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Sample records for envelope protein vp51a

  1. Different evolutionary patterns of classical swine fever virus envelope proteins.

    PubMed

    Li, Yan; Yang, Zexiao; Zhang, Mingwang

    2016-03-01

    Classical swine fever virus (CSFV) is the causative agent of classical swine fever, which is a highly contagious disease of the domestic pig as well as wild boar. The proteins E(rns), E1, and E2 are components of the viral envelope membrane. They are also implicated in virus attachment and entry, replication, and (or) anti-immune response. Here, we studied the genetic variations of these envelope proteins in the evolution of CSFV. The results reveal that the envelope proteins underwent different evolutionary fates. In E(rns) and E1, but not E2, a number of amino acid sites experienced functional divergence. Furthermore, the diversification in E(rns) and E1 was generally episodic because the divergence-related changes of E1 only occurred with the separation of 2 major groups of CSFV and that of E(rns) took place with the division of 1 major group. The major divergence-related sites of E(rns) are located on one of the substrate-binding regions of the RNase domain and C-terminal extension. These functional domains have been reported to block activation of the innate immune system and attachment and entry into host cells, respectively. Our results may shed some light on the divergent roles of the envelope proteins. PMID:26911308

  2. Thioaptamers Targeting Dengue Virus Type-2 Envelope Protein Domain III

    PubMed Central

    Gandham, Sai Hari A.; Volk, David E.; Rao, Lokesh G. L.; Neerathilingam, Muniasamy; Gorenstein, David G.

    2014-01-01

    Thioaptamers targeting the dengue-2 virus (DENV-2) envelope protein domain III (EDIII) were developed. EDIII, which contains epitopes for binding neutralizing antibodies, is the putative host-receptor binding domain and is thus an attractive target for development of vaccines, anti-viral therapeutic and diagnostic agents. Thioaptamer DENTA-1 bound to DENV-2 EDIII adjacent to a known neutralizing antibody binding site with a dissociation constant of 154 nM. PMID:25261724

  3. Inactivation of enveloped virus by laser-driven protein aggregation

    NASA Astrophysics Data System (ADS)

    Tsen, Shaw-Wei D.; Chapa, Travis; Beatty, Wandy; Tsen, Kong-Thon; Yu, Dong; Achilefu, Samuel

    2012-12-01

    Ultrafast lasers in the visible and near-infrared range have emerged as a potential new method for pathogen reduction of blood products and pharmaceuticals. However, the mechanism of enveloped virus inactivation by this method is unknown. We report the inactivation as well as the molecular and structural effects caused by visible (425 nm) femtosecond laser irradiation on murine cytomegalovirus (MCMV), an enveloped, double-stranded DNA virus. Our results show that laser irradiation (1) caused a 5-log reduction in MCMV titer, (2) did not cause significant changes to the global structure of MCMV virions including membrane and capsid, as assessed by electron microscopy, (3) produced no evidence of double-strand breaks or crosslinking in MCMV genomic DNA, and (4) caused selective aggregation of viral capsid and tegument proteins. We propose a model in which ultrafast laser irradiation induces partial unfolding of viral proteins by disrupting hydrogen bonds and/or hydrophobic interactions, leading to aggregation of closely associated viral proteins and inactivation of the virus. These results provide new insight into the inactivation of enveloped viruses by visible femtosecond lasers at the molecular level, and help pave the way for the development of a new ultrafast laser technology for pathogen reduction.

  4. Inactivation of enveloped virus by laser-driven protein aggregation.

    PubMed

    Tsen, Shaw-Wei D; Chapa, Travis; Beatty, Wandy; Tsen, Kong-Thon; Yu, Dong; Achilefu, Samuel

    2012-12-01

    Ultrafast lasers in the visible and near-infrared range have emerged as a potential new method for pathogen reduction of blood products and pharmaceuticals. However, the mechanism of enveloped virus inactivation by this method is unknown. We report the inactivation as well as the molecular and structural effects caused by visible (425 nm) femtosecond laser irradiation on murine cytomegalovirus (MCMV), an enveloped, double-stranded DNA virus. Our results show that laser irradiation (1) caused a 5-log reduction in MCMV titer, (2) did not cause significant changes to the global structure of MCMV virions including membrane and capsid, as assessed by electron microscopy, (3) produced no evidence of double-strand breaks or crosslinking in MCMV genomic DNA, and (4) caused selective aggregation of viral capsid and tegument proteins. We propose a model in which ultrafast laser irradiation induces partial unfolding of viral proteins by disrupting hydrogen bonds and/or hydrophobic interactions, leading to aggregation of closely associated viral proteins and inactivation of the virus. These results provide new insight into the inactivation of enveloped viruses by visible femtosecond lasers at the molecular level, and help pave the way for the development of a new ultrafast laser technology for pathogen reduction. PMID:23224114

  5. Coronavirus envelope (E) protein remains at the site of assembly.

    PubMed

    Venkatagopalan, Pavithra; Daskalova, Sasha M; Lopez, Lisa A; Dolezal, Kelly A; Hogue, Brenda G

    2015-04-01

    Coronaviruses (CoVs) assemble at endoplasmic reticulum Golgi intermediate compartment (ERGIC) membranes and egress from cells in cargo vesicles. Only a few molecules of the envelope (E) protein are assembled into virions. The role of E in morphogenesis is not fully understood. The cellular localization and dynamics of mouse hepatitis CoV A59 (MHV) E protein were investigated to further understanding of its role during infection. E protein localized in the ERGIC and Golgi with the amino and carboxy termini in the lumen and cytoplasm, respectively. E protein does not traffic to the cell surface. MHV was genetically engineered with a tetracysteine tag at the carboxy end of E. Fluorescence recovery after photobleaching (FRAP) showed that E is mobile in ERGIC/Golgi membranes. Correlative light electron microscopy (CLEM) confirmed the presence of E in Golgi cisternae. The results provide strong support that E proteins carry out their function(s) at the site of budding/assembly. PMID:25726972

  6. Actin Interacts with Dengue Virus 2 and 4 Envelope Proteins

    PubMed Central

    Jitoboam, Kunlakanya; Phaonakrop, Narumon; Libsittikul, Sirikwan; Thepparit, Chutima; Roytrakul, Sittiruk; Smith, Duncan R.

    2016-01-01

    Dengue virus (DENV) remains a significant public health problem in many tropical and sub-tropical countries worldwide. The DENV envelope (E) protein is the major antigenic determinant and the protein that mediates receptor binding and endosomal fusion. In contrast to some other DENV proteins, relatively few cellular interacting proteins have been identified. To address this issue a co-immuoprecipitation strategy was employed. The predominant co-immunoprecipitating proteins identified were actin and actin related proteins, however the results suggested that actin was the only bona fide interacting partner. Actin was shown to interact with the E protein of DENV 2 and 4, and the interaction between actin and DENV E protein was shown to occur in a truncated DENV consisting of only domains I and II. Actin was shown to decrease during infection, but this was not associated with a decrease in gene transcription. Actin-related proteins also showed a decrease in expression during infection that was not transcriptionally regulated. Cytoskeletal reorganization was not observed during infection, suggesting that the interaction between actin and E protein has a cell type specific component. PMID:27010925

  7. Protein folding in the cell envelope of Escherichia coli.

    PubMed

    De Geyter, Jozefien; Tsirigotaki, Alexandra; Orfanoudaki, Georgia; Zorzini, Valentina; Economou, Anastassios; Karamanou, Spyridoula

    2016-01-01

    While the entire proteome is synthesized on cytoplasmic ribosomes, almost half associates with, localizes in or crosses the bacterial cell envelope. In Escherichia coli a variety of mechanisms are important for taking these polypeptides into or across the plasma membrane, maintaining them in soluble form, trafficking them to their correct cell envelope locations and then folding them into the right structures. The fidelity of these processes must be maintained under various environmental conditions including during stress; if this fails, proteases are called in to degrade mislocalized or aggregated proteins. Various soluble, diffusible chaperones (acting as holdases, foldases or pilotins) and folding catalysts are also utilized to restore proteostasis. These responses can be general, dealing with multiple polypeptides, with functional overlaps and operating within redundant networks. Other chaperones are specialized factors, dealing only with a few exported proteins. Several complex machineries have evolved to deal with binding to, integration in and crossing of the outer membrane. This complex protein network is responsible for fundamental cellular processes such as cell wall biogenesis; cell division; the export, uptake and degradation of molecules; and resistance against exogenous toxic factors. The underlying processes, contributing to our fundamental understanding of proteostasis, are a treasure trove for the development of novel antibiotics, biopharmaceuticals and vaccines. PMID:27573113

  8. Coronavirus envelope (E) protein remains at the site of assembly

    SciTech Connect

    Venkatagopalan, Pavithra; Daskalova, Sasha M.; Lopez, Lisa A.; Dolezal, Kelly A.; Hogue, Brenda G.

    2015-04-15

    Coronaviruses (CoVs) assemble at endoplasmic reticulum Golgi intermediate compartment (ERGIC) membranes and egress from cells in cargo vesicles. Only a few molecules of the envelope (E) protein are assembled into virions. The role of E in morphogenesis is not fully understood. The cellular localization and dynamics of mouse hepatitis CoV A59 (MHV) E protein were investigated to further understanding of its role during infection. E protein localized in the ERGIC and Golgi with the amino and carboxy termini in the lumen and cytoplasm, respectively. E protein does not traffic to the cell surface. MHV was genetically engineered with a tetracysteine tag at the carboxy end of E. Fluorescence recovery after photobleaching (FRAP) showed that E is mobile in ERGIC/Golgi membranes. Correlative light electron microscopy (CLEM) confirmed the presence of E in Golgi cisternae. The results provide strong support that E proteins carry out their function(s) at the site of budding/assembly. - Highlights: • Mouse hepatitis coronavirus (MHV-CoV) E protein localizes in the ERGIC and Golgi. • MHV-CoV E does not transport to the cell surface. • MHV-CoV can be genetically engineered with a tetracysteine tag appended to E. • First FRAP and correlative light electron microscopy of a CoV E protein. • Live-cell imaging shows that E is mobile in ERGIC/Golgi membranes.

  9. Crystal Structure of the Japanese Encephalitis Virus Envelope Protein

    SciTech Connect

    Luca, Vincent C.; AbiMansour, Jad; Nelson, Christopher A.; Fremont, Daved H.

    2012-03-13

    Japanese encephalitis virus (JEV) is the leading global cause of viral encephalitis. The JEV envelope protein (E) facilitates cellular attachment and membrane fusion and is the primary target of neutralizing antibodies. We have determined the 2.1-{angstrom} resolution crystal structure of the JEV E ectodomain refolded from bacterial inclusion bodies. The E protein possesses the three domains characteristic of flavivirus envelopes and epitope mapping of neutralizing antibodies onto the structure reveals determinants that correspond to the domain I lateral ridge, fusion loop, domain III lateral ridge, and domain I-II hinge. While monomeric in solution, JEV E assembles as an antiparallel dimer in the crystal lattice organized in a highly similar fashion as seen in cryo-electron microscopy models of mature flavivirus virions. The dimer interface, however, is remarkably small and lacks many of the domain II contacts observed in other flavivirus E homodimers. In addition, uniquely conserved histidines within the JEV serocomplex suggest that pH-mediated structural transitions may be aided by lateral interactions outside the dimer interface in the icosahedral virion. Our results suggest that variation in dimer structure and stability may significantly influence the assembly, receptor interaction, and uncoating of virions.

  10. Envelope protein palmitoylations are crucial for murine coronavirus assembly.

    PubMed

    Boscarino, Joseph A; Logan, Hillary L; Lacny, Jason J; Gallagher, Thomas M

    2008-03-01

    The coronavirus assembly process encloses a ribonucleoprotein genome into vesicles containing the lipid-embedded proteins S (spike), E (envelope), and M (membrane). This process depends on interactions with membranes that may involve palmitoylation, a common posttranslational lipidation of cysteine residues. To determine whether specific palmitoylations influence coronavirus assembly, we introduced plasmid DNAs encoding mouse hepatitis coronavirus (MHV) S, E, M, and N (nucleocapsid) into 293T cells and found that virus-like particles (VLPs) were robustly assembled and secreted into culture medium. Palmitate adducts predicted on cysteines 40, 44, and 47 of the 83-residue E protein were then evaluated by constructing mutant cDNAs with alanine or glycine codon substitutions at one or more of these positions. Triple-substituted proteins (E.Ts) lacked palmitate adducts. Both native E and E.T proteins localized at identical perinuclear locations, and both copurified with M proteins, but E.T was entirely incompetent for VLP production. In the presence of the E.T proteins, the M protein subunits accumulated into detergent-insoluble complexes that failed to secrete from cells, while native E proteins mobilized M into detergent-soluble secreted forms. Many of these observations were corroborated in the context of natural MHV infections, with native E, but not E.T, complementing debilitated recombinant MHVs lacking E. Our findings suggest that palmitoylations are essential for E to act as a vesicle morphogenetic protein and further argue that palmitoylated E proteins operate by allowing the primary coronavirus assembly subunits to assume configurations that can mobilize into secreted lipid vesicles and virions. PMID:18184706

  11. Nucleocytoplasmic transport of nucleocapsid proteins of enveloped RNA viruses

    PubMed Central

    Wulan, Wahyu N.; Heydet, Deborah; Walker, Erin J.; Gahan, Michelle E.; Ghildyal, Reena

    2015-01-01

    Most viruses with non-segmented single stranded RNA genomes complete their life cycle in the cytoplasm of infected cells. However, despite undergoing replication in the cytoplasm, the structural proteins of some of these RNA viruses localize to the nucleus at specific times in the virus life cycle, primarily early in infection. Limited evidence suggests that this enhances successful viral replication by interfering with or inhibiting the host antiviral response. Nucleocapsid proteins of RNA viruses have a well-established, essential cytoplasmic role in virus replication and assembly. Intriguingly, nucleocapsid proteins of some RNA viruses also localize to the nucleus/nucleolus of infected cells. Their nuclear function is less well understood although significant advances have been made in recent years. This review will focus on the nucleocapsid protein of cytoplasmic enveloped RNA viruses, including their localization to the nucleus/nucleolus and function therein. A greater understanding of the nuclear localization of nucleocapsid proteins has the potential to enhance therapeutic strategies as it can be a target for the development of live-attenuated vaccines or antiviral drugs. PMID:26082769

  12. Structural changes of envelope proteins during alphavirus fusion

    SciTech Connect

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

    2010-12-08

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

  13. Mechanisms of Oxidative Protein Folding in the Bacterial Cell Envelope

    PubMed Central

    2010-01-01

    Abstract Disulfide-bond formation is important for the correct folding of a great number of proteins that are exported to the cell envelope of bacteria. Bacterial cells have evolved elaborate systems to promote the joining of two cysteines to form a disulfide bond and to repair misoxidized proteins. In the past two decades, significant advances have occurred in our understanding of the enzyme systems (DsbA, DsbB, DsbC, DsbG, and DsbD) used by the gram-negative bacterium Escherichia coli to ensure that correct pairs of cysteines are joined during the process of protein folding. However, a number of fundamental questions about these processes remain, especially about how they occur inside the cell. In addition, recent recognition of the increasing diversity among bacteria in the disulfide bond–forming capacity and in the systems for introducing disulfide bonds into proteins is raising new questions. We review here the marked progress in this field and discuss important questions that remain for future studies. Antioxid. Redox Signal. 13, 1231–1246. PMID:20367276

  14. Structure of Yellow Fever Virus Envelope Protein Domain III

    PubMed Central

    Volk, David E.; May, Fiona J.; Gandham, Sai H. A.; Anderson, Anjenique; Von Lindern, Jana J.; Beasley, David W. C.; Barrett, Alan D. T.; Gorenstein, David G.

    2009-01-01

    The structure of recombinant domain III of the envelope protein (rED3) of yellow fever virus (YFV), containing the major neutralization site, was determined using NMR spectroscopy. The amino acid sequence and structure of the YFV-rED3 shows differences from ED3s of other mosquito-borne flaviviruses; in particular, the partially surface-exposed BC loop where methionine-304 and valine-324 were identified as being critical for the structure of the loop. Variations in the structure and surface chemistry of ED3 between flaviviruses affect neutralization sites and may affect host cell receptor interactions and play a role in the observed variations in viral pathogenesis and tissue tropism. PMID:19818466

  15. 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

  16. Several novel nuclear envelope transmembrane proteins identified in skeletal muscle have cytoskeletal associations.

    PubMed

    Wilkie, Gavin S; Korfali, Nadia; Swanson, Selene K; Malik, Poonam; Srsen, Vlastimil; Batrakou, Dzmitry G; de las Heras, Jose; Zuleger, Nikolaj; Kerr, Alastair R W; Florens, Laurence; Schirmer, Eric C

    2011-01-01

    Nuclear envelopes from liver and a neuroblastoma cell line have previously been analyzed by proteomics; however, most diseases associated with the nuclear envelope affect muscle. To determine whether muscle has unique nuclear envelope proteins, rat skeletal muscle nuclear envelopes were prepared and analyzed by multidimensional protein identification technology. Many novel muscle-specific proteins were identified that did not appear in previous nuclear envelope data sets. Nuclear envelope residence was confirmed for 11 of these by expression of fusion proteins and by antibody staining of muscle tissue cryosections. Moreover, transcript levels for several of the newly identified nuclear envelope transmembrane proteins increased during muscle differentiation using mouse and human in vitro model systems. Some of these proteins tracked with microtubules at the nuclear surface in interphase cells and accumulated at the base of the microtubule spindle in mitotic cells, suggesting they may associate with complexes that connect the nucleus to the cytoskeleton. The finding of tissue-specific proteins in the skeletal muscle nuclear envelope proteome argues the importance of analyzing nuclear envelopes from all tissues linked to disease and suggests that general investigation of tissue differences in organellar proteomes might yield critical insights. PMID:20876400

  17. Several Novel Nuclear Envelope Transmembrane Proteins Identified in Skeletal Muscle Have Cytoskeletal Associations*

    PubMed Central

    Wilkie, Gavin S.; Korfali, Nadia; Swanson, Selene K.; Malik, Poonam; Srsen, Vlastimil; Batrakou, Dzmitry G.; de las Heras, Jose; Zuleger, Nikolaj; Kerr, Alastair R. W.; Florens, Laurence; Schirmer, Eric C.

    2011-01-01

    Nuclear envelopes from liver and a neuroblastoma cell line have previously been analyzed by proteomics; however, most diseases associated with the nuclear envelope affect muscle. To determine whether muscle has unique nuclear envelope proteins, rat skeletal muscle nuclear envelopes were prepared and analyzed by multidimensional protein identification technology. Many novel muscle-specific proteins were identified that did not appear in previous nuclear envelope data sets. Nuclear envelope residence was confirmed for 11 of these by expression of fusion proteins and by antibody staining of muscle tissue cryosections. Moreover, transcript levels for several of the newly identified nuclear envelope transmembrane proteins increased during muscle differentiation using mouse and human in vitro model systems. Some of these proteins tracked with microtubules at the nuclear surface in interphase cells and accumulated at the base of the microtubule spindle in mitotic cells, suggesting they may associate with complexes that connect the nucleus to the cytoskeleton. The finding of tissue-specific proteins in the skeletal muscle nuclear envelope proteome argues the importance of analyzing nuclear envelopes from all tissues linked to disease and suggests that general investigation of tissue differences in organellar proteomes might yield critical insights. PMID:20876400

  18. Green Fluorescent Protein-Tagged Retroviral Envelope Protein for Analysis of Virus-Cell Interactions

    PubMed Central

    Spitzer, Dirk; Dittmar, Kurt E. J.; Rohde, Manfred; Hauser, Hansjörg; Wirth, Dagmar

    2003-01-01

    Fluorescent retroviral envelope (Env) proteins were developed for direct visualization of viral particles. By fusing the enhanced green fluorescent protein (eGFP) to the N terminus of the amphotropic 4070A envelope protein, extracellular presentation of eGFP was achieved. Viruses incorporated the modified Env protein and efficiently infected cells. We used the GFP-tagged viruses for staining retrovirus receptor-positive cells, thereby circumventing indirect labeling techniques. By generating cells which conditionally expressed the GFP-tagged Env protein, we could confirm an inverse correlation between retroviral Env expression and infectivity (superinfection). eGFP-tagged virus particles are suitable for monitoring the dynamics of virus-cell interactions. PMID:12719600

  19. Dual Protein Localization to the Envelope and Thylakoid Membranes Within the Chloroplast.

    PubMed

    Klasek, Laura; Inoue, Kentaro

    2016-01-01

    The chloroplast houses various metabolic processes essential for plant viability. This organelle originated from an ancestral cyanobacterium via endosymbiosis and maintains the three membranes of its progenitor. Among them, the outer envelope membrane functions mainly in communication with cytoplasmic components while the inner envelope membrane houses selective transport of various metabolites and the biosynthesis of several compounds, including membrane lipids. These two envelope membranes also play essential roles in import of nuclear-encoded proteins and in organelle division. The third membrane, the internal membrane system known as the thylakoid, houses photosynthetic electron transport and chemiosmotic phosphorylation. The inner envelope and thylakoid membranes share similar lipid composition. Specific targeting pathways determine their defined proteomes and, thus, their distinct functions. Nonetheless, several proteins have been shown to exist in both the envelope and thylakoid membranes. These proteins include those that play roles in protein transport, tetrapyrrole biosynthesis, membrane dynamics, or transport of nucleotides or inorganic phosphate. In this review, we summarize the current knowledge about proteins localized to both the envelope and thylakoid membranes in the chloroplast, discussing their roles in each membrane and potential mechanisms of their dual localization. Addressing the unanswered questions about these dual-localized proteins should help advance our understanding of chloroplast development, protein transport, and metabolic regulation. PMID:26944623

  20. OOCYTE ENVELOPE PROTEINS AND VITELLOGENIN IN MALE SHEEPHEAD MINNOW EXPOSED TO ESTRADIOL

    EPA Science Inventory

    Oocyte Envelope Proteins and Vitellogenin Expression in Male Sheepshead Minnows Exposed to Estradiol (Abstract). To be presented at the 22nd Annual Meeting of the Society of Environmental Toxicology and Chemistry: Changing Environmental Awareness: Societal Concerns and Scientifi...

  1. Structure and biological properties of solubilized envelope proteins of Bordetella pertussis.

    PubMed Central

    Robinson, A; Hawkins, D C

    1983-01-01

    The structure and biological properties of solubilized envelope proteins of Bordetella pertussis have been examined. Several envelope proteins were found to be specific for phase I strains of B. pertussis and could be isolated by selective detergent extraction. These proteins had molecular weights of 90,000, 86,000, 81,000, 33,000, 31,000, and 30,000 and were reduced or absent in envelope preparations from Bordetella bronchiseptica, Bordetella parapertussis, or phase IV strains of B. pertussis. When the envelope preparations from phase I B. pertussis were assayed in the mouse intracerebral protection test they were found to be highly protective, and there was a strong correlation between the protective potency and the lymphocytosis-promoting factor (LPF) content of different preparations. Treatment with glutaraldehyde reduced the LPF activity, toxicity, and protective potency of the envelope extracts. Similarly affinity chromatography of envelope proteins on columns of haptoglobin coupled to Sepharose 4B reduced both the LPF content and the protective potency. The addition of a small amount of purified LPF to the haptoglobin-treated proteins restored the protective potency. The LPF by itself was nonprotective, indicating a potentiating role of LPF in the mouse intracerebral challenge test. Images PMID:6299946

  2. Canine distemper virus envelope protein interactions modulated by hydrophobic residues in the fusion protein globular head.

    PubMed

    Avila, Mislay; Khosravi, Mojtaba; Alves, Lisa; Ader-Ebert, Nadine; Bringolf, Fanny; Zurbriggen, Andreas; Plemper, Richard K; Plattet, Philippe

    2015-01-15

    Membrane fusion for morbillivirus cell entry relies on critical interactions between the viral fusion (F) and attachment (H) envelope glycoproteins. Through extensive mutagenesis of an F cavity recently proposed to contribute to F's interaction with the H protein, we identified two neighboring hydrophobic residues responsible for severe F-to-H binding and fusion-triggering deficiencies when they were mutated in combination. Since both residues reside on one side of the F cavity, the data suggest that H binds the F globular head domain sideways. PMID:25355896

  3. Sequence analysis and protein import studies of an outer chloroplast envelope polypeptide.

    PubMed Central

    Salomon, M; Fischer, K; Flügge, U I; Soll, J

    1990-01-01

    A chloroplast outer envelope membrane protein was cloned and sequenced and from the sequence it was possible to deduce a polypeptide of 6.7 kDa. It has only one membrane-spanning region; the C terminus extends into the cytosol, whereas the N terminus is exposed to the space between the two envelope membranes. The protein was synthesized in an in vitro transcription-translation system to study its routing into isolated chloroplasts. The import studies revealed that the 6.7-kDa protein followed a different and heretofore undescribed translocation pathway in the respect that (i) it does not have a cleavable transit sequence, (ii) it does not require ATP hydrolysis for import, and (iii) protease-sensitive components that are responsible for recognition of precursor proteins destined for the inside of the chloroplasts are not involved in routing the 6.7-kDa polypeptide to the outer chloroplast envelope. Images PMID:2377616

  4. Nuclear envelope-associated endosomes deliver surface proteins to the nucleus.

    PubMed

    Chaumet, Alexandre; Wright, Graham D; Seet, Sze Hwee; Tham, Keit Min; Gounko, Natalia V; Bard, Frederic

    2015-01-01

    Endocytosis directs molecular cargo along three main routes: recycling to the cell surface, transport to the Golgi apparatus or degradation in endolysosomes. Pseudomonas exotoxin A (PE) is a bacterial protein that typically traffics to the Golgi and then the endoplasmic reticulum before translocating to the cytosol. Here we show that a substantial fraction of internalized PE is also located in nuclear envelope-associated endosomes (NAE), which display limited mobility, exhibit a propensity to undergo fusion and readily discharge their contents into the nuclear envelope. Electron microscopy and protein trapping in the nucleus indicate that NAE mediate PE transfer into the nucleoplasm. RNAi screening further revealed that NAE-mediated transfer depends on the nuclear envelope proteins SUN1 and SUN2, as well as the Sec61 translocon complex. These data reveal a novel endosomal route from the cell surface to the nucleoplasm that facilitates the accumulation of extracellular and cell surface proteins in the nucleus. PMID:26356418

  5. A Betabaculovirus-Encoded gp64 Homolog Codes for a Functional Envelope Fusion Protein

    PubMed Central

    Ardisson-Araújo, Daniel M. P.; Melo, Fernando L.; Clem, Rollie J.; Wolff, José L. C.

    2015-01-01

    The GP64 envelope fusion protein is a hallmark of group I alphabaculoviruses. However, the Diatraea saccharalis granulovirus genome sequence revealed the first betabaculovirus species harboring a gp64 homolog (disa118). In this work, we have shown that this homolog encodes a functional envelope fusion protein and could enable the infection and fusogenic abilities of a gp64-null prototype baculovirus. Therefore, GP64 may complement or may be in the process of replacing F protein activity in this virus lineage. PMID:26537678

  6. A Betabaculovirus-Encoded gp64 Homolog Codes for a Functional Envelope Fusion Protein.

    PubMed

    Ardisson-Araújo, Daniel M P; Melo, Fernando L; Clem, Rollie J; Wolff, José L C; Ribeiro, Bergmann M

    2016-02-01

    The GP64 envelope fusion protein is a hallmark of group I alphabaculoviruses. However, the Diatraea saccharalis granulovirus genome sequence revealed the first betabaculovirus species harboring a gp64 homolog (disa118). In this work, we have shown that this homolog encodes a functional envelope fusion protein and could enable the infection and fusogenic abilities of a gp64-null prototype baculovirus. Therefore, GP64 may complement or may be in the process of replacing F protein activity in this virus lineage. PMID:26537678

  7. Shrimp arginine kinase being a binding protein of WSSV envelope protein VP31

    NASA Astrophysics Data System (ADS)

    Ma, Cuiyan; Gao, Qiang; Liang, Yan; Li, Chen; Liu, Chao; Huang, Jie

    2016-03-01

    Viral entry into the host is the earliest stage of infection in the viral life cycle in which attachment proteins play a key role. VP31 (WSV340/WSSV396), an envelope protein of white spot syndrome virus (WSSV), contains an Arg-Gly-Asp (RGD) peptide domain known as a cellular attachment site. At present, the process of VP31 interacting with shrimp host cells has not been explored. Therefore, the VP31 gene was cloned into pET30a (+), expressed in Escherichia coli strain BL21 and purified with immobilized metal ion affinity chromatography. Four gill cellular proteins of shrimp (Fenneropenaeus chinensis) were pulled down by an affinity column coupled with recombinant VP31 (rVP31), and the amino acid sequences were identified with MALDI-TOF/TOF mass spectrometry. Hemocyanin, beta-actin, arginine kinase (AK), and an unknown protein were suggested as the putative VP31 receptor proteins. SDS-PAGE showed that AK is the predominant binding protein of VP31. An i n vitro binding activity experiment indicated that recombinant AK's (rAK) binding activity with rVP31 is comparable to that with the same amount of WSSV. These results suggested that AK, as a member of the phosphagen kinase family, plays a role in WSSV infection. This is the first evidence showing that AK is a binding protein of VP31. Further studies on this topic will elucidate WSSV infection mechanism in the future.

  8. Functional incorporation of green fluorescent protein into hepatitis B virus envelope particles

    SciTech Connect

    Lambert, Carsten; Thome, Nicole; Kluck, Christoph J.; Prange, Reinhild . E-mail: prange@mail.uni-mainz.de

    2004-12-05

    The envelope of hepatitis B virus (HBV), containing the L, M, and S proteins, is essential for virus entry and maturation. For direct visualization of HBV, we determined whether envelope assembly could accommodate the green fluorescent protein (GFP). While the C-terminal addition of GFP to S trans-dominant negatively inhibited empty envelope particle secretion, the N-terminal GFP fusion to S (GFP.S) was co-integrated into the envelope, giving rise to fluorescent particles. Microscopy and topogenesis analyses demonstrated that the proper intracellular distribution and folding of GFP.S, required for particle export were rescued by interprotein interactions with wild-type S. Thereby, a dual location of GFP, inside and outside the envelope, was observed. GFP.S was also efficiently packaged into the viral envelope, and these GFP-tagged virions retained the capacity for attachment to HBV receptor-positive cells in vitro. Together, GFP-tagged virions should be suitable to monitor HBV uptake and egress in live hepatocytes.

  9. The Novel Nuclear Envelope Protein KAKU4 Modulates Nuclear Morphology in Arabidopsis[W

    PubMed Central

    Goto, Chieko; Tamura, Kentaro; Fukao, Yoichiro; Shimada, Tomoo; Hara-Nishimura, Ikuko

    2014-01-01

    In animals, the nuclear lamina is a fibrillar meshwork on the inner surface of the nuclear envelope, composed of coiled-coil lamin proteins and lamin binding membrane proteins. Plants also have a meshwork on the inner surface of the nuclear envelope, but little is known about its composition other than the presence of members of the CROWDED NUCLEI (CRWN) protein family, possible plant lamin analogs. Here, we describe a candidate lamina component, based on two Arabidopsis thaliana mutants (kaku2 and kaku4) with aberrant nuclear morphology. The responsible gene in kaku2 encodes CRWN1, and the responsible gene in kaku4 encodes a plant-specific protein of unknown function (KAKU4) that physically interacts with CRWN1 and its homolog CRWN4. Immunogold labeling revealed that KAKU4 localizes at the inner nuclear membrane. KAKU4 deforms the nuclear envelope in a dose-dependent manner, in association with nuclear membrane invagination and stack formation. The KAKU4-dependent nuclear envelope deformation was enhanced by overaccumulation of CRWN1, although KAKU4 can deform the nuclear envelope even in the absence of CRWN1 and/or CRWN4. Together, these results suggest that plants have evolved a unique lamina-like structure to modulate nuclear shape and size. PMID:24824484

  10. Arterivirus Minor Envelope Proteins Are a Major Determinant of Viral Tropism in Cell Culture

    PubMed Central

    Tian, Debin; Wei, Zuzhang; Zevenhoven-Dobbe, Jessika C.; Liu, Runxia; Tong, Guangzhi

    2012-01-01

    Arteriviruses are enveloped positive-strand RNA viruses for which the attachment proteins and cellular receptors have remained largely controversial. Arterivirus particles contain at least eight envelope proteins, an unusually large number among RNA viruses. These appear to segregate into three groups: major structural components (major glycoprotein GP5 and membrane protein [M]), minor glycoproteins (GP2a, GP3, and GP4), and small hydrophobic proteins (E and the recently discovered ORF5a protein). Biochemical studies previously suggested that the GP5-M heterodimer of porcine reproductive and respiratory syndrome virus (PRRSV) interacts with porcine sialoadhesin (pSn) in porcine alveolar macrophages (PAM). However, another study proposed that minor protein GP4, along with GP2a, interacts with CD163, another reported cellular receptor for PRRSV. In this study, we provide genetic evidence that the minor envelope proteins are the major determinant of arterivirus entry into cultured cells. A PRRSV infectious cDNA clone was equipped with open reading frames (ORFs) encoding minor envelope and E proteins of equine arteritis virus (EAV), the only known arterivirus displaying a broad tropism in cultured cells. Although PRRSV and EAV are only distantly related and utilize diversified transcription-regulating sequences (TRSs), a viable chimeric progeny virus was rescued. Strikingly, this chimeric virus (vAPRRS-EAV2ab34) acquired the broad in vitro cell tropism of EAV, demonstrating that the minor envelope proteins play a critical role as viral attachment proteins. We believe that chimeric arteriviruses of this kind will be a powerful tool for further dissection of the arterivirus replicative cycle, including virus entry, subgenomic RNA synthesis, and virion assembly. PMID:22258262

  11. Computational analysis reveal inhibitory action of nimbin against dengue viral envelope protein.

    PubMed

    Lavanya, P; Ramaiah, Sudha; Anbarasu, Anand

    2015-12-01

    Dengue has emerged to be global health problem worldwide. Hence there is an immediate need to adopt new strategies in the development of effective anti-dengue drugs. Extracts from the leaves of Azadirachta indica has been traditionally used in folk medicine for viral infections. In the present study we report the anti-viral potency of nimbin, the active compound from the neem leaf extract against the envelope protein of dengue virus. Progression of viral entry into the host cell is facilitated by the envelope protein of dengue virus, suggesting; it as an effective anti-viral target. Nimbin is found to be effective against the envelope protein of all four types of dengue virus (dengue 1-4), which is evident from our in silico analysis. Our findings suggest the clinical importance of nimbin, which can serve as effective lead compound for further analysis. PMID:26645034

  12. Co-evolution analysis to predict protein-protein interactions within influenza virus envelope.

    PubMed

    Mintaev, Ramil R; Alexeevski, Andrei V; Kordyukova, Larisa V

    2014-04-01

    Interactions between integral membrane proteins hemagglutinin (HA), neuraminidase (NA), M2 and membrane-associated matrix protein M1 of influenza A virus are thought to be crucial for assembly of functionally competent virions. We hypothesized that the amino acid residues located at the interface of two different proteins are under physical constraints and thus probably co-evolve. To predict co-evolving residue pairs, the EvFold ( http://evfold.org ) program searching the (nontransitive) Direct Information scores was applied for large samplings of amino acid sequences from Influenza Research Database ( http://www.fludb.org/ ). Having focused on the HA, NA, and M2 cytoplasmic tails as well as C-terminal domain of M1 (being the less conserved among the protein domains) we captured six pairs of correlated positions. Among them, there were one, two, and three position pairs for HA-M2, HA-M1, and M2-M1 protein pairs, respectively. As expected, no co-varying positions were found for NA-HA, NA-M1, and NA-M2 pairs obviously due to high conservation of the NA cytoplasmic tail. The sum of frequencies calculated for two major amino acid patterns observed in pairs of correlated positions was up to 0.99 meaning their high to extreme evolutionary sustainability. Based on the predictions a hypothetical model of pair-wise protein interactions within the viral envelope was proposed. PMID:24712535

  13. The Nuclear Envelope Protein, LAP1B, Is a Novel Protein Phosphatase 1 Substrate

    PubMed Central

    Santos, Mariana; Rebelo, Sandra; Van Kleeff, Paula J. M.; Kim, Connie E.; Dauer, William T.; Fardilha, Margarida; da Cruz e Silva, Odete A.; da Cruz e Silva, Edgar F.

