32 residues from the N terminus. This N-terminal membrane insertion is similar to that of influenza virus neuraminidase and the recently reported structures of HN proteins of Sendai virus and simian virus 5. Images PMID:3003381

  20. 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.

  1. Processing, fusogenicity, virion incorporation and CXCR4-binding activity of a feline immunodeficiency virus envelope glycoprotein lacking the two conserved N-glycosylation sites at the C-terminus of the V3 domain.

    PubMed

    González, Silvia A; Affranchino, José L

    2016-07-01

    The process of feline immunodeficiency virus (FIV) entry into its target cells is initiated by the association of the surface (SU) subunit of the viral envelope glycoprotein (Env) with the cellular receptors CD134 and CXCR4. This event is followed by the fusion of the viral and cellular membranes, which is mediated by the transmembrane (TM) subunit of Env. We and others have previously demonstrated that the V3 domain of the SU subunit of Env is essential for CXCR4 binding. Of note, there are two contiguous and highly conserved potential N-glycosylation sites ((418)NST(420) and (422)NLT(424)) located at the C-terminal side of the V3 domain. We therefore decided to study the relevance for Env functions of these N-glycosylation motifs and found that disruption of both of them by introducing the N418Q/N422Q double amino acid substitution drastically impairs Env processing into the SU and TM subunits. Moreover, the simultaneous mutation of these N-glycosylation sites prevents Env incorporation into virions and Env-mediated cell-to-cell fusion. Notably, a recombinant soluble version of the SU glycoprotein carrying the double amino acid replacement N418Q/N422Q at the V3 C-terminal side binds to CXCR4 with an efficiency similar to that of wild-type SU. PMID:27020572

  2. 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

  3. 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

  4. 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

  5. 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.

  6. Complete Protection of Mice against Lethal Murine Cytomegalovirus Challenge by Immunization with DNA Vaccines Encoding Envelope Glycoprotein Complex III Antigens gH, gL and gO

    PubMed Central

    Wang, Huadong; Huang, Chaoyang; Dong, Jinrong; Yao, Yanfeng; Xie, Zhenyuan; Liu, Xueying; Zhang, Wenjie; Fang, Fang; Chen, Ze

    2015-01-01

    Human cytomegalovirus infects the majority of humanity which may lead to severe morbidity and mortality in newborns and immunocompromised adults. Humoral and cellular immunity are critical for controlling CMV infection. HCMV envelope glycoprotein complexes (gC I, II, III) represent major antigenic targets of antiviral immune responses. The gCIII complex is comprised of three glycoproteins, gH, gL, and gO. In the present study, DNA vaccines expressing the murine cytomegalovirus homologs of the gH, gL, and gO proteins were evaluated for protection against lethal MCMV infection in the mouse model. The results demonstrated that gH, gL, or gO single gene immunization could not yet offer good protection, whereas co-vaccination strategy apparently showed effects superior to separate immunization. Twice immunization with gH/gL/gO pDNAs could provide mice complete protection against lethal salivary gland-derived MCMV (SG-MCMV) challenge, while thrice immunization with pgH/pgL, pgH/pgO or pgL/pgO could not provide full protection. Co-vaccination with gH, gL and gO pDNAs elicited robust neutralizing antibody and cellular immune responses. Moreover, full protection was also achieved by simply passive immunization with anti-gH/gL/gO sera. These data demonstrated that gCIII complex antigens had fine immunogenicity and might be a promising candidate for the development of HCMV vaccines. PMID:25803721

  7. 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.

  8. 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

  9. 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

  10. Development of an infectious surrogate hepatitis C virus based on a recombinant vesicular stomatitis virus expressing hepatitis C virus envelope glycoproteins and green fluorescent protein.

    PubMed

    Okuma, Kazu; Fukagawa, Koji; Tateyama, Seiji; Kohma, Takuya; Mochida, Keiko; Hiyoshi, Masateru; Takahama, Youichi; Hamaguchi, Yukio; Hirose, Kunitaka; Buonocore, Linda; Rose, John K; Mizuochi, Toshiaki; Hamaguchi, Isao

    2015-01-01

    To develop surrogate viruses for hepatitis C virus (HCV), we previously produced recombinant vesicular stomatitis viruses (rVSVs) lacking glycoprotein G but instead expressing chimeric HCV E1/E2 fused to G. These rVSVs were not infectious in HCV-susceptible hepatoma cells. In this study, to develop an infectious surrogate HCV based on an rVSV (vesicular stomatitis virus [VSV]/HCV), we generated a novel rVSV encoding the native E1/E2 (H77 strain) and green fluorescent protein (GFP) instead of G. Here, we showed that this VSV/HCV efficiently infected human hepatoma cells, including Huh7 human hepatoma cells, expressed GFP in these cells, and propagated, but did not do so in nonsusceptible BHK-21 cells. The infectivity of VSV/HCV, measured as the number of foci of GFP-positive cells, was specifically reduced by the addition of chimpanzee anti-HCV serum, anti-E2 antibody, or anti-CD81 antibody to the cultures. When sera obtained from HCV-infected or uninfected patients were added, infection was selectively inhibited only by the sera of HCV-infected patients. These data together suggest that this infectious GFP-expressing VSV/HCV could be a useful tool for studying the mechanisms of HCV entry into cells and for assessing potential inhibitors of viral entry, including neutralizing antibodies. PMID:25672345

  11. 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

  12. 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.