    2013-01-01

    Protein phosphatase 1 (PP1) binding proteins are quintessential regulators, determining substrate specificity and defining subcellular localization and activity of the latter. Here, we describe a novel PP1 binding protein, the nuclear membrane protein lamina associated polypeptide 1B (LAP1B), which interacts with the DYT1 dystonia protein torsinA. The PP1 binding domain in LAP1B was here identified as the REVRF motif at amino acids 55-59. The LAP1B:PP1 complex can be immunoprecipitated from cells in culture and rat cortex and the complex was further validated by yeast co-transformations and blot overlay assays. PP1, which is enriched in the nucleus, binds to the N-terminal nuclear domain of LAP1B, as shown by immunocolocalization and domain specific binding studies. PP1 dephosphorylates LAP1B, confirming the physiological relevance of this interaction. These findings place PP1 at a key position to participate in the pathogenesis of DYT1 dystonia and related nuclear envelope-based diseases. PMID:24116158

  14. Distinct Pathways Mediate the Sorting of Tail-Anchored Proteins to the Plastid Outer Envelope

    PubMed Central

    Dhanoa, Preetinder K.; Richardson, Lynn G. L.; Smith, Matthew D.; Gidda, Satinder K.; Henderson, Matthew P. A.; Andrews, David W.; Mullen, Robert T.

    2010-01-01

    Background Tail-anchored (TA) proteins are a distinct class of membrane proteins that are sorted post-translationally to various organelles and function in a number of important cellular processes, including redox reactions, vesicular trafficking and protein translocation. While the molecular targeting signals and pathways responsible for sorting TA proteins to their correct intracellular destinations in yeasts and mammals have begun to be characterized, relatively little is known about TA protein biogenesis in plant cells, especially for those sorted to the plastid outer envelope. Methodology/Principal Findings Here we investigated the biogenesis of three plastid TA proteins, including the 33-kDa and 34-kDa GTPases of the translocon at the outer envelope of chloroplasts (Toc33 and Toc34) and a novel 9-kDa protein of unknown function that we define here as an outer envelope TA protein (OEP9). Using a combination of in vivo and in vitro assays we show that OEP9 utilizes a different sorting pathway than that used by Toc33 and Toc34. For instance, while all three TA proteins interact with the cytosolic OEP chaperone/receptor, AKR2A, the plastid targeting information within OEP9 is distinct from that within Toc33 and Toc34. Toc33 and Toc34 also appear to differ from OEP9 in that their insertion is dependent on themselves and the unique lipid composition of the plastid outer envelope. By contrast, the insertion of OEP9 into the plastid outer envelope occurs in a proteinaceous-dependent, but Toc33/34-independent manner and membrane lipids appear to serve primarily to facilitate normal thermodynamic integration of this TA protein. Conclusions/Significance Collectively, the results provide evidence in support of at least two sorting pathways for plastid TA outer envelope proteins and shed light on not only the complex diversity of pathways involved in the targeting and insertion of proteins into plastids, but also the molecular mechanisms that underlie the delivery of TA

  15. Characterization of the VP39 envelope protein from Singapore grouper iridovirus.

    PubMed

    Zhang, Honglian; Zhou, Sheng; Xia, Liqun; Huang, Xiaohong; Huang, Youhua; Cao, Jianhao; Qin, Qiwei

    2015-12-01

    Singapore grouper iridovirus (SGIV) is a major pathogen that causes heavy economic losses to the grouper aquaculture industry in China and Southeast Asian countries. In the present study, a viral envelope protein, VP39, encoded by SGIV ORF39L, was identified and characterized. SGIV ORF39L was found in all sequenced iridoviruses and is now considered to be a core gene of the family Iridoviridae. ORF39L was classified as a late gene during in vitro infection using reverse transcription-polymerase chain reaction, western blotting, and a drug inhibition analysis. An indirect immunofluorescence assay revealed that the VP39 protein was confined to the cytoplasm, especially at viral assembly sites. Western blot and matrix-assisted laser desorption/ionization-time of flight tandem mass spectrometry analyses suggested that VP39 is an envelope protein. Immunogold electron microscopy further confirmed that VP39 is a viral envelope protein. Furthermore, a mouse anti-VP39 polyclonal antibody exhibited SGIV-neutralizing activity in vitro, suggesting that VP39 is involved in SGIV infection. Taken together, the current data suggest that VP39 represents a conserved envelope protein of iridoviruses that contributes to viral infection. PMID:26524136

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

    SciTech Connect

    Apte, Swapna; Sanders, David Avram

    2010-09-15

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

  17. Crystal Structure of Major Envelope Protein VP24 from White Spot Syndrome Virus

    PubMed Central

    Sun, Lifang; Su, Yintao; Zhao, Yanhe; Fu, Zheng-qing; Wu, Yunkun

    2016-01-01

    White spot syndrome virus (WSSV) is one of the major and most serious pathogen in the shrimp industry. As one of the most abundant envelope protein, VP24 acts as a core protein interacting with other structure proteins and plays an important role in virus assembly and infection. Here, we have presented the crystal structure of VP24 from WSSV. In the structure, VP24 consists of a nine-stranded β–barrel fold with mostly antiparallel β-strands, and the loops extending out the β–barrel at both N-terminus and C-terminus, which is distinct to those of the other two major envelope proteins VP28 and VP26. Structural comparison of VP24 with VP26 and VP28 reveals opposite electrostatic surface potential properties of them. These structural differences could provide insight into their differential functional mechanisms and roles for virus assembly and infection. Moreover, the structure reveals a trimeric assembly, suggesting a likely natural conformation of VP24 in viral envelope. Therefore, in addition to confirming the evolutionary relationship among the three abundant envelope proteins of WSSV, our structural studies also facilitate a better understanding of the molecular mechanism underlying special roles of VP24 in WSSV assembly and infection. PMID:27572278

  18. Sortases and the Art of Anchoring Proteins to the Envelopes of Gram-Positive Bacteria

    PubMed Central

    Marraffini, Luciano A.; DeDent, Andrea C.; Schneewind, Olaf

    2006-01-01

    The cell wall envelopes of gram-positive bacteria represent a surface organelle that not only functions as a cytoskeletal element but also promotes interactions between bacteria and their environment. Cell wall peptidoglycan is covalently and noncovalently decorated with teichoic acids, polysaccharides, and proteins. The sum of these molecular decorations provides bacterial envelopes with species- and strain-specific properties that are ultimately responsible for bacterial virulence, interactions with host immune systems, and the development of disease symptoms or successful outcomes of infections. Surface proteins typically carry two topogenic sequences, i.e., N-terminal signal peptides and C-terminal sorting signals. Sortases catalyze a transpeptidation reaction by first cleaving a surface protein substrate at the cell wall sorting signal. The resulting acyl enzyme intermediates between sortases and their substrates are then resolved by the nucleophilic attack of amino groups, typically provided by the cell wall cross bridges of peptidoglycan precursors. The surface protein linked to peptidoglycan is then incorporated into the envelope and displayed on the microbial surface. This review focuses on the mechanisms of surface protein anchoring to the cell wall envelope by sortases and the role that these enzymes play in bacterial physiology and pathogenesis. PMID:16524923

  19. Phytochrome regulates GTP-binding protein activity in the envelope of pea nuclei

    NASA Technical Reports Server (NTRS)

    Clark, G. B.; Memon, A. R.; Thompson, G. A. Jr; Roux, S. J.

    1993-01-01

    Three GTP-binding proteins with apparent molecular masses of 27, 28 and 30 kDa have been detected in isolated nuclei of etiolated pea plumules. After LDS-PAGE and transfer to nitrocellulose these proteins bind [32P]GTP in the presence of excess ATP, suggesting that they are monomeric G proteins. When nuclei are disrupted, three proteins co-purify with the nuclear envelope fraction and are highly enriched in this fraction. The level of [32P]GTP-binding for all three protein bands is significantly increased when harvested pea plumules are irradiated by red light, and this effect is reversed by far-red light. The results indicate that GTP-binding activity associated with the nuclear envelope of plant cells is photoreversibly regulated by the pigment phytochrome.

  20. The nuclear envelope LEM-domain protein emerin

    PubMed Central

    Berk, Jason M; Tifft, Kathryn E; Wilson, Katherine L

    2013-01-01

    Emerin, a conserved LEM-domain protein, is among the few nuclear membrane proteins for which extensive basic knowledge—biochemistry, partners, functions, localizations, posttranslational regulation, roles in development and links to human disease—is available. This review summarizes emerin and its emerging roles in nuclear “lamina” structure, chromatin tethering, gene regulation, mitosis, nuclear assembly, development, signaling and mechano-transduction. We also highlight many open questions, exploration of which will be critical to understand how this intriguing nuclear membrane protein and its “family” influence the genome. PMID:23873439

  1. TMEM120A and B: Nuclear Envelope Transmembrane Proteins Important for Adipocyte Differentiation

    PubMed Central

    Batrakou, Dzmitry G.; de las Heras, Jose I.; Czapiewski, Rafal; Mouras, Rabah; Schirmer, Eric C.

    2015-01-01

    Recent work indicates that the nuclear envelope is a major signaling node for the cell that can influence tissue differentiation processes. Here we present two nuclear envelope trans-membrane proteins TMEM120A and TMEM120B that are paralogs encoded by the Tmem120A and Tmem120B genes. The TMEM120 proteins are expressed preferentially in fat and both are induced during 3T3-L1 adipocyte differentiation. Knockdown of one or the other protein altered expression of several genes required for adipocyte differentiation, Gata3, Fasn, Glut4, while knockdown of both together additionally affected Pparg and Adipoq. The double knockdown also increased the strength of effects, reducing for example Glut4 levels by 95% compared to control 3T3-L1 cells upon pharmacologically induced differentiation. Accordingly, TMEM120A and B knockdown individually and together impacted on adipocyte differentiation/metabolism as measured by lipid accumulation through binding of Oil Red O and coherent anti-Stokes Raman scattering microscopy (CARS). The nuclear envelope is linked to several lipodystrophies through mutations in lamin A; however, lamin A is widely expressed. Thus it is possible that the TMEM120A and B fat-specific nuclear envelope transmembrane proteins may play a contributory role in the tissue-specific pathology of this disorder or in the wider problem of obesity. PMID:26024229

  2. Envelope protein complexes of Mycobacterium avium subsp. paratuberculosis and their antigenicity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of Johne’s disease, a chronic enteric disease of ruminant animals. In the present study, blue native PAGE electrophoresis and 2D SDS-PAGE were used to separate MAP envelope protein complexes, followed by mass spectrometry (MS) ...

  3. 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...

  4. Characterization of the fusion core in zebrafish endogenous retroviral envelope protein

    SciTech Connect

    Shi, Jian; Zhang, Huaidong; Gong, Rui; Xiao, Gengfu

    2015-05-08

    Zebrafish endogenous retrovirus (ZFERV) is the unique endogenous retrovirus in zebrafish, as yet, containing intact open reading frames of its envelope protein gene in zebrafish genome. Similarly, several envelope proteins of endogenous retroviruses in human and other mammalian animal genomes (such as syncytin-1 and 2 in human, syncytin-A and B in mouse) were identified and shown to be functional in induction of cell–cell fusion involved in placental development. ZFERV envelope protein (Env) gene appears to be also functional in vivo because it is expressible. After sequence alignment, we found ZFERV Env shares similar structural profiles with syncytin and other type I viral envelopes, especially in the regions of N- and C-terminal heptad repeats (NHR and CHR) which were crucial for membrane fusion. We expressed the regions of N + C protein in the ZFERV Env (residues 459–567, including predicted NHR and CHR) to characterize the fusion core structure. We found N + C protein could form a stable coiled-coil trimer that consists of three helical NHR regions forming a central trimeric core, and three helical CHR regions packing into the grooves on the surface of the central core. The structural characterization of the fusion core revealed the possible mechanism of fusion mediated by ZFERV Env. These results gave comprehensive explanation of how the ancient virus infects the zebrafish and integrates into the genome million years ago, and showed a rational clue for discovery of physiological significance (e.g., medicate cell–cell fusion). - Highlights: • ZFERV Env shares similar structural profiles with syncytin and other type I viral envelopes. • The fusion core of ZFERV Env forms stable coiled-coil trimer including three NHRs and three CHRs. • The structural mechanism of viral entry mediated by ZFERV Env is disclosed. • The results are helpful for further discovery of physiological function of ZFERV Env in zebrafish.

  5. Chloroplast envelope protein targeting fidelity is independent of cytosolic components in dual organelle assays

    PubMed Central

    Kriechbaumer, Verena; Abell, Ben M.

    2012-01-01

    The general mechanisms of intracellular protein targeting are well established, and depend on a targeting sequence in the protein, which is recognized by a targeting factor. Once a membrane protein is delivered to the correct organelle its targeting sequence can be recognized by receptors and a translocase, leading to membrane insertion. However, the relative contribution of each step for generating fidelity and efficiency of the overall process has not been systematically addressed. Here, we use tail-anchored (TA) membrane proteins in cell-free competitive targeting assays to chloroplasts to show that targeting can occur efficiently and with high fidelity in the absence of all cytosolic components, suggesting that chloroplast envelope protein targeting is primarily dependent on events at the outer envelope. Efficiency of targeting was increased by the addition of complete cytosol, and by Hsp70 or Hsp90, depending on the protein, but none of these cytosolic components influenced the fidelity of targeting. Our results suggest that the main role of targeting factors in chloroplast localization is to increase targeting efficiency by maintaining recognition competency at the outer envelope. PMID:22783268

  6. Transcription and identification of an envelope protein gene (p22) from shrimp white spot syndrome virus.

    PubMed

    Zhang, Xiaobo; Huang, Canhua; Xu, Xun; Hew, Choy L

    2002-02-01

    White spot syndrome virus (WSSV) is one of the most virulent pathogens causing high mortality in shrimp. In the present study, an open reading frame (termed the p22 gene) was revealed from a WSSV cDNA library. The gene was expressed as a fusion protein with glutathione S-transferase (GST) in Escherichia coli and purified. Specific antibody was raised using the purified fusion protein (GST-P22). Temporal analysis showed that the p22 gene was a late gene. After binding between purified WSSV virions and anti-GST-P22 IgG followed by labelling with gold-labelled secondary antibody, the gold particles, under a transmission electron microscope, could be found along the outer envelope of WSSV virions. This experiment suggests that the p22 gene encodes an envelope protein of the virus. PMID:11807241

  7. Reduction of a 4q35-encoded nuclear envelope protein in muscle differentiation

    SciTech Connect

    Ostlund, Cecilia; Guan, Tinglu; Figlewicz, Denise A.; Hays, Arthur P.; Worman, Howard J.; Gerace, Larry; Schirmer, Eric C.

    2009-11-13

    Muscular dystrophy and peripheral neuropathy have been linked to mutations in genes encoding nuclear envelope proteins; however, the molecular mechanisms underlying these disorders remain unresolved. Nuclear envelope protein p19A is a protein of unknown function encoded by a gene at chromosome 4q35. p19A levels are significantly reduced in human muscle as cells differentiate from myoblasts to myotubes; however, its levels are not similarly reduced in all differentiation systems tested. Because 4q35 has been linked to facioscapulohumeral muscular dystrophy (FSHD) and some adjacent genes are reportedly misregulated in the disorder, levels of p19A were analyzed in muscle samples from patients with FSHD. Although p19A was increased in most cases, an absolute correlation was not observed. Nonetheless, p19A downregulation in normal muscle differentiation suggests that in the cases where its gene is inappropriately re-activated it could affect muscle differentiation and contribute to disease pathology.

  8. Characteristics of Epstein-Barr Virus Envelope Protein gp42

    PubMed Central

    Shaw, Pamela L.; Kirschner, Austin N.; Jardetzky, Theodore S.; Longnecker, Richard

    2010-01-01

    Epstein-Barr virus (EBV) glycoprotein 42 (gp42) is a membrane protein essential for fusion and entry of EBV into host B-lymphocytes. Gp42 is a member of the protein fold family C-type lectin or lectin-like domains (CLECT or CTLD) and specifically is classified as a natural-killer receptor (NKR)- like CLECT. Literature review and phylogenetic comparison show that EBV gp42 shares a common structure with other NKR-like CLECTs and possibly with many viral CTLDs, but does not appear to exhibit some common binding characteristics of many CTLDs, such as features required for calcium binding. The flexible N-terminal region adjacent to the CTLD fold is important for binding to other EBV glycoproteins and for a cleavage site that is necessary for infection of host cells. From structural studies of gp42 unbound and bound to receptor and extensive mutational analysis, a general model of how gp42 triggers membrane fusion utilizing both the flexible N-terminal region and the CTLD domain has emerged. PMID:20162447

  9. Analysis of Pseudomonas aeruginosa Cell Envelope Proteome by Capture of Surface-Exposed Proteins on Activated Magnetic Nanoparticles

    PubMed Central

    Vecchietti, Davide; Di Silvestre, Dario; Miriani, Matteo; Bonomi, Francesco; Marengo, Mauro; Bragonzi, Alessandra; Cova, Lara; Franceschi, Eleonora; Mauri, Pierluigi; Bertoni, Giovanni

    2012-01-01

    We report on specific magneto-capturing followed by Multidimensional Protein Identification Technology (MudPIT) for the analysis of surface-exposed proteins of intact cells of the bacterial opportunistic pathogen Pseudomonas aeruginosa. The magneto-separation of cell envelope fragments from the soluble cytoplasmic fraction allowed the MudPIT identification of the captured and neighboring proteins. Remarkably, we identified 63 proteins captured directly by nanoparticles and 67 proteins embedded in the cell envelope fragments. For a high number of proteins, our analysis strongly indicates either surface exposure or localization in an envelope district. The localization of most identified proteins was only predicted or totally unknown. This novel approach greatly improves the sensitivity and specificity of the previous methods, such as surface shaving with proteases that was also tested on P. aeruginosa. The magneto-capture procedure is simple, safe, and rapid, and appears to be well-suited for envelope studies in highly pathogenic bacteria. PMID:23226459

  10. In vitro and in vivo screening for novel essential cell-envelope proteins in Pseudomonas aeruginosa

    PubMed Central

    Fernández-Piñar, Regina; Lo Sciuto, Alessandra; Rossi, Alice; Ranucci, Serena; Bragonzi, Alessandra; Imperi, Francesco

    2015-01-01

    The Gram-negative bacterium Pseudomonas aeruginosa represents a prototype of multi-drug resistant opportunistic pathogens for which novel therapeutic options are urgently required. In order to identify new candidates as potential drug targets, we combined large-scale transposon mutagenesis data analysis and bioinformatics predictions to retrieve a set of putative essential genes which are conserved in P. aeruginosa and predicted to encode cell envelope or secreted proteins. By generating unmarked deletion or conditional mutants, we confirmed the in vitro essentiality of two periplasmic proteins, LptH and LolA, responsible for lipopolysaccharide and lipoproteins transport to the outer membrane respectively, and confirmed that they are important for cell envelope stability. LptH was also found to be essential for P. aeruginosa ability to cause infection in different animal models. Conversely, LolA-depleted cells appeared only partially impaired in pathogenicity, indicating that this protein likely plays a less relevant role during bacterial infection. Finally, we ruled out any involvement of the other six proteins under investigation in P. aeruginosa growth, cell envelope stability and virulence. Besides proposing LptH as a very promising drug target in P. aeruginosa, this study confirms the importance of in vitro and in vivo validation of potential essential genes identified through random transposon mutagenesis. PMID:26621210

  11. Restoration of flagellar clockwise rotation in bacterial envelopes by insertion of the chemotaxis protein CheY.

    PubMed Central

    Ravid, S; Matsumura, P; Eisenbach, M

    1986-01-01

    When cells of the bacterium Salmonella typhimurium are incubated with penicillin and lysed in a dilute buffer, flagellated cytoplasm-free envelopes are formed. When the envelopes are tethered to glass by their flagella and then energized, some of them spin. The direction of rotation of wild-type envelopes is exclusively counterclockwise (CCW). We perturbed this system by including in the lysis medium (and hence in the envelopes) the chemotaxis protein CheY. As a result, some of the envelopes rotated exclusively clockwise (CW). The fraction of envelopes that did so increased with the concentration of CheY; at a concentration of 48 microM (pH 8), all functional envelopes spun CW. The fraction also increased with the pH of the lysis medium in the range of 6.6-8.4. The results were the same in the presence or absence of intracellular Ca2+. Reconstituted envelopes failed to respond to chemotactic stimuli. None of them changed the direction of their rotation. However, when the intracellular pH was lowered to 6.6 or below, envelopes that spun CW stopped rotating, while envelopes that spun CCW continued to rotate. This phenomenon was reversible. We conclude that CheY per se, without any additional free cytoplasmic mediators, interacts with a switch at the base of the flagellum to cause CW rotation. PMID:3532103

  12. Two-dimensional polyacylamide gel electrophoresis of envelope proteins of Escherichia coli.

    PubMed

    Johnson, W C; Silhavy, T J; Boos, W

    1975-03-01

    A method of separating envelope proteins by two-dimensional polyacrylamide gel electrophoresis is described. Escherichia coli envelopes (inner and outer membranes) were prepared by French pressing and washed by repeated centrifugation. Membrane proteins were solubilized with guanidine thiocyanate and were dialyzed against urea prior to two-dimensional electrophoretic analysis. The slab gel apparatus and conditions were similar to the technique developed by Metz and Bogorad (1974) for the separation of ribosomal proteins. This separation occurs in 8 M urea for the first dimension and in 0.2% sodium dodecyl sulfate for the second dimension. The technique separates about 70 different membrane proteins in a highly reproducible fashion according to both intrinsic charge and molecular weight. Some examples of alterations in the membrane protein pattern are demonstrated. These alterations are caused by a mutation affecting a sugar transport system and by growth in the presence of D-fucose, inducer of the transport system. A further example of membrane protein changes introduced by growth at the nonpermissive temperature of a temperature-sensitive cell division mutant is shown. Finally, it is demonstrated that the major outer membrane component of Escherichia coli K-12 contains more than four proteins of similar molecular weight. PMID:803821

  13. Energetic cost of protein import across the envelope membranes of chloroplasts.

    PubMed

    Shi, Lan-Xin; Theg, Steven M

    2013-01-15

    Chloroplasts are the organelles of green plants in which light energy is transduced into chemical energy, forming ATP and reduced carbon compounds upon which all life depends. The expenditure of this energy is one of the central issues of cellular metabolism. Chloroplasts contain ~3,000 proteins, among which less than 100 are typically encoded in the plastid genome. The rest are encoded in the nuclear genome, synthesized in the cytosol, and posttranslationally imported into the organelle in an energy-dependent process. We report here a measurement of the amount of ATP hydrolyzed to import a protein across the chloroplast envelope membranes--only the second complete accounting of the cost in Gibbs free energy of protein transport to be undertaken. Using two different precursors prepared by three distinct techniques, we show that the import of a precursor protein into chloroplasts is accompanied by the hydrolysis of ~650 ATP molecules. This translates to a ΔG(protein) (transport) of some 27,300 kJ/mol protein imported. We estimate that protein import across the plastid envelope membranes consumes ~0.6% of the total light-saturated energy output of the organelle. PMID:23277572

  14. Mutational analyses of fs(1)Ya, an essential, developmentally regulated, nuclear envelope protein in Drosophila

    SciTech Connect

    Liu, Jun; Song, Kiwon; Wolfner, M.F.

    1995-12-01

    The fs(1)Ya protein (YA) is an essential, maternally encoded, nuclear lamina protein that is under both developmental and cell cycle control. A strong Ya mutation results in early arrest of embryos. To define the function of YA in the nuclear envelope during early embryonic development, we characterized the phenotypes of four Ya mutant alleles and determined their molecular lesions. Ya mutant embryos arrest with abnormal nuclear envelopes prior to the first mitotic division; a proportion of embryos from two leaky Ya mutants proceed beyond this but arrest after several abnormal divisions. Ya unfertilized eggs contain nuclei of different sizes and condensation states, apparently due to abnormal fusion of the meiotic products immediately after meiosis. Lamin is localized at the periphery of the uncondensed nuclei in these eggs. These results suggest that Ya function is required during and after egg maturation to facilitate proper chromatin condensation, rather than to allow a lamin-containing nuclear envelope to form. Two leaky Ya alleles that partially complement have lesions at opposite ends of the YA protein, suggesting that the N- and C-termini are important for YA function might interact with itself either directly or indirectly. 27 refs., 6 figs.

  15. Energetic cost of protein import across the envelope membranes of chloroplasts

    PubMed Central

    Shi, Lan-Xin; Theg, Steven M.

    2013-01-01

    Chloroplasts are the organelles of green plants in which light energy is transduced into chemical energy, forming ATP and reduced carbon compounds upon which all life depends. The expenditure of this energy is one of the central issues of cellular metabolism. Chloroplasts contain ∼3,000 proteins, among which less than 100 are typically encoded in the plastid genome. The rest are encoded in the nuclear genome, synthesized in the cytosol, and posttranslationally imported into the organelle in an energy-dependent process. We report here a measurement of the amount of ATP hydrolyzed to import a protein across the chloroplast envelope membranes—only the second complete accounting of the cost in Gibbs free energy of protein transport to be undertaken. Using two different precursors prepared by three distinct techniques, we show that the import of a precursor protein into chloroplasts is accompanied by the hydrolysis of ∼650 ATP molecules. This translates to a ΔGprotein transport of some 27,300 kJ/mol protein imported. We estimate that protein import across the plastid envelope membranes consumes ∼0.6% of the total light-saturated energy output of the organelle. PMID:23277572

  16. Western Blot Detection of Human Anti-Chikungunya Virus Antibody with Recombinant Envelope 2 Protein.

    PubMed

    Yang, Zhaoshou; Lee, Jihoo; Ahn, Hye-Jin; Chong, Chom-Kyu; Dias, Ronaldo F; Nam, Ho-Woo

    2016-04-01

    Chikungunya virus (CHIKV), a tropical pathogen, has re-emerged and has massive outbreaks abruptly all over the world. Containing many dominant epitopes, the envelope E2 protein of CHIKV has been explored for the vaccination or diagnosis. In the present study, the antigenicity of a recombinant expressed intrinsically disorder domain (IUD) of E2 was tested for the detection of the antibody against CHIKV through western blot method. The gene of the IUD of E2 was inserted into 2 different vectors and expressed as recombinant GST-E2 and recombinant MBP-E2 fusion protein, respectively. Two kinds of fusion proteins were tested with 30 CHIKV patient sera and 30 normal sera, respectively. Both proteins were detected by 25 patients sera (83.3%) and 1 normal serum (3.3%). This test showed a relatively high sensitivity and very high specificity of the recombinant E2 proteins to be used as diagnostic antigens against CHIKV infection. PMID:27180586

  17. Western Blot Detection of Human Anti-Chikungunya Virus Antibody with Recombinant Envelope 2 Protein

    PubMed Central

    Yang, Zhaoshou; Lee, Jihoo; Ahn, Hye-Jin; Chong, Chom-Kyu; Dias, Ronaldo F.; Nam, Ho-Woo

    2016-01-01

    Chikungunya virus (CHIKV), a tropical pathogen, has re-emerged and has massive outbreaks abruptly all over the world. Containing many dominant epitopes, the envelope E2 protein of CHIKV has been explored for the vaccination or diagnosis. In the present study, the antigenicity of a recombinant expressed intrinsically disorder domain (IUD) of E2 was tested for the detection of the antibody against CHIKV through western blot method. The gene of the IUD of E2 was inserted into 2 different vectors and expressed as recombinant GST-E2 and recombinant MBP-E2 fusion protein, respectively. Two kinds of fusion proteins were tested with 30 CHIKV patient sera and 30 normal sera, respectively. Both proteins were detected by 25 patients sera (83.3%) and 1 normal serum (3.3%). This test showed a relatively high sensitivity and very high specificity of the recombinant E2 proteins to be used as diagnostic antigens against CHIKV infection. PMID:27180586

  18. In Situ Detection of Interactions Between Nuclear Envelope Proteins and Partners.

    PubMed

    Barateau, Alice; Buendia, Brigitte

    2016-01-01

    Proximity ligation assay (PLA) appears as a quick and easy technique to visualize within fixed cells the occurrence and in situ distribution of protein complexes. PLA has been validated to detect protein-protein interactions within the nuclear compartment. Here, we describe a protocol which allows the detection of interactions between A-type nuclear lamins and either LEM-domain proteins (such as emerin, integrated within the inner nuclear membrane, and LAP2α which accumulates within the nucleoplasm) or gene regulatory factors (e.g., the transcription factor SREBP1). The distinct amounts and patterns of PLA signals obtained for various complexes highlight the pertinence of using PLA to reveal in situ where and to which extent nuclear envelope proteins bind specific partners. PMID:27147040

  19. Repairing oxidized proteins in the bacterial envelope using respiratory chain electrons.

    PubMed

    Gennaris, Alexandra; Ezraty, Benjamin; Henry, Camille; Agrebi, Rym; Vergnes, Alexandra; Oheix, Emmanuel; Bos, Julia; Leverrier, Pauline; Espinosa, Leon; Szewczyk, Joanna; Vertommen, Didier; Iranzo, Olga; Collet, Jean-François; Barras, Frédéric

    2015-12-17

    The reactive species of oxygen and chlorine damage cellular components, potentially leading to cell death. In proteins, the sulfur-containing amino acid methionine is converted to methionine sulfoxide, which can cause a loss of biological activity. To rescue proteins with methionine sulfoxide residues, living cells express methionine sulfoxide reductases (Msrs) in most subcellular compartments, including the cytosol, mitochondria and chloroplasts. Here we report the identification of an enzymatic system, MsrPQ, repairing proteins containing methionine sulfoxide in the bacterial cell envelope, a compartment particularly exposed to the reactive species of oxygen and chlorine generated by the host defence mechanisms. MsrP, a molybdo-enzyme, and MsrQ, a haem-binding membrane protein, are widely conserved throughout Gram-negative bacteria, including major human pathogens. MsrPQ synthesis is induced by hypochlorous acid, a powerful antimicrobial released by neutrophils. Consistently, MsrPQ is essential for the maintenance of envelope integrity under bleach stress, rescuing a wide series of structurally unrelated periplasmic proteins from methionine oxidation, including the primary periplasmic chaperone SurA. For this activity, MsrPQ uses electrons from the respiratory chain, which represents a novel mechanism to import reducing equivalents into the bacterial cell envelope. A remarkable feature of MsrPQ is its capacity to reduce both rectus (R-) and sinister (S-) diastereoisomers of methionine sulfoxide, making this oxidoreductase complex functionally different from previously identified Msrs. The discovery that a large class of bacteria contain a single, non-stereospecific enzymatic complex fully protecting methionine residues from oxidation should prompt a search for similar systems in eukaryotic subcellular oxidizing compartments, including the endoplasmic reticulum. PMID:26641313

  20. Repairing oxidized proteins in the bacterial envelope using respiratory chain electrons

    PubMed Central

    Henry, Camille; Agrebi, Rym; Vergnes, Alexandra; Oheix, Emmanuel; Bos, Julia; Leverrier, Pauline; Espinosa, Leon; Szewczyk, Joanna; Vertommen, Didier; Iranzo, Olga; Collet, Jean-François; Barras, Frédéric

    2015-01-01

    The reactive species of oxygen (ROS) and chlorine (RCS) damage cellular components, potentially leading to cell death. In proteins, the sulfur-containing amino acid methionine (Met) is converted to methionine sulfoxide (Met-O), which can cause a loss of biological activity. To rescue proteins with Met-O residues, living cells express methionine sulfoxide reductases (Msrs) in most subcellular compartments, including the cytosol, mitochondria and chloroplasts 1-3. Here, we report the identification of an enzymatic system, MsrPQ, repairing Met-O containing proteins in the bacterial cell envelope, a compartment particularly exposed to the ROS and RCS generated by the host defense mechanisms. MsrP, a molybdo-enzyme, and MsrQ, a heme-binding membrane protein, are widely conserved throughout Gram-negative bacteria, including major human pathogens. MsrPQ synthesis is induced by hypochlorous acid (HOCl), a powerful antimicrobial released by neutrophils. Consistently, MsrPQ is essential for the maintenance of envelope integrity under bleach stress, rescuing a wide series of structurally unrelated periplasmic proteins from Met oxidation, including the primary periplasmic chaperone SurA. For this activity, MsrPQ uses electrons from the respiratory chain, which represents a novel mechanism to import reducing equivalents into the bacterial cell envelope. A remarkable feature of MsrPQ is its capacity to reduce both R- and S- diastereoisomers of Met-O, making this oxidoreductase complex functionally different from previously identified Msrs. The discovery that a large class of bacteria contain a single, non-stereospecific enzymatic complex fully protecting Met residues from oxidation should prompt search for similar systems in eukaryotic subcellular oxidizing compartments, including the endoplasmic reticulum (ER). PMID:26641313

  1. Glycolipid analyses of light-harvesting chlorosomes from envelope protein mutants of Chlorobaculum tepidum.

    PubMed

    Tsukatani, Yusuke; Mizoguchi, Tadashi; Thweatt, Jennifer; Tank, Marcus; Bryant, Donald A; Tamiaki, Hitoshi

    2016-06-01

    Chlorosomes are large and efficient light-harvesting organelles in green photosynthetic bacteria, and they characteristically contain large numbers of bacteriochlorophyll c, d, or e molecules. Self-aggregated bacteriochlorophyll pigments are surrounded by a monolayer envelope membrane comprised of glycolipids and Csm proteins. Here, we analyzed glycolipid compositions of chlorosomes from the green sulfur bacterium Chlorobaculum tepidum mutants lacking one, two, or three Csm proteins by HPLC equipped with an evaporative light-scattering detector. The ratio of monogalactosyldiacylglyceride (MGDG) to rhamnosylgalactosyldiacylglyceride (RGDG) was smaller in chlorosomes from mutants lacking two or three proteins in CsmC/D/H motif family than in chlorosomes from the wild-type, whereas chlorosomes lacking CsmIJ showed relatively less RGDG than MGDG. The results suggest that the CsmC, CsmD, CsmH, and other chlorosome proteins are involved in organizing MGDG and RGDG and thereby affect the size and shape of the chlorosome. PMID:26869354

  2. Inhibition of hepatitis C virus infection by DNA aptamer against envelope protein.

    PubMed

    Yang, Darong; Meng, Xianghe; Yu, Qinqin; Xu, Li; Long, Ying; Liu, Bin; Fang, Xiaohong; Zhu, Haizhen

    2013-10-01

    Hepatitis C virus (HCV) envelope protein (E1E2) is essential for virus binding to host cells. Aptamers have been demonstrated to have strong promising applications in drug development. In the current study, a cDNA fragment encoding the entire E1E2 gene of HCV was cloned. E1E2 protein was expressed and purified. Aptamers for E1E2 were selected by the method of selective evolution of ligands by exponential enrichment (SELEX), and the antiviral actions of the aptamers were examined. The mechanism of their antiviral activity was investigated. The data show that selected aptamers for E1E2 specifically recognize the recombinant E1E2 protein and E1E2 protein from HCV-infected cells. CD81 protein blocks the binding of aptamer E1E2-6 to E1E2 protein. Aptamers against E1E2 inhibit HCV infection in an infectious cell culture system although they have no effect on HCV replication in a replicon cell line. Beta interferon (IFN-β) and IFN-stimulated genes (ISGs) are not induced in virus-infected hepatocytes with aptamer treatment, suggesting that E1E2-specific aptamers do not induce innate immunity. E2 protein is essential for the inhibition of HCV infection by aptamer E1E2-6, and the aptamer binding sites are located in E2. Q412R within E1E2 is the major resistance substitution identified. The data indicate that an aptamer against E1E2 exerts its antiviral effects through inhibition of virus binding to host cells. Aptamers against E1E2 can be used with envelope protein to understand the mechanisms of HCV entry and fusion. The aptamers may hold promise for development as therapeutic drugs for hepatitis C patients. PMID:23877701

  3. Inhibition of Hepatitis C Virus Infection by DNA Aptamer against Envelope Protein

    PubMed Central

    Yang, Darong; Meng, Xianghe; Yu, Qinqin; Xu, Li; Long, Ying; Liu, Bin; Fang, Xiaohong

    2013-01-01

    Hepatitis C virus (HCV) envelope protein (E1E2) is essential for virus binding to host cells. Aptamers have been demonstrated to have strong promising applications in drug development. In the current study, a cDNA fragment encoding the entire E1E2 gene of HCV was cloned. E1E2 protein was expressed and purified. Aptamers for E1E2 were selected by the method of selective evolution of ligands by exponential enrichment (SELEX), and the antiviral actions of the aptamers were examined. The mechanism of their antiviral activity was investigated. The data show that selected aptamers for E1E2 specifically recognize the recombinant E1E2 protein and E1E2 protein from HCV-infected cells. CD81 protein blocks the binding of aptamer E1E2-6 to E1E2 protein. Aptamers against E1E2 inhibit HCV infection in an infectious cell culture system although they have no effect on HCV replication in a replicon cell line. Beta interferon (IFN-β) and IFN-stimulated genes (ISGs) are not induced in virus-infected hepatocytes with aptamer treatment, suggesting that E1E2-specific aptamers do not induce innate immunity. E2 protein is essential for the inhibition of HCV infection by aptamer E1E2-6, and the aptamer binding sites are located in E2. Q412R within E1E2 is the major resistance substitution identified. The data indicate that an aptamer against E1E2 exerts its antiviral effects through inhibition of virus binding to host cells. Aptamers against E1E2 can be used with envelope protein to understand the mechanisms of HCV entry and fusion. The aptamers may hold promise for development as therapeutic drugs for hepatitis C patients. PMID:23877701

  4. Molecular docking analyses of Avicennia marinaderived phytochemicals against white spot syndrome virus (WSSV) envelope protein-VP28

    PubMed Central

    Sahu, Sunil Kumar; Kathiresan, Kandasamy; Singh, Reena; Senthilraja, Poomalai

    2012-01-01

    White spot syndrome (WSS) is one of the most common and most disastrous diseases of shrimp worldwide. It causes up to 100% mortality within 3 to 4 days in commercial shrimp farms, resulting in large economic losses to the shrimp farming industry. VP28 envelope protein of WSSV is reported to play a key role in the systemic infection in shrimps. Considering the most sombre issue of viral disease in cultivated shrimp, the present study was undertaken to substantiate the inhibition potential of Avicennia marinaderived phytochemicals against the WSSV envelope protein VP28. Seven A. marina-derived phytochemicals namely stigmasterol, triterpenoid, betulin, lupeol, avicenol-A, betulinic acid and quercetin were docked against the WSSV protein VP28 by using Argus lab molecular docking software. The chemical structures of the phytochemicals were retrieved from Pubchem database and generated from SMILES notation. Similarly the protein structure of the envelope protein was obtained from protein data bank (PDB-ID: 2ED6). Binding sites were predicted by using ligand explorer software. Among the phytochemicals screened, stigmasterol, lupeol and betulin showed the best binding exhibiting the potential to block VP28 envelope protein of WSSV, which could possibly inhibit the attachment of WSSV to the host species. Further experimental studies will provide a clear understanding on the mode of action of these phytochemicals individually or synergistically against WSSV envelope protein and can be used as an inhibitory drug to reduce white spot related severe complications in crustaceans. PMID:23144547

  5. Nuclear Envelope Protein SUN2 Promotes Cyclophilin-A-Dependent Steps of HIV Replication

    PubMed Central

    Lahaye, Xavier; Satoh, Takeshi; Gentili, Matteo; Cerboni, Silvia; Silvin, Aymeric; Conrad, Cécile; Ahmed-Belkacem, Abdelhakim; Rodriguez, Elisa C.; Guichou, Jean-François; Bosquet, Nathalie; Piel, Matthieu; Le Grand, Roger; King, Megan C.; Pawlotsky, Jean-Michel; Manel, Nicolas

    2016-01-01

    Summary During the early phase of replication, HIV reverse transcribes its RNA and crosses the nuclear envelope while escaping host antiviral defenses. The host factor Cyclophilin A (CypA) is essential for these steps and binds the HIV capsid; however, the mechanism underlying this effect remains elusive. Here, we identify related capsid mutants in HIV-1, HIV-2, and SIVmac that are restricted by CypA. This antiviral restriction of mutated viruses is conserved across species and prevents nuclear import of the viral cDNA. Importantly, the inner nuclear envelope protein SUN2 is required for the antiviral activity of CypA. We show that wild-type HIV exploits SUN2 in primary CD4+ T cells as an essential host factor that is required for the positive effects of CypA on reverse transcription and infection. Altogether, these results establish essential CypA-dependent functions of SUN2 in HIV infection at the nuclear envelope. PMID:27149839

  6. Identification of unique SUN-interacting nuclear envelope proteins with diverse functions in plants.

    PubMed

    Zhou, Xiao; Graumann, Katja; Wirthmueller, Lennart; Jones, Jonathan D G; Meier, Iris

    2014-06-01

    Although a plethora of nuclear envelope (NE) transmembrane proteins (NETs) have been identified in opisthokonts, plant NETs are largely unknown. The only known NET homologues in plants are Sad1/UNC-84 (SUN) proteins, which bind Klarsicht/ANC-1/Syne-1 homology (KASH) proteins. Therefore, de novo identification of plant NETs is necessary. Based on similarities between opisthokont KASH proteins and the only known plant KASH proteins, WPP domain-interacting proteins, we used a computational method to identify the KASH subset of plant NETs. Ten potential plant KASH protein families were identified, and five candidates from four of these families were verified for their NE localization, depending on SUN domain interaction. Of those, Arabidopsis thaliana SINE1 is involved in actin-dependent nuclear positioning in guard cells, whereas its paralogue SINE2 contributes to innate immunity against an oomycete pathogen. This study dramatically expands our knowledge of plant KASH proteins and suggests that plants and opisthokonts have recruited different KASH proteins to perform NE regulatory functions. PMID:24891605

  7. Nuclear envelope breakdown induced by herpes simplex virus type 1 involves the activity of viral fusion proteins

    SciTech Connect

    Maric, Martina; Haugo, Alison C.; Dauer, William; Johnson, David; Roller, Richard J.

    2014-07-15

    Herpesvirus infection reorganizes components of the nuclear lamina usually without loss of integrity of the nuclear membranes. We report that wild-type HSV infection can cause dissolution of the nuclear envelope in transformed mouse embryonic fibroblasts that do not express torsinA. Nuclear envelope breakdown is accompanied by an eight-fold inhibition of virus replication. Breakdown of the membrane is much more limited during infection with viruses that lack the gB and gH genes, suggesting that breakdown involves factors that promote fusion at the nuclear membrane. Nuclear envelope breakdown is also inhibited during infection with virus that does not express UL34, but is enhanced when the US3 gene is deleted, suggesting that envelope breakdown may be enhanced by nuclear lamina disruption. Nuclear envelope breakdown cannot compensate for deletion of the UL34 gene suggesting that mixing of nuclear and cytoplasmic contents is insufficient to bypass loss of the normal nuclear egress pathway. - Highlights: • We show that wild-type HSV can induce breakdown of the nuclear envelope in a specific cell system. • The viral fusion proteins gB and gH are required for induction of nuclear envelope breakdown. • Nuclear envelope breakdown cannot compensate for deletion of the HSV UL34 gene.

  8. Coat as a Dagger: The Use of Capsid Proteins to Perforate Membranes during Non-Enveloped DNA Viruses Trafficking

    PubMed Central

    Bilkova, Eva; Forstova, Jitka; Abrahamyan, Levon

    2014-01-01

    To get access to the replication site, small non-enveloped DNA viruses have to cross the cell membrane using a limited number of capsid proteins, which also protect the viral genome in the extracellular environment. Most of DNA viruses have to reach the nucleus to replicate. The capsid proteins involved in transmembrane penetration are exposed or released during endosomal trafficking of the virus. Subsequently, the conserved domains of capsid proteins interact with cellular membranes and ensure their efficient permeabilization. This review summarizes our current knowledge concerning the role of capsid proteins of small non-enveloped DNA viruses in intracellular membrane perturbation in the early stages of infection. PMID:25055856

  9. Functional dissection of the Moloney murine leukemia virus envelope protein gp70.

    PubMed

    Bae, Y; Kingsman, S M; Kingsman, A J

    1997-03-01

    The envelope protein of Moloney murine leukemia virus (Mo-MLV) is a complex glycoprotein that mediates receptor binding and entry via fusion with cell membranes. By using a series of substitution mutations and truncations in the Mo-MLV external envelope surface protein gp70, we have identified regions important for these processes. Firstly, truncations of gp70 revealed that the minimal continuous receptor-binding region is amino acids 9 to 230, in broad agreement with other studies. Secondly, within this region there are two key basic amino acids, Arg-83 and Arg-95, that are essential for receptor binding and may interact with a negatively charged residue(s) or with the pi electrons of the aromatic ring on a hydrophobic residue(s) in the basic amino acid transporter protein that is the Mo-MLV ecotropic receptor. Finally, we showed that outside the minimal receptor-binding region at amino acids 2 to 8, there is a region that is essential for postbinding fusion events. PMID:9032341

  10. Accurate and Efficient Resolution of Overlapping Isotopic Envelopes in Protein Tandem Mass Spectra

    PubMed Central

    Xiao, Kaijie; Yu, Fan; Fang, Houqin; Xue, Bingbing; Liu, Yan; Tian, Zhixin

    2015-01-01

    It has long been an analytical challenge to accurately and efficiently resolve extremely dense overlapping isotopic envelopes (OIEs) in protein tandem mass spectra to confidently identify proteins. Here, we report a computationally efficient method, called OIE_CARE, to resolve OIEs by calculating the relative deviation between the ideal and observed experimental abundance. In the OIE_CARE method, the ideal experimental abundance of a particular overlapping isotopic peak (OIP) is first calculated for all the OIEs sharing this OIP. The relative deviation (RD) of the overall observed experimental abundance of this OIP relative to the summed ideal value is then calculated. The final individual abundance of the OIP for each OIE is the individual ideal experimental abundance multiplied by 1 + RD. Initial studies were performed using higher-energy collisional dissociation tandem mass spectra on myoglobin (with direct infusion) and the intact E. coli proteome (with liquid chromatographic separation). Comprehensive data at the protein and proteome levels, high confidence and good reproducibility were achieved. The resolving method reported here can, in principle, be extended to resolve any envelope-type overlapping data for which the corresponding theoretical reference values are available. PMID:26439836

  11. A Single Herpesvirus Protein Can Mediate Vesicle Formation in the Nuclear Envelope*

    PubMed Central

    Lorenz, Michael; Vollmer, Benjamin; Unsay, Joseph D.; Klupp, Barbara G.; García-Sáez, Ana J.; Mettenleiter, Thomas C.; Antonin, Wolfram

    2015-01-01

    Herpesviruses assemble capsids in the nucleus and egress by unconventional vesicle-mediated trafficking through the nuclear envelope. Capsids bud at the inner nuclear membrane into the nuclear envelope lumen. The resulting intralumenal vesicles fuse with the outer nuclear membrane, delivering the capsids to the cytoplasm. Two viral proteins are required for vesicle formation, the tail-anchored pUL34 and its soluble interactor, pUL31. Whether cellular proteins are involved is unclear. Using giant unilamellar vesicles, we show that pUL31 and pUL34 are sufficient for membrane budding and scission. pUL34 function can be bypassed by membrane tethering of pUL31, demonstrating that pUL34 is required for pUL31 membrane recruitment but not for membrane remodeling. pUL31 can inwardly deform membranes by oligomerizing on their inner surface to form buds that constrict to vesicles. Therefore, a single viral protein can mediate all events necessary for membrane budding and abscission. PMID:25605719

  12. Role of protein disulfide isomerase and other thiol-reactive proteins in HIV-1 envelope protein-mediated fusion

    SciTech Connect

    Ou Wu . E-mail: wou@niaid.nih.gov; Silver, Jonathan . E-mail: jsilver@nih.gov

    2006-07-05

    Cell-surface protein disulfide isomerase (PDI) has been proposed to promote disulfide bond rearrangements in HIV-1 envelope protein (Env) that accompany Env-mediated fusion. We evaluated the role of PDI in ways that have not been previously tested by downregulating PDI with siRNA and by overexpressing wild-type or variant forms of PDI in transiently and stably transfected cells. These manipulations, as well as treatment with anti-PDI antibodies, had only small effects on infection or cell fusion mediated by NL4-3 or AD8 strains of HIV-1. However, the cell-surface thiol-reactive reagent 5, 5'-dithiobis(2-nitrobenzoic acid) (DTNB) had a much stronger inhibitory effect in our system, suggesting that cell-surface thiol-containing molecules other than PDI, acting alone or in concert, have a greater effect than PDI on HIV-1 Env-mediated fusion. We evaluated one such candidate, thioredoxin, a PDI family member reported to reduce a labile disulfide bond in CD4. We found that the ability of thioredoxin to reduce the disulfide bond in CD4 is enhanced in the presence of HIV-1 Env gp120 and that thioredoxin also reduces disulfide bonds in gp120 directly in the absence of CD4. We discuss the implications of these observations for identification of molecules involved in disulfide rearrangements in Env during fusion.

  13. The small envelope protein of porcine reproductive and respiratory syndrome virus possesses ion channel protein-like properties

    SciTech Connect

    Lee, Changhee; Yoo, Dongwan . E-mail: dyoo@uoguelph.ca

    2006-11-10

    The small envelope (E) protein of porcine reproductive and respiratory syndrome virus (PRRSV) is a hydrophobic 73 amino acid protein encoded in the internal open reading frame (ORF) of the bicistronic mRNA2. As a first step towards understanding the biological role of E protein during PRRSV replication, E gene expression was blocked in a full-length infectious clone by mutating the ATG translational initiation to GTG, such that the full-length mutant genomic clone was unable to synthesize the E protein. DNA transfection of PRRSV-susceptible cells with the E gene knocked-out genomic clone showed the absence of virus infectivity. P129-{delta}E-transfected cells however produced virion particles in the culture supernatant, and these particles contained viral genomic RNA, demonstrating that the E protein is essential for PRRSV infection but dispensable for virion assembly. Electron microscopy suggests that the P129-{delta}E virions assembled in the absence of E had a similar appearance to the wild-type particles. Strand-specific RT-PCR demonstrated that the E protein-negative, non-infectious P129-{delta}E virus particles were able to enter cells but further steps of replication were interrupted. The entry of PRRSV has been suggested to be via receptor-mediated endocytosis, and lysomotropic basic compounds and known ion-channel blocking agents both inhibited PRRSV replication effectively during the uncoating process. The expression of E protein in Escherichia coli-mediated cell growth arrests and increased the membrane permeability. Cross-linking experiments in cells infected with PRRSV or transfected with E gene showed that the E protein was able to form homo-oligomers. Taken together, our data suggest that the PRRSV E protein is likely an ion-channel protein embedded in the viral envelope and facilitates uncoating of virus and release of the genome in the cytoplasm.

  14. The HERV-K Human Endogenous Retrovirus Envelope Protein Antagonizes Tetherin Antiviral Activity

    PubMed Central

    Lemaître, Cécile; Harper, Francis; Pierron, Gérard

    2014-01-01

    ABSTRACT Endogenous retroviruses are the remnants of past retroviral infections that are scattered within mammalian genomes. In humans, most of these elements are old degenerate sequences that have lost their coding properties. The HERV-K(HML2) family is an exception: it recently amplified in the human genome and corresponds to the most active proviruses, with some intact open reading frames and the potential to encode viral particles. Here, using a reconstructed consensus element, we show that HERV-K(HML2) proviruses are able to inhibit Tetherin, a cellular restriction factor that is active against most enveloped viruses and acts by keeping the viral particles attached to the cell surface. More precisely, we identify the Envelope protein (Env) as the viral effector active against Tetherin. Through immunoprecipitation experiments, we show that the recognition of Tetherin is mediated by the surface subunit of Env. Similar to Ebola glycoprotein, HERV-K(HML2) Env does not mediate Tetherin degradation or cell surface removal; therefore, it uses a yet-undescribed mechanism to inactivate Tetherin. We also assessed all natural complete alleles of endogenous HERV-K(HML2) Env described to date for their ability to inhibit Tetherin and found that two of them (out of six) can block Tetherin restriction. However, due to their recent amplification, HERV-K(HML2) elements are extremely polymorphic in the human population, and it is likely that individuals will not all possess the same anti-Tetherin potential. Because of Tetherin's role as a restriction factor capable of inducing innate immune responses, this could have functional consequences for individual responses to infection. IMPORTANCE Tetherin, a cellular protein initially characterized for its role against HIV-1, has been proven to counteract numerous enveloped viruses. It blocks the release of viral particles from producer cells, keeping them tethered to the cell surface. Several viruses have developed strategies to

  15. Proteomic analysis of shrimp white spot syndrome viral proteins and characterization of a novel envelope protein VP466.

    PubMed

    Huang, Canhua; Zhang, Xiaobo; Lin, Qingsong; Xu, Xun; Hu, Zhihong; Hew, Choy-L

    2002-03-01

    White spot syndrome virus (WSSV) is at present one of the major pathogens in shrimp culture worldwide. The complete genome of this virus has been sequenced recently. To identify the structural and functional proteins of WSSV, the purified virions were separated by SDS-PAGE. Twenty-four protein bands were excised, in-gel digested with trypsin, and subjected to matrix-assisted laser desorption ionization-time of flight mass spectrometry and electrospray ionization tandem mass spectrometry, respectively. Eighteen proteins matching the open reading frames of WSSV genome were identified. Except for three known structural proteins and collagen, the functions of the remaining 14 proteins were unknown. Temporal analysis revealed that all the genes were transcribed in the late stage of WSSV infection except for vp121. Of the newly identified proteins, VP466 (derived from band 16) was further characterized. The cDNA encoding VP466 was expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein. Specific antibody was generated with the purified GST-VP466 fusion protein. Western blot showed that the mouse anti-GST-VP466 antibody bound specifically to a 51-kDa protein of WSSV. Immunogold labeling revealed that VP466 protein is a component of the viral envelope. Results in this investigation thus proved the effectiveness of proteomic approaches for discovering new proteins of WSSV. PMID:12096122

  16. Inner nuclear envelope protein SUN1 plays a prominent role in mammalian mRNA export

    PubMed Central

    Li, Ping; Noegel, Angelika A.

    2015-01-01

    Nuclear export of messenger ribonucleoproteins (mRNPs) through the nuclear pore complex (NPC) can be roughly classified into two forms: bulk and specific export, involving an nuclear RNA export factor 1 (NXF1)-dependent pathway and chromosome region maintenance 1 (CRM1)-dependent pathway, respectively. SUN proteins constitute the inner nuclear envelope component of the linker of nucleoskeleton and cytoskeleton (LINC) complex. Here, we show that mammalian cells require SUN1 for efficient nuclear mRNP export. The results indicate that both SUN1 and SUN2 interact with heterogeneous nuclear ribonucleoprotein (hnRNP) F/H and hnRNP K/J. SUN1 depletion inhibits the mRNP export, with accumulations of both hnRNPs and poly(A)+RNA in the nucleus. Leptomycin B treatment indicates that SUN1 functions in mammalian mRNA export involving the NXF1-dependent pathway. SUN1 mediates mRNA export through its association with mRNP complexes via a direct interaction with NXF1. Additionally, SUN1 associates with the NPC through a direct interaction with Nup153, a nuclear pore component involved in mRNA export. Taken together, our results reveal that the inner nuclear envelope protein SUN1 has additional functions aside from being a central component of the LINC complex and that it is an integral component of the mammalian mRNA export pathway suggesting a model whereby SUN1 recruits NXF1-containing mRNP onto the nuclear envelope and hands it over to Nup153. PMID:26476453

  17. The selective biotin tagging and thermolysin proteolysis of chloroplast outer envelope proteins reveals information on protein topology and association into complexes

    PubMed Central

    Hardré, Hélène; Kuhn, Lauriane; Albrieux, Catherine; Jouhet, Juliette; Michaud, Morgane; Seigneurin-Berny, Daphné; Falconet, Denis; Block, Maryse A.; Maréchal, Eric

    2014-01-01

    The understanding of chloroplast function requires the precise localization of proteins in each of its sub-compartments. High-sensitivity mass spectrometry has allowed the inventory of proteins in thylakoid, stroma, and envelope fractions. Concerning membrane association, proteins can be either integral or peripheral or even soluble proteins bound transiently to a membrane complex. We sought a method providing information at the surface of the outer envelope membrane (OEM), based on specific tagging with biotin or proteolysis using thermolysin, a non-membrane permeable protease. To evaluate this method, envelope, thylakoid, and stroma proteins were separated by two-dimensional electrophoresis and analyzed by immunostaining and mass spectrometry. A short selection of proteins associated to the chloroplast envelope fraction was checked after superficial treatments of intact chloroplasts. We showed that this method could allow the characterization of OEM embedded proteins facing the cytosol, as well as peripheral and soluble proteins associated via tight or lose interactions. Some stromal proteins were associated with biotinylated spots and analyzes are still needed to determine whether polypeptides were tagged prior import or if they co-migrated with OEM proteins. This method also suggests that some proteins associated with the inner envelope membrane (IEM) might need the integrity of a trans-envelope (IEM–OEM) protein complex (e.g., division ring-forming components) or at least an intact OEM partner. Following this evaluation, proteomic analyzes should be refined and the putative role of inter-membrane space components stabilizing trans-envelope complexes demonstrated. For future comprehensive studies, perspectives include the dynamic analyses of OEM proteins and IEM–OEM complexes in various physiological contexts and using virtually any other purified membrane organelle. PMID:24999344

  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. Envelope Proteins of White Spot Syndrome Virus (WSSV) Interact with Litopenaeus vannamei Peritrophin-Like Protein (LvPT)

    PubMed Central

    Xie, Shijun; Zhang, Xiaojun; Zhang, Jiquan; Li, Fuhua; Xiang, Jianhai

    2015-01-01

    White spot syndrome virus (WSSV) is a major pathogen in shrimp cultures. The interactions between viral proteins and their receptors on the surface of cells in a frontier target tissue are crucial for triggering an infection. In this study, a yeast two-hybrid (Y2H) library was constructed using cDNA obtained from the stomach and gut of Litopenaeus vannamei, to ascertain the role of envelope proteins in WSSV infection. For this purpose, VP37 was used as the bait in the Y2H library screening. Forty positive clones were detected after screening. The positive clones were analyzed and discriminated, and two clones belonging to the peritrophin family were subsequently confirmed as genuine positive clones. Sequence analysis revealed that both clones could be considered as the same gene, LV-peritrophin (LvPT). Co-immunoprecipitation confirmed the interaction between LvPT and VP37. Further studies in the Y2H system revealed that LvPT could also interact with other WSSV envelope proteins such as VP32, VP38A, VP39B, and VP41A. The distribution of LvPT in tissues revealed that LvPT was mainly expressed in the stomach than in other tissues. In addition, LvPT was found to be a secretory protein, and its chitin-binding ability was also confirmed. PMID:26692362

  20. Nuclear Pore Basket Proteins Are Tethered to the Nuclear Envelope and Can Regulate Membrane Curvature

    PubMed Central

    Mészáros, Noémi; Cibulka, Jakub; Mendiburo, Maria Jose; Romanauska, Anete; Schneider, Maren; Köhler, Alwin

    2015-01-01

    Summary Nuclear pore complexes (NPCs) are selective transport channels embedded in the nuclear envelope. The cylindrical NPC core forms a protein coat lining a highly curved membrane opening and has a basket-like structure appended to the nucleoplasmic side. How NPCs interact with lipids, promoting membrane bending and NPC integrity, is poorly understood. Here we show that the NPC basket proteins Nup1 and Nup60 directly induce membrane curvature by amphipathic helix insertion into the lipid bilayer. In a cell-free system, both Nup1 and Nup60 transform spherical liposomes into highly curved membrane structures. In vivo, high levels of the Nup1/Nup60 amphipathic helices cause deformation of the yeast nuclear membrane, whereas adjacent helical regions contribute to anchoring the basket to the NPC core. Basket amphipathic helices are functionally linked to distinct transmembrane nucleoporins of the NPC core, suggesting a key contribution to the membrane remodeling events that underlie NPC assembly. PMID:25942622

  1. Enhanced proliferation of primary rat type II pneumocytes by Jaagsiekte sheep retrovirus envelope protein

    SciTech Connect

    Johnson, Chassidy; Jahid, Sohail; Voelker, Dennis R.; Fan Hung

    2011-04-10

    Jaagsiekte sheep retrovirus (JSRV) is the causative agent of a contagious lung cancer in sheep. The envelope protein (Env) is the oncogene, as it can transform cell lines in culture and induce tumors in animals, although the mechanisms for transformation are not yet clear because a system to perform transformation assays in differentiated type II pneumocytes does not exist. In this study we report culture of primary rat type II pneumocytes in conditions that favor prolonged expression of markers for type II pneumocytes. Env-expressing cultures formed more colonies that were larger in size and were viable for longer periods of time compared to vector control samples. The cells that remained in culture longer were confirmed to be derived from type II pneumocytes because they expressed surfactant protein C, cytokeratin, displayed alkaline phosphatase activity and were positive for Nile red. This system will be useful to study JSRV Env in the targets of transformation.

  2. The Flavivirus Precursor Membrane-Envelope Protein Complex: Structure and Maturation

    SciTech Connect

    Li, Long; Lok, Shee-Mei; Yu, I-Mei; Zhang, Ying; Kuhn, Richard J.; Chen, Jue; Rossmann, Michael G.

    2008-09-17

    Many viruses go through a maturation step in the final stages of assembly before being transmitted to another host. The maturation process of flaviviruses is directed by the proteolytic cleavage of the precursor membrane protein (prM), turning inert virus into infectious particles. We have determined the 2.2 angstrom resolution crystal structure of a recombinant protein in which the dengue virus prM is linked to the envelope glycoprotein E. The structure represents the prM-E heterodimer and fits well into the cryo-electron microscopy density of immature virus at neutral pH. The pr peptide {beta}-barrel structure covers the fusion loop in E, preventing fusion with host cell membranes. The structure provides a basis for identifying the stages of its pH-directed conformational metamorphosis during maturation, ending with release of pr when budding from the host.

  3. Proteomic analysis of Brucella abortus cell envelope and identification of immunogenic candidate proteins for vaccine development.

    PubMed

    Connolly, Joseph P; Comerci, Diego; Alefantis, Timothy G; Walz, Alexander; Quan, Marian; Chafin, Ryan; Grewal, Paul; Mujer, Cesar V; Ugalde, Rodolfo A; DelVecchio, Vito G

    2006-07-01

    Brucella abortus is the etiologic agent of bovine brucellosis and causes a chronic disease in humans known as undulant fever. In livestock the disease is characterized by abortion and sterility. Live, attenuated vaccines such as S19 and RB51 have been used to control the spread of the disease in animals; however, they are considered unsafe for human use and they induce abortion in pregnant cattle. For the development of a safer and equally efficacious vaccine, immunoproteomics was utilized to identify novel candidate proteins from B. abortus cell envelope (CE). A total of 163 proteins were identified using 2-DE with MALDI-TOF MS and LC-MS/MS. Some of the major protein components include outer-membrane protein (OMP) 25, OMP31, Omp2b porin, and 60 kDa chaperonin GroEL. 2-DE Western blot analyses probed with antiserum from bovine and a human patient infected with Brucella identified several new immunogenic proteins such as fumarate reductase flavoprotein subunit, F0F1-type ATP synthase alpha subunit, and cysteine synthase A. The elucidation of the immunome of B. abortus CE identified a number of candidate proteins for developing vaccines against Brucella infection in bovine and humans. PMID:16739129

  4. Oral and parenteral immunization of chickens (Gallus gallus) against West Nile virus with recombinant envelope protein

    USGS Publications Warehouse

    Fassbinder-Orth, C. A.; Hofmeister, E.K.; Weeks-Levy, C.; Karasov, W.H.

    2009-01-01

    West Nile virus (WNV) causes morbidity and mortality in humans, horses, and in more than 315 bird species in North America. Currently approved WNV vaccines are designed for parenteral administration and, as yet, no effective oral WNV vaccines have been developed. WNV envelope (E) protein is a highly antigenic protein that elicits the majority of virus-neutralizing antibodies during a WNV immune response. Leghorn chickens were given three vaccinations (each 2 wk apart) of E protein orally (20 ??g or 100 ??g/dose), of E protein intramuscularly (IM, 20 ??g/dose), or of adjuvant only (control group) followed by a WNV challenge. Viremias were measured post-WNV infection, and three new enzyme-linked immunosorbent assays were developed for quantifying IgM, IgY, and IgA-mediated immune response of birds following WNV infection. WNV viremia levels were significantly lower in the IM group than in both oral groups and the control group. Total WNV E protein-specific IgY production was significantly greater, and WNV nonstructural 1-specific IgY was significantly less, in the IM group compared to all other treatment groups. The results of this study indicate that IM vaccination of chickens with E protein is protective against WNV infection and results in a significantly different antibody production profile as compared to both orally vaccinated and nonvaccinated birds. ?? 2009 American Association of Avian Pathologists.

  5. Contribution of Protein and Lipid Components to the Salt Response of Envelopes of an Extremely Halophilic Bacterium1

    PubMed Central

    Kushner, D. J.; Onishi, H.

    1966-01-01

    Kushner, D. J. (National Research Council, Ottawa, Ontario, Canada), and H. Onishi. Contribution of protein and lipid components to the salt response of envelopes of an extremely halophilic bacterium. J. Bacteriol. 91:653–660. 1966.—Removal of protein from envelopes of Halobacterium cutirubrum by peptic digestion left residues that required little or no salt for stability. The salt requirement of envelopes was also lowered by incubation in 0.1 m MgCl2, and could be lowered even further by digestion with trypsin or chymotrypsin in 0.1 m MgCl2. Dissolution of envelopes in low salt concentrations made their protein more susceptible to attack by these and other proteolytic enzymes. Removal of lipids raised the requirement for divalent cations, particularly for Mg++; it slightly increased the Na+ requirement and did not affect the requirement for K+. It was concluded that the requirement for high salt concentrations in extreme halophiles is due to mutual repulsion between negatively charged groups on proteins rather than to repulsion between negatively charged phosphate groups on the lipids. The latter act primarily as sites on which divalent cations, especially Mg++ which is required in high concentrations by growing cells, are bound. In this manner, the phosphate groups support envelope structure. PMID:5327362

  6. Severe acute respiratory syndrome coronavirus envelope protein ion channel activity promotes virus fitness and pathogenesis.