  13. 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.

  14. 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.

  15. A monoclonal antibody to CD4 domain 2 blocks soluble CD4-induced conformational changes in the envelope glycoproteins of human immunodeficiency virus type 1 (HIV-1) and HIV-1 infection of CD4+ cells.

    PubMed Central

    Moore, J P; Sattentau, Q J; Klasse, P J; Burkly, L C

    1992-01-01

    The murine monoclonal antibody (MAb) 5A8, which is reactive with domain 2 of CD4, blocks human immunodeficiency virus type 1 (HIV-1) infection and syncytium formation of CD4+ cells (L. C. Burkly, D. Olson, R. Shapiro, G. Winkler, J. J. Rosa, D. W. Thomas, C. Williams, and P. Chisholm, J. Immunol., in press). Here we show that, in contrast to the CD4 domain 1 MAb 6H10, 5A8 and its Fab fragment do not block soluble CD4 (sCD4) binding to virions, whereas they do inhibit sCD4-induced exposure of cryptic epitopes on gp41 and dissociation of gp120 from virions. Two other MAbs, OKT4 and L120, which are reactive with domains 3 and 4 of CD4, have little or no effect on HIV-1 infection, syncytium formation, or sCD4-induced conformational changes in the envelope glycoproteins. The mechanisms of action of 5A8 and 6H10 can be further distinguished in syncytium inhibition assays: 6H10 blocks competitively, while 5A8 does not. We opine that 5A8 blocks HIV-1 infection and fusion by interfering with conformational changes in gp120/gp41 and/or CD4 that are necessary for virus-cell fusion. Images PMID:1378510

  16. 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

  17. 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

  18. Attenuation of Chikungunya Virus Vaccine Strain 181/Clone 25 Is Determined by Two Amino Acid Substitutions in the E2 Envelope Glycoprotein

    PubMed Central

    Gorchakov, Rodion; Wang, Eryu; Leal, Grace; Forrester, Naomi L.; Plante, Kenneth; Rossi, Shannan L.; Partidos, Charalambos D.; Adams, A. Paige; Seymour, Robert L.; Weger, James; Borland, Erin M.; Sherman, Michael B.; Powers, Ann M.; Osorio, Jorge E.

    2012-01-01

    Chikungunya virus (CHIKV) is the mosquito-borne alphavirus that is the etiologic agent of massive outbreaks of arthralgic febrile illness that recently affected millions of people in Africa and Asia. The only CHIKV vaccine that has been tested in humans, strain 181/clone 25, is a live-attenuated derivative of Southeast Asian human isolate strain AF15561. The vaccine was immunogenic in phase I and II clinical trials; however, it induced transient arthralgia in 8% of the vaccinees. There are five amino acid differences between the vaccine and its parent, as well as five synonymous mutations, none of which involves cis-acting genome regions known to be responsible for replication or packaging. To identify the determinants of attenuation, we therefore tested the five nonsynonymous mutations by cloning them individually or in different combinations into infectious clones derived from two wild-type (WT) CHIKV strains, La Reunion and AF15561. Levels of virulence were compared with those of the WT strains and the vaccine strain in two different murine models: infant CD1 and adult A129 mice. An attenuated phenotype indistinguishable from that of the 181/clone 25 vaccine strain was obtained by the simultaneous expression of two E2 glycoprotein substitutions, with intermediate levels of attenuation obtained with the single E2 mutations. The other three amino acid mutations, in nsP1, 6K, and E1, did not have a detectable effect on CHIKV virulence. These results indicate that the attenuation of strain 181/clone 25 is mediated by two point mutations, explaining the phenotypic instability observed in human vaccinees and also in our studies. PMID:22457519

  19. 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.

  20. Impact of HIV-1 Backbone on Neutralization Sensitivity: Neutralization Profiles of Heterologous Envelope Glycoproteins Expressed in Native Subtype C and CRF01_AE Backbone

    PubMed Central

    Sanders-Buell, Eric; Wesberry, Maggie; Towle, Teresa; Pillis, Devin M.; Molnar, Sebastian; McLinden, Robert; Edmonds, Tara; Hirsch, Ivan; O’Connell, Robert; McCutchan, Francine E.; Montefiori, David C.; Ochsenbauer, Christina; Kappes, John C.; Kim, Jerome H.; Polonis, Victoria R.; Tovanabutra, Sodsai

    2013-01-01

    Standardized assays to assess vaccine and antiviral drug efficacy are critical for the development of protective HIV-1 vaccines and drugs. These immune assays will be advanced by the development of standardized viral stocks, such as HIV-1 infectious molecular clones (IMC), that i) express a reporter gene, ii) are representative of globally diverse subtypes and iii) are engineered to easily exchange envelope (env) genes for expression of sequences of interest. Thus far, a subtype B IMC backbone expressing Renilla luciferase (LucR), and into which the ectodomain of heterologous env coding sequences can be expressed has been successfully developed but as execution of HIV-1 vaccine efficacy trials shifts increasingly to non-subtype B epidemics (Southern African and Southeast Asia), non-subtype B HIV-1 reagents are needed to support vaccine development. Here we describe two IMCs derived from subtypes C and CRF01_AE HIV-1 primary isolates expressing LucR (IMC.LucR) that were engineered to express heterologous gp160 Envs. 18 constructs expressing various subtypes C and CRF01_AE Envs, mostly acute, in subtype-matched and –unmatched HIV backbones were tested for functionality and neutralization sensitivity. Our results suggest a possible effect of non-env HIV-1 genes on the interaction of Env and neutralizing antibodies and highlight the need to generate a library of IMCs representative of the HIV-1 subtype spectrum to be used as standardized neutralization assay reagents for assessing HIV-1 vaccine efficacy. PMID:24312165

  1. 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

  2. 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.