    PubMed

    Nieto-Torres, Jose L; DeDiego, Marta L; Verdiá-Báguena, Carmina; Jimenez-Guardeño, Jose M; Regla-Nava, Jose A; Fernandez-Delgado, Raul; Castaño-Rodriguez, Carlos; Alcaraz, Antonio; Torres, Jaume; Aguilella, Vicente M; Enjuanes, Luis

    2014-05-01

    Deletion of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) envelope (E) gene attenuates the virus. E gene encodes a small multifunctional protein that possesses ion channel (IC) activity, an important function in virus-host interaction. To test the contribution of E protein IC activity in virus pathogenesis, two recombinant mouse-adapted SARS-CoVs, each containing one single amino acid mutation that suppressed ion conductivity, were engineered. After serial infections, mutant viruses, in general, incorporated compensatory mutations within E gene that rendered active ion channels. Furthermore, IC activity conferred better fitness in competition assays, suggesting that ion conductivity represents an advantage for the virus. Interestingly, mice infected with viruses displaying E protein IC activity, either with the wild-type E protein sequence or with the revertants that restored ion transport, rapidly lost weight and died. In contrast, mice infected with mutants lacking IC activity, which did not incorporate mutations within E gene during the experiment, recovered from disease and most survived. Knocking down E protein IC activity did not significantly affect virus growth in infected mice but decreased edema accumulation, the major determinant of acute respiratory distress syndrome (ARDS) leading to death. Reduced edema correlated with lung epithelia integrity and proper localization of Na+/K+ ATPase, which participates in edema resolution. Levels of inflammasome-activated IL-1β were reduced in the lung airways of the animals infected with viruses lacking E protein IC activity, indicating that E protein IC function is required for inflammasome activation. Reduction of IL-1β was accompanied by diminished amounts of TNF and IL-6 in the absence of E protein ion conductivity. All these key cytokines promote the progression of lung damage and ARDS pathology. In conclusion, E protein IC activity represents a new determinant for SARS-CoV virulence. PMID:24788150

  7. Severe Acute Respiratory Syndrome Coronavirus Envelope Protein Ion Channel Activity Promotes Virus Fitness and Pathogenesis

    PubMed Central

    Nieto-Torres, Jose L.; DeDiego, Marta L.; Verdiá-Báguena, Carmina; Jimenez-Guardeño, Jose M.; Regla-Nava, Jose A.; Fernandez-Delgado, Raul; Castaño-Rodriguez, Carlos; Alcaraz, Antonio; Torres, Jaume; Aguilella, Vicente M.; Enjuanes, Luis

    2014-01-01

    Deletion of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) envelope (E) gene attenuates the virus. E gene encodes a small multifunctional protein that possesses ion channel (IC) activity, an important function in virus-host interaction. To test the contribution of E protein IC activity in virus pathogenesis, two recombinant mouse-adapted SARS-CoVs, each containing one single amino acid mutation that suppressed ion conductivity, were engineered. After serial infections, mutant viruses, in general, incorporated compensatory mutations within E gene that rendered active ion channels. Furthermore, IC activity conferred better fitness in competition assays, suggesting that ion conductivity represents an advantage for the virus. Interestingly, mice infected with viruses displaying E protein IC activity, either with the wild-type E protein sequence or with the revertants that restored ion transport, rapidly lost weight and died. In contrast, mice infected with mutants lacking IC activity, which did not incorporate mutations within E gene during the experiment, recovered from disease and most survived. Knocking down E protein IC activity did not significantly affect virus growth in infected mice but decreased edema accumulation, the major determinant of acute respiratory distress syndrome (ARDS) leading to death. Reduced edema correlated with lung epithelia integrity and proper localization of Na+/K+ ATPase, which participates in edema resolution. Levels of inflammasome-activated IL-1β were reduced in the lung airways of the animals infected with viruses lacking E protein IC activity, indicating that E protein IC function is required for inflammasome activation. Reduction of IL-1β was accompanied by diminished amounts of TNF and IL-6 in the absence of E protein ion conductivity. All these key cytokines promote the progression of lung damage and ARDS pathology. In conclusion, E protein IC activity represents a new determinant for SARS-CoV virulence. PMID:24788150

  8. Immunogenicity of a novel tetravalent vaccine formulation with four recombinant lipidated dengue envelope protein domain IIIs in mice

    PubMed Central

    Chiang, Chen-Yi; Pan, Chien-Hsiung; Chen, Mei-Yu; Hsieh, Chun-Hsiang; Tsai, Jy-Ping; Liu, Hsueh-Hung; Liu, Shih-Jen; Chong, Pele; Leng, Chih-Hsiang; Chen, Hsin-Wei

    2016-01-01

    We developed a novel platform to express high levels of recombinant lipoproteins with intrinsic adjuvant properties. Based on this technology, our group developed recombinant lipidated dengue envelope protein domain IIIs as vaccine candidates against dengue virus. This work aims to evaluate the immune responses in mice to the tetravalent formulation. We demonstrate that 4 serotypes of recombinant lipidated dengue envelope protein domain III induced both humoral and cellular immunity against all 4 serotypes of dengue virus on the mixture that formed the tetravalent formulation. Importantly, the immune responses induced by the tetravalent formulation in the absence of the exogenous adjuvant were functional in clearing the 4 serotypes of dengue virus in vivo. We affirm that the tetravalent formulation of recombinant lipidated dengue envelope protein domain III is a potential vaccine candidate against dengue virus and suggest further detailed studies of this formulation in nonhuman primates. PMID:27470096

  9. Immunogenicity of a novel tetravalent vaccine formulation with four recombinant lipidated dengue envelope protein domain IIIs in mice.

    PubMed

    Chiang, Chen-Yi; Pan, Chien-Hsiung; Chen, Mei-Yu; Hsieh, Chun-Hsiang; Tsai, Jy-Ping; Liu, Hsueh-Hung; Liu, Shih-Jen; Chong, Pele; Leng, Chih-Hsiang; Chen, Hsin-Wei

    2016-01-01

    We developed a novel platform to express high levels of recombinant lipoproteins with intrinsic adjuvant properties. Based on this technology, our group developed recombinant lipidated dengue envelope protein domain IIIs as vaccine candidates against dengue virus. This work aims to evaluate the immune responses in mice to the tetravalent formulation. We demonstrate that 4 serotypes of recombinant lipidated dengue envelope protein domain III induced both humoral and cellular immunity against all 4 serotypes of dengue virus on the mixture that formed the tetravalent formulation. Importantly, the immune responses induced by the tetravalent formulation in the absence of the exogenous adjuvant were functional in clearing the 4 serotypes of dengue virus in vivo. We affirm that the tetravalent formulation of recombinant lipidated dengue envelope protein domain III is a potential vaccine candidate against dengue virus and suggest further detailed studies of this formulation in nonhuman primates. PMID:27470096

  10. Structural, Antigenic, and Evolutionary Characterizations of the Envelope Protein of Newly Emerging Duck Tembusu Virus

    PubMed Central

    Huang, Bing; Ma, Xiuli; Li, Yufeng; Yuan, Xiaoyuan; Qin, Zhuoming; Wang, Dan; Chakravarty, Suvobrata; Li, Feng; Song, Minxun; Sun, Huaichang

    2013-01-01

    Since the first reported cases of ducks infected with a previously unknown flavivirus in eastern China in April 2010, the virus, provisionally designated Duck Tembusu Virus (DTMUV), has spread widely in domestic ducks in China and caused significant economic losses to poultry industry. In this study, we examined in detail structural, antigenic, and evolutionary properties of envelope (E) proteins of six DTMUV isolates spanning 2010–2012, each being isolated from individual farms with different geographical locations where disease outbreaks were documented. Structural analysis showed that E proteins of DTMUV and its closely related flavivirus (Japanese Encephalitis Virus) shared a conserved array of predicted functional domains and motifs. Among the six DTMUV strains, mutations were observed only at thirteen amino acid positions across three separate domains of the E protein. Interestingly, these genetic polymorphisms resulted in no detectable change in viral neutralization properties as demonstrated in a serum neutralization assay. Furthermore, phylogenetic analysis of the nucleotide sequences of the E proteins showed that viruses evolved into two distinct genotypes, termed as DTMUV.I and DTMUV.II, with II emerging as the dominant genotype. New findings described here shall give insights into the antigenicity and evolution of this new pathogen and provide guidance for further functional studies of the E protein for which no effective vaccine has yet been developed. PMID:23990944

  11. Identification of the Receptor Binding Domain of the Mouse Mammary Tumor Virus Envelope Protein

    PubMed Central

    Zhang, Yuanming; Rassa, John C.; deObaldia, Maria Elena; Albritton, Lorraine M.; Ross, Susan R.

    2003-01-01

    Mouse mammary tumor virus (MMTV) is a betaretrovirus that infects rodent cells and uses mouse transferrin receptor 1 for cell entry. To characterize the interaction of MMTV with its receptor, we aligned the MMTV envelope surface (SU) protein with that of Friend murine leukemia virus (F-MLV) and identified a putative receptor-binding domain (RBD) that included a receptor binding sequence (RBS) of five amino acids and a heparin-binding domain (HBD). Mutation of the HBD reduced virus infectivity, and soluble heparan sulfate blocked infection of cells by wild-type pseudovirus. Interestingly, some but not all MMTV-like elements found in primary and cultured human breast cancer cell lines, termed h-MTVs, had sequence alterations in the putative RBS. Single substitution of one of the amino acids found in an h-MTV RBS variant in the RBD of MMTV, Phe40 to Ser, did not alter species tropism but abolished both virus binding to cells and infectivity. Neutralizing anti-SU monoclonal antibodies also recognized a glutathione S-transferase fusion protein that contained the five-amino-acid RBS region from MMTV. The critical Phe40 residue is located on a surface of the MMTV RBD model that is distant from and may be structurally more rigid than the region of F-MLV RBD that contains its critical binding site residues. This suggests that, in contrast to other murine retroviruses, binding to its receptor may result in few or no changes in MMTV envelope protein conformation. PMID:12970432

  12. Infectious Entry Pathway Mediated by the Human Endogenous Retrovirus K Envelope Protein

    PubMed Central

    Robinson, Lindsey R.

    2016-01-01

    ABSTRACT Endogenous retroviruses (ERVs), the majority of which exist as degraded remnants of ancient viruses, comprise approximately 8% of the human genome. The youngest human ERVs (HERVs) belong to the HERV-K(HML-2) subgroup and were endogenized within the past 1 million years. The viral envelope protein (ENV) facilitates the earliest events of endogenization (cellular attachment and entry), and here, we characterize the requirements for HERV-K ENV to mediate infectious cell entry. Cell-cell fusion assays indicate that a minimum of two events are required for fusion, proteolytic processing by furin-like proteases and exposure to acidic pH. We generated an infectious autonomously replicating recombinant vesicular stomatitis virus (VSV) in which the glycoprotein was replaced by HERV-K ENV. HERV-K ENV imparts an endocytic entry pathway that requires dynamin-mediated membrane scission and endosomal acidification but is distinct from clathrin-dependent or macropinocytic uptake pathways. The lack of impediments to the replication of the VSV core in eukaryotic cells allowed us to broadly survey the HERV-K ENV-dictated tropism. Unlike extant betaretroviral envelopes, which impart a narrow species tropism, we found that HERV-K ENV mediates broad tropism encompassing cells from multiple mammalian and nonmammalian species. We conclude that HERV-K ENV dictates an evolutionarily conserved entry pathway and that the restriction of HERV-K to primate genomes reflects downstream stages of the viral replication cycle. IMPORTANCE Approximately 8% of the human genome is of retroviral origin. While many of those viral genomes have become inactivated, some copies of the most recently endogenized human retrovirus, HERV-K, can encode individual functional proteins. Here, we characterize the envelope protein (ENV) of the virus to define how it mediates infection of cells. We demonstrate that HERV-K ENV undergoes a proteolytic processing step and triggers membrane fusion in response to

  13. Porcine reproductive and respiratory syndrome virus envelope (E) protein interacts with mitochondrial proteins and induces apoptosis.

    PubMed

    Pujhari, Sujit; Zakhartchouk, Alexander N

    2016-07-01

    Porcine reproductive and respiratory syndrome virus (PRRSV) causes significant economic losses for the swine industry worldwide. The PRRSV E protein, encoded by ORF 2b, is one of the non-glycosylated minor structural proteins. In this study, we present evidence for the interaction of the E protein with mitochondrial proteins ATP5A (part of ATP synthase complex), prohibitin, and ADP/ATP translocase. We additionally demonstrate partial mitochondrial localization of the E protein in transfected cells. To functionally investigate these interactions, we infected MARC-145 cells with PRRSV or alphavirus replicon particles (VRPs) expressing PRRSV E protein. In infected cells, production of ATP was significantly reduced. The E protein also induced apoptosis by activating caspase-3, which results in PARP cleavage. Taken together, these data suggest that the PRRSV E protein interacts with mitochondrial proteins and induces apoptosis by inhibiting ATP production. PMID:27068165

  14. Cell envelope of Bordetella pertussis: immunological and biochemical analyses and characterization of a major outer membrane porin protein

    SciTech Connect

    Armstrong, S.K.

    1986-01-01

    Surface molecules of Bordetella pertussis which may be important in metabolism, pathogenesis, and immunity to whooping cough were examined using cell fractionation and /sup 125/I cell surface labeling. Antigenic envelope proteins were examined by immunofluorescence microscopy and Western blotting procedures using monoclonal antibodies and convalescent sera. A surface protein with a high M/sub r/, missing in a mutant lacking the filamentous hemagglutinin, was identified in virulent Bordetella pertussis but was absent in virulent B. pertussis strains. At least three envelope proteins were found only in virulent B. pertussis strains and were absent or diminished in avirulent and most phenotypically modulated strains. Transposon-induced mutants unable to produce hemolysin, dermonecrotic toxin, pertussis toxin, and filamentous hemagglutinin also lacked these three envelope proteins, confirming that virulence-associated envelope proteins were genetically regulated with other virulence-associated traits. Two dimensional gel electrophoresis revealed at least five heat modifiable proteins which migrated as higher or lower M/sub r/ moieties if solubilized at 25/sup 0/C instead of 100/sup 0/C.

  15. The 32-kilodalton envelope protein of vaccinia virus synthesized in Escherichia coli binds with specificity to cell surfaces.

    PubMed Central

    Lai, C F; Gong, S C; Esteban, M

    1991-01-01

    The nature of interaction between vaccinia virus and the surface of host cells as the first step in virus infection is undefined. A 32-kDa virus envelope protein has been identified as a cell surface binding protein (J.-S. Maa, J. F. Rodriguez, and M. Esteban, J. Biol. Chem. 265:1569-1577, 1990). To carry out studies on the structure-function relationship of this protein, the 32-kDa protein was obtained from Escherichia coli cells harboring the expression plasmid pT7Ek32. The recombinant polypeptide was found to have structural properties similar to those of the native virus envelope protein. Binding studies of 125I-labeled 32-kDa protein to cultured cells of various origins revealed that the E. coli-produced 32-kDa protein exhibited selectivity, specificity, and saturability. Scatchard analysis indicated about 4.5 x 10(4) sites per cell with a high affinity (Kd = 1.8 x 10(-9) M), suggesting interaction of the 32-kDa protein with a specific receptor. The availability of large quantities of the 32-kDa virus protein in bacteria will permit further structural and functional studies of this virus envelope protein and facilitate identification of the specific cell surface receptor. Images PMID:1985213

  16. Dystonin/Bpag1 is a necessary endoplasmic reticulum/nuclear envelope protein in sensory neurons

    SciTech Connect

    Young, Kevin G.; Kothary, Rashmi

    2008-09-10

    Dystonin/Bpag1 proteins are cytoskeletal linkers whose loss of function in mice results in a hereditary sensory neuropathy with a progressive loss of limb coordination starting in the second week of life. These mice, named dystonia musculorum (dt), succumb to the disease and die of unknown causes prior to sexual maturity. Previous evidence indicated that cytoskeletal defects in the axon are a primary cause of dt neurodegeneration. However, more recent data suggests that other factors may be equally important contributors to the disease process. In the present study, we demonstrate perikaryal defects in dorsal root ganglion (DRG) neurons at stages preceding the onset of loss of limb coordination in dt mice. Abnormalities include alterations in endoplasmic reticulum (ER) chaperone protein expression, indicative of an ER stress response. Dystonin in sensory neurons localized in association with the ER and nuclear envelope (NE). A fusion protein ofthe dystonin-a2 isoform, which harbors an N-terminal transmembrane domain, associated with and reorganized the ER in cell culture. This isoform also interacts with the NE protein nesprin-3{alpha}, but not nesprin-3{beta}. Defects in dt mice, as demonstrated here, may ultimately result in pathogenesis involving ER dysfunction and contribute significantly to the dt phenotype.

  17. Docking studies towards exploring antiviral compounds against envelope protein of yellow fever virus.

    PubMed

    Umamaheswari, Amineni; Kumar, Manne Muni; Pradhan, Dibyabhaba; Marisetty, Hemanthkumar

    2011-03-01

    Yellow fever is among one of the most lethal viral diseases for which approved antiviral therapies were yet to be discovered. Herein, functional assignment of complete YFV proteome was done through support vector machine. Major envelope (E) protein that mediates entry of YFV into host cell was selected as a potent molecular target. Three dimensional structure of the molecular target was predicted using Modeller9v7. The model was optimized in Maestro9.0 applying OPLS AA force field and was evaluated using PROCHECK, ProSA, ProQ and Profile 3D. The BOG pocket residues Val48, Glu197, Thr200, Ile204, Thr265, Thr268 and Gly278 were located in YFV E protein using SiteMap2.3. More than one million compounds of Ligandinfo Meta database were explored using a computational virtual screening protocol targeting BOG pocket of the E protein. Finally, ten top ranked lead molecules with strong binding affinity to BOG pocket of YFV E protein were identified based on XP Gscore. Drug likeliness and comparative bioactivity analysis for these leads using QikProp3.2 had shown that these molecules would have the potential to act as better drug. Thus, the 10 lead molecules suggested in the present study would be of interest as promising starting point for designing antiviral compound against yellow fever. PMID:21369890

  18. The dengue virus type 2 envelope protein fusion peptide is essential for membrane fusion

    SciTech Connect

    Huang, Claire Y.-H.; Butrapet, Siritorn; Moss, Kelly J.; Childers, Thomas; Erb, Steven M.; Calvert, Amanda E.; Silengo, Shawn J.; Kinney, Richard M.; Blair, Carol D.; Roehrig, John T.

    2010-01-20

    The flaviviral envelope (E) protein directs virus-mediated membrane fusion. To investigate membrane fusion as a requirement for virus growth, we introduced 27 unique mutations into the fusion peptide of an infectious cDNA clone of dengue 2 virus and recovered seven stable mutant viruses. The fusion efficiency of the mutants was impaired, demonstrating for the first time the requirement for specific FP AAs in optimal fusion. Mutant viruses exhibited different growth kinetics and/or genetic stabilities in different cell types and adult mosquitoes. Virus particles could be recovered following RNA transfection of cells with four lethal mutants; however, recovered viruses could not re-infect cells. These viruses could enter cells, but internalized virus appeared to be retained in endosomal compartments of infected cells, thus suggesting a fusion blockade. Mutations of the FP also resulted in reduced virus reactivity with flavivirus group-reactive antibodies, confirming earlier reports using virus-like particles.

  19. Plasticity of a critical antigenic determinant in the West Nile virus NY99 envelope protein domain III.

    PubMed

    Plante, Jessica A; Torres, Maricela; Huang, Claire Y-H; Beasley, David W C

    2016-09-01

    West Nile virus (WNV) is a mosquito-borne flavivirus that causes febrile illness, encephalitis, and occasionally death in humans. The envelope protein is the main component of the WNV virion surface, and domain III of the envelope protein (EIII) is both a putative receptor binding domain and a target of highly specific, potently neutralizing antibodies. Envelope E-332 (E-332) is known to have naturally occurring variation and to be a key determinant of neutralization for anti-EIII antibodies. A panel of viruses containing all possible amino acid substitutions at E-332 was constructed. E-332 was found to be highly tolerant of mutation, and almost all of these changes had large impacts on antigenicity of EIII but only limited effects on growth or virulence phenotypes. PMID:27284640

  20. Mutations within a putative cysteine loop of the transmembrane protein of an attenuated immunodeficiency-inducing feline leukemia virus variant inhibit envelope protein processing.

    PubMed Central

    Burns, C C; Poss, M L; Thomas, E; Overbaugh, J

    1995-01-01

    A replication-defective feline leukemia virus molecular clone, 61B, has been shown to cause immunodeficiency in cats and cytopathicity in T cells after a long latency period when coinfected with a minimally pathogenic helper virus (J. Overbaugh, E. A. Hoover, J. I. Mullins, D. P. W. Burns, L. Rudensey, S. L. Quackenbush, V. Stallard, and P. R. Donahue, Virology 188:558-569, 1992). The long-latency phenotype of 61B has been mapped to four mutations in the extracellular domain of the envelope transmembrane protein, and we report here that these mutations cause a defect in envelope protein processing. Immunoprecipitation analyses demonstrated that the 61B gp85 envelope precursor was produced but that further processing to generate the surface protein (SU/gp70) and the transmembrane protein (TM/p15E) did not occur. The 61B precursor was not expressed on the cell surface and appeared to be retained in the endoplasmic reticulum or Golgi apparatus. Two of the four 61B-specific amino acid changes are located within a putative cysteine loop in a region of TM that is conserved among retroviruses. Introduction of these two amino acid changes into a replication-competent highly cytopathic virus resulted in the production of noninfectious virus that exhibited an envelope-protein-processing defect. This analysis suggests that mutations in a conserved region within a putative cysteine loop affect retroviral envelope protein maturation and viral infectivity. PMID:7884859

  1. Expression of Leukemia-Associated Nup98 Fusion Proteins Generates an Aberrant Nuclear Envelope Phenotype

    PubMed Central

    Fahrenkrog, Birthe; Martinelli, Valérie; Nilles, Nadine; Fruhmann, Gernot; Chatel, Guillaume; Juge, Sabine; Sauder, Ursula; Di Giacomo, Danika; Mecucci, Cristina; Schwaller, Jürg

    2016-01-01

    Chromosomal translocations involving the nucleoporin NUP98 have been described in several hematopoietic malignancies, in particular acute myeloid leukemia (AML). In the resulting chimeric proteins, Nup98's N-terminal region is fused to the C-terminal region of about 30 different partners, including homeodomain (HD) transcription factors. While transcriptional targets of distinct Nup98 chimeras related to immortalization are relatively well described, little is known about other potential cellular effects of these fusion proteins. By comparing the sub-nuclear localization of a large number of Nup98 fusions with HD and non-HD partners throughout the cell cycle we found that while all Nup98 chimeras were nuclear during interphase, only Nup98-HD fusion proteins exhibited a characteristic speckled appearance. During mitosis, only Nup98-HD fusions were concentrated on chromosomes. Despite the difference in localization, all tested Nup98 chimera provoked morphological alterations in the nuclear envelope (NE), in particular affecting the nuclear lamina and the lamina-associated polypeptide 2α (LAP2α). Importantly, such aberrations were not only observed in transiently transfected HeLa cells but also in mouse bone marrow cells immortalized by Nup98 fusions and in cells derived from leukemia patients harboring Nup98 fusions. Our findings unravel Nup98 fusion-associated NE alterations that may contribute to leukemogenesis. PMID:27031510

  2. Boosting of HIV-1 Neutralizing Antibody Responses by a Distally Related Retroviral Envelope Protein

    PubMed Central

    Uchtenhagen, Hannes; Schiffner, Torben; Bowles, Emma; Heyndrickx, Leo; LaBranche, Celia; Applequist, Steven E.; Jansson, Marianne; De Silva, Thushan; Back, Jaap Willem; Achour, Adnane; Scarlatti, Gabriella; Fomsgaard, Anders; Montefiori, David; Stewart-Jones, Guillaume; Spetz, Anna-Lena

    2014-01-01

    Our knowledge of the binding sites for neutralizing antibodies (NAbs) that recognize a broad range of HIV-1 strains (bNAb) has substantially increased in recent years. However, gaps remain in our understanding of how to focus B-cell responses to vulnerable conserved sites within the HIV-1 envelope glycoprotein (Env). Here we report an immunization strategy composed of a trivalent HIV-1 (clade B envs) DNA prime, followed by a SIVmac239 gp140 Env protein boost that aimed to focus the immune response to structurally conserved parts of the HIV-1 and SIV Envs. Heterologous NAb titres, primarily to tier 1 HIV-1 isolates, elicited during the trivalent HIV-1 env prime, were significantly increased by the SIVmac239 gp140 protein boost in rabbits. Epitope mapping of antibody binding reactivity revealed preferential recognition of the C1, C2, V2, V3 and V5 regions. These results provide a proof of concept that a distally related retroviral SIV Env protein boost can increase pre-existing NAb responses against HIV-1. PMID:24829409

  3. Duck tembusu virus and its envelope protein induce programmed cell death.

    PubMed

    Shaozhou, Wulin; Li, Chenxi; Zhang, Qingshan; Meng, Runzhe; Gao, Youlan; Liu, Hongyu; Bai, Xiaofei; Chen, Yuhuan; Liu, Ming; Liu, Siguo; Zhang, Yun

    2015-08-01

    The cytopathic effect produced in cells infected with duck tembusu virus (DTMUV) suggests that this emerging virus may induce apoptosis in primary cultures of duck embryo fibroblasts (DEF). Here, we present evidence that DTMUV infection of cultured cells activates apoptosis and that the ability of DTMUV to induce apoptosis is not restricted to cell type because DTMUV-induced apoptosis in duck and mammalian host cells. We further investigated which viral components induce apoptosis in DTMUV-infected host cells. The major envelope glycoprotein (E) was investigated for its apoptotic activities in expressed cells. Transient expression of the E protein alone triggered apoptosis in DEF, Vero, and BHK cells. Expression of the E protein resulted in activation of caspase-3-like proteases in cultured cells. These results indicate that infection of cells with DTMUV or expression of DTMUV E protein alone induces apoptosis, providing the basis for future to define the molecules that play key roles in the fate of DTMUV-infected cells. PMID:26056013

  4. SUN proteins facilitate the removal of membranes from chromatin during nuclear envelope breakdown

    PubMed Central

    Turgay, Yagmur; Champion, Lysie; Balazs, Csaba; Held, Michael; Toso, Alberto; Gerlich, Daniel W.; Meraldi, Patrick

    2014-01-01

    SUN proteins reside in the inner nuclear membrane and form complexes with KASH proteins of the outer nuclear membrane that connect the nuclear envelope (NE) to the cytoskeleton. These complexes have well-established functions in nuclear anchorage and migration in interphase, but little is known about their involvement in mitotic processes. Our analysis demonstrates that simultaneous depletion of human SUN1 and SUN2 delayed removal of membranes from chromatin during NE breakdown (NEBD) and impaired the formation of prophase NE invaginations (PNEIs), similar to microtubule depolymerization or down-regulation of the dynein cofactors NudE/EL. In addition, overexpression of dominant-negative SUN and KASH constructs reduced the occurrence of PNEI, indicating a requirement for functional SUN–KASH complexes in NE remodeling. Codepletion of SUN1/2 slowed cell proliferation and resulted in an accumulation of morphologically defective and disoriented mitotic spindles. Quantification of mitotic timing revealed a delay between NEBD and chromatin separation, indicating a role of SUN proteins in bipolar spindle assembly and mitotic progression. PMID:24662567

  5. Human endogenous retrovirus envelope proteins target dendritic cells to suppress T-cell activation.

    PubMed

    Hummel, Jonas; Kämmerer, Ulrike; Müller, Nora; Avota, Elita; Schneider-Schaulies, Sibylle

    2015-06-01

    Though mostly defective, human endogenous retroviruses (HERV) can retain open reading frames, which are especially expressed in the placenta. There, the envelope (env) proteins of HERV-W (Syncytin-1), HERV-FRD (Syncytin-2), and HERV-K (HML-2) were implicated in tolerance against the semi-allogenic fetus. Here, we show that the known HERV env-binding receptors ASCT-1 and -2 and MFSD2 are expressed by DCs and T-cells. When used as effectors in coculture systems, CHO cells transfected to express Syncytin-1, -2, or HML-2 did not affect T-cell expansion or overall LPS-driven phenotypic DC maturation, however, promoted release of IL-12 and TNF-α rather than IL-10. In contrast, HERV env expressing choriocarcinoma cell lines suppressed T-cell proliferation and LPS-induced TNF-α and IL-12 release, however, promoted IL-10 accumulation, indicating that these effects might not rely on HERV env interactions. However, DCs conditioned by choriocarcinoma, but also transgenic CHO cells failed to promote allogenic T-cell expansion. This was associated with a loss of DC/T-cell conjugate frequencies, impaired Ca(2+) mobilization, and aberrant patterning of f-actin and tyrosine phosphorylated proteins in T-cells. Altogether, these findings suggest that HERV env proteins target T-cell activation indirectly by modulating the stimulatory activity of DCs. PMID:25752285

  6. The role of HCV e2 protein glycosylation in functioning of virus envelope proteins in insect and Mammalian cells.

    PubMed

    Orlova, O V; Drutsa, V L; Spirin, P V; Prasolov, V S; Rubtsov, P M; Kochetkov, S N; Beljelarskaya, S N

    2015-01-01

    The hepatitis C virus (HCV) envelope proteins E1 and E2, being virion components, are involved in the formation of infectious particles in infected cells. The detailed structure of the infectious particle of HCV remains poorly understood. Moreover, the virion assembly and release of virions by the cell are the least understood processes. It is believed that virion properties depend on glycosylation of the virus envelope proteins in a cell, while glycansat several glycosylation sites of these proteins play a pivotal role in protein functioning and the HCV life cycle. N-glycans of glycoproteins can influence viral particle formation, virus binding to cell surface, and HCV pathogenesis. We studied the effect of glycans on the folding ofthe E2 glycoprotein, formation of functional glycoprotein complexes and virus particles in insect and mammalian cells. In order to investigate these processes, point mutations of the N-glycosylation sites of HCV protein E2 (genotype 1b strain 274933RU) were generated and the mutant proteins were further analyzed in the baculovirus expression system. Elimination of the single glycosylation sites of the E2 glycoprotein, except for the N6 site, did not affect its synthesis efficiency in Sf9 insect cells, while the electrophoretic mobility of mutant proteins increased in proportion to the decrease in the number of glycosylation sites. The level of synthesis of HCV glycoprotein E2 in human HEK293T cells depended on the presence of glycans at the N1 and N8 glycosylation sites in contrast to Sf9 cells. At the same time, elimination of glycans at the N1, N2, and N10 sites led to the accumulation of unproductive E1E2 dimers as aggregates and productive assembly suppression of virus-like particles both in insect and mammalian cells. In addition, elimination of single glycosylation sites of HCV E2 had no impact on the RNA synthesis of structural proteins and formation of virus-like particles in insect and mammalian cells. PMID:25927005

  7. Transmembrane protein TMEM170A is a newly discovered regulator of ER and nuclear envelope morphogenesis in human cells

    PubMed Central

    Christodoulou, Andri; Santarella-Mellwig, Rachel; Santama, Niovi

    2016-01-01

    ABSTRACT The mechanism of endoplasmic reticulum (ER) morphogenesis is incompletely understood. ER tubules are shaped by the reticulons (RTNs) and DP1/Yop1p family members, but the mechanism of ER sheet formation is much less clear. Here, we characterize TMEM170A, a human transmembrane protein, which localizes in ER and nuclear envelope membranes. Silencing or overexpressing TMEM170A in HeLa K cells alters ER shape and morphology. Ultrastructural analysis reveals that downregulation of TMEM170A specifically induces tubular ER formation, whereas overexpression of TMEM170A induces ER sheet formation, indicating that TMEM170A is a newly discovered ER-sheet-promoting protein. Additionally, downregulation of TMEM170A alters nuclear shape and size, decreases the density of nuclear pore complexes (NPCs) in the nuclear envelope and causes either a reduction in inner nuclear membrane (INM) proteins or their relocalization to the ER. TMEM170A interacts with RTN4, a member of the reticulon family; simultaneous co-silencing of TMEM170A and RTN4 rescues ER, NPC and nuclear-envelope-related phenotypes, implying that the two proteins have antagonistic effects on ER membrane organization, and nuclear envelope and NPC formation. PMID:26906412

  8. Iron transport in Mycobacterium smegmatis: occurrence of iron-regulated envelope proteins as potential receptors for iron uptake.

    PubMed

    Hall, R M; Sritharan, M; Messenger, A J; Ratledge, C

    1987-08-01

    Cell-envelope fractions were isolated from the rapidly growing saprophyte Mycobacterium smegmatis following growth in glycerol/asparagine medium under both iron-limited (0.02 microgram Fe ml-1) and iron-sufficient (2.0 to 4.0 micrograms Fe ml-1) conditions. Examination of these preparations by SDS-PAGE demonstrated the production of at least four additional proteins when iron was limiting. These iron-regulated envelope proteins (IREPs) were ascribed apparent molecular masses of 180 kDa (protein I), 84 kDa (protein II), 29 kDa (protein III) and 25 kDa (protein IV). All four proteins were present in both cell-wall and membrane preparations but spheroplast preparations were devoid of the 29 kDa protein. Attempts at labelling the proteins with 55FeCl3 or 55Fe-exochelin, the siderophore for iron uptake, were unsuccessful, though this was attributed to the denatured state of the proteins following electrophoresis. Antibodies were raised to each of the four proteins: the one raised to protein III inhibited exochelin-mediated iron uptake into iron-deficiently grown cells by 70% but was ineffective against iron uptake into iron-sufficiently grown cells. As exochelin is taken up into both types of cells by a similar process, protein III may not be a simple receptor for iron uptake though the results imply some function connected with this process. The role of the other IREPs is less certain. PMID:3127539

  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. Structures of the Zika Virus Envelope Protein and Its Complex with a Flavivirus Broadly Protective Antibody.