  3. Phosphorylation of the multidrug resistance associated glycoprotein.

    PubMed

    Mellado, W; Horwitz, S B

    1987-11-01

    Drug-resistant cell lines derived from the mouse macrophage-like cell line J774.2 express the multidrug resistance 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 MgCl2 was enhanced a minimum of 2-fold by 10 microM 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-32P]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. PMID:3427052

  4. Characterization of germline antibody libraries from human umbilical cord blood and selection of monoclonal antibodies to viral envelope glycoproteins: Implications for mechanisms of immune evasion and design of vaccine immunogens.

    PubMed

    Chen, Weizao; Streaker, Emily D; Russ, Daniel E; Feng, Yang; Prabakaran, Ponraj; Dimitrov, Dimiter S

    2012-01-27

    We have previously observed that all known HIV-1 broadly neutralizing antibodies (bnAbs) are highly divergent from germline antibodies in contrast to bnAbs against Hendra virus, Nipah virus and SARS coronavirus (SARS CoV). We have hypothesized that because the germline antibodies are so different from the mature HIV-1-specific bnAbs they may not bind the epitopes of the mature antibodies and provided the first evidence to support this hypothesis by using individual putative germline-like predecessor antibodies. To further validate the hypothesis and understand initial immune responses to different viruses, two phage-displayed human cord blood-derived IgM libraries were constructed which contained mostly germline antibodies or antibodies with very low level of somatic hypermutations. They were panned against different HIV-1 envelope glycoproteins (Envs), SARS CoV protein receptor-binding domain (RBD), and soluble Hendra virus G protein (sG). Despite a high sequence and combinatorial diversity observed in the cord blood-derived IgM antibody repertoire, no enrichment for binders of Envs was observed in contrast to considerable specific enrichments produced with panning against RBD and sG; one of the selected monoclonal antibodies (against the RBD) was of high (nM) affinity with only few somatic mutations. These results further support and expand our initial hypothesis for fundamental differences in immune responses leading to elicitation of bnAbs against HIV-1 compared to SARS CoV and Hendra virus. HIV-1 uses a strategy to minimize or eliminate strong binding of germline antibodies to its Env; in contrast, SARS CoV and Hendra virus, and perhaps other viruses causing acute infections, can bind germline antibody or minimally somatically mutated antibodies with relatively high affinity which could be one of the reasons for the success of sG and RBD as vaccine immunogens. PMID:22226962

  5. Antibodies to envelope glycoprotein of dengue virus during the natural course of infection are predominantly cross-reactive and recognize epitopes containing highly conserved residues at the fusion loop of domain II.

    PubMed

    Lai, Chih-Yun; Tsai, Wen-Yang; Lin, Su-Ru; Kao, Chuan-Liang; Hu, Hsien-Ping; King, Chwan-Chuen; Wu, Han-Chung; Chang, Gwong-Jen; Wang, Wei-Kung

    2008-07-01

    The antibody response to the envelope (E) glycoprotein of dengue virus (DENV) is known to play a critical role in both protection from and enhancement of disease, especially after primary infection. However, the relative amounts of homologous and heterologous anti-E antibodies and their epitopes remain unclear. In this study, we examined the antibody responses to E protein as well as to precursor membrane (PrM), capsid, and nonstructural protein 1 (NS1) of four serotypes of DENV by Western blot analysis of DENV serotype 2-infected patients with different disease severity and immune status during an outbreak in southern Taiwan in 2002. Based on the early-convalescent-phase sera tested, the rates of antibody responses to PrM and NS1 proteins were significantly higher in patients with secondary infection than in those with primary infection. A blocking experiment and neutralization assay showed that more than 90% of anti-E antibodies after primary infection were cross-reactive and nonneutralizing against heterologous serotypes and that only a minor proportion were type specific, which may account for the type-specific neutralization activity. Moreover, the E-binding activity in sera of 10 patients with primary infection was greatly reduced by amino acid replacements of three fusion loop residues, tryptophan at position 101, leucine at position 107, and phenylalanine at position 108, but not by replacements of those outside the fusion loop of domain II, suggesting that the predominantly cross-reactive anti-E antibodies recognized epitopes involving the highly conserved residues at the fusion loop of domain II. These findings have implications for our understanding of the pathogenesis of dengue and for the future design of subunit vaccine against DENV as well. PMID:18448542

  6. 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

  7. Cross-Reactive HIV-1 Neutralizing Monoclonal Antibodies Selected by Screening of an Immune Human Phage Library against an Envelope Glycoprotein (gp140) Isolated from a Patient (R2) with Broadly HIV-1 Neutralizing Antibodies

    PubMed Central

    Choudhry, Vidita; Zhang, Mei-Yun; Sidorov, Igor A.; Louise, John M.; Harris, Ilia; Dimitrov, Antony S.; Bouma, Peter; Cham, Fatim; Choudhary, Anil; Rybak, Susanna M.; Fouts, Timothy; Montefiori, David C.; Broder, Christopher C.; Quinnan, Gerald V.; Dimitrov, Dimiter S.

    2009-01-01

    Elicitation of broadly cross-reactive neutralizing antibodies (bcnAbs) in HIV infections is rare. To test the hypothesis that such antibodies could be elicited by HIV envelope glycoproteins (Envs) with unusual immunogenic properties and to identify novel bcnAbs, we used a soluble Env ectodomain (gp140) from a donor (R2) with high level of bcnAbs as an antigen for panning of an immune phage-displayed antibody library. The panning with the R2 Env resulted in significantly higher number of cross-reactive antibody clones than by using Envs from two other isolates (89.6 and IIIB). Two of the identified human monoclonal antibodies (hmAbs), m22 and m24, had sequences, neutralizing and binding activities similar or identical to those of the gp120-specific bcnAbs m18 and m14. The use of the R2 Env but not other Envs for panning resulted in the identification of a novel gp41-specific hmAb, m46. For several of the tested HIV-1 primary isolates its potency on molar basis was comparable to that of T20. It inhibited entry of primary isolates from different clades with an increased activity for cell lines with low CCR5 surface concentrations. The m46 neutralizing activity against a panel of clade C isolates was significantly higher in an assay based on peripheral blood mononuclear cells (4 out of 5 isolates were neutralized with an IC50 in the range from 1.5 to 25 μg/ml) than in an assay based on a cell line with relatively high concentration of cell-surface associated CCR5. In contrast to 2F5 and Z13, this antibody did not bind to denatured gp140 and gp41-derived peptides indicating a conformational nature of its epitope. It bound to a 5-helix bundle but not to N-heptad repeat coiled coils and a 6-helix bundle construct indicating contribution of both gp41 heptad repeats to its epitope and to a possible mechanism of neutralization. These results indicate that the R2 Env may contain unique exposed conserved epitopes that could contribute to its ability to elicit broadly cross

  8. Seroreactive recombinant herpes simplex virus type 2-specific glycoprotein G.

    PubMed Central

    Parkes, D L; Smith, C M; Rose, J M; Brandis, J; Coates, S R

    1991-01-01

    The herpes simplex virus type 2 (HSV-2) genome codes for an envelope protein, glycoprotein G (gG), which contains predominantly type 2-specific epitopes. A portion of this gG gene has been expressed as a fusion protein in Escherichia coli. Expression was regulated by a lambda phage pL promoter. The 60,000-molecular-weight recombinant protein was purified by ion-exchange chromatography. Amino acid sequence analysis confirmed the N terminus of the purified protein. Mice immunized with recombinant gG developed antibodies reactive with native HSV-2 protein, but not with HSV-1 protein, in an indirect immunofluorescence assay. The serological activity of this purified recombinant gG protein was evaluated by immunoblot assay. This protein was reactive with an HSV-2 gG monoclonal antibody. It was also reactive with HSV-2 rabbit antiserum but not with HSV-1 rabbit antiserum. Of 15 patient serum samples known to have antibody to HSV-2, 14 were reactive with this recombinant type 2-specific gG protein, and none of 15 HSV antibody-negative patient serum samples showed reactivity. In agreement with the expected prevalence of HSV-2 infection, 27.6% of 134 serum samples from random normal individuals had antibodies reactive with recombinant gG. This recombinant gG protein may be of value in detecting HSV-2-specific antibody responses in patients infected with HSV-2. Images PMID:1653787

  9. Glycoprotein Analysis

    NASA Astrophysics Data System (ADS)

    Fernandes, Daryl; Spencer, Daniel

    This chapter provides an overview of practical methods for glycosylation analysis of glycoprotein therapeutics . The topics include glycoprofiling methods for glycoforms, monosaccharides (neutral and N-acetylated species as well as sialic acids), oligosaccharides (chemical and enzymatic methods for glycan release, post-release purification, labeling and derivatization, different types of glycan HPLC and MS), and glycosylation site profiling.

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. 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.

  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

    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.

  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. 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.