    PubMed

    Dai, Lianpan; Song, Jian; Lu, Xishan; Deng, Yong-Qiang; Musyoki, Abednego Moki; Cheng, Huijun; Zhang, Yanfang; Yuan, Yuan; Song, Hao; Haywood, Joel; Xiao, Haixia; Yan, Jinghua; Shi, Yi; Qin, Cheng-Feng; Qi, Jianxun; Gao, George F

    2016-05-11

    Zika virus (ZIKV), a mosquito-borne flavivirus, is a current global public health concern. The flavivirus envelope (E) glycoprotein is responsible for virus entry and represents a major target of neutralizing antibodies for other flaviviruses. Here, we report the structures of ZIKV E protein at 2.0 Å and in complex with a flavivirus broadly neutralizing murine antibody 2A10G6 at 3.0 Å. ZIKV-E resembles all the known flavivirus E structures but contains a unique, positively charged patch adjacent to the fusion loop region of the juxtaposed monomer, which may influence host attachment. The ZIKV-E-2A10G6 complex structure reveals antibody recognition of a highly conserved fusion loop. 2A10G6 binds to ZIKV-E with high affinity in vitro and neutralizes currently circulating ZIKV strains in vitro and in mice. The E protein fusion loop epitope represents a potential candidate for therapeutic antibodies against ZIKV. PMID:27158114

  11. Baculovirus Envelope Protein ODV-E66 Is a Novel Chondroitinase with Distinct Substrate Specificity*

    PubMed Central

    Sugiura, Nobuo; Setoyama, Yuka; Chiba, Mie; Kimata, Koji; Watanabe, Hideto

    2011-01-01

    Chondroitin sulfate is a linear polysaccharide of alternating d-glucuronic acid and N-acetyl-d-galactosamine residues with sulfate groups at various positions of the sugars. It interacts with and regulates cytokine and growth factor signal transduction, thus influencing development, organ morphogenesis, inflammation, and infection. We found chondroitinase activity in medium conditioned by baculovirus-infected insect cells and identified a novel chondroitinase. Sequence analysis revealed that the enzyme was a truncated form of occlusion-derived virus envelope protein 66 (ODV-E66) of Autographa californica nucleopolyhedrovirus. The enzyme was a novel chondroitin lyase with distinct substrate specificity. The enzyme was active over a wide range of pH (pH 4–9) and temperature (30–60 °C) and was unaffected by divalent metal ions. The ODV-E66 truncated protein digested chondroitin most efficiently followed by chondroitin 6-sulfate. It degraded hyaluronan to a minimal extent but did not degrade dermatan sulfate, heparin, and N-acetylheparosan. Further analysis using chemo-enzymatically synthesized substrates revealed that the enzyme specifically acted on glucuronate residues in non-sulfated and chondroitin 6-sulfate structures but not in chondroitin 4-sulfate structures. These results suggest that this chondroitinase is useful for detailed structural and compositional analysis of chondroitin sulfate, preparation of specific chondroitin oligosaccharides, and study of baculovirus infection mechanism. PMID:21715327

  12. 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.

  13. 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

  14. Structural Organization of Baculovirus Occlusion Bodies and Protective Role of Multilayered Polyhedron Envelope Protein.

    PubMed

    Sajjan, Dayanand B; Hinchigeri, Shivayogeppa B

    2016-03-01

    Baculoviruses are the ingenious insect pathogens. Outside the host, baculovirus occlusion bodies (OB) provide stability to occlusion-derived viruses (ODV) embedded within. The OB is an organized structure, chiefly composed of proteins namely polyhedrin, polyhedron envelope protein (PEP) and P10. Currently, the structural organization of OB is poorly understood and the role of OB proteins in conferring the stability to ODV is unknown. Here we have shown that the assembly of polyhedrin unit cells into an OB is a rapid process; the PEP forms in multiple layers; the PEP layers predominantly contribute to ODV viability. Full-grown OBs (n = 36) were found to be 4.0 ± 1.0 µm in diameter and possessed a peculiar geometry of a truncated rhombic dodecahedron. The atomic force microscopy (AFM) study on the structure of OBs at different stages of growth in insect cells revealed polyhedrin assembly and thickness of PEP layers. The thickness of PEP layers at 53 h post-transfection (hpt) ranged from 56 to 80 nm. Mature PEP layers filled up approximately one third of the OB volume. The size of ODV nucleocapsid was found to be 433 ± 10 nm in length. The zeta potential and particle size distribution study of viruses revealed the protective role of PEP layers. The presence of a multilayered PEP confers a viable advantage to the baculoviruses compared to single-layered PEP. Thus, these findings may help in developing PEP layer-based biopolymers for protein-based nanodevices, nanoelectrodes and more stable biopesticides. PMID:26787118

  15. Nucleotide sequence variation of the envelope protein gene identifies two distinct genotypes of yellow fever virus.

    PubMed Central

    Chang, G J; Cropp, B C; Kinney, R M; Trent, D W; Gubler, D J

    1995-01-01

    The evolution of yellow fever virus over 67 years was investigated by comparing the nucleotide sequences of the envelope (E) protein genes of 20 viruses isolated in Africa, the Caribbean, and South America. Uniformly weighted parsimony algorithm analysis defined two major evolutionary yellow fever virus lineages designated E genotypes I and II. E genotype I contained viruses isolated from East and Central Africa. E genotype II viruses were divided into two sublineages: IIA viruses from West Africa and IIB viruses from America, except for a 1979 virus isolated from Trinidad (TRINID79A). Unique signature patterns were identified at 111 nucleotide and 12 amino acid positions within the yellow fever virus E gene by signature pattern analysis. Yellow fever viruses from East and Central Africa contained unique signatures at 60 nucleotide and five amino acid positions, those from West Africa contained unique signatures at 25 nucleotide and two amino acid positions, and viruses from America contained such signatures at 30 nucleotide and five amino acid positions in the E gene. The dissemination of yellow fever viruses from Africa to the Americas is supported by the close genetic relatedness of genotype IIA and IIB viruses and genetic evidence of a possible second introduction of yellow fever virus from West Africa, as illustrated by the TRINID79A virus isolate. The E protein genes of American IIB yellow fever viruses had higher frequencies of amino acid substitutions than did genes of yellow fever viruses of genotypes I and IIA on the basis of comparisons with a consensus amino acid sequence for the yellow fever E gene. The great variation in the E proteins of American yellow fever virus probably results from positive selection imposed by virus interaction with different species of mosquitoes or nonhuman primates in the Americas. PMID:7637022

  16. Expression, Purification, Crystallization of Two Major Envelope Proteins from White Spot Syndrome Virus

    SciTech Connect

    Tang,X.; Hew, C.

    2007-01-01

    White spot syndrome virus (WSSV) is a major virulent pathogen known to infect penaeid shrimp and other crustaceans. VP26 and VP28, two major envelope proteins from WSSV, have been identified and overexpressed in Escherichia coli. In order to facilitate purification and crystallization, predicted N-terminal transmembrane regions of approximately 35 amino acids have been truncated from both VP26 and VP28. Truncated VP26 and VP28 and their corresponding SeMet-labelled proteins were purified and the SeMet proteins were crystallized by the hanging-drop vapor-diffusion method. Crystals of SeMet-labelled VP26 were obtained using a reservoir consisting of 0.1 M citric acid pH 3.5, 3.0 M sodium chloride and 1%(w/v) polyethylene glycol 3350, whereas SeMet VP28 was crystallized using a reservoir solution consisting of 25% polyethylene glycol 8000, 0.2 M calcium acetate, 0.1 M Na HEPES pH 7.5 and 1.5%(w/v) 1,2,3-heptanetriol. Crystals of SeMet-labelled VP26 diffract to 2.2 {angstrom} resolution and belong to space group R32, with unit-cell parameters a = b = 73.92, c = 199.31 {angstrom}. SeMet-labelled VP28 crystallizes in space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 105.33, b = 106.71, c = 200.37 {angstrom}, and diffracts to 2.0 {angstrom} resolution.

  17. Expression, purification and crystallization of two major envelope proteins from white spot syndrome virus

    SciTech Connect

    Tang, Xuhua; Hew, Choy Leong

    2007-07-01

    The crystallization of the N-terminal transmembrane region-truncated VP26 and VP28 of white spot syndrome virus is described. White spot syndrome virus (WSSV) is a major virulent pathogen known to infect penaeid shrimp and other crustaceans. VP26 and VP28, two major envelope proteins from WSSV, have been identified and overexpressed in Escherichia coli. In order to facilitate purification and crystallization, predicted N-terminal transmembrane regions of approximately 35 amino acids have been truncated from both VP26 and VP28. Truncated VP26 and VP28 and their corresponding SeMet-labelled proteins were purified and the SeMet proteins were crystallized by the hanging-drop vapour-diffusion method. Crystals of SeMet-labelled VP26 were obtained using a reservoir consisting of 0.1 M citric acid pH 3.5, 3.0 M sodium chloride and 1%(w/v) polyethylene glycol 3350, whereas SeMet VP28 was crystallized using a reservoir solution consisting of 25% polyethylene glycol 8000, 0.2 M calcium acetate, 0.1 M Na HEPES pH 7.5 and 1.5%(w/v) 1,2,3-heptanetriol. Crystals of SeMet-labelled VP26 diffract to 2.2 Å resolution and belong to space group R32, with unit-cell parameters a = b = 73.92, c = 199.31 Å. SeMet-labelled VP28 crystallizes in space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 105.33, b = 106.71, c = 200.37 Å, and diffracts to 2.0 Å resolution.

  18. Further analysis of the involvement of the envelope anion channel PIRAC in chloroplast protein import.

    PubMed

    van den Wijngaard, P W; Demmers, J A; Thompson, S J; Wienk, H L; de Kruijff, B; Vredenberg, W J

    2000-06-01

    The ability of preferredoxin to inactivate a 50-pS anion channel of the chloroplast inner membrane in the presence of an energy source was investigated using single-channel recordings. It was found that preferredoxin cannot inactivate the channel when GTP is the only energy source present. From this it is concluded that the precursor has to interact with the, translocon of the inner membrane of chloroplasts (Tic) complex to be able to inactivate the 50-pS anion channel. The ability of two mutants of preferredoxin with deletions in their transit sequence to inactivate the channel was also tested. Both mutants have been shown to have a similar binding affinity for the chloroplast envelope, but only one is able to fully translocate. The mutants were both able to inactivate the channel in a similar manner. From this it is concluded that full translocation is not necessary for the inactivation of the channel. It is also shown that preferredoxin is capable of inactivating the 50-pS anion channel in the chloroplast-attached configuration as was previously found in the inside-out configuration. From this it is concluded that stromal factors do not influence the protein-import-induced inactivation of the 50-pS anion channel of the chloroplast inner membrane. Finally the effect of the anion channel blocker 4, 4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) on the channel activity and on protein import was investigated. It was found that DIDS blocked the channel. Furthermore the addition of the channel blocker reduces the efficiency of import to 52%. This leads to the conclusion that correct functioning of the channel is important for protein import. PMID:10849000

  19. Surface Proteins of Gram-Positive Bacteria and Mechanisms of Their Targeting to the Cell Wall Envelope

    PubMed Central

    Navarre, William Wiley; Schneewind, Olaf

    1999-01-01

    The cell wall envelope of gram-positive bacteria is a macromolecular, exoskeletal organelle that is assembled and turned over at designated sites. The cell wall also functions as a surface organelle that allows gram-positive pathogens to interact with their environment, in particular the tissues of the infected host. All of these functions require that surface proteins and enzymes be properly targeted to the cell wall envelope. Two basic mechanisms, cell wall sorting and targeting, have been identified. Cell well sorting is the covalent attachment of surface proteins to the peptidoglycan via a C-terminal sorting signal that contains a consensus LPXTG sequence. More than 100 proteins that possess cell wall-sorting signals, including the M proteins of Streptococcus pyogenes, protein A of Staphylococcus aureus, and several internalins of Listeria monocytogenes, have been identified. Cell wall targeting involves the noncovalent attachment of proteins to the cell surface via specialized binding domains. Several of these wall-binding domains appear to interact with secondary wall polymers that are associated with the peptidoglycan, for example teichoic acids and polysaccharides. Proteins that are targeted to the cell surface include muralytic enzymes such as autolysins, lysostaphin, and phage lytic enzymes. Other examples for targeted proteins are the surface S-layer proteins of bacilli and clostridia, as well as virulence factors required for the pathogenesis of L. monocytogenes (internalin B) and Streptococcus pneumoniae (PspA) infections. In this review we describe the mechanisms for both sorting and targeting of proteins to the envelope of gram-positive bacteria and review the functions of known surface proteins. PMID:10066836

  20. [Envelope protein of Jaagsiekte sheep retrovious expressed in NIH3T3 cells promotes cell proliferation].

    PubMed

    DU, Fangyuan; Chen, Dayong; Zhang, Yufei; Sun, Xiaolin; Guo, Wenqing; Liu, Shuying

    2016-09-01

    Objective To explore the influence of the exogenous Jaagsiekte sheep retrovious (exJSRV) envelope protein (Env) on NIH3T3 cell proliferation. Methods A recombinant plasmid pcDNA4/myc-His/exJSRV- env carrying exJSRV- env gene was constructed, and then the correctness of the recombinant plasmid was identified by PCR, restriction enzyme digestion and sequencing. The recombinant plasmid pcDNA4/myc-His/exJSRV- env was transiently transfected into NIH3T3 cells by Lipofectamine(TM) LTX. After the transfection of the recombinant plasmid, the expression of exJSRV- env was detected by reverse transcription PCR and Western blotting. The effect of Env on cell proliferation was investigated by CCK-8 assay and plate colony formation assay. Results The recombinant eukaryotic expression plasmid containing exJSRV- env was successfully constructed as identified by PCR, restriction enzyme identification and sequencing. After the recombinant plasmid was transiently transfected into NIH3T3 cells, reverse transcription PCR and Western blotting showed the expression of exJSRV- env , and Env promoted NIH3T3 cell proliferation significantly. Conclusion JSRV Env was expressed successfully in the NIH3T3 cells and promoted the proliferation of NIH3T3 cells. PMID:27609573

  1. Crystal Structures of Major Envelope Proteins VP26 and VP28 from White Spot Syndrome Virus Shed Light on Their Evolutionary Relationship

    SciTech Connect

    Tang,X.; Wu, J.; Sivaraman, J.; Hew, C.

    2007-01-01

    White spot syndrome virus (WSSV) is a virulent pathogen known to infect various crustaceans. It has bacilliform morphology with a tail-like appendage at one end. The envelope consists of four major proteins. Envelope structural proteins play a crucial role in viral infection and are believed to be the first molecules to interact with the host. Here, we report the localization and crystal structure of major envelope proteins VP26 and VP28 from WSSV at resolutions of 2.2 and 2.0 {angstrom}, respectively. These two proteins alone account for approximately 60% of the envelope, and their structures represent the first two structural envelope proteins of WSSV. Structural comparisons among VP26, VP28, and other viral proteins reveal an evolutionary relationship between WSSV envelope proteins and structural proteins from other viruses. Both proteins adopt {beta}-barrel architecture with a protruding N-terminal region. We have investigated the localization of VP26 and VP28 using immunoelectron microscopy. This study suggests that VP26 and VP28 are located on the outer surface of the virus and are observed as a surface protrusion in the WSSV envelope, and this is the first convincing observation for VP26. Based on our studies combined with the literature, we speculate that the predicted N-terminal transmembrane region of VP26 and VP28 may anchor on the viral envelope membrane, making the core {beta}-barrel protrude outside the envelope, possibly to interact with the host receptor or to fuse with the host cell membrane for effective transfer of the viral infection. Furthermore, it is tempting to extend this host interaction mode to other structural viral proteins of similar structures. Our finding has the potential to extend further toward drug and vaccine development against WSSV.

  2. The intracellular production and secretion of HIV-1 envelope protein in the methylotrophic yeast Pichia pastoris.

    PubMed

    Scorer, C A; Buckholz, R G; Clare, J J; Romanos, M A

    1993-12-22

    The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein, gp120 (ENV), is required in large quantities for immunological studies and as a potential vaccine component. We have expressed the DNA encoding gp120 in a highly efficient expression system based on the methylotrophic yeast, Pichia pastoris. The native gene was found to contain a sequence which resembled a Saccharomyces cerevisiae polyadenylation consensus and acted as a premature polyadenylation site in P. pastoris, resulting in the production of truncated mRNA. As full-length mRNA was produced in S. cerevisiae, this indicates differences in mRNA 3'-end formation between the two yeasts. Inactivation of this site by site-directed mutagenesis revealed several additional fortuitous polyadenylation sites within the gene. We have designed and constructed a 69%-synthetic gene with increased G + C content which overcomes this transcriptional problem, giving rise to full-length mRNA. High levels of intracellular, insoluble, unglycosylated ENV were produced [1.25 mg/ml in high-density (2 x 10(10) cells per ml) fermentations]. ENV also was secreted from P. pastoris using the S. cerevisiae alpha-factor prepro secretion leader and the S. cerevisiae invertase signal sequence. However, a high proportion of the secreted product was found to be hyperglycosylated, in contrast to other foreign proteins secreted from P. pastoris. There also was substantial proteolytic degradation, but this was minimized by maintaining a low pH on induction. Insoluble, yeast-derived ENV proteins are being considered as vaccine antigens and the P. pastoris system offers an efficient method of production. PMID:8293993

  3. Nuclear envelope proteins modulate proliferation of vascular smooth muscle cells during cyclic stretch application

    PubMed Central

    Qi, Ying-Xin; Yao, Qing-Ping; Huang, Kai; Shi, Qian; Zhang, Ping; Wang, Guo-Liang; Han, Yue; Bao, Han; Wang, Lu; Li, Hai-Peng; Shen, Bao-Rong; Wang, Yingxiao; Chien, Shu; Jiang, Zong-Lai

    2016-01-01

    Cyclic stretch is an important inducer of vascular smooth muscle cell (VSMC) proliferation, which is crucial in vascular remodeling during hypertension. However, the molecular mechanism remains unclear. We studied the effects of emerin and lamin A/C, two important nuclear envelope proteins, on VSMC proliferation in hypertension and the underlying mechano-mechanisms. In common carotid artery of hypertensive rats in vivo and in cultured cells subjected to high (15%) cyclic stretch in vitro, VSMC proliferation was increased significantly, and the expression of emerin and lamin A/C was repressed compared with normotensive or normal (5%) cyclic stretch controls. Using targeted siRNA to mimic the repressed expression of emerin or lamin A/C induced by 15% stretch, we found that VSMC proliferation was enhanced under static and 5%-stretch conditions. Overexpression of emerin or lamin A/C reversed VSMC proliferation induced by 15% stretch. Hence, emerin and lamin A/C play critical roles in suppressing VSMC hyperproliferation induced by hyperstretch. ChIP-on-chip and MOTIF analyses showed that the DNAs binding with emerin contain three transcription factor motifs: CCNGGA, CCMGCC, and ABTTCCG; DNAs binding with lamin A/C contain the motifs CVGGAA, GCCGCYGC, and DAAGAAA. Protein/DNA array proved that altered emerin or lamin A/C expression modulated the activation of various transcription factors. Furthermore, accelerating local expression of emerin or lamin A/C reversed cell proliferation in the carotid artery of hypertensive rats in vivo. Our findings establish the pathogenetic role of emerin and lamin A/C repression in stretch-induced VSMC proliferation and suggest mechanobiological mechanism underlying this process that involves the sequence-specific binding of emerin and lamin A/C to specific transcription factor motifs. PMID:27114541

  4. Identification and characterization of a prawn white spot syndrome virus gene that encodes an envelope protein VP31

    SciTech Connect

    Li Li; Xie Xixian; Yang Feng . E-mail: mbiotech@public.xm.fj.cn

    2005-09-15

    Based on a combination of SDS-PAGE and mass spectrometry, a protein with an apparent molecular mass of 31 kDa (termed as VP31) was identified from purified shrimp white spot syndrome virus (WSSV) envelope fraction. The resulting amino acid (aa) sequence matched an open reading frame (WSV340) of the WSSV genome. This ORF contained 783 nucleotides (nt), encoding 261 aa. A fragment of WSV340 was expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein with a 6His-tag, and then specific antibody was raised. Western blot analysis and the immunoelectron microscope method (IEM) confirmed that VP31 was present exclusively in the viral envelope fraction. The neutralization experiment suggested that VP31 might play an important role in WSSV infectivity.

  5. Expression, immunogenicity and diagnostic value of envelope proteins from an Egyptian hepatitis C virus isolate.

    PubMed

    Shawky, Heba; Maghraby, Amany S; Solliman, Mohei El-Din; El-Mokadem, Mehreshan T; Sherif, Mohamed M; Arafa, Azza; Bahgat, Mahmoud M

    2015-04-01

    The present work aimed at 1) characterization of the E1 and E2 proteins (HCV-E) from an Egyptian hepatitis C virus genotype 4a (HCV-4a) isolate at the molecular and immunological level, 2) in silico identification of the B- and T-cell epitopes responsible for the immunogenicity of HCV-E, and 3) evaluation of the diagnostic potential of both the recombinant HCV-E and antibodies raised using mammalian expression constructs encoding the protein. The region encoding the E1 and E2 proteins was amplified by RT-PCR from RNA isolated from blood of a human infected with HCV-4 and cloned into the pSC-TA plasmid, and the sequence was verified and used to construct a neighbor-joining phylogenetic tree. The translated nucleotide sequence was used to predict the HCV-E secondary structure using the PREDICT-PROTEIN server and PSI-PRED. A 3D model of HCV-E was generated using the online tool 3Dpro. B- and T-cell epitopes were predicted using the online tools BCPred and Epijen v1.0, respectively. The HCV-E-encoding sequence was later subcloned into the mammalian expression plasmid pQE, and the constructs that were generated were used to immunize mice in the absence and presence of adjuvants of plant origin. The maximum sequence identity obtained by nucleotide and protein BLAST analysis with previously published HCV-E sequences was 85 and 77 %, respectively. The B-cell epitope CFTPSPVVV at position 203 and the T-cell epitope ALSTGLIHL at position 380 were found to be highly conserved among all HCV genotypes. Both ELISA and Western blotting experiments on crude and purified recombinant HCV envelope proteins using mouse antisera raised using the HCV-E mammalian expression construct confirmed the specific antigenicity of the expressed protein. The antibodies raised in mice using the HCV-E-encoding construct could efficiently capture circulating antigens in patients' sera with good sensitivity that correlated with liver enzyme levels (r = 0.4052, P < 0.0001 for ALT; r = -0.5439, P = 0

  6. Species-specific sequences of abalone lysin, the sperm protein that creates a hole in the egg envelope.

    PubMed Central

    Vacquier, V D; Carner, K R; Stout, C D

    1990-01-01

    Abalone eggs are contained within a rigid, elevated vitelline envelope through which the sperm must pass before reaching the egg cell membrane. Abalone spermatozoa possess an acrosomal protein called lysin that creates a hole in the egg vitelline envelope by a nonenzymatic mechanism. Lysins from two species of abalone, termed pink and red, which share the same habitat, exhibit species specificity in the dissolution of isolated egg envelopes. Cloning and sequencing the cDNAs for pink and red abalone lysins reveal transcript lengths of approximately 660 nucleotides. The open reading frames of 465 (pink) and 462 (red) nucleotides show a 13% difference. The 3' untranslated regions before the poly(A) tails are 170 (pink) and 165 (red) nucleotides long and differ from each other by about 7%. The protein sequences show nearly identical signal sequences of 18 amino acids for both lysins. The mature protein is 137 amino acids in the pink abalone and 136 in the red abalone; the two mature lysins differ in 29 of 137 amino acids (21%). The most variable region, which may account for lysin's species specificity, is at the NH2 terminus, where 11 of the 15 amino acids differ between the two species. Predictions of secondary structure indicate that both lysins contain four homologous amphiphilic alpha-helices. Images PMID:2377618

  7. Analogs of LDL Receptor Ligand Motifs in Dengue Envelope and Capsid Proteins as Potential Codes for Cell Entry

    PubMed Central

    Guevara, Juan; Romo, Jamie; McWhorter, Troy; Guevara, Natalia Valentinova

    2016-01-01

    It is established that cell entry of low density lipoprotein particles (LLPs) containing Apo B100 and Apo E is mediated by receptors and GAGs. Receptor ligand motifs, XBBBXXBX, XBBXBX, and ΨBΨXB, and mono- and bipartite NLS sequences are abundant in Apo E and Apo B100 as well as in envelope and capsid proteins of Dengue viruses 1–4 (DENV1–4). Synthetic, fluorescence-labeled peptides of sequences in DENV2 envelope protein, and DENV3 capsid that include these motifs were used to conduct a qualitative assessment of cell binding and entry capacity using HeLa cells. DENV2 envelope peptide, Dsp2EP, 0564Gly-Gly0595, was shown to bind and remain at the cell surface. In contrast, DENV3 capsid protein peptide, Dsp3CP, 0002Asn-Gln0028, readily enters HeLa cells and accumulates at discrete loci in the nucleus. FITC-labeled dengue synthetic peptides colocalize with Low Density Lipoprotein-CM-DiI and Apo E-CM-DiI to a degree that suggests that Dengue viruses may utilize cell entry pathways used by LLPs. PMID:27123468

  8. Equine Tetherin Blocks Retrovirus Release and Its Activity Is Antagonized by Equine Infectious Anemia Virus Envelope Protein

    PubMed Central

    Yin, Xin; Hu, Zhe; Gu, Qinyong; Wu, Xingliang; Zheng, Yong-Hui; Wei, Ping

    2014-01-01

    Human tetherin is a host restriction factor that inhibits replication of enveloped viruses by blocking viral release. Tetherin has an unusual topology that includes an N-terminal cytoplasmic tail, a single transmembrane domain, an extracellular domain, and a C-terminal glycosylphosphatidylinositol anchor. Tetherin is not well conserved across species, so it inhibits viral replication in a species-specific manner. Thus, studies of tetherin activities from different species provide an important tool for understanding its antiviral mechanism. Here, we report cloning of equine tetherin and characterization of its antiviral activity. Equine tetherin shares 53%, 40%, 36%, and 34% amino acid sequence identity with feline, human, simian, and murine tetherins, respectively. Like the feline tetherin, equine tetherin has a shorter N-terminal domain than human tetherin. Equine tetherin is localized on the cell surface and strongly blocks human immunodeficiency virus type 1 (HIV-1), simian immunodeficiency virus (SIV), and equine infectious anemia virus (EIAV) release from virus-producing cells. The antiviral activity of equine tetherin is neutralized by EIAV envelope protein, but not by the HIV-1 accessory protein Vpu, which is a human tetherin antagonist, and EIAV envelope protein does not counteract human tetherin. These results shed new light on our understanding of the species-specific tetherin antiviral mechanism. PMID:24227834

  9. Targeted gene transfer to lymphocytes using murine leukaemia virus vectors pseudotyped with spleen necrosis virus envelope proteins.

    PubMed

    Engelstädter, M; Buchholz, C J; Bobkova, M; Steidl, S; Merget-Millitzer, H; Willemsen, R A; Stitz, J; Cichutek, K

    2001-08-01

    In contrast to murine leukaemia virus (MLV)-derived vector systems, vector particles derived from the avian spleen necrosis virus (SNV) have been successfully targeted to subsets of human cells by envelope modification with antibody fragments (scFv). However, an in vivo application of the SNV vector system in gene transfer protocols is hampered by its lack of resistance against human complement. To overcome this limitation we established pseudotyping of MLV vector particles produced in human packaging cell lines with the SNV envelope (Env) protein. Three variants of SNV Env proteins differing in the length of their cytoplasmic domains were all efficiently incorporated into MLV core particles. These pseudotype particles infected the SNV permissive cell line D17 at titers of up to 10(5) IU/ml. A stable packaging cell line (MS4) of human origin released MLV(SNV) pseudotype vectors that were resistant against human complement inactivation. To redirect their tropism to human T cells, MS4 cells were transfected with the expression gene encoding the scFv 7A5 in fusion with the transmembrane domain (TM) of the SNV Env protein, previously shown to retarget SNV vector particles to human lymphocytes. MLV(SNV-7A5)-vector particles released from these cells were selectively infectious for human T cell lines. The data provide a proof of principle for targeting MLV-derived vectors to subpopulations of human cells through pseudotyping with SNV targeting envelopes. PMID:11509952

  10. Exogenous hepatitis B virus envelope proteins induce endoplasmic reticulum stress: involvement of cannabinoid axis in liver cancer cells

    PubMed Central

    Montalbano, Roberta; Honrath, Birgit; Wissniowski, Thaddeus Till; Elxnat, Moritz; Roth, Silvia; Ocker, Matthias; Quint, Karl; Churin, Yuri; Roederfeld, Martin; Schroeder, Dirk; Glebe, Dieter; Roeb, Elke; Fazio, Pietro Di

    2016-01-01

    HBV represents the most common chronic viral infection and major cause of hepatocellular carcinoma (HCC), although its exact role in liver tumorigenesis is unclear. Massive storage of the small (SHBs), middle (MHBs) and large surface (LHBs) HBV envelope proteins leads to cell stress and sustained inflammatory responses. Cannabinoid (CB) system is involved in the pathogenesis of liver diseases, stimulating acute and chronic inflammation, liver damage and fibrogenesis; it triggers endoplasmic reticulum (ER) stress response. The aim of our work was to investigate the activation of ER stress pathway after ectopic HBV envelope proteins expression, in liver cancer cells, and the role exerted by CB receptors. PCR, immunofluorescence and western blotting showed that exogenous LHBs and MHBs induce a clear ER stress response in Huh-7 cells expressing CB1 receptor. Up-regulation of the chaperone BiP/GRP78 (Binding Immunoglobulin Protein/Glucose-Regulated Protein 78) and of the transcription factor CHOP/GADD153 (C/EBP Homologous Protein/Growth Arrest and DNA Damage inducible gene 153), phosphorylation of PERK (PKR-like ER Kinase) and eIF2α (Eukaryotic Initiation Factor 2α) and splicing of XBP1 (X-box binding protein 1) was observed. CB1−/− HepG2 cells did not show any ER stress activation. Inhibition of CB1 receptor counteracted BiP expression in transfected Huh-7 and in HBV+ PLC/PRF/5 cells; whereas no effect was observed in HBV− HLF cells. These results suggest that HBV envelope proteins are able to induce the ER stress pathway. CB1 expression is directly correlated with ER stress function. Further investigations are needed to clarify the involvement of cannabinoid in HCC progression after HBV infection. PMID:26967385

  11. In silico analysis of an envelope domain III-based multivalent fusion protein as a potential dengue vaccine candidate

    PubMed Central

    2016-01-01

    Purpose Dengue virus infection is now a global problem. Currently, there is no licensed vaccine or proven antiviral treatment against this virus. All four serotypes (1-4) of dengue virus can infect human. An effective dengue vaccine should be tetravalent to induce protective immune responses against all four serotypes. Most of dengue vaccine candidates are monovalent, or in the form of physically mixed multivalent formulations. Recently envelope protein domain III of virus is considered as a vaccine candidate, which plays critical roles in the most important viral activities. Development of a tetravalent protein subunit vaccine is very important for equal induction of immune system and prevention of unbalanced immunity. Here, we have presented and used a rational approach to design a tetravalent dengue vaccine candidate. Materials and Methods We designed a multi domain antigen by fusing four consensus domain III sequences together with appropriate hydrophobic linkers and used several types of bioinformatics software and neural networks to predict structural and immunological properties of the designed tetravalent antigen. Results We designed a tetravalent protein (EDIIIF) based on domain III of dengue virus envelope protein. According to the results of the bioinformatics analysis, the constructed models for EDIIIF protein were structurally stable and potentially immunogenic. Conclusion The designed tetravalent protein can be considered as a potential dengue vaccine candidate. The presented approach can be used for rational design and in silico evaluation of chimeric dengue vaccine candidates. PMID:26866023

  12. Dengue-4 envelope domain III fused twice within the meningococcal P64k protein carrier induces partial protection in mice.

    PubMed

    Lazo, Laura; Zulueta, Aída; Hermida, Lisset; Blanco, Aracelys; Sánchez, Jorge; Valdés, Iris; Gil, Lázaro; López, Carlos; Romero, Yaremis; Guzmán, María G; Guillén, Gerardo

    2009-04-01

    A vaccine against dengue virus must be able to induce an effective and equivalent immune response to the four viral serotypes; however, some studies have revealed that DEN4 (dengue-virus serotype 4) induces a weaker immune response than the others in quadrivalent (tetravalent') formulations. We have previously reported the protective capacity, in a viral encephalitis murine model, of fusion protein P64k-envelope domain III of DEN1, DEN2 and DEN3. We also reported that the P64k protein can be used as a carrier in two different positions: the insertion following the first 45 amino acids and the fusion at the C-terminus. Considering the low immunogenicity described for DEN4, in the present study we obtained a novel chimaeric protein by inserting two dengue-4 envelope domains III in both sites of P64k (PD24), and hence increasing the presence of the virus in the final construct. After expression in Escherichia coli and semipurification, the protein exhibited a pattern of high molecular mass and was well recognized by human and murine polyclonal antibodies. The protein was finally evaluated in mice, Al(OH)(3) being employed as the adjuvant. Even though the animals exhibited low levels of antiviral antibodies, the recombinant protein induced significant protection against lethal challenge with dengue-4 virus. PMID:18636968

  13. Immunogenicity and protective efficacy of the E. coli-expressed domain III of Japanese encephalitis virus envelope protein in mice.

    PubMed

    Alka; Bharati, Kaushik; Malik, Y P S; Vrati, Sudhanshu

    2007-12-01

    Domain III of Japanese encephalitis virus (JEV) envelope protein (E-DIII) was synthesized in E. coli as a fusion protein containing maltose-binding protein (MBP-E-DIII) or six contiguous histidine residues (His-E-DIII) at its N-terminus. MBP-E-DIII was found both in the soluble as well as the insoluble fraction of the bacterial lysate, while His-E-DIII was found exclusively in the inclusion bodies. These purified proteins were examined in mice for their immunogenicity in presence of an aluminium hydroxide based-adjuvant Alhydrogel and Freund's adjuvant. While both proteins generated anti-JEV antibodies that neutralized JEV activity in vitro, His-E-DIII generated higher antibody titers than MBP-E-DIII. Mice immunized with His-E-DIII in presence of Alhydrogel generated antibody titers similar to those induced by the commercial vaccine and protected mice against lethal JEV challenge. PMID:17377815

  14. Structural protein 4.1R is integrally involved in nuclear envelope protein localization, centrosome–nucleus association and transcriptional signaling

    PubMed Central

    Meyer, Adam J.; Almendrala, Donna K.; Go, Minjoung M.; Krauss, Sharon Wald

    2011-01-01

    The multifunctional structural protein 4.1R is required for assembly and maintenance of functional nuclei but its nuclear roles are unidentified. 4.1R localizes within nuclei, at the nuclear envelope, and in cytoplasm. Here we show that 4.1R, the nuclear envelope protein emerin and the intermediate filament protein lamin A/C co-immunoprecipitate, and that 4.1R-specific depletion in human cells by RNA interference produces nuclear dysmorphology and selective mislocalization of proteins from several nuclear subcompartments. Such 4.1R-deficiency causes emerin to partially redistribute into the cytoplasm, whereas lamin A/C is disorganized at nuclear rims and displaced from nucleoplasmic foci. The nuclear envelope protein MAN1, nuclear pore proteins Tpr and Nup62, and nucleoplasmic proteins NuMA and LAP2α also have aberrant distributions, but lamin B and LAP2β have normal localizations. 4.1R-deficient mouse embryonic fibroblasts show a similar phenotype. We determined the functional effects of 4.1R-deficiency that reflect disruption of the association of 4.1R with emerin and A-type lamin: increased nucleus–centrosome distances, increased β-catenin signaling, and relocalization of β-catenin from the plasma membrane to the nucleus. Furthermore, emerin- and lamin-A/C-null cells have decreased nuclear 4.1R. Our data provide evidence that 4.1R has important functional interactions with emerin and A-type lamin that impact upon nuclear architecture, centrosome–nuclear envelope association and the regulation of β-catenin transcriptional co-activator activity that is dependent on β-catenin nuclear export. PMID:21486941

  15. Refolding, crystallization and preliminary X-ray structural studies of the West Nile virus envelope (E) protein domain III

    SciTech Connect

    Yuan, Fang; Lou, Zhiyong; Li, Xiaofeng; Chen, Yu Wai; Bell, John I.; Rao, Zihe; Gao, George F.