  1. 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. PMID:27018820

  2. Functional processing and secretion of Chikungunya virus E1 and E2 glycoproteins in insect cells

    PubMed Central

    2011-01-01

    Background Chikungunya virus (CHIKV) is a mosquito-borne, arthrogenic Alphavirus that causes large epidemics in Africa, South-East Asia and India. Recently, CHIKV has been transmitted to humans in Southern Europe by invading and now established Asian tiger mosquitoes. To study the processing of envelope proteins E1 and E2 and to develop a CHIKV subunit vaccine, C-terminally his-tagged E1 and E2 envelope glycoproteins were produced at high levels in insect cells with baculovirus vectors using their native signal peptides located in CHIKV 6K and E3, respectively. Results Expression in the presence of either tunicamycin or furin inhibitor showed that a substantial portion of recombinant intracellular E1 and precursor E3E2 was glycosylated, but that a smaller fraction of E3E2 was processed by furin into mature E3 and E2. Deletion of the C-terminal transmembrane domains of E1 and E2 enabled secretion of furin-cleaved, fully processed E1 and E2 subunits, which could then be efficiently purified from cell culture fluid via metal affinity chromatography. Confocal laser scanning microscopy on living baculovirus-infected Sf21 cells revealed that full-length E1 and E2 translocated to the plasma membrane, suggesting similar posttranslational processing of E1 and E2, as in a natural CHIKV infection. Baculovirus-directed expression of E1 displayed fusogenic activity as concluded from syncytia formation. CHIKV-E2 was able to induce neutralizing antibodies in rabbits. Conclusions Chikungunya virus glycoproteins could be functionally expressed at high levels in insect cells and are properly glycosylated and cleaved by furin. The ability of purified, secreted CHIKV-E2 to induce neutralizing antibodies in rabbits underscores the potential use of E2 in a subunit vaccine to prevent CHIKV infections. PMID:21762510

  3. 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.

  4. Baculoviral display of the green fluorescent protein and rubella virus envelope proteins.

    PubMed

    Mottershead, D; van der Linden, I; von Bonsdorff, C H; Keinänen, K; Oker-Blom, C

    1997-09-29

    The ability to display heterologous proteins and peptides on the surface of different types of bacteriophage has proven extremely useful in protein structure/function studies. To display such proteins in a eucaryotic environment, we have produced a vector allowing for fusion of proteins to the amino-terminus of the Autographa californica nuclear polyhedrosis virus (AcNPV) major envelope glycoprotein, gp64. Such fusion proteins incorporate into the baculoviral virion and display the FLAG epitope tag. We have further produced recombinant baculoviruses displaying the green fluorescent protein (GFP) and the rubella virus envelope proteins, E1 and E2. The incorporation of the GFPgp64, E1gp64, and E2gp64 fusion proteins into the baculovirus particle was demonstrated by western blot analysis of purified budded virus. This is the first report of the display of the GFP protein or the individual rubella virus spike proteins on the surface of an enveloped virus. Such a eucaryotic viral display system may be useful for the display of proteins dependent on glycosylation for activity and for targeting of recombinant baculoviruses to novel host cell types as a gene transfer vehicle. PMID:9325155

  5. 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.

  6. Interaction of Sindbis virus glycoproteins during morphogenesis.

    PubMed Central

    Jones, K J; Scupham, R K; Pfeil, J A; Wan, K; Sagik, B P; Bose, H R

    1977-01-01

    In cells infected with the Sindbis temperature-sensitive mutants ts-23 and ts-10 (complementation group D), which contain a defect in the envelope glycoprotein E1, the precursor polypeptide PE2 is not cleaved to the envelope glycoprotein E2 at the nonpermissive temperature. This defect is phenotypically identical to the defect observed in the complementation group E mutant, ts-20. The lesion in ts-23 is reversible upon shift to permissive temperature, whereas that of ts-10 is not. Antiserum against whole virus, E1, or E2 also prevents the cleavage of PE2 in cells infected with wild-type Sindbis virus. Because the cleavage of PE2 is inhibited by the lesion in mutants that are genotypically distinct and by anti-E1 or -E2 serum, it appears that PE2 and E1 exist as a complex in the membrane of the infected cell. Images PMID:833949

  7. 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

  8. 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.

  9. 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

  10. Glycosylation Engineering of Glycoproteins

    NASA Astrophysics Data System (ADS)

    Sadamoto, Reiko; Nishimura, Shin-Ichiro

    Naturally occurring glycosylation of glycoproteins varies in glycosylation site and in the number and structure of glycans. The engineering of well-defined glycoproteins is an important technology for the preparation of pharmaceutically relevant glycoproteins and in the study of the relationship between glycans and proteins on a structure-function level. In pharmaceutical applications of glycoproteins, the presence of terminal sialic acids on glycans is particularly important for the in vivo circulatory half life, since sialic acid-terminated glycans are not recognized by asialoglycoprotein receptors. Therefore, there have been a number of attempts to control or modify cellular metabolism toward the expression of glycoproteins with glycosylation profiles similar to that of human glycoproteins. In this chapter, recent methods for glycoprotein engineering in various cell culture systems (mammalian cells, plant, yeast, and E. coli) and advances in the chemical approach to glycoprotein formation are described.

  11. 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.

  12. 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.

  13. Purifying Water by Imbibition

    NASA Technical Reports Server (NTRS)

    Lawton, E. A.

    1986-01-01

    Concept for purifying water uses absorbent material to remove organic substances. Entire bulk of material employed, not just surface. Proposed purification process uses inexpensive equipment and low energy. Material is methyl acrylate polymer. Material cheap and regenerated by rinsing with methanol or by allowing absorbed compounds to evaporate from it.

  14. Gas purifier unit

    SciTech Connect

    Hawryluk, J.

    1984-09-18

    A liquid contact type gas purifier unit in which gases to be treated are passed upwardly through a bath cleansing fluid to impart a circular and radially outward movement alone a central perforate support plate, the periphery of which has a circular rim with an adjacent collecting and reciprocating trough to provide for an even outward flow of cleansing fluid and stability of flow of the interacting fluid and gas streams and wider latitude of operable gas and pressure and flows.

  15. 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.