    2005-04-01

    Domain III of the West Nile virus envelope protein, the putative receptor-binding domain, was reported to be intrinsically unstable and has defied previous crystallization attempts. It has now been purified from inclusion bodies by protein refolding and was crystallized using the hanging-drop vapour-diffusion method at 291 K. Domain III of the West Nile virus envelope protein, the putative receptor-binding domain, is a major virion-surface determinant for virulence. This protein was reported to be intrinsically unstable and has defied previous crystallization attempts. It has now been purified from inclusion bodies by protein refolding and was crystallized using the hanging-drop vapour-diffusion method at 291 K. The crystals belong to space group P222{sub 1}, with unit-cell parameters a = 52.6, b = 59.7, c = 95.0 Å. A complete data set was collected to 2.8 Å at 100 K with Cu Kα X-rays from a rotating-anode generator.

  16. Murine Monoclonal Antibodies for Antigenic Discrimination of HIV-1 Envelope Proteins

    PubMed Central

    Sealy, Robert E.; Jones, Bart G.; Surman, Sherri L.; Branum, Kristen; Howlett, Nanna M.; Flynn, Patricia M.

    2016-01-01

    Abstract In the influenza virus field, antibody reagents from research animals have been instrumental in the characterization of antigenically distinct hemagglutinin and neuraminidase membrane molecules. These small animal reagents continue to support the selection of components for inclusion in human influenza virus vaccines. Other cocktail vaccines against variant pathogens (e.g., polio virus, pneumococcus) are similarly designed to represent variant antigens, as defined by antibody reactivity patterns. However, a vaccine cocktail comprising diverse viral membrane antigens defined in this way has not yet been advanced to a clinical efficacy study in the HIV-1 field. In this study, we describe the preparation of mouse antibodies specific for HIV-1 gp140 or gp120 envelope molecules. Our experiments generated renewable reagents able to discriminate HIV-1 envelopes from one another. Monoclonals yielded more precise discriminatory capacity against their respective immunogens than did a small panel of polyclonal human sera derived from recently HIV-1-infected patients. Perhaps these and other antibody reagents will ultimately support high-throughput cartography studies with which antigenically-distinct envelope immunogens may be formulated into a successful HIV-1 envelope cocktail vaccine. PMID:26544795

  17. Human Immunodeficiency Virus Type 1 Subtype B Ancestral Envelope Protein Is Functional and Elicits Neutralizing Antibodies in Rabbits Similar to Those Elicited by a Circulating Subtype B Envelope

    PubMed Central

    Doria-Rose, N. A.; Learn, G. H.; Rodrigo, A. G.; Nickle, D. C.; Li, F.; Mahalanabis, M.; Hensel, M. T.; McLaughlin, S.; Edmonson, P. F.; Montefiori, D.; Barnett, S. W.; Haigwood, N. L.; Mullins, J. I.

    2005-01-01

    Human immunodeficiency virus type 1 (HIV-1) is a difficult target for vaccine development, in part because of its ever-expanding genetic diversity and attendant capacity to escape immunologic recognition. Vaccine efficacy might be improved by maximizing immunogen antigenic similarity to viruses likely to be encountered by vaccinees. To this end, we designed a prototype HIV-1 envelope vaccine using a deduced ancestral state for the env gene. The ancestral state reconstruction method was shown to be >95% accurate by computer simulation and 99.8% accurate when estimating the known inoculum used in an experimental infection study in rhesus macaques. Furthermore, the deduced ancestor gene differed from the set of sequences used to derive the ancestor by an average of 12.3%, while these latter sequences were an average of 17.3% different from each other. A full-length ancestral subtype B HIV-1 env gene was constructed and shown to produce a glycoprotein of 160 kDa that bound and fused with cells expressing the HIV-1 coreceptor CCR5. This Env was also functional in a virus pseudotype assay. When either gp160- or gp140-expressing plasmids and recombinant gp120 were used to immunize rabbits in a DNA prime-protein boost regimen, the artificial gene induced immunoglobulin G antibodies capable of weakly neutralizing heterologous primary HIV-1 strains. The results were similar for rabbits immunized in parallel with a natural isolate, HIV-1 SF162. Further design efforts to better present conserved neutralization determinants are warranted. PMID:16103173

  18. Type B lamins remain associated with the integral nuclear envelope protein p58 during mitosis: implications for nuclear reassembly.

    PubMed Central

    Meier, J; Georgatos, S D

    1994-01-01

    p58 (also referred to as the lamin B receptor) is an integral membrane protein of the nuclear envelope known to form a multimeric complex with the lamins and other nuclear proteins during interphase. To examine the fate of this complex during mitosis, we have investigated the partitioning and the molecular interactions of p58 in dividing chicken hepatoma (DU249) cells. Using confocal microscopy and double immunolabelling, we show here that lamins B1 and B2 co-localize with p58 during all phases of mitosis and co-assemble around reforming nuclei. A close juxtaposition of p58/lamin B-containing vesicles and chromosomes is already detectable in metaphase; however, p58 and lamin reassembly proceeds slowly and is completed in late telophase--G1. Flotation of mitotic membranes in sucrose density gradients and analysis of mitotic vesicles by immunoelectron microscopy confirms that p58 and most of the type B lamins reside in the same compartment. Co-immunoprecipitation of both proteins by affinity-purified anti-p58 antibodies shows that they are physically associated in the context of a mitotic p58 'sub-complex'. This sub-assembly does not include the type A lamins which are fully solubilized during mitosis. Our data provide direct, in vivo and in vitro evidence that the majority of type B lamins remain connected to nuclear membrane 'receptors' during mitosis. The implications of these findings in nuclear envelope reassembly are discussed below. Images PMID:8168487

  19. MAF1, a novel plant protein interacting with matrix attachment region binding protein MFP1, is located at the nuclear envelope.

    PubMed Central

    Gindullis, F; Peffer, N J; Meier, I

    1999-01-01

    The interaction of chromatin with the nuclear matrix via matrix attachment region (MAR) DNA is considered to be of fundamental importance for chromatin organization in all eukaryotic cells. MAR binding filament-like protein 1 (MFP1) from tomato is a novel plant protein that specifically binds to MAR DNA. Its filament protein-like structure makes it a likely candidate for a structural component of the nuclear matrix. MFP1 is located at nuclear matrix-associated, specklelike structures at the nuclear envelope. Here, we report the identification of a novel protein that specifically interacts with MFP1 in yeast two-hybrid and in vitro binding assays. MFP1 associated factor 1 (MAF1) is a small, soluble, serine/threonine-rich protein that is ubiquitously expressed and has no similarity to known proteins. MAF1, like MFP1, is located at the nuclear periphery and is a component of the nuclear matrix. These data suggest that MFP1 and MAF1 are in vivo interaction partners and that both proteins are components of a nuclear substructure, previously undescribed in plants, that connects the nuclear envelope and the internal nuclear matrix. PMID:10488241

  20. A Visual Screen of a Gfp-Fusion Library Identifies a New Type of Nuclear Envelope Membrane Protein

    PubMed Central

    Rolls, Melissa M.; Stein, Pascal A.; Taylor, Stephen S.; Ha, Edward; McKeon, Frank; Rapoport, Tom A.

    1999-01-01

    The nuclear envelope (NE) is a distinct subdomain of the ER, but few membrane components have been described that are specific to it. We performed a visual screen in tissue culture cells to identify proteins targeted to the NE. This approach does not require assumptions about the nature of the association with the NE or the physical separation of NE and ER. We confirmed that screening a library of fusions to the green fluorescent protein can be used to identify proteins targeted to various subcompartments of mammalian cells, including the NE. With this approach, we identified a new NE membrane protein, named nurim. Nurim is a multispanning membrane protein without large hydrophilic domains that is very tightly associated with the nucleus. Unlike the known NE membrane proteins, it is neither associated with nuclear pores, nor targeted like lamin-associated membrane proteins. Thus, nurim is a new type of NE membrane protein that is localized to the NE by a distinct mechanism. PMID:10402458

  1. Identification of the Lymantria dispar Nucleopolyhedrovirus Envelope Fusion Protein Provides Evidence for a Phylogenetic Division of the Baculoviridae†

    PubMed Central

    Pearson, Margot N.; Groten, Christoph; Rohrmann, George F.

    2000-01-01

    The complete genome sequences of a number of diverse members of the Baculoviridae including both nucleopolyhedroviruses (NPVs) and granuloviruses (GVs) revealed that they lack a homolog of GP64, the envelope fusion protein of the budded form of Autographa californica multinucleocapsid NPV (AcMNPV) and its close relatives. Computer-assisted analyses of the genome of one of these viruses, Lymantria dispar MNPV (LdMNPV), revealed a single open reading frame (ld130) whose product had the predicted properties of a membrane protein. Characterization of the localization of the products of the full-length ld130 gene and of an ld130-enhanced green fluorescent protein gene (egfp) fusion using both immunofluorescence and fluorescence microscopy revealed that LD130 accumulates at the plasma membranes of cells infected with LdMNPV or transfected with ld130-egfp. In addition, cells transfected with either ld130 or ld130-egfp or infected with wild-type virus undergo membrane fusion at pH 5. Western blot analyses indicate that LD130 is present in infected cells as an 83-kDa protein and is also present in budded virions as a protein doublet containing bands of 81 and 83 kDa. Tunicamycin treatment of infected cells resulted in an immunoreactive band of about 72 kDa, indicating that LD130 is N-glycosylated. Whereas the distribution of gp64 appears to be confined to a relatively closely related group of NPVs, homologs of ld130 are present in a diverse number of both NPVs and GVs. This suggests that LD130 may be the primordial baculovirus envelope fusion protein. PMID:10846096

  2. Potent in vitro antiviral activity of Cistus incanus extract against HIV and Filoviruses targets viral envelope proteins

    PubMed Central

    Rebensburg, Stephanie; Helfer, Markus; Schneider, Martha; Koppensteiner, Herwig; Eberle, Josef; Schindler, Michael; Gürtler, Lutz; Brack-Werner, Ruth

    2016-01-01

    Novel therapeutic options are urgently needed to improve global treatment of virus infections. Herbal products with confirmed clinical safety features are attractive starting material for the identification of new antiviral activities. Here we demonstrate that Cistus incanus (Ci) herbal products inhibit human immunodeficiency virus (HIV) infections in vitro. Ci extract inhibited clinical HIV-1 and HIV-2 isolates, and, importantly, a virus isolate with multiple drug resistances, confirming broad anti-HIV activity. Antiviral activity was highly selective for virus particles, preventing primary attachment of the virus to the cell surface and viral envelope proteins from binding to heparin. Bioassay-guided fractionation indicated that Ci extract contains numerous antiviral compounds and therefore has favorably low propensity to induce virus resistance. Indeed, no resistant viruses emerged during 24 weeks of continuous propagation of the virus in the presence of Ci extracts. Finally, Ci extracts also inhibited infection by virus particles pseudotyped with Ebola and Marburg virus envelope proteins, indicating that antiviral activity of Ci extract extends to emerging viral pathogens. These results demonstrate that Ci extracts show potent and broad in vitro antiviral activity against viruses that cause life-threatening diseases in humans and are promising sources of agents that target virus particles. PMID:26833261

  3. Potent in vitro antiviral activity of Cistus incanus extract against HIV and Filoviruses targets viral envelope proteins.

    PubMed

    Rebensburg, Stephanie; Helfer, Markus; Schneider, Martha; Koppensteiner, Herwig; Eberle, Josef; Schindler, Michael; Gürtler, Lutz; Brack-Werner, Ruth

    2016-01-01

    Novel therapeutic options are urgently needed to improve global treatment of virus infections. Herbal products with confirmed clinical safety features are attractive starting material for the identification of new antiviral activities. Here we demonstrate that Cistus incanus (Ci) herbal products inhibit human immunodeficiency virus (HIV) infections in vitro. Ci extract inhibited clinical HIV-1 and HIV-2 isolates, and, importantly, a virus isolate with multiple drug resistances, confirming broad anti-HIV activity. Antiviral activity was highly selective for virus particles, preventing primary attachment of the virus to the cell surface and viral envelope proteins from binding to heparin. Bioassay-guided fractionation indicated that Ci extract contains numerous antiviral compounds and therefore has favorably low propensity to induce virus resistance. Indeed, no resistant viruses emerged during 24 weeks of continuous propagation of the virus in the presence of Ci extracts. Finally, Ci extracts also inhibited infection by virus particles pseudotyped with Ebola and Marburg virus envelope proteins, indicating that antiviral activity of Ci extract extends to emerging viral pathogens. These results demonstrate that Ci extracts show potent and broad in vitro antiviral activity against viruses that cause life-threatening diseases in humans and are promising sources of agents that target virus particles. PMID:26833261

  4. The cell envelope-associated protein, LytR, regulates the cysteine protease SpeB in Streptococcus pyogenes.

    PubMed

    Minami, Masaaki; Ichikawa, Mariko; Ohta, Michio; Hasegawa, Tadao

    2012-05-01

    The LytR family of cell envelope-associated transcriptional attenuators in bacteria has been brought into focus of scientific interest on the expression of various virulence factors, as well as bacterial cell envelope maintenance. However, this protein of Streptococcus pyogenes has been only described as cell surface-associated protein, and its function is completely unknown. We created lytR mutant strains from two independent S. pyogenes strains to analyze the function of LytR. The protease assay in culture supernatant showed that lytR mutant had the higher cysteine protease activity than wild-type. Two-dimensional gel electrophoresis and western blotting analysis revealed that the amount of cysteine protease, SpeB in lytR mutant was more compared with that in wild-type. The level of speB mRNA in lytR mutant also increased compared with that of wild-type. The membrane integrity and potential in lytR mutant also were decreased compared with that of wild-type. Murine infection model showed that less survival was detected in mice inoculated with lytR mutant than that with wild-type, and the size of wound lesion of mice with lytR mutant was larger than that with wild-type. Our data suggest that the lytR regulates the expression of SpeB in S. pyogenes with relation to membrane integrity. PMID:22515297

  5. Dengue virus envelope domain III protein based on a tetravalent antigen secreted from insect cells: Potential use for serological diagnosis.

    PubMed

    Niu, Guoyu; Pang, Zheng; Guan, Chun; Qi, Jun; Li, Dexin

    2015-04-01

    In the present study, we developed a tetravalent protein by connecting the receptor-binding envelope domain III (EDIII) of the four dengue virus serotypes in the order of D1-D3-D4-D2. Using a baculovirus expression system, the protein was secreted into the supernatant of infected sf9 cells in a stable form with preserved native conformation. Using immobilized affinity chromatography, the recombinant EDIII (rEDIII) protein was purified with a yield of 300μg per 10(6) cells. The purity and reactivity of the protein were determined via SDS-PAGE and Western blot respectively. A MAC-ELISA method based on the secreted rEDIII protein was subsequently established and evaluated using a panel of pre-characterized dengue IgM-positive and -negative human sera. We obtained a specificity of 100% and sensitivity of 93% using this method. Our data collectively suggest that the secreted tetravalent rEDIII protein has potential utility in the diagnosis of dengue virus infections. PMID:25697685

  6. The mechanism of inactivation of a 50-pS envelope anion channel during chloroplast protein import.

    PubMed Central

    van den Wijngaard, P W; Dabney-Smith, C; Bruce, B D; Vredenberg, W J

    1999-01-01

    The mechanism of import-competent precursor protein-induced inactivation of a 50-pS anion channel of the chloroplast envelope is investigated using single-channel recordings. The inactivation by precursor protein is the result of the induction of a long-lived closed state of the channel. The mean duration of this state does not depend on precursor concentration. From this it can be concluded that the protein import related anion channel enters the inactive state less frequently when the precursor concentration is lowered, but that the time spent in this state remains the same. Furthermore, it was found that the presence of precursor protein also decreases the mean durations of preexisting open and closed states of the channel. This decrease is found to be dependent on the precursor concentration. From this it is concluded that there is a direct interaction between the precursor protein and a protein complex of which the channel is a constituent. The mean duration of the precursor-induced long-lived closed state does not depend on the length of the translocation-competent precursor. This suggests that the duration of import is independent of precursor length. PMID:10585937

  7. FlaF is a β-sandwich protein that anchors the archaellum in the archaeal cell envelope by binding the S-layer protein

    SciTech Connect

    Banerjee, Ankan; Tsai, Chi -Lin; Chaudhury, Paushali; Tripp, Patrick; Arvai, Andrew  S.; Ishida, Justin  P.; Tainer, John  A.; Albers, Sonja -Verena

    2015-05-01

    Archaea employ the archaellum, a type IV pilus-like nanomachine, for swimming motility. In the crenarchaeon Sulfolobus acidocaldarius, the archaellum consists of seven proteins: FlaB/X/G/F/H/I/J. FlaF is conserved and essential for archaellum assembly but no FlaF structures exist. Here, we truncated the FlaF N terminus and solved 1.5-Å and 1.65-Å resolution crystal structures of this monotopic membrane protein. Structures revealed an N-terminal α-helix and an eight-strand β-sandwich, immunoglobulin-like fold with striking similarity to S-layer proteins. Crystal structures, X-ray scattering, and mutational analyses suggest dimer assembly is needed for in vivo function. The sole cell envelope component of S. acidocaldarius is a paracrystalline S-layer, and FlaF specifically bound to S-layer protein, suggesting that its interaction domain is located in the pseudoperiplasm with its N-terminal helix in the membrane. From these data, FlaF may act as the previously unknown archaellum stator protein that anchors the rotating archaellum to the archaeal cell envelope.

  8. FlaF is a β-sandwich protein that anchors the archaellum in the archaeal cell envelope by binding the S-layer protein

    DOE PAGESBeta

    Banerjee, Ankan; Tsai, Chi -Lin; Chaudhury, Paushali; Tripp, Patrick; Arvai, Andrew  S.; Ishida, Justin  P.; Tainer, John  A.; Albers, Sonja -Verena

    2015-05-01

    Archaea employ the archaellum, a type IV pilus-like nanomachine, for swimming motility. In the crenarchaeon Sulfolobus acidocaldarius, the archaellum consists of seven proteins: FlaB/X/G/F/H/I/J. FlaF is conserved and essential for archaellum assembly but no FlaF structures exist. Here, we truncated the FlaF N terminus and solved 1.5-Å and 1.65-Å resolution crystal structures of this monotopic membrane protein. Structures revealed an N-terminal α-helix and an eight-strand β-sandwich, immunoglobulin-like fold with striking similarity to S-layer proteins. Crystal structures, X-ray scattering, and mutational analyses suggest dimer assembly is needed for in vivo function. The sole cell envelope component of S. acidocaldarius is amore » paracrystalline S-layer, and FlaF specifically bound to S-layer protein, suggesting that its interaction domain is located in the pseudoperiplasm with its N-terminal helix in the membrane. From these data, FlaF may act as the previously unknown archaellum stator protein that anchors the rotating archaellum to the archaeal cell envelope.« less

  9. FlaF Is a β-Sandwich Protein that Anchors the Archaellum in the Archaeal Cell Envelope by Binding the S-Layer Protein

    PubMed Central

    Banerjee, Ankan; Tsai, Chi-Lin; Chaudhury, Paushali; Tripp, Patrick; Arvai, Andrew S.; Ishida, Justin P.; Tainer, John A.; Albers, Sonja-Verena

    2015-01-01

    Summary Archaea employ the archaellum, a type IV pilus-like nanomachine, for swimming motility. In the crenarchaeon Sulfolobus acidocaldarius, the archaellum consists of seven proteins: FlaB/X/G/F/H/I/J. FlaF is conserved and essential for archaellum assembly but no FlaF structures exist. Here, we truncated the FlaF N terminus and solved 1.5-Å and 1.65-Å resolution crystal structures of this monotopic membrane protein. Structures revealed an N-terminal α-helix and an eight-strand β-sandwich, immunoglobulin-like fold with striking similarity to S-layer proteins. Crystal structures, X-ray scattering, and mutational analyses suggest dimer assembly is needed for in vivo function. The sole cell envelope component of S. acidocaldarius is a paracrystalline S-layer, and FlaF specifically bound to S-layer protein, suggesting that its interaction domain is located in the pseudoperiplasm with its N-terminal helix in the membrane. From these data, FlaF may act as the previously unknown archaellum stator protein that anchors the rotating archaellum to the archaeal cell envelope. PMID:25865246

  10. Information for targeting to the chloroplastic inner envelope membrane is contained in the mature region of the maize Bt1-encoded protein

    SciTech Connect

    Li, H.M.; Sullivan, T.D.; Keegstra, K.

    1992-09-15

    Based on the protein sequence deduced from a cDNA clone, it has been proposed that the maize Bt1 locus encodes an amyloplast membrane metabolite translocator protein. The present work provides further evidence for this hypothesis by showing that the gene product of Bt1 could be imported into chloroplasts in vitro and processed to lower molecular weight mature proteins. More importantly, the imported mature proteins were localized to the inner envelope membrane, where metabolite tranlocators are located in plastids. In addition, the location of information for targeting to the inner membrane was investigated by constructing and analyzing the import of chimeric precursor proteins. A chimeric protein with the transit peptide of the precursor to the small subunit of ribulose-1,5-bisphosphate carboxylase fused to the mature region of the Bt1-encoded protein was targeted to the inner envelope membrane of chloroplasts. Moreover, a chimeric protein with the transit peptide of the Bt1-encoded protein fused to the mature protein of the light-harvesting chlorophyll a/b binding protein was targeted to the thylakoid. These results indicate that the transit peptide of the Bt1-encoded protein functions primarily as a stromal targeting sequence. The information for targeting to the chloroplastic inner envelope membrane is contained in the mature region of the protein.

  11. Immunoadhesins Containing Pre-S Domains of Hepatitis B Virus Large Envelope Protein Are Secreted and Inhibit Virus Infection▿

    PubMed Central

    Chai, Ning; Gudima, Severin; Chang, Jinhong; Taylor, John

    2007-01-01

    Hepatitis B virus (HBV) replication produces three envelope proteins (L, M, and S) that have a common C terminus. L, the largest, contains a domain, pre-S1, not present on M. Similarly M contains a domain, pre-S2, not present on S. The pre-S1 region has important functions in the HBV life cycle. Thus, as an approach to studying these roles, the pre-S1 and/or pre-S2 sequences of HBV (serotype adw2, genotype A) were expressed as N-terminal fusions to the Fc domain of a rabbit immunoglobulin G chain. Such proteins, known as immunoadhesins (IA), were highly expressed following transfection of cultured cells and, when the pre-S1 region was present, >80% were secreted. The IA were myristoylated at a glycine penultimate to the N terminus, although mutation studies showed that this modification was not needed for secretion. As few as 30 amino acids from the N terminus of pre-S1 were both necessary and sufficient to drive secretion of IA. Even expression of pre-S1 plus pre-S2, in the absence of an immunoglobulin chain, led to efficient secretion. Overall, these studies demonstrate an unexpected ability of the N terminus of pre-S1 to promote protein secretion. In addition, some of these secreted IA, at nanomolar concentrations, inhibited infection of primary human hepatocytes either by hepatitis delta virus (HDV), a subviral agent that uses HBV envelope proteins, or HBV. These IA have potential to be part of antiviral therapies against chronic HDV and HBV, and may help understand the attachment and entry mechanisms used by these important human pathogens. PMID:17329331

  12. The PDZ-Binding Motif of Severe Acute Respiratory Syndrome Coronavirus Envelope Protein Is a Determinant of Viral Pathogenesis

    PubMed Central

    Jimenez-Guardeño, Jose M.; Nieto-Torres, Jose L.; DeDiego, Marta L.; Regla-Nava, Jose A.; Fernandez-Delgado, Raul; Castaño-Rodriguez, Carlos; Enjuanes, Luis

    2014-01-01

    A recombinant severe acute respiratory syndrome coronavirus (SARS-CoV) lacking the envelope (E) protein is attenuated in vivo. Here we report that E protein PDZ-binding motif (PBM), a domain involved in protein-protein interactions, is a major determinant of virulence. Elimination of SARS-CoV E protein PBM by using reverse genetics caused a reduction in the deleterious exacerbation of the immune response triggered during infection with the parental virus and virus attenuation. Cellular protein syntenin was identified to bind the E protein PBM during SARS-CoV infection by using three complementary strategies, yeast two-hybrid, reciprocal coimmunoprecipitation and confocal microscopy assays. Syntenin redistributed from the nucleus to the cell cytoplasm during infection with viruses containing the E protein PBM, activating p38 MAPK and leading to the overexpression of inflammatory cytokines. Silencing of syntenin using siRNAs led to a decrease in p38 MAPK activation in SARS-CoV infected cells, further reinforcing their functional relationship. Active p38 MAPK was reduced in lungs of mice infected with SARS-CoVs lacking E protein PBM as compared with the parental virus, leading to a decreased expression of inflammatory cytokines and to virus attenuation. Interestingly, administration of a p38 MAPK inhibitor led to an increase in mice survival after infection with SARS-CoV, confirming the relevance of this pathway in SARS-CoV virulence. Therefore, the E protein PBM is a virulence domain that activates immunopathology most likely by using syntenin as a mediator of p38 MAPK induced inflammation. PMID:25122212

  13. N-glycosylation of the premembrane protein of Japanese encephalitis virus is critical for folding of the envelope protein and assembly of virus-like particles.

    PubMed

    Zai, J; Mei, L; Wang, C; Cao, S; Fu, Z F; Chen, H; Song, Y

    2013-01-01

    Premembrane (prM) and envelope (E) proteins, the major structural proteins of Japanese encephalitis virus (JEV) each contain single potential N-glycosylation site. In this study, the role of N-glycosylation of these proteins on their folding and activity were investigated. Three mutant prM and/or E (prM-E) genes lacking N-glycosylation sites were generated by site-directed mutagenesis. The effects of the N-glycan on folding, secretion and cytotoxicity of mutant proteins were determined by comparison with their wild type (wt) counterparts. Removal of N-glycan from the prM protein resulted in a complete misfolding of the E protein and failure to form virus-like particles (VLPs). A similar removal of N-glycan from the E protein led to a low efficiency of its folding and VLPs formation. The secretion and cytotoxicity of the E protein was also markedly impaired in case the glycosylation sites in the prM or E or both proteins were removed. These results suggest that the N-glycosylation of the prM protein is critical to the folding of the E protein, which makes it pivotal in the cytotoxicity of JEV particles and their production. PMID:23530821

  14. The scaffolding protein IQGAP1 co-localizes with actin at the cytoplasmic face of the nuclear envelope: implications for cytoskeletal regulation

    PubMed Central

    Johnson, Michael A.

    2012-01-01

    IQGAP1 is an important cytoskeletal regulator, known to act at the plasma membrane to bundle and cap actin filaments, and to tether the cortical actin meshwork to microtubules via plus-end binding proteins. Here we describe the novel subcellular localization of IQGAP1 at the cytoplasmic face of the nuclear envelope, where it co-located with F-actin. The IQGAP1 and F-actin staining overlapped that of microtubules at the nuclear envelope, revealing a pattern strikingly similar to that observed at the plasma membrane. In detergent-extracted cells IQGAP1 was retained at cytoskeletal structures at the nuclear envelope. This finding has new implications for involvement of IQGAP1 in cell polarization and migration events and potentially in cell cycle-associated nuclear envelope assembly/disassembly. PMID:22964981

  15. Role for a Zinc Finger Protein (Zfp111) in Transformation of 208F Rat Fibroblasts by Jaagsiekte Sheep Retrovirus Envelope Protein

    PubMed Central

    Hsu, Tom; Phung, An; Choe, Kevin; Kim, Jung Woo

    2015-01-01

    ABSTRACT The native envelope gene (env) of Jaagsiekte sheep retrovirus (JSRV) also acts as an oncogene. To investigate the mechanism of transformation, we performed yeast 2-hybrid screening for cellular proteins that interact with Env. Among several candidates, we identified mouse or rat zinc finger protein 111 (zfp111). The interaction between Env and Zfp111 was confirmed through in vivo coimmunoprecipitation assays. Knockdown of endogenous Zfp111 caused a decrease in cell transformation by JSRV Env, while overexpression of Zfp111 increased overall Env transformation, supporting a role for Zfp111 in Env transformation. Knockdown of Zfp111 had no effect on the growth rate of parental rat 208F cells, while it decreased the proliferation rate of JSRV-transformed 208F cells, suggesting that JSRV-transformed cells became dependent on Zfp111. In addition, Zfp111 preferentially bound to a higher-mobility form of JSRV Env that has not been described previously. The higher-mobility form of Env (P70env) was found exclusively in the nuclear fraction, and size of its polypeptide backbone was the same as that of the cytoplasmic Env polyprotein (Pr80env). The differences in glycosylation between the two versions of Env were characterized. These results identify a novel cellular protein, Zfp111, that binds to the JSRV Env protein, and this binding plays a role in Env transformation. These results indicate that JSRV transformation also involves proteins and interactions in the nucleus. IMPORTANCE The envelope protein (Env) of Jaagsiekte sheep retrovirus (JSRV) is an oncogene, but its mechanism of cell transformation is still unclear. Here we identified seven candidate cellular proteins that can interact with JSRV Env by yeast two-hybrid screening. This study focused on one of the seven candidates, zinc finger protein 111 (Zfp111). Zfp111 was shown to interact with JSRV Env in cells and to be involved in JSRV transformation. Moreover, coexpression of JSRV Env and Zfp111 led to the

  16. Unusual Reversible Oligomerization of Unfolded Dengue Envelope Protein Domain 3 at High Temperatures and Its Abolition by a Point Mutation.

    PubMed

    Saotome, Tomonori; Nakamura, Shigeyoshi; Islam, Mohammad M; Nakazawa, Akiko; Dellarole, Mariano; Arisaka, Fumio; Kidokoro, Shun-Ichi; Kuroda, Yutaka

    2016-08-16

    We report differential scanning calorimetry (DSC) experiments between 10 and 120 °C of Dengue 4 envelope protein domain 3 (DEN4 ED3), a small 107-residue monomeric globular protein domain. The thermal unfolding of DEN4 ED3 was fully reversible and exhibited two peculiar endothermic peaks. AUC (analytical ultracentrifugation) experiments at 25 °C indicated that DEN4 ED3 was monomeric. Detailed thermodynamic analysis indicated that the two endothermic peaks separated with an increasing protein concentration, and global fitting of the DSC curves strongly suggested the presence of unfolded tetramers at temperatures around 80-90 °C, which dissociated to unfolded monomers at even higher temperatures. To further characterize this rare thermal unfolding process, we designed and constructed a DEN4 ED3 variant that would unfold according to a two-state model, typical of globular proteins. We thus substituted Val 380, the most buried residue at the dimeric interface in the protein crystal, with less hydrophobic amino acids (Ala, Ser, Thr, Asn, and Lys). All variants showed a single heat absorption peak, typical of small globular proteins. In particular, the DSC thermogram of DEN4 V380K indicated a two-state reversible thermal unfolding independent of protein concentration, indicating that the high-temperature oligomeric state was successfully abolished by a single mutation. These observations confirmed the standard view that small monomeric globular proteins undergo a two-state unfolding. However, the reversible formation of unfolded oligomers at high temperatures is a truly new phenomenon, which was fully inhibited by an accurately designed single mutation. PMID:27433922

  17. Insights into the Function of YciM, a Heat Shock Membrane Protein Required To Maintain Envelope Integrity in Escherichia coli

    PubMed Central

    Nicolaes, Valérie; El Hajjaji, Hayat; Davis, Rebecca M.; Van der Henst, Charles; Depuydt, Matthieu; Leverrier, Pauline; Aertsen, Abram; Haufroid, Vincent; Ollagnier de Choudens, Sandrine; De Bolle, Xavier; Ruiz, Natividad

    2013-01-01

    The cell envelope of Gram-negative bacteria is an essential organelle that is important for cell shape and protection from toxic compounds. Proteins involved in envelope biogenesis are therefore attractive targets for the design of new antibacterial agents. In a search for new envelope assembly factors, we screened a collection of Escherichia coli deletion mutants for sensitivity to detergents and hydrophobic antibiotics, a phenotype indicative of defects in the cell envelope. Strains lacking yciM were among the most sensitive strains of the mutant collection. Further characterization of yciM mutants revealed that they display a thermosensitive growth defect on low-osmolarity medium and that they have a significantly altered cell morphology. At elevated temperatures, yciM mutants form bulges containing cytoplasmic material and subsequently lyse. We also discovered that yciM genetically interacts with envC, a gene encoding a regulator of the activity of peptidoglycan amidases. Altogether, these results indicate that YciM is required for envelope integrity. Biochemical characterization of the protein showed that YciM is anchored to the inner membrane via its N terminus, the rest of the protein being exposed to the cytoplasm. Two CXXC motifs are present at the C terminus of YciM and serve to coordinate a redox-sensitive iron center of the rubredoxin type. Both the N-terminal membrane anchor and the C-terminal iron center of YciM are important for function. PMID:24187084

  18. Crystallization and X-ray diffraction analysis of chondroitin lyase from baculovirus: envelope protein ODV-E66

    PubMed Central

    Kawaguchi, Yoshirou; Sugiura, Nobuo; Onishi, Momo; Kimata, Koji; Kimura, Makoto; Kakuta, Yoshimitu

    2012-01-01

    Baculovirus envelope protein ODV-E66 (67–704), in which the N-terminal 66 amino acids are truncated, is a chondroitin lyase. It digests chondroitin and chondroitin 6-sulfate efficiently, but does not digest chondroitin 4-sulfate. This unique characteristic is useful for the preparation of specific chondroitin oligosaccharides and for investigation of the mechanism of baculovirus infection. ODV-E66 (67–704) was crystallized; the crystal diffracted to 1.8 Å resolution and belonged to space group P62 or P64, with unit-cell parameters a = b = 113.5, c = 101.5 Å. One molecule is assumed to be present per asymmetric unit, which gives a Matthews coefficient of 2.54 Å3 Da−1. PMID:22297996

  19. A mutation in the envelope protein fusion loop attenuates mouse neuroinvasiveness of the NY99 strain of West Nile virus

    SciTech Connect

    Zhang Shuliu; Li Li; Woodson, Sara E.; Huang, Claire Y.-H.; Kinney, Richard M.; Barrett, Alan D.T. ||||; Beasley, David W.C. |||. E-mail: d.beasley@utmb.edu

    2006-09-15

    Substitutions were engineered individually and in combinations at the fusion loop, receptor-binding domain and a stem-helix structure of the envelope protein of a West Nile virus strain, NY99, and their effects on mouse virulence and presentation of epitopes recognized by monoclonal antibodies (MAbs) were assessed. A single substitution within the fusion loop (L107F) attenuated mouse neuroinvasiveness of NY99. No substitutions attenuated NY99 neurovirulence. The L107F mutation also abolished binding of a non-neutralizing MAb, 3D9, whose epitope had not been previously identified. MAb 3D9 was subsequently shown to be broadly cross-reactive with other flaviviruses, consistent with binding near the highly conserved fusion loop.

  20. The immunosuppressive domain of the transmembrane envelope protein gp41 of HIV-1 binds to human monocytes and B cells.

    PubMed

    Mühle, Michael; Kroniger, Tobias; Hoffmann, Kerstin; Denner, Joachim

    2016-06-01

    The induction of the acquired immunodeficiency syndrome by the human immunodeficiency virus-1 (HIV-1) is a complex process which is not yet understood in full detail. Still open is the question whether the highly conserved so-called immunosuppressive (Isu) domain in the transmembrane envelope (TM) protein gp41 of HIV-1 is actively participating in immunopathogenesis. Inactivated virus particles, recombinant gp41 and peptides corresponding to the Isu domain have been reported to inhibit lymphocyte proliferation, as well as to alter cytokine release and gene expression. Here we demonstrate, using fluorescence-activated cell sorting and competition experiments, that homopolymers of the Isu peptide of HIV-1 are binding specifically to human peripheral blood mononuclear cells, mainly to monocytes and B cells. These data suggest that a putative receptor might be involved in the immunomodulatory effects observed previously. PMID:26754765

  1. Immunogenicity and protective efficacy of recombinant major envelope protein (rH3L) of buffalopox virus in animal models.

    PubMed

    Kumar, Amit; Yogisharadhya, Revanaiah; Venkatesan, Gnanavel; Bhanuprakash, Veerakyathappa; Shivachandra, Sathish Bhadravati

    2016-02-01

    Buffalopox virus, a zoonotic Indian vaccinia-like virus, is responsible for contagious disease affecting mainly buffaloes, cattle and humans. H3L gene, encoding for an immunodominant major envelope protein of intracellular mature virion of orthopoxviruses, is highly conserved and found to elicit neutralizing antibodies. Therefore in the present study, the immunogenicity and protective efficacy of the recombinant H3L protein of buffalopox virus in laboratory animal models has been evaluated. A partial H3L gene encoding for the C-terminal truncated ectodomain of H3L protein (1M to I280) of BPXV-Vij/96 strain was cloned, over-expressed and purified as histidine-tagged fusion protein (50 kDa) from Escherichia coli using Ni-NTA affinity chromatography. The purified rH3L protein was further used for active immunization of guinea pig (250 μg/dose) and adult mice (10 μg and 50 μg/dose) with or without adjuvants (alum, Freund's Complete Adjuvant and CpG). Subsequently, a gradual increase in antigen specific serum IgG as well as neutralizing antibody titres measured by using indirect-ELISA and serum neutralization test respectively, was noted in both guinea pigs and mouse models. Suckling mice immunized passively with anti-H3L serum showed 80% pre-exposure prophylaxis upon challenge with virulent buffalopox virus strain. An indirect-ELISA based on rH3L protein showed no cross-reactivity with hyperimmune sera against sheeppox virus (SPPV), goatpox virus (GTPV), orf virus (ORFV), foot- and- mouth disease virus (FMDV), peste des petits ruminants virus (PPRV) and bluetongue virus (BTV) during the course of study. The study highlights the potential utility of rH3L protein as a safer prophylactic and diagnostic reagent for buffalopox. PMID:26723250

  2. Cell- and Protein-Directed Glycosylation of Native Cleaved HIV-1 Envelope

    PubMed Central

    Pritchard, Laura K.; Harvey, David J.; Bonomelli, Camille

    2015-01-01

    ABSTRACT The gp120/gp41 HIV-1 envelope glycoprotein (Env) is highly glycosylated, with up to 50% of its mass consisting of N-linked glycans. This dense carbohydrate coat has emerged as a promising vaccine target, with its glycans acting as epitopes for a number of potent and broadly neutralizing antibodies (bnAbs). Characterizing the glycan structures present on native HIV-1 Env is thus a critical goal for the design of Env immunogens. In this study, we used a complementary, multistep approach involving ion mobility mass spectrometry and high-performance liquid chromatography to comprehensively characterize the glycan structures present on HIV-1 gp120 produced in peripheral blood mononuclear cells (PBMCs). The capacity of different expression systems, including pseudoviral particles and recombinant cell surface trimers, to reproduce native-like glycosylation was then assessed. A population of oligomannose glycans on gp120 was reproduced across all expression systems, supporting this as an intrinsic property of Env that can be targeted for vaccine design. In contrast, Env produced in HEK 293T cells failed to accurately reproduce the highly processed complex-type glycan structures observed on PBMC-derived gp120, and in particular the precise linkage of sialic acid residues that cap these glycans. Finally, we show that unlike for gp120, the glycans decorating gp41 are mostly complex-type sugars, consistent with the glycan specificity of bnAbs that target this region. These findings provide insights into the glycosylation of native and recombinant HIV-1 Env and can be used to inform strategies for immunogen design and preparation. IMPORTANCE Development of an HIV vaccine is desperately needed to control new infections, and elicitation of HIV bnAbs will likely be an important component of an effective vaccine. Increasingly, HIV bnAbs are being identified that bind to the N-linked glycans coating the HIV envelope glycoproteins gp120 and gp41, highlighting them as

  3. The BRCA1-binding protein BRAP2 can act as a cytoplasmic retention factor for nuclear and nuclear envelope-localizing testicular proteins.

    PubMed

    Davies, Rebecca G; Wagstaff, Kylie M; McLaughlin, Eileen A; Loveland, Kate L; Jans, David A

    2013-12-01

    Regulation of nuclear protein import is central to many cellular processes such as development, with a key mechanism being factors that retain cargoes in the cytoplasm that normally localize in the nucleus. The breast cancer antigen BRCA1-binding protein BRAP2 has been reported as a novel negative regulator of nuclear import of various nuclear localization signal (NLS)-containing viral and cellular proteins, but although implicated in differentiation pathways and highly expressed in tissues including testis, the gamut of targets for BRAP2 action in a developmental context is unknown. As a first step towards defining the BRAP2 interactome, we performed a yeast-2-hybrid screen to identify binding partners of BRAP2 in human testis. Here we report characterization for the first time of three of these: the high mobility group (HMG)-box-domain-containing chromatin component HMG20A, nuclear mitotic apparatus protein NuMA1 and synaptic nuclear envelope protein SYNE2. Co-immunoprecipitation experiments indicate association of BRAP2 with HMG20A, NuMA1, and SYNE2 in testis, underlining the physiological relevance of the interactions, with immunohistochemistry showing that where BRAP2 is co-expressed with HMG20A and NuMA1, both are present in the cytoplasm, in contrast to their nuclear localization in other testicular cell types. Importantly, quantitative confocal microscopic analysis of cultured cells indicates that ectopic expression of BRAP2 inhibits nuclear localization of HMG20A and NuMA1, and prevents nuclear envelope accumulation of SYNE2, the first report of BRAP2 altering localization of a non-nuclear protein. These results imply for the first time that BRAP2 may have an important role in modulating subcellular localization during testicular development. PMID:23707952

  4. Membrane bound Indian clade C HIV-1 envelope antigen induces antibodies to diverse and conserved epitopes upon DNA prime/protein boost in rabbits.

    PubMed

    Rangasamy, Sneha Priya; Menon, Veena; Dhopeshwarkar, Priyanka; Pal, Ranajit; Vaniambadi, Kalyanaraman S; Mahalingam, Sundarasamy

    2016-05-01

    The partial success of RV144 human clinical trial demonstrated that ALVAC prime/envelope protein boost vaccine regimen may represent a promising strategy for the development of an effective HIV-1 vaccine. Our earlier study demonstrated that a trimeric HIV-1 envelope gp145 from an Indian clade C isolate elicited cross clade neutralizing antibodies primarily towards Tier 1 isolates. In the present study, we examined the immunogenicity of DNA prime/envelope protein boost vaccine in rabbits using gp160 DNA of the Indian clade C isolate with various cytoplasmic tail truncations and trimeric gp145 protein. Cytoplasmic tail mutants of gp160 exposed epitopes that reacted strongly with a number of broadly neutralizing human monoclonal antibodies against HIV-1. Overall, envelope specific titers were found to be similar in all rabbit groups with higher pseudovirus neutralization in protein only immunized rabbits. The complete linear epitope mapping of rabbit immune sera revealed strong binding to C1, C2, V3, C3 and C4 domains of gp145. Importantly, reactivity of gp41 ecto-domain peptides was observed in DNA prime/protein boost sera but not in the sera of rabbits immunized with protein alone. Moreover, membrane anchored but not soluble envelope encoding DNA immunization elicited antibodies against linear epitopes on the conserved gp41 ecto-domain. Together, these results suggest that priming with DNA encoding cytoplasmic domains of Env alters the quality of antibodies elicited following protein boost and hence may be utilized to generate protective immunity by HIV-1 vaccine. PMID:27032514

  5. GB virus type C envelope protein E2 elicits antibodies that react with a cellular antigen on HIV-1 particles and neutralize diverse HIV-1 isolates.

    PubMed

    Mohr, Emma L; Xiang, Jinhua; McLinden, James H; Kaufman, Thomas M; Chang, Qing; Montefiori, David C; Klinzman, Donna; Stapleton, Jack T

    2010-10-01

    Broadly neutralizing Abs to HIV-1 are well described; however, identification of Ags that elicit these Abs has proven difficult. Persistent infection with GB virus type C (GBV-C) is associated with prolonged survival in HIV-1-infected individuals, and among those without HIV-1 viremia, the presence of Ab to GBV-C glycoprotein E2 is also associated with survival. GBV-C E2 protein inhibits HIV-1 entry, and an antigenic peptide within E2 interferes with gp41-induced membrane perturbations in vitro, suggesting the possibility of structural mimicry between GBV-C E2 protein and HIV-1 particles. Naturally occurring human and experimentally induced GBV-C E2 Abs were examined for their ability to neutralize infectious HIV-1 particles and HIV-1-enveloped pseudovirus particles. All GBV-C E2 Abs neutralized diverse isolates of HIV-1 with the exception of rabbit anti-peptide Abs raised against a synthetic GBV-C E2 peptide. Rabbit anti-GBV-C E2 Abs neutralized HIV-1-pseudotyped retrovirus particles but not HIV-1-pseudotyped vesicular stomatitis virus particles, and E2 Abs immune-precipitated HIV-1 gag particles containing the vesicular stomatitis virus type G envelope, HIV-1 envelope, GBV-C envelope, or no viral envelope. The Abs did not neutralize or immune-precipitate mumps or yellow fever viruses. Rabbit GBV-C E2 Abs inhibited HIV attachment to cells but did not inhibit entry following attachment. Taken together, these data indicate that the GBV-C E2 protein has a structural motif that elicits Abs that cross-react with a cellular Ag present on retrovirus particles, independent of HIV-1 envelope glycoproteins. The data provide evidence that a heterologous viral protein can induce HIV-1-neutralizing Abs. PMID:20826757

  6. Identification of a binding site of the human immunodeficiency virus envelope protein gp120 to neuronal-specific tubulin.

    PubMed

    Avdoshina, Valeria; Taraballi, Francesca; Dedoni, Simona; Corbo, Claudia; Paige, Mikell; Saygideğer Kont, Yasemin; Üren, Aykut; Tasciotti, Ennio; Mocchetti, Italo

    2016-04-01

    Human immunodeficiency virus-1 (HIV) promotes synaptic simplification and neuronal apoptosis, and causes neurological impairments termed HIV-associated neurological disorders. HIV-associated neurotoxicity may be brought about by acute and chronic mechanisms that still remain to be fully characterized. The HIV envelope glycoprotein gp120 causes neuronal degeneration similar to that observed in HIV-associated neurocognitive disorders subjects. This study was undertaken to discover novel mechanisms of gp120 neurotoxicity that could explain how the envelope protein promotes neurite pruning. Gp120 has been shown to associate with various intracellular organelles as well as microtubules in neurons. We then analyzed lysates of neurons exposed to gp120 with liquid chromatography mass spectrometry for potential protein interactors. We found that one of the proteins interacting with gp120 is tubulin β-3 (TUBB3), a major component of neuronal microtubules. We then tested the hypothesis that gp120 binds to neuronal microtubules. Using surface plasmon resonance, we confirmed that gp120 binds with high affinity to neuronal-specific TUBB3. We have also identified the binding site of gp120 to TUBB3. We then designed a small peptide (Helix-A) that displaced gp120 from binding to TUBB3. To determine whether this peptide could prevent gp120-mediated neurotoxicity, we cross-linked Helix-A to mesoporous silica nanoparticles (Helix-A nano) to enhance the intracellular delivery of the peptide. We then tested the neuroprotective property of Helix-A nano against three strains of gp120 in rat cortical neurons. Helix-A nano prevented gp120-mediated neurite simplification as well as neuronal loss. These data propose that gp120 binding to TUBB3 could be another mechanism of gp120 neurotoxicity. We propose a novel direct mechanism of human immunodeficiency virus neurotoxicity. Our data show that the viral protein gp120 binds to neuronal specific tubulin β-3 and blocks microtubule transport

  7. Human Cytomegalovirus Nuclear Egress Proteins Ectopically Expressed in the Heterologous Environment of Plant Cells are Strictly Targeted to the Nuclear Envelope

    PubMed Central

    Lamm, Christian E.; Link, Katrin; Wagner, Sabrina; Milbradt, Jens; Marschall, Manfred; Sonnewald, Uwe

    2016-01-01

    In all eukaryotic cells, the nucleus forms a prominent cellular compartment containing the cell’s nuclear genome. Although structurally similar, animal and plant nuclei differ substantially in details of their architecture. One example is the nuclear lamina, a layer of tightly interconnected filament proteins (lamins) underlying the nuclear envelope of metazoans. So far no orthologous lamin genes could be detected in plant genomes and putative lamin-like proteins are only poorly described in plants. To probe for potentially conserved features of metazoan and plant nuclear envelopes, we ectopically expressed the core nuclear egress proteins of human cytomegalovirus pUL50 and pUL53 in plant cells. pUL50 localizes to the inner envelope of metazoan nuclei and recruits the nuclear localized pUL53 to it, forming heterodimers. Upon expression in plant cells, a very similar localization pattern of both proteins could be determined. Notably, pUL50 is specifically targeted to the plant nuclear envelope in a rim-like fashion, a location to which coexpressed pUL53 becomes strictly corecruited from its initial nucleoplasmic distribution. Using pUL50 as bait in a yeast two-hybrid screening, the cytoplasmic re-initiation supporting protein RISP could be identified. Interaction of pUL50 and RISP could be confirmed by coexpression and coimmunoprecipitation in mammalian cells and by confocal laser scanning microscopy in plant cells, demonstrating partial pUL50-RISP colocalization in areas of the nuclear rim and other intracellular compartments. Thus, our study provides strong evidence for conserved structural features of plant and metazoan nuclear envelops and identifies RISP as a potential pUL50-interacting plant protein. PMID:26978388

  8. Human Cytomegalovirus Nuclear Egress Proteins Ectopically Expressed in the Heterologous Environment of Plant Cells are Strictly Targeted to the Nuclear Envelope.

    PubMed

    Lamm, Christian E; Link, Katrin; Wagner, Sabrina; Milbradt, Jens; Marschall, Manfred; Sonnewald, Uwe

    2016-01-01

    In all eukaryotic cells, the nucleus forms a prominent cellular compartment containing the cell's nuclear genome. Although structurally similar, animal and plant nuclei differ substantially in details of their architecture. One example is the nuclear lamina, a layer of tightly interconnected filament proteins (lamins) underlying the nuclear envelope of metazoans. So far no orthologous lamin genes could be detected in plant genomes and putative lamin-like proteins are only poorly described in plants. To probe for potentially conserved features of metazoan and plant nuclear envelopes, we ectopically expressed the core nuclear egress proteins of human cytomegalovirus pUL50 and pUL53 in plant cells. pUL50 localizes to the inner envelope of metazoan nuclei and recruits the nuclear localized pUL53 to it, forming heterodimers. Upon expression in plant cells, a very similar localization pattern of both proteins could be determined. Notably, pUL50 is specifically targeted to the plant nuclear envelope in a rim-like fashion, a location to which coexpressed pUL53 becomes strictly corecruited from its initial nucleoplasmic distribution. Using pUL50 as bait in a yeast two-hybrid screening, the cytoplasmic re-initiation supporting protein RISP could be identified. Interaction of pUL50 and RISP could be confirmed by coexpression and coimmunoprecipitation in mammalian cells and by confocal laser scanning microscopy in plant cells, demonstrating partial pUL50-RISP colocalization in areas of the nuclear rim and other intracellular compartments. Thus, our study provides strong evidence for conserved structural features of plant and metazoan nuclear envelops and identifies RISP as a potential pUL50-interacting plant protein. PMID:26978388

  9. Characterization of Chloroplast Protein Import without Tic56, a Component of the 1-Megadalton Translocon at the Inner Envelope Membrane of Chloroplasts1

    PubMed Central

    Köhler, Daniel; Montandon, Cyril; Hause, Gerd; Majovsky, Petra; Kessler, Felix; Baginsky, Sacha; Agne, Birgit

    2015-01-01

    We report on the characterization of Tic56, a unique component of the recently identified 1-MD translocon at the inner envelope membrane of chloroplasts (TIC) in Arabidopsis (Arabidopsis thaliana) comprising Tic20, Tic100, and Tic214. We isolated Tic56 by copurification with Tandem Affinity Purification-tagged Toc159 in the absence of precursor protein, indicating spontaneous and translocation-independent formation of the translocon at the outer envelope membrane of chloroplasts (TOC) and TIC supercomplexes. Tic56 mutant plants have an albino phenotype and are unable to grow without an external carbon source. Using specific enrichment of protein amino termini, we analyzed the tic56-1 and plastid protein import2 (toc159) mutants to assess the in vivo import capacity of plastids in mutants of an outer and inner envelope component of the anticipated TOC-TIC supercomplex. In both mutants, we observed processing of several import substrates belonging to various pathways. Our results suggest that despite the severe developmental defects, protein import into Tic56-deficient plastids is functional to a considerable degree, indicating the existence of alternative translocases at the inner envelope membrane. PMID:25588737

  10. Production of dengue virus envelope protein domain III-based antigens in tobacco chloroplasts using inducible and constitutive expression systems.

    PubMed

    Gottschamel, Johanna; Lössl, Andreas; Ruf, Stephanie; Wang, Yanliang; Skaugen, Morten; Bock, Ralph; Clarke, Jihong Liu

    2016-07-01

    Dengue fever is a disease in many parts of the tropics and subtropics and about half the world's population is at risk of infection according to the World Health Organization. Dengue is caused by any of the four related dengue virus serotypes DEN-1, -2, -3 and -4, which are transmitted to people by Aedes aegypti mosquitoes. Currently there is only one vaccine (Dengvaxia(®)) available (limited to a few countries) on the market since 2015 after half a century's intensive efforts. Affordable and accessible vaccines against dengue are hence still urgently needed. The dengue envelop protein domain III (EDIII), which is capable of eliciting serotype-specific neutralizing antibodies, has become the focus for subunit vaccine development. To contribute to the development of an accessible and affordable dengue vaccine, in the current study we have used plant-based vaccine production systems to generate a dengue subunit vaccine candidate in tobacco. Chloroplast genome engineering was applied to express serotype-specific recombinant EDIII proteins in tobacco chloroplasts using both constitutive and ethanol-inducible expression systems. Expression of a tetravalent antigen fusion construct combining EDIII polypeptides from all four serotypes was also attempted. Transplastomic EDIII-expressing tobacco lines were obtained and homoplasmy was verified by Southern blot analysis. Northern blot analyses showed expression of EDIII antigen-encoding genes. EDIII protein accumulation levels varied for the different recombinant EDIII proteins and the different expression systems, and reached between 0.8 and 1.6 % of total cellular protein. Our study demonstrates the suitability of the chloroplast compartment as a production site for an EDIII-based vaccine candidate against dengue fever and presents a Gateway(®) plastid transformation vector for inducible transgene expression. PMID:27116001

  11. Interactions of human lymphoblasts with targeted vesicles containing Sendai virus envelope proteins

    SciTech Connect

    Sechoy, O.; Vidal, M.; Philippot, J.R. ); Bienvenue, A. )

    1989-11-01

    The authors studied the internalization of targeted fusogenic liposome content to leukemic T cells (CEM) in vitro. They describe a method for the covalent coupling of T101 antibody to the surface of liposomes and the incorporation of fusogenic viral protein into the liposome membrane. Hygromycin B, an impermeant inhibitor of protein synthesis, was encapsulated in the targeted fusogenic liposomes and delivered directly to the cytoplasm of leukemic T cells by fusion between the two membranes. The cytotoxic effect was measured by ({sup 3H})thymidine incorporation. They show that CEM are rapidly and specifically killed by the drug encapsulated in the targeted fusogenic liposomes. This effect is due to the binding of the liposome by means of the antibody and then to the fusion of the liposome with the targeted cell membrane, mediated by F protein.

  12. A Mutation in the Human Immunodeficiency Virus Type 1 Gag Protein Destabilizes the Interaction of the Envelope Protein Subunits gp120 and gp41

    PubMed Central

    Davis, Melody R.; Jiang, Jiyang; Zhou, Jing; Freed, Eric O.; Aiken, Christopher

    2006-01-01

    The Gag protein of human immunodeficiency virus type 1 (HIV-1) associates with the envelope protein complex during virus assembly. The available evidence indicates that this interaction involves recognition of the gp41 cytoplasmic tail (CT) by the matrix protein (MA) region of Pr55Gag. Here we show that substitution of Asp for Leu at position 49 (L49D) in MA results in a specific reduction in particle-associated gp120 without affecting the levels of gp41. Mutant virions were markedly reduced in single-cycle infectivity despite a relatively modest defect in fusion with target cells. Studies with HIV-1 particles containing decreased levels of envelope proteins suggested that the L49D mutation also inhibits a postentry step in infection. Truncation of the gp41 tail, or pseudotyping by vesicular stomatitis virus glycoprotein, restored both the fusion and infectivity of L49D mutant virions to wild-type levels. Truncation of gp41 also resulted in equivalent levels of gp120 on particles with and without the MA mutation and enhanced the replication of the L49D mutant virus in T cells. The impaired fusion and infectivity of L49D mutant particles were also complemented by a single point mutation in the gp41 CT that disrupted the tyrosine-containing endocytic motif. Our results suggest that an altered interaction between the MA domain of Gag and the gp41 cytoplasmic tail leads to dissociation of gp120 from gp41 during HIV-1 particle assembly, thus resulting in impaired fusion and infectivity. PMID:16474147

  13. Crystal Structure of Dengue Virus Type 1 Envelope Protein in the Postfusion Conformation and Its Implications for Membrane Fusion

    SciTech Connect

    Nayak, Vinod; Dessau, Moshe; Kucera, Kaury; Anthony, Karen; Ledizet, Michel; Modis, Yorgo

    2009-07-31

    Dengue virus relies on a conformational change in its envelope protein, E, to fuse the viral lipid membrane with the endosomal membrane and thereby deliver the viral genome into the cytosol. We have determined the crystal structure of a soluble fragment E (sE) of dengue virus type 1 (DEN-1). The protein is in the postfusion conformation even though it was not exposed to a lipid membrane or detergent. At the domain I-domain III interface, 4 polar residues form a tight cluster that is absent in other flaviviral postfusion structures. Two of these residues, His-282 and His-317, are conserved in flaviviruses and are part of the 'pH sensor' that triggers the fusogenic conformational change in E, at the reduced pH of the endosome. In the fusion loop, Phe-108 adopts a distinct conformation, forming additional trimer contacts and filling the bowl-shaped concavity observed at the tip of the DEN-2 sE trimer.

  14. Recombinant Envelope-Proteins with Mutations in the Conserved Fusion Loop Allow Specific Serological Diagnosis of Dengue-Infections.

    PubMed

    Rockstroh, Alexandra; Barzon, Luisa; Pacenti, Monia; Palù, Giorgio; Niedrig, Matthias; Ulbert, Sebastian

    2015-11-01

    Dengue virus (DENV) is a mosquito-borne flavivirus and a major international public health concern in many tropical and sub-tropical areas worldwide. DENV is divided into four major serotypes, and infection with one serotype leads to immunity against the same, but not the other serotypes. The specific diagnosis of DENV-infections via antibody-detection is problematic due to the high degree of cross-reactivity displayed by antibodies against related flaviviruses, such as West Nile virus (WNV), Yellow Fever virus (YFV) or Tick-borne encephalitis virus (TBEV). Especially in areas where several flaviviruses co-circulate or in the context of vaccination e.g. against YFV or TBEV, this severely complicates diagnosis and surveillance. Most flavivirus cross-reactive antibodies are produced against the highly conserved fusion loop (FL) domain in the viral envelope (E) protein. We generated insect-cell derived recombinant E-proteins of the four DENV-serotypes which contain point mutations in the FL domain. By using specific mixtures of these mutant antigens, cross-reactivity against heterologous flaviviruses was strongly reduced, enabling sensitive and specific diagnosis of the DENV-infected serum samples in IgG and IgM-measurements. These results have indications for the development of serological DENV-tests with improved specificity. PMID:26565964

  15. Fragments of Target Cells are Internalized into Retroviral Envelope Protein-Expressing Cells during Cell-Cell Fusion by Endocytosis

    PubMed Central

    Izumida, Mai; Kamiyama, Haruka; Suematsu, Takashi; Honda, Eri; Koizumi, Yosuke; Yasui, Kiyoshi; Hayashi, Hideki; Ariyoshi, Koya; Kubo, Yoshinao

    2016-01-01

    Retroviruses enter into host cells by fusion between viral and host cell membranes. Retroviral envelope glycoprotein (Env) induces the membrane fusion, and also mediates cell-cell fusion. There are two types of cell-cell fusions induced by the Env protein. Fusion-from-within is induced by fusion between viral fusogenic Env protein-expressing cells and susceptible cells, and virions induce fusion-from-without by fusion between adjacent cells. Although entry of ecotropic murine leukemia virus (E-MLV) requires host cell endocytosis, the involvement of endocytosis in cell fusion is unclear. By fluorescent microscopic analysis of the fusion-from-within, we found that fragments of target cells are internalized into Env-expressing cells. Treatment of the Env-expressing cells with an endocytosis inhibitor more significantly inhibited the cell fusion than that of the target cells, indicating that endocytosis in Env-expressing cells is required for the cell fusion. The endocytosis inhibitor also attenuated the fusion-from-without. Electron microscopic analysis suggested that the membrane fusion resulting in fusion-from-within initiates in endocytic membrane dents. This study shows that two types of the viral cell fusion both require endocytosis, and provides the cascade of fusion-from-within. PMID:26834711

  16. Recombinant Envelope-Proteins with Mutations in the Conserved Fusion Loop Allow Specific Serological Diagnosis of Dengue-Infections

    PubMed Central

    Rockstroh, Alexandra; Barzon, Luisa; Pacenti, Monia; Palù, Giorgio; Niedrig, Matthias; Ulbert, Sebastian

    2015-01-01

    Dengue virus (DENV) is a mosquito-borne flavivirus and a major international public health concern in many tropical and sub-tropical areas worldwide. DENV is divided into four major serotypes, and infection with one serotype leads to immunity against the same, but not the other serotypes. The specific diagnosis of DENV-infections via antibody-detection is problematic due to the high degree of cross-reactivity displayed by antibodies against related flaviviruses, such as West Nile virus (WNV), Yellow Fever virus (YFV) or Tick-borne encephalitis virus (TBEV). Especially in areas where several flaviviruses co-circulate or in the context of vaccination e.g. against YFV or TBEV, this severely complicates diagnosis and surveillance. Most flavivirus cross-reactive antibodies are produced against the highly conserved fusion loop (FL) domain in the viral envelope (E) protein. We generated insect-cell derived recombinant E-proteins of the four DENV-serotypes which contain point mutations in the FL domain. By using specific mixtures of these mutant antigens, cross-reactivity against heterologous flaviviruses was strongly reduced, enabling sensitive and specific diagnosis of the DENV-infected serum samples in IgG and IgM-measurements. These results have indications for the development of serological DENV-tests with improved specificity. PMID:26565964

  17. Advanced Paramagnetic Resonance Spectroscopies of Iron-Sulfur Proteins: Electron Nuclear Double Resonance (ENDOR) and Electron Spin Echo Envelope Modulation (ESEEM)

    PubMed Central

    Cutsail, George E.; Telser, Joshua; Hoffman, Brian M.

    2015-01-01

    The advanced electron paramagnetic resonance (EPR) techniques, electron nuclear double resonance (ENDOR) and electron spin echo envelope modulation (ESEEM) spectroscopies, provide unique insights into the structure, coordination chemistry, and biochemical mechanism of Nature’s widely distributed iron-sulfur cluster (FeS) proteins. This review describes the ENDOR and ESEEM techniques and then provides a series of case studies on their application to a wide variety of FeS proteins including ferredoxins, nitrogenase, and radical SAM enzymes. PMID:25686535

  18. Nuclear envelope morphology constrains diffusion and promotes asymmetric protein segregation in closed mitosis

    PubMed Central

    Boettcher, Barbara; Marquez-Lago, Tatiana T.; Bayer, Mathias; Weiss, Eric L.

    2012-01-01

    During vegetative growth, Saccharomyces cerevisiae cells divide asymmetrically: the mother cell buds to produce a smaller daughter cell. This daughter asymmetrically inherits the transcription factor Ace2, which activates daughter-specific transcriptional programs. In this paper, we investigate when and how this asymmetry is established and maintained. We show that Ace2 asymmetry is initiated in the elongated, but undivided, anaphase nucleus. At this stage, the nucleoplasm was highly compartmentalized; little exchange was observed for nucleoplasmic proteins between mother and bud. Using photobleaching and in silico modeling, we show that diffusion barriers compartmentalize the nuclear membranes. In contrast, the behavior of proteins in the nucleoplasm is well explained by the dumbbell shape of the anaphase nucleus. This compartmentalization of the nucleoplasm promoted Ace2 asymmetry in anaphase nuclei. Thus, our data indicate that yeast cells use the process of closed mitosis and the morphological constraints associated with it to asymmetrically segregate nucleoplasmic components. PMID:22711697

  19. Contributions of herpes simplex virus type 1 envelope proteins to entry by endocytosis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Herpes simplex virus (HSV) proteins specifically required for endocytic entry but not direct penetration have not been identified. HSVs deleted of gE, gG, gI, gJ, gM, UL45, or Us9 entered cells via either pH-dependent or pH-independent endocytosis and were inactivated by mildly acidic pH. Thus, the ...

  20. Generation and characterization of potential dengue vaccine candidates based on domain III of the envelope protein and the capsid protein of the four serotypes of dengue virus.

    PubMed

    Suzarte, Edith; Marcos, Ernesto; Gil, Lázaro; Valdés, Iris; Lazo, Laura; Ramos, Yassel; Pérez, Yusleidi; Falcón, Viviana; Romero, Yaremis; Guzmán, María G; González, Sirenia; Kourí, Juan; Guillén, Gerardo; Hermida, Lisset

    2014-07-01

    Dengue is currently one of the most important arthropod-borne diseases, causing up to 25,000 deaths annually. There is currently no vaccine to prevent dengue virus infection, which needs a tetravalent vaccine approach. In this work, we describe the cloning and expression in Escherichia coli of envelope domain III-capsid chimeric proteins (DIIIC) of the four dengue serotypes as a tetravalent dengue vaccine candidate that is potentially able to generate humoral and cellular immunity. The recombinant proteins were purified to more than 85 % purity and were recognized by anti-dengue mouse and human sera. Mass spectrometry analysis verified the identity of the proteins and the correct formation of the intracatenary disulfide bond in the domain III region. The chimeric DIIIC proteins were also serotype-specific, and in the presence of oligonucleotides, they formed aggregates that were visible by electron microscopy. These results support the future use of DIIIC recombinant chimeric proteins in preclinical studies in mice for assessing their immunogenicity and efficacy. PMID:24420159

  1. Monoclonal antibody-escape variant of dengue virus serotype 1: Genetic composition and envelope protein expression.

    PubMed

    Chem, Y K; Chua, K B; Malik, Y; Voon, K

    2015-06-01

    Monoclonal antibody-escape variant of dengue virus type 1 (MabEV DEN-1) was discovered and isolated in an outbreak of dengue in Klang Valley, Malaysia from December 2004 to March 2005. This study was done to investigate whether DEN152 (an isolate of MabEV DEN-1) is a product of recombination event or not. In addition, the non-synonymous mutations that correlate with the monoclonal antibody-escape variant were determined in this study. The genomes of DEN152 and two new DEN-1 isolates, DENB04 and DENK154 were completely sequenced, aligned, and compared. Phylogenetic tree was plotted and the recombination event on DEN152 was investigated. DEN152 is sub-grouped under genotype I and is closely related genetically to a DEN-1 isolated in Japan in 2004. DEN152 is not a recombinant product of any parental strains. Four amino acid substitutions were unique only to DEN 152. These amino acid substitutions were (Ser)[326](Leu), (Ser)[340](Leu) at the deduced E protein, (Ile)[250](Thr) at NS1 protein, and (Thr)[41](Ser) at NS5 protein. Thus, DEN152 is an isolate of the emerging monoclonal antibody-escape variant DEN-1 that escaped diagnostic laboratory detection. PMID:26691263

  2. 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

  3. Human Immunodeficiency Virus Type 1 Particles Pseudotyped with Envelope Proteins That Fuse at Low pH No Longer Require Nef for Optimal Infectivity

    PubMed Central

    Chazal, Nathalie; Singer, Gregory; Aiken, Christopher; Hammarskjöld, Marie-Louise; Rekosh, David

    2001-01-01

    We have investigated the effects of Nef on infectivity in the context of various viral envelope proteins. These experiments were performed with a minimal vector system where Nef is the only accessory protein present. Our results support the hypothesis that the route of entry influences the ability of Nef to enhance human immunodeficiency virus (HIV) infectivity. We show that HIV particles pseudotyped with Ebola virus glycoprotein or vesicular stomatitis virus glycoprotein (VSV-G), which fuse at low pH, do not require Nef for optimal infectivity. In contrast, Nef significantly enhances the infectivity of virus particles that contain envelope proteins that fuse at neutral pH (CCR5-dependent HIV Env, CXCR4-dependent HIV Env, or amphotropic murine leukemia virus Env). In addition, our results demonstrate that virus particles containing mixed CXCR4-dependent HIV and VSV-G envelope proteins show a conditional requirement for Nef for optimal infectivity, depending on which protein is allowed to facilitate entry. PMID:11264394

  4. Ultrastructural Analysis and Identification of Envelope Proteins of “Candidatus Chloracidobacterium thermophilum” Chlorosomes▿

    PubMed Central

    Garcia Costas, Amaya M.; Tsukatani, Yusuke; Romberger, Steven P.; Oostergetel, Gert T.; Boekema, Egbert J.; Golbeck, John H.; Bryant, Donald A.