  16. 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. PMID:24783808

  17. 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

  18. 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

  19. 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

  20. 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.

  1. Native and recombinant herpes simplex virus type 1 envelope proteins induce human immune T-lymphocyte responses.

    PubMed

    Torseth, J W; Cohen, G H; Eisenberg, R J; Berman, P W; Lasky, L A; Cerini, C P; Heilman, C J; Kerwar, S; Merigan, T C

    1987-05-01

    The abilities of whole herpes simplex virus type 1 (HSV-1) antigen (HSV-ag) and purified HSV-1 native and recombinant envelope proteins to stimulate in vitro T-lymphocyte responses were compared in patients with recurrent herpes labialis. Immunochemically purified preparations of native glycoproteins B, C, and D (ngB, ngC, ngD) from cultured HSV-1 as well as expressed recombinant plasmid preparations of gD (rgD-1t, rgD-45K) elicited lymphocyte proliferation (LT) and production of gamma interferon (IFN-gamma) and interleukin-2 (IL-2) only in seropositive individuals. The IFN-gamma induced by rgD-1t correlated with the time to the next herpetic lesion in 19 volunteers followed to recurrence (r = 0.69, P less than 0.008), although the magnitude and frequency of LT and IFN-gamma responses were lower with either recombinant or native purified antigens than with the whole-virus antigen. Combinations of ngB plus ngD or ngB plus ngC plus ngD stimulated more IFN-gamma, equivalent to whole-virus-antigen responses. Recombinant-derived human IL-2 also specifically increased LT and IFN-gamma responses in antigen-driven cultures. ngD stimulated IL-2 and LT responses similar to those of whole-virus antigen and higher than those of ngC. HSV-ag and ngB induced significantly higher titers of total IFN than could be accounted for by IFN-gamma; this was not seen for the other antigens, which induced only IFN-gamma. HSV-ag-driven Leu 2a-, plastic-nonadherent blood cells, unlike whole peripheral blood mononuclear cells, showed evidence of an increase and then a decline in the frequency of HSV-responsive cells after a lesion recurrence. These studies suggest that HSV-1 envelope proteins are capable of stimulating an immune T-helper-cell response which is associated with the prevention of human herpes simplex lesion recurrence. Although the whole virus probably contains additional important antigens, increasing concentrations or combinations of certain purified glycoproteins or the

  2. Purification of a Trypanosoma cruzi trypomastigote 60-kilodalton surface glycoprotein that primes and activates murine lymphocytes.

    PubMed Central

    Villalta, F; Lima, M F; Howard, S A; Zhou, L; Ruiz-Ruano, A

    1992-01-01

    We have purified a glycoprotein with a relative molecular mass of 60 kDa and present on the surface of Trypanosoma cruzi trypomastigotes and studied its ability to prime and stimulate the proliferation of murine spleen cells. T. cruzi trypomastigote membrane proteins were separated by preparative isoelectrofocusing. A trypomastigote 60-kDa surface protein with an isoelectric point of 4.2 was enriched by chromatofocusing and was readily purified in native form to homogeneity by gel filtration on a Superose column by use of a fast protein liquid chromatography system. Biotinylated wheat germ agglutinin, Ricinus communis agglutinin, and Datura stramonium agglutinin bound to blots containing the purified trypomastigote 60-kDa surface protein, indicating that this protein was glycosylated. The purified trypomastigote 60-kDa glycoprotein was recognized by antibodies produced during human infection, and immunoglobulin G against the purified glycoprotein immunoprecipitated a biotinylated 60-kDa molecule from the surface of trypomastigotes but not epimastigotes. Specific immunoglobulin G against the 60-kDa glycoprotein also increased the uptake of trypomastigotes and promoted parasite killing by macrophages. The purified 60-kDa glycoprotein was able to specifically activate primed lymphocytes, since there was a significant increase in [3H]thymidine incorporation by spleen cells obtained from CBA mice primed with this glycoprotein, with respect to control values. Furthermore, the 60-kDa glycoprotein did not stimulate unprimed spleen cells, indicating that the lymphoproliferation induced by this glycoprotein was specific and was not due to polyclonal activation. Our findings indicate that this T. cruzi trypomastigote 60-kDa surface glycoprotein primes and activates lymphocytes, which could lead to a beneficial immune response in the host. Images PMID:1639469

  3. Histidine-rich glycoprotein inhibits contact activation of blood coagulation.

    PubMed

    Vestergaard, A B; Andersen, H F; Magnusson, S; Halkier, T

    1990-12-01

    Histidine-rich glycoprotein has been purified from bovine plasma employing two different purification procedures. The first procedure was one-step ion-exchange chromatography using phosphocellulose, while the second procedure involved fractionation using polyethyleneglycol 6000 followed by column chromatography employing CM-Sepharose and heparin-Sepharose. The effect of purified bovine histidine-rich glycoprotein on the contact activation of blood coagulation was studied in human plasma by using as activating surface either an ellagic acid-phospholipid suspension (Cephotest) or sulfatide. Contact activation was monitored by the generation of amidolytic activity towards a synthetic chromogenic substrate (S-2302) for factor XIIa and plasma kallikrein. Bovine histidine-rich glycoprotein inhibits the contact activation induced by both of these activating surfaces. PMID:2084959

  4. 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.

  5. 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

  6. 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,...

  7. Human immunodeficiency virus glycoprotein (gp120) induction of monocyte arachidonic acid metabolites and interleukin 1.

    PubMed Central

    Wahl, L M; Corcoran, M L; Pyle, S W; Arthur, L O; Harel-Bellan, A; Farrar, W L

    1989-01-01

    This study reports on the direct effect of the envelope glycoprotein (gp120) of the human immunodeficiency virus type 1 (HIV-1) on human monocyte function. Addition of preparations of purified gp120 from the HIV-1 to human monocytes resulted in the production of interleukin 1 (IL-1) and arachidonic acid metabolites from the cyclooxygenase and lipoxygenase pathways. Quantification of prostaglandin E2 (PGE2) and IL-1 revealed an increase in both mediators with 50 ng of gp120 per ml and an increase of 12- and 30- to 40-fold with 200-400 ng of gp120 per ml, respectively. Unlike native gp120, the recombinant nonglycosylated gp120 fragments PB1-RF and PB1-IIIB, as well as one of the core structural proteins of HIV-1, p24, did not increase arachidonic acid metabolism or IL-1 activity. Cytofluorometric analysis revealed that gp120 blocked the binding of OKT4A to the CD4 on monocytes, whereas OKT4 binding was unaffected. Involvement of the CD4 in signal transduction was further demonstrated by the ability of OKT4 and OKT4A monoclonal antibodies to increase monocyte PGE2, IL-1 activity, and nanogram amounts of IL-1 beta. PMID:2536171

  8. Stereopsis from contrast envelopes.

    PubMed

    Langley, K; Fleet, D J; Hibbard, P B

    1999-07-01

    We report two experiments concerning the site of the principal nonlinearity in second-order stereopsis. The first exploits the asymmetry in perceiving transparency with second-order stimuli found by Langley et al. (1998) (Proceedings of the Royal Society of London B, 265, 1837-1845) i.e. the product of a positive-valued contrast envelope and a mean-zero carrier grating can be seen transparently only when the disparities are consistent with the envelope appearing in front of the carrier. We measured the energy at the envelope frequencies that must be added in order to negate this asymmetry. We report that this amplitude can be predicted from the envelope sidebands and not from the magnitude of compressive pre-cortical nonlinearities measured by other researchers. In the second experiment, contrast threshold elevations were measured for the discrimination of envelope disparities following adaptation to sinusoidal gratings. It is reported that perception of the envelope's depth was affected most when the adapting grating was similar (in orientation and frequency) to the carrier, rather than to the contrast envelope. These results suggest that the principal nonlinearity in second-order stereopsis is cortical, occurring after orientation- and frequency-selective linear filtering. PMID:10367053

  9. Structures and Functions of Pestivirus Glycoproteins: Not Simply Surface Matters.

    PubMed

    Wang, Fun-In; Deng, Ming-Chung; Huang, Yu-Liang; Chang, Chia-Yi

    2015-07-01

    Pestiviruses, which include economically important animal pathogens such as bovine viral diarrhea virus and classical swine fever virus, possess three envelope glycoproteins, namely Erns, E1, and E2. This article discusses the structures and functions of these glycoproteins and their effects on viral pathogenicity in cells in culture and in animal hosts. E2 is the most important structural protein as it interacts with cell surface receptors that determine cell tropism and induces neutralizing antibody and cytotoxic T-lymphocyte responses. All three glycoproteins are involved in virus attachment and entry into target cells. E1-E2 heterodimers are essential for viral entry and infectivity. Erns is unique because it possesses intrinsic ribonuclease (RNase) activity that can inhibit the production of type I interferons and assist in the development of persistent infections. These glycoproteins are localized to the virion surface; however, variations in amino acids and antigenic structures, disulfide bond formation, glycosylation, and RNase activity can ultimately affect the virulence of pestiviruses in animals. Along with mutations that are driven by selection pressure, antigenic differences in glycoproteins influence the efficacy of vaccines and determine the appropriateness of the vaccines that are currently being used in the field. PMID:26131960

  10. The solar envelope

    NASA Technical Reports Server (NTRS)

    Burlaga, L. F.

    1971-01-01

    Processes which occur within the region between approximately 2 solar radii and 25 solar radii, which is called the solar envelope and the effect on the solar wind as seen at 1 AU are discussed. In the envelope the wind speed becomes supersonic and super-Alfvenic, the magnetic energy density is larger than the flow energy density, and the magnetic energy density is much larger than the thermal energy density. Large azimuthal gradients in the bulk speed are expected in the envelope, but the stream interactions near the outer edge of the envelope are probably relatively small. Cosmic ray observations suggest the presence of hydromagnetic waves in the envelope. The collisionless damping of such waves could heat protons out to approximately 25 solar radii and thereby cause an increase in V and T sub p consistent with the observed T sub p -V relation. A mechanism which couples protons and electrons would also heat and accelerate the wind. Alfven waves can accelerate the wind in the envelope without necessarily causing heating of protons; the Lorentz force might have a similar effect.

  11. [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

  12. Immunogenic Display of Purified Chemically Cross-Linked HIV-1 Spikes

    PubMed Central

    Leaman, Daniel P.; Lee, Jeong Hyun; Ward, Andrew B.