    2011-01-01

    Chlorosomes are sac-like, light-harvesting organelles that characteristically contain very large numbers of bacteriochlorophyll (BChl) c, d, or e molecules. These antenna structures occur in chlorophototrophs belonging to some members of the Chlorobi and Chloroflexi phyla and are also found in a recently discovered member of the phylum Acidobacteria, “Candidatus Chloracidobacterium thermophilum.” “Ca. Chloracidobacterium thermophilum” is the first aerobic organism discovered to possess chlorosomes as light-harvesting antennae for phototrophic growth. Chlorosomes were isolated from “Ca. Chloracidobacterium thermophilum” and subjected to electron microscopic, spectroscopic, and biochemical analyses. The chlorosomes of “Ca. Chloracidobacterium thermophilum” had an average size of ∼100 by 30 nm. Cryo-electron microscopy showed that the BChl c molecules formed folded or twisted, sheet-like structures with a lamellar spacing of ∼2.3 nm. Unlike the BChls in the chlorosomes of the green sulfur bacterium Chlorobaculum tepidum, concentric cylindrical nanotubes were not observed. Chlorosomes of “Ca. Chloracidobacterium thermophilum” contained a homolog of CsmA, the BChl a-binding, baseplate protein; CsmV, a protein distantly related to CsmI, CsmJ, and CsmX of C. tepidum, which probably binds a single [2Fe-2S] cluster; and five unique polypeptides (CsmR, CsmS, CsmT, CsmU, and a type II NADH dehydrogenase homolog). Although “Ca. Chloracidobacterium thermophilum” is an aerobe, energy transfer among the BChls in these chlorosomes was very strongly quenched in the presence of oxygen (as measured by quenching of fluorescence emission). The combined analyses showed that the chlorosomes of “Ca. Chloracidobacterium thermophilum” possess a number of unique features but also share some properties with the chlorosomes found in anaerobic members of other phyla. PMID:21965575

  5. Envelope protein of parasitic wasp symbiont virus, polydnavirus, protects the wasp eggs from cellular immune reactions by the host insect.

    PubMed

    Hayakawa, Y; Yazaki, K

    1997-06-15

    Cotesia kariyai polydnavirus (CkPDV) virions are present in the oviducts of C. kariyai wasp and are injected with eggs into the hemocoel of the host armyworm Pseudaletia separata larvae during parasitization. Evidence that the presence of polydnavirus particles on the surface of the wasp eggs may be essential for prevention of cellular immune reactions by the host hemocytes was obtained by isolating an immunoevasive factor from CkPDV virions. The purified proteinaceous factor protects foreign materials from adhesion and encapsulation by hemocytes of the host P. separata larvae but not by those of common cutworm Spodoptera litura larvae which is an incompatible host for the C. kariyai wasp. Purification procedures consisted of extraction with ethanol/trifluoroacetic acid and reverse-phase high performance liquid chromatography. A factor with a molecular mass of approximately 50 kDa is demonstrated to be present on the envelope of CkPDV virion by immunoelectronmicroscopic observations. Furthermore, immunoreactive proteins are found in plasma of the armyworm larvae but not in the common cutworm larvae, indicating that only the natural host of C. kariyai wasp shares a similar epitope with CkPDV. The sequence of 23 amino acid residues at the amino terminus of the factor was determined to be Ile-Ser-Val-Glu-Asn-Val-Xaa-Thr-Thr-Gly-Ile-Phe-Leu-Asp-Ser-Gly-Glu-Xaa- Val- Pro-Tyr-Ala-Thr-Lys-Pro. PMID:9219544

  6. 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

  7. Induction of protective antibodies against dengue virus by tetravalent DNA immunization of mice with domain III of the envelope protein.

    PubMed

    Mota, Javier; Acosta, Maribel; Argotte, Rocio; Figueroa, Raymunda; Méndez, Armando; Ramos, Celso

    2005-05-16

    Dengue fever is a growing public health concern around the world and despite vaccine development efforts, there are currently no effective dengue vaccines. In the present study we report the induction of protective antibodies against dengue virus by DNA immunization with domain III (DIII) region of the envelope protein (E) in a mouse model. The DIII region of all four dengue virus serotypes were cloned separately into pcDNA 3 plasmid. Protein expression was tested in COS-7 cells. Each plasmid, or a tetravalent combination, were used to immunize BALB/c mice by intramuscular route. Presence of specific antibodies was evaluated by ELISA, and neutralizing antibodies were tested using a cytopathogenic effect (CPE) inhibition assay in BHK-21 cells, as well as in newborn mice challenged intracranially with dengue 2 virus. Mice immunized with individual DIII constructs or the tetravalent formulation developed antibodies against each corresponding dengue serotype. Antibody titers by ELISA were similar for all serotypes and no significant differences were observed when boosters were administered, although antibody responses were dose-dependent. CPE inhibition assays using Den-2 virus showed neutralization titers of 1:10 in mice immunized with individual DIII plasmid or those immunized with the tetravalent formulations. 43% of newborn mice challenged with Den-2 in combination with sera from mice immunized with Den-2 DIII plasmid were protected, whereas sera from mice immunized with the tetravalent formulation conferred 87% protection. Our results suggest that DIII can be used as a tetravalent DNA formulation to induce neutralizing and protective antibodies against dengue virus. PMID:15837370

  8. Use of envelope domain III protein for detection and differentiation of flaviviruses in the Free State Province, South Africa.

    PubMed

    Mathengtheng, Lehlohonolo; Burt, Felicity J

    2014-04-01

    The presence of the mosquito-borne flavivirus species West Nile virus (WNV) and Wesselsbron virus (WESSV) in southern Africa is well established; however, their true prevalence remains unknown. To date, the presence of tick-borne flaviviruses has not been confirmed in this region. Serological assays using reagents that can be handled in a biosafety level 2 or lower facility were developed and evaluated for the detection and differentiation of tick- and mosquito-borne flaviviruses in the Free State province of South Africa. A total of 2393 serum samples from a variety of species including humans, cattle, and sheep were tested using Kunjin virus (KUNV) cell lysate antigen for the detection of anti-flavivirus antibodies in an indirect immunoglobulin G (IgG) enzyme-linked immonosorbent assay (ELISA). To further differentiate positive reactors on the KUNV assay for antibodies against tick- or mosquito-borne flaviviruses, recombinant envelope domain III (r-EDIII) proteins of Langat virus (LGTV), WNV, and WESSV were expressed in a bacterial expression system and used in ELISA. A total of 722 samples were positive using the KUNV assay, of which 71, 457, and 431 were positive using the r-LGTVEDIII, r-WNVEDIII, and r-WESSVEDIII assays, respectively. A total of 70 samples were reactive using the KUNV assay but not using any of the other assays, suggesting that there are possibly other flaviviruses circulating in the Free State province for which specific r-EDIII assays were not available. Collectively, the results suggest a strong presence of flaviviruses co-circulating in the Free State province with an abundance of mosquito-borne flaviviruses. There is evidence suggesting the presence of tick-borne flaviviruses, but it has yet to be confirmed. The EDIII protein is a useful tool that can be used in the detection and differentiation of flaviviruses in resource-limited laboratories, but virus neutralization assays are suggested for accurate confirmation of results. PMID

  9. Advanced paramagnetic resonance spectroscopies of iron-sulfur proteins: Electron nuclear double resonance (ENDOR) and electron spin echo envelope modulation (ESEEM).

    PubMed

    Cutsail, George E; Telser, Joshua; Hoffman, Brian M

    2015-06-01

    The advanced electron paramagnetic resonance (EPR) techniques, electron nuclear double resonance (ENDOR) and electron spin echo envelope modulation (ESEEM) spectroscopies, provide unique insights into the structure, coordination chemistry, and biochemical mechanism of nature's widely distributed iron-sulfur cluster (FeS) proteins. This review describes the ENDOR and ESEEM techniques and then provides a series of case studies on their application to a wide variety of FeS proteins including ferredoxins, nitrogenase, and radical SAM enzymes. This article is part of a Special Issue entitled: Fe/S proteins: Analysis, structure, function, biogenesis and diseases. PMID:25686535

  10. Envelope formation is blocked by mutation of a sequence related to the HKD phospholipid metabolism motif in the vaccinia virus F13L protein.

    PubMed

    Roper, R L; Moss, B

    1999-02-01

    The outer envelope of the extracellular form of vaccinia virus is derived from Golgi membranes that have been modified by the insertion of specific viral proteins, of which the major component is the 37-kDa, palmitylated, nonglycosylated product of the F13L gene. The F13L protein contains a variant of the HKD (His-Lys-Asp) motif, which is conserved in numerous enzymes of phospholipid metabolism. Vaccinia virus mutants with a conservative substitution of either the K (K314R) or the D (D319E) residue of the F13L protein formed only tiny plaques similar to those produced by an F13L deletion mutant, were unable to produce extracellular enveloped virions, and failed to mediate low-pH-induced fusion of infected cells. Membrane-wrapped forms of intracellular virus were rarely detected in electron microscopic images of cells infected with either of the mutants. Western blotting and pulse-chase experiments demonstrated that the D319E protein was less stable than either the K314R or wild-type F13L protein. Most striking, however, was the failure of either of the two mutated proteins to concentrate in the Golgi compartment. Palmitylation, oleation, and partitioning of the F13L protein in Triton X-114 detergent were unaffected by the K314R substitution. These results indicated that the F13L protein must retain the K314 and D319 for it to localize in the Golgi compartment and function in membrane envelopment of vaccinia virus. PMID:9882312

  11. The occlusion-derived virus envelope protein ODV-E56 is required for optimal oral infectivity but is not essential for virus binding and fusion

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The Autographa californica multiple nucleopolyhedrovirus (AcMNPV) odv-e56 gene encodes an occlusion-derived virus (ODV)-specific envelope protein, ODV-E56. To determine the role of ODV-E56 in oral infectivity, we produced recombinant EGFP-expressing AcMNPV clones (Ac69GFP-e56lacZ and AcIEGFP-e56lac...

  12. Yellow fever virus envelope protein expressed in insect cells is capable of syncytium formation in lepidopteran cells and could be used for immunodetection of YFV in human sera

    PubMed Central

    2011-01-01

    Background Yellow fever is an haemorrhagic disease caused by a virus that belongs to the genus Flavivirus (Flaviviridae family) and is transmitted by mosquitoes. Among the viral proteins, the envelope protein (E) is the most studied one, due to its high antigenic potencial. Baculovirus are one of the most popular and efficient eukaryotic expression system. In this study a recombinant baculovirus (vSynYFE) containing the envelope gene (env) of the 17D vaccine strain of yellow fever virus was constructed and the recombinant protein antigenicity was tested. Results Insect cells infected with vSynYFE showed syncytium formation, which is a cytopathic effect characteristic of flavivirus infection and expressed a polypeptide of around 54 kDa, which corresponds to the expected size of the recombinant E protein. Furthermore, the recombinant E protein expression was also confirmed by fluorescence microscopy of vSynYFE-infected insect cells. Total vSynYFE-infected insect extracts used as antigens detected the presence of antibodies for yellow fever virus in human sera derived from yellow fever-infected patients in an immunoassay and did not cross react with sera from dengue virus-infected patients. Conclusions The E protein expressed by the recombinant baculovirus in insect cells is antigenically similar to the wild protein and it may be useful for different medical applications, from improved diagnosis of the disease to source of antigens for the development of a subunit vaccine. PMID:21619598

  13. Characterization of the TolB-Pal trans-envelope complex from Xylella fastidiosa reveals a dynamic and coordinated protein expression profile during the biofilm development process.

    PubMed

    Santos, Clelton A; Janissen, Richard; Toledo, Marcelo A S; Beloti, Lilian L; Azzoni, Adriano R; Cotta, Monica A; Souza, Anete P

    2015-10-01

    The intriguing roles of the bacterial Tol-Pal trans-envelope protein complex range from maintenance of cell envelope integrity to potential participation in the process of cell division. In this study, we report the characterization of the XfTolB and XfPal proteins of the Tol-Pal complex of Xylella fastidiosa. X. fastidiosa is a major plant pathogen that forms biofilms inside xylem vessels, triggering the development of diseases in important cultivable plants around the word. Based on functional complementation experiments in Escherichia coli tolB and pal mutant strains, we confirmed the role of xftolB and xfpal in outer membrane integrity. In addition, we observed a dynamic and coordinated protein expression profile during the X. fastidiosa biofilm development process. Using small-angle X-ray scattering (SAXS), the low-resolution structure of the isolated XfTolB-XfPal complex in solution was solved for the first time. Finally, the localization of the XfTolB and XfPal polar ends was visualized via immunofluorescence labeling in vivo during bacterial cell growth. Our results highlight the major role of the components of the cell envelope, particularly the TolB-Pal complex, during the different phases of bacterial biofilm development. PMID:26049080

  14. Analyses of inter- and intra-patient variation in the V3 loop of the HIV-1 envelope protein

    SciTech Connect

    Korber, B.; Myers, G. ); Wolinsky, S. . Medical School)

    1991-09-17

    The third hypervariable domain of the HIV-1 gp120 envelope protein (V3) has been the focus of intensive sequencing efforts. To date, nearly one thousand V3 loop sequences have been stored in the HIV sequence database. Studies have revealed that the V3 loop elicits potent type-specific immune responses, and that it plays a significant role in cell tropism and fusion . The immunogenic tip of the loop can serve as a type-specific neutralizing antibody epitope, as well as a cytotoxic T-cell epitope. A helper T-cell epitope that lies within the amino terminal half of the V3 loop has also been characterized. Despite the richness of the immunologic response to this region, its potential for variation makes it an elusive target for vaccine design. Analyses of sibling sequence sets (sets of viral sequences derived from one person) show that multiple forms of the immunogenic tip of the loop are found within most HIV-1 infected individuals. Viral V3 sequences obtained from epidemiologically unlinked individuals from North America and Europe show extensive variation. However, some amino acid positions distributed throughout the V3 loop are highly conserved, and there is also conservation of the charge class of amino acid able to occupy certain positions relative to the tip of the loop. By contrast, the sequences obtained from many countries throughout the African continent reveal that V3 is a remarkably fluid region with few absolute constraints on the nature of the amino acids that can occupy most positions in the loop. The high degree of heterogeneity in this region is particularly striking in view of its contribution to biologically important viral functions.

  15. Generation of Monoclonal Antibodies against Dengue Virus Type 4 and Identification of Enhancing Epitopes on Envelope Protein

    PubMed Central

    Tang, Chung-Tao; Liao, Mei-Ying; Chiu, Chien-Yu; Shen, Wen-Fan; Chiu, Chiung-Yi; Cheng, Ping-Chang; Chang, Gwong-Jen J.; Wu, Han-Chung

    2015-01-01

    The four serotypes of dengue virus (DENV1-4) pose a serious threat to global health. Cross-reactive and non-neutralizing antibodies enhance viral infection, thereby exacerbating the disease via antibody-dependent enhancement (ADE). Studying the epitopes targeted by these enhancing antibodies would improve the immune responses against DENV infection. In order to investigate the roles of antibodies in the pathogenesis of dengue, we generated a panel of 16 new monoclonal antibodies (mAbs) against DENV4. Using plaque reduction neutralization test (PRNT), we examined the neutralizing activity of these mAbs. Furthermore, we used the in vitro and in vivo ADE assay to evaluate the enhancement of DENV infection by mAbs. The results indicate that the cross-reactive and poorly neutralizing mAbs, DD11-4 and DD18-5, strongly enhance DENV1-4 infection of K562 cells and increase mortality in AG129 mice. The epitope residues of these enhancing mAbs were identified using virus-like particle (VLP) mutants. W212 and E26 are the epitope residues of DD11-4 and DD18-5, respectively. In conclusion, we generated and characterized 16 new mAbs against DENV4. DD11-4 and D18-5 possessed non-neutralizing activities and enhanced viral infection. Moreover, we identified the epitope residues of enhancing mAbs on envelope protein. These results may provide useful information for development of safe dengue vaccine. PMID:26309127

  16. Transmitted/Founder and Chronic HIV-1 Envelope Proteins Are Distinguished by Differential Utilization of CCR5

    PubMed Central

    Parker, Zahra F.; Iyer, Shilpa S.; Wilen, Craig B.; Parrish, Nicholas F.; Chikere, Kelechi C.; Lee, Fang-Hua; Didigu, Chuka A.; Berro, Reem; Klasse, Per Johan; Lee, Benhur; Moore, John P.; Shaw, George M.

    2013-01-01

    Infection by HIV-1 most often results from the successful transmission and propagation of a single virus variant, termed the transmitted/founder (T/F) virus. Here, we compared the attachment and entry properties of envelope (Env) glycoproteins from T/F and chronic control (CC) viruses. Using a panel of 40 T/F and 47 CC Envs, all derived by single genome amplification, we found that 52% of clade C and B CC Envs exhibited partial resistance to the CCR5 antagonist maraviroc (MVC) on cells expressing high levels of CCR5, while only 15% of T/F Envs exhibited this same property. Moreover, subtle differences in the magnitude with which MVC inhibited infection on cells expressing low levels of CCR5, including primary CD4+ T cells, were highly predictive of MVC resistance when CCR5 expression levels were high. These results are consistent with previous observations showing a greater sensitivity of T/F Envs to MVC inhibition on cells expressing very high levels of CCR5 and indicate that CC Envs are often capable of recognizing MVC-bound CCR5, albeit inefficiently on cells expressing physiologic levels of CCR5. When CCR5 expression levels are high, this phenotype becomes readily detectable. The utilization of drug-bound CCR5 conformations by many CC Envs was seen with other CCR5 antagonists, with replication-competent viruses, and did not obviously correlate with other phenotypic traits. The striking ability of clade C and B CC Envs to use MVC-bound CCR5 relative to T/F Envs argues that the more promiscuous use of CCR5 by these Env proteins is selected against at the level of virus transmission and is selected for during chronic infection. PMID:23269796

  17. Natural selection of adaptive mutations in non-structural genes increases trans-encapsidation of hepatitis C virus replicons lacking envelope protein genes.

    PubMed

    Fournier, Carole; Helle, François; Descamps, Véronique; Morel, Virginie; François, Catherine; Dedeurwaerder, Sarah; Wychowski, Czeslaw; Duverlie, Gilles; Castelain, Sandrine

    2013-05-01

    A trans-packaging system for hepatitis C virus (HCV) replicons lacking envelope glycoproteins was developed. The replicons were efficiently encapsidated into infectious particles after expression in trans of homologous HCV envelope proteins under the control of an adenoviral vector. Interestingly, expression in trans of core or core, p7 and NS2 with envelope proteins did not enhance trans-encapsidation. Expression of heterologous envelope proteins, in the presence or absence of heterologous core, p7 and NS2, did not rescue single-round infectious particle production. To increase the titre of homologous, single-round infectious particles in our system, successive cycles of trans-encapsidation and infection were performed. Four cycles resulted in a 100-fold increase in the yield of particles. Sequence analysis revealed a total of 16 potential adaptive mutations in two independent experiments. Except for a core mutation in one experiment, all the mutations were located in non-structural regions mainly in NS5A (four in domain III and two near the junction with the NS5B gene). Reverse genetics studies suggested that D2437A and S2443T adaptive mutations, which are located at the NS5A-B cleavage site did not affect viral replication, but enhanced the single-round infectious particles assembly only in trans-encapsidation model. In conclusion, our trans-encapsidation system enables the production of HCV single-round infectious particles. This system is adaptable and can positively select variants. The adapted variants promote trans-encapsidation and should constitute a valuable tool in the development of replicon-based HCV vaccines. PMID:23288424

  18. Gypsy transposition correlates with the production of a retroviral envelope-like protein under the tissue-specific control of the Drosophila flamenco gene.

    PubMed

    Pélisson, A; Song, S U; Prud'homme, N; Smith, P A; Bucheton, A; Corces, V G

    1994-09-15

    Gypsy displays striking similarities to vertebrate retroviruses, including the presence of a yet uncharacterized additional open reading frame (ORF3) and the recent evidence for infectivity. It is mobilized with high frequency in the germline of the progeny of females homozygous for the flamenco permissive mutation. We report the characterization of a gypsy subgenomic ORF3 RNA encoding typical retroviral envelope proteins. In females, env expression is strongly repressed by one copy of the non-permissive allele of flamenco. A less dramatic reduction in the accumulation of other transcripts and retrotranscripts is also observed. These effects correlate well with the inhibition of gypsy transposition in the progeny of these females, and are therefore likely to be responsible for this phenomenon. The effects of flamenco on gypsy expression are apparently restricted to the somatic follicle cells that surround the maternal germline. Moreover, permissive follicle cells display a typically polarized distribution of gypsy RNAs and envelope proteins, both being mainly accumulated at the apical pole, close to the oocyte. We propose a model suggesting that gypsy germinal transposition might occur only in individuals that have maternally inherited enveloped gypsy particles due to infection of the maternal germline by the soma. PMID:7925283

  19. Generation of recombinant monoclonal antibodies to study structure-function of envelope protein VP28 of white spot syndrome virus from shrimp

    SciTech Connect

    Wang Yuzhen; Zhang Xiaohua; Yuan Li; Xu Tao; Rao Yu; Li Jia; Dai Heping

    2008-08-08

    White spot syndrome virus (WSSV) is a major pathogen in shrimp aquaculture. VP28 is one of the most important envelope proteins of WSSV. In this study, a recombinant antibody library, as single-chain fragment variable (scFv) format, displayed on phage was constructed using mRNA from spleen cells of mice immunized with full-length VP28 expressed in Escherichia coli. After several rounds of panning, six scFv antibodies specifically binding to the epitopes in the N-terminal, middle, and C-terminal regions of VP28, respectively, were isolated from the library. Using these scFv antibodies as tools, the epitopes in VP28 were located on the envelope of the virion by immuno-electron microscopy. Neutralization assay with these antibodies in vitro suggested that these epitopes may not be the attachment site of WSSV to host cell receptor. This study provides a new way to investigate the structure and function of the envelope proteins of WSSV.

  20. Autographa californica Multiple Nucleopolyhedrovirus orf132 Encodes a Nucleocapsid-Associated Protein Required for Budded-Virus and Multiply Enveloped Occlusion-Derived Virus Production

    PubMed Central

    Yang, Ming; Wang, Shuo; Yue, Xiu-Li

    2014-01-01

    ABSTRACT Autographa californica multiple nucleopolyhedrovirus orf132 (named ac132) has homologs in all genome-sequenced group I nucleopolyhedroviruses. Its role in the viral replication cycle is unknown. In this study, ac132 was shown to express a protein of around 28 kDa, which was determined to be associated with the nucleocapsids of both occlusion-derived virus and budded virus. Confocal microscopy showed that AC132 protein appeared in central region of the nucleus as early as 12 h postinfection with the virus. It formed a ring zone at the periphery of the nucleus by 24 h postinfection. To investigate its role in virus replication, ac132 was deleted from the viral genome by using a bacmid system. In the Sf9 cell culture transfected by the ac132 knockout bacmid, infection was restricted to single cells, and the titer of infectious budded virus was reduced to an undetectable level. However, viral DNA replication and the expression of late genes vp39 and odv-e25 and a reporter gene under the control of the very late gene p10 promoter were unaffected. Electron microscopy showed that nucleocapsids, virions, and occlusion bodies were synthesized in the cells transfected by an ac132 knockout bacmid, but the formation of the virogenic stroma and occlusion bodies was delayed, the numbers of enveloped nucleocapsids were reduced, and the occlusion bodies contained mainly singly enveloped nucleocapsids. AC132 was found to interact with envelope protein ODV-E18 and the viral DNA-binding protein P6.9. The data from this study suggest that ac132 possibly plays an important role in the assembly and envelopment of nucleocapsids. IMPORTANCE To our knowledge, this is the first report on a functional analysis of ac132. The data presented here demonstrate that ac132 is required for production of the budded virus and multiply enveloped occlusion-derived virus of Autographa californica multiple nucleopolyhedrovirus. This article reveals unique phenotypic changes induced by ac132

  1. The surface envelope protein gene region of equine infectious anemia virus is not an important determinant of tropism in vitro.

    PubMed Central

    Perry, S T; Flaherty, M T; Kelley, M J; Clabough, D L; Tronick, S R; Coggins, L; Whetter, L; Lengel, C R; Fuller, F

    1992-01-01

    Virulent, wild-type equine infectious anemia virus (EIAV) is restricted in one or more early steps in replication in equine skin fibroblast cells compared with cell culture-adapted virus, which is fully competent for replication in this cell type. We compared the sequences of wild-type EIAV and a full-length infectious proviral clone of the cell culture-adapted EIAV and found that the genomes were relatively well conserved with the exception of the envelope gene region, which showed extensive sequence differences. We therefore constructed several wild-type and cell culture-adapted virus chimeras to examine the role of the envelope gene in replication in different cell types in vitro. Unlike wild-type virus, which is restricted by an early event(s) for replication in equine dermis cells, the wild-type outer envelope gene chimeras are replication competent in this cell type. We conclude that even though there are extensive sequence differences between wild-type and cell culture-adapted viruses in the surface envelope gene region, this domain is not a determinant of the differing in vitro cell tropisms. Images PMID:1318398

  2. Comparative Proteomics of Chloroplasts Envelopes from Bundle Sheath and Mesophyll Chloroplasts Reveals Novel Membrane Proteins with a Possible Role in C4-Related Metabolite Fluxes and Development

    PubMed Central

    Manandhar-Shrestha, K.; Tamot, B.; Pratt, E. P. S.; Saitie, S.; Bräutigam, A.; Weber, A. P. M.; Hoffmann-Benning, Susanne

    2013-01-01

    As the world population grows, our need for food increases drastically. Limited amounts of arable land lead to a competition between food and fuel crops, while changes in the global climate may impact future crop yields. Thus, a second “green revolution” will need a better understanding of the processes essential for plant growth and development. One approach toward the solution of this problem is to better understand regulatory and transport processes in C4 plants. C4 plants display an up to 10-fold higher apparent CO2 assimilation and higher yields while maintaining high water use efficiency. This requires differential regulation of mesophyll (M) and bundle sheath (BS) chloroplast development as well as higher metabolic fluxes of photosynthetic intermediates between cells and particularly across chloroplast envelopes. While previous analyses of overall chloroplast membranes have yielded significant insight, our comparative proteomics approach using enriched BS and M chloroplast envelopes of Zea mays allowed us to identify 37 proteins of unknown function that have not been seen in these earlier studies. We identified 280 proteins, 84% of which are known/predicted to be present in chloroplasts. Seventy-four percent have a known or predicted membrane association. Twenty-one membrane proteins were 2–15 times more abundant in BS cells, while 36 of the proteins were more abundant in M chloroplast envelopes. These proteins could represent additional candidates of proteins essential for development or metabolite transport processes in C4 plants. RT-PCR confirmed differential expression of 13 candidate genes. Chloroplast association for seven proteins was confirmed using YFP/GFP labeling. Gene expression of four putative transporters was examined throughout the leaf and during the greening of leaves. Genes for a PIC-like protein and an ER-AP-like protein show an early transient increase in gene expression during the transition to light. In addition, PIC gene

  3. The Use of Two-Photon FRET-FLIM to Study Protein Interactions During Nuclear Envelope Fusion In Vivo and In Vitro.

    PubMed

    Byrne, Richard D; Larijani, Banafshé; Poccia, Dominic L

    2016-01-01

    FRET-FLIM techniques have wide application in the study of protein and protein-lipid interactions in cells. We have pioneered an imaging platform for accurate detection of functional states of proteins and their interactions in fixed cells. This platform, two-site-amplified Förster resonance energy transfer (a-FRET), allows greater signal generation while retaining minimal noise thus enabling application of fluorescence lifetime imaging microscopy (FLIM) to be routinely deployed in different types of cells and tissue. We have used the method described here, time-resolved FRET monitored by two-photon FLIM, to demonstrate the direct interaction of Phospholipase Cγ (PLCγ) by Src Family Kinase 1 (SFK1) during nuclear envelope formation and during male and female pronuclear membrane fusion in fertilized sea urchin eggs. We describe here a generic method that can be applied to monitor any proteins of interest. PMID:27147038

  4. A Fungal Sarcolemmal Membrane-Associated Protein (SLMAP) Homolog Plays a Fundamental Role in Development and Localizes to the Nuclear Envelope, Endoplasmic Reticulum, and Mitochondria

    PubMed Central

    Nordzieke, Steffen; Zobel, Thomas; Fränzel, Benjamin; Wolters, Dirk A.

    2014-01-01

    Sarcolemmal membrane-associated protein (SLMAP) is a tail-anchored protein involved in fundamental cellular processes, such as myoblast fusion, cell cycle progression, and chromosomal inheritance. Further, SLMAP misexpression is associated with endothelial dysfunctions in diabetes and cancer. SLMAP is part of the conserved striatin-interacting phosphatase and kinase (STRIPAK) complex required for specific signaling pathways in yeasts, filamentous fungi, insects, and mammals. In filamentous fungi, STRIPAK was initially discovered in Sordaria macrospora, a model system for fungal differentiation. Here, we functionally characterize the STRIPAK subunit PRO45, a homolog of human SLMAP. We show that PRO45 is required for sexual propagation and cell-to-cell fusion and that its forkhead-associated (FHA) domain is essential for these processes. Protein-protein interaction studies revealed that PRO45 binds to STRIPAK subunits PRO11 and SmMOB3, which are also required for sexual propagation. Superresolution structured-illumination microscopy (SIM) further established that PRO45 localizes to the nuclear envelope, endoplasmic reticulum, and mitochondria. SIM also showed that localization to the nuclear envelope requires STRIPAK subunits PRO11 and PRO22, whereas for mitochondria it does not. Taken together, our study provides important insights into fundamental roles of the fungal SLMAP homolog PRO45 and suggests STRIPAK-related and STRIPAK-unrelated functions. PMID:25527523

  5. Characterization of a monoclonal antibody to a novel glycan-dependent epitope in the V1/V2 domain of the HIV-1 envelope protein, gp120.

    PubMed

    Doran, Rachel C; Morales, Javier F; To, Briana; Morin, Trevor J; Theolis, Richard; O'Rourke, Sara M; Yu, Bin; Mesa, Kathryn A; Berman, Phillip W

    2014-11-01

    Recent studies have described several broadly neutralizing monoclonal antibodies (bN-mAbs) that recognize glycan-dependent epitopes (GDEs) in the HIV-1 envelope protein, gp120. These were recovered from HIV-1 infected subjects, and several (e.g., PG9, PG16, CH01, CH03) target glycans in the first and second variable (V1/V2) domain of gp120. The V1/V2 domain is thought to play an important role in conformational masking, and antibodies to the V1/V2 domain were recently identified as the only immune response that correlated with protection in the RV144 HIV-1 vaccine trial. While the importance of antibodies to polymeric glycans is well established for vaccines targeting bacterial diseases, the importance of antibodies to glycans in vaccines targeting HIV has only recently been recognized. Antibodies to GDEs may be particularly significant in HIV vaccines based on gp120, where 50% of the molecular mass of the envelope protein is contributed by N-linked carbohydrate. However, few studies have reported antibodies to GDEs in humans or animals immunized with candidate HIV-1 vaccines. In this report, we describe the isolation of a mouse mAb, 4B6, after immunization with the extracellular domain of the HIV-1 envelope protein, gp140. Epitope mapping using glycopeptide fragments and in vitro mutagenesis showed that binding of this antibody depends on N-linked glycosylation at asparagine N130 (HXB2 numbering) in the gp120 V1/V2 domain. Our results demonstrate that, in addition to natural HIV-1 infection, immunization with recombinant proteins can elicit antibodies to the GDEs in the V1/V2 domain of gp120. Although little is known regarding conditions that favor antibody responses to GDEs, our studies demonstrate that these antibodies can arise from a short-term immunization regimen. Our results suggest that antibodies to GDEs are more common than previously suspected, and that further analysis of antibody responses to the HIV-1 envelope protein will lead to the discovery of

  6. A soluble envelope protein of endogenous retrovirus (FeLIX) present in serum of domestic cats mediates infection of a pathogenic variant of feline leukemia virus.

    PubMed

    Sakaguchi, Shoichi; Shojima, Takayuki; Fukui, Daisuke; Miyazawa, Takayuki

    2015-03-01

    T-lymphotropic feline leukemia virus (FeLV-T), a highly pathogenic variant of FeLV, induces severe immunosuppression in cats. FeLV-T is fusion defective because in its PHQ motif, a gammaretroviral consensus motif in the N terminus of an envelope protein, histidine is replaced with aspartate. Infection by FeLV-T requires FeLIX, a truncated envelope protein encoded by an endogenous FeLV, for transactivation of infectivity and Pit1 for binding FeLIX. Although Pit1 is present in most tissues in cats, the expression of FeLIX is limited to certain cells in lymphoid organs. Therefore, the host cell range of FeLV-T was thought to be restricted to cells expressing FeLIX. However, because FeLIX is a soluble factor and is expressed constitutively in lymphoid organs, we presumed it to be present in blood and evaluated its activities in sera of various mammalian species using a pseudotype assay. We demonstrated that cat serum has FeLIX activity at a functional level, suggesting that FeLIX is present in the blood and that FeLV-T may be able to infect cells expressing Pit1 regardless of the expression of FeLIX in vivo. In addition, FeLIX activities in sera were detected only in domestic cats and not in other feline species tested. To our knowledge, this is the first report to prove that a large amount of truncated envelope protein of endogenous retrovirus is circulating in the blood to facilitate the infection of a pathogenic exogenous retrovirus. PMID:25395593

  7. New developments in an old strategy: heterologous vector primes and envelope protein boosts in HIV vaccine design.

    PubMed

    Musich, Thomas; Robert-Guroff, Marjorie

    2016-08-01

    Prime/boost vaccination strategies for HIV/SIV vaccine development have been used since the early 1990s and have become an established method for eliciting cell and antibody mediated immunity. Here