    2015-01-01

    ABSTRACT HIV-1 envelope glycoprotein (Env) spikes are prime vaccine candidates, at least in principle, but suffer from instability, molecular heterogeneity and a low copy number on virions. We anticipated that chemical cross-linking of HIV-1 would allow purification and molecular characterization of trimeric Env spikes, as well as high copy number immunization. Broadly neutralizing antibodies bound tightly to all major quaternary epitopes on cross-linked spikes. Covalent cross-linking of the trimer also stabilized broadly neutralizing epitopes, although surprisingly some individual epitopes were still somewhat sensitive to heat or reducing agent. Immunodepletion using non-neutralizing antibodies to gp120 and gp41 was an effective method for removing non-native-like Env. Cross-linked spikes, purified via an engineered C-terminal tag, were shown by negative stain EM to have well-ordered, trilobed structure. An immunization was performed comparing a boost with Env spikes on virions to spikes cross-linked and captured onto nanoparticles, each following a gp160 DNA prime. Although differences in neutralization did not reach statistical significance, cross-linked Env spikes elicited a more diverse and sporadically neutralizing antibody response against Tier 1b and 2 isolates when displayed on nanoparticles, despite attenuated binding titers to gp120 and V3 crown peptides. Our study demonstrates display of cross-linked trimeric Env spikes on nanoparticles, while showing a level of control over antigenicity, purity and density of virion-associated Env, which may have relevance for Env based vaccine strategies for HIV-1. IMPORTANCE The envelope spike (Env) is the target of HIV-1 neutralizing antibodies, which a successful vaccine will need to elicit. However, native Env on virions is innately labile, as well as heterogeneously and sparsely displayed. We therefore stabilized Env spikes using a chemical cross-linker and removed non-native Env by immunodepletion with non

  13. 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.

  14. 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

  15. 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.

  16. 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.

  17. 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.

  18. 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. PMID:27155240

  19. 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...

  20. 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.

  1. Purification and in vitro-phospholabeling of secretory envelope proteins E1 and E2 of hepatitis C virus expressed in insect cells.

    PubMed

    Hüssy, P; Schmid, G; Mous, J; Jacobsen, H

    1996-11-01

    The putative envelope glycoproteins of hepatitis C virus (HCV), E1 and E2, were expressed as recombinant, secretory proteins in Sf9 insect cells through infection with recombinant baculoviruses. The influenza virus hemagglutinin signal sequence (HASS) was inserted upstream of the HCV-cDNAs in order to effect secretion. Furthermore, a hexa-histidine tag for purification on a Ni(2+)-nitrilotriacetic acid (Ni(2+)-NTA) column and a protein kinase A (PKA) recognition sequence for in vitro-phospholabeling were fused upstream of the HCV-cDNA. E1- and E2 proteins lacking their carboxy-terminal, hydrophobic sequence were produced by baculovirus-infected insect cells in bioreactors of 23 1. The medium was concentrated and proteins were purified under native conditions on Ni(2+)-NTA columns. Purified proteins could be phospholabeled in vitro using the catalytic subunit of protein kinase. A isolated from bovine heart and gamma-[32P]ATP. Labeled E1 and E2 proteins expressed in insect cells could be immunoprecipitated with sera from HCV-infected patients. Co-expression of these E1 and E2 proteins led to the formation of E1-E2 complexes within the insect cell and to secretion of these complexes into the medium. PMID:8896240

  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. 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.

  4. 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.

  5. Process for purifying geothermal steam

    SciTech Connect

    Li, C.T.

    1980-04-01

    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 of solvent extraction. The process is suitable for the purification of steam from geothermal sources which may also contain other noncondensable gases.

  6. 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.

  7. 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.

  8. 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

  9. Distinct structural rearrangements of the VSV glycoprotein drive membrane fusion

    PubMed Central

    Libersou, Sonia; Albertini, Aurélie A.V.; Ouldali, Malika; Maury, Virginie; Maheu, Christine; Raux, Hélène; de Haas, Felix; Roche, Stéphane

    2010-01-01

    The entry of enveloped viruses into cells requires the fusion of viral and cellular membranes, driven by conformational changes in viral glycoproteins. Many studies have shown that fusion involves the cooperative action of a large number of these glycoproteins, but the underlying mechanisms are unknown. We used electron microscopy and tomography to study the low pH–induced fusion reaction catalyzed by vesicular stomatitis virus glycoprotein (G). Pre- and post-fusion crystal structures were observed on virions at high and low pH, respectively. Individual fusion events with liposomes were also visualized. Fusion appears to be driven by two successive structural rearrangements of G at different sites on the virion. Fusion is initiated at the flat base of the particle. Glycoproteins located outside the contact zone between virions and liposomes then reorganize into regular arrays. We suggest that the formation of these arrays, which have been shown to be an intrinsic property of the G ectodomain, induces membrane constraints, achieving the fusion reaction. PMID:20921141

  10. STS-8 postal Stamp envelope

    NASA Technical Reports Server (NTRS)

    1983-01-01

    STS-8 postal Stamp envelope with Challenger insignia, USA eagle stamp, 25th NASA anniversary stamp. The envelope is stamped with various postmarks, one saying Kennedy Space Center, Fl., another saying 'Returned to earth, Edwards AFB, CA'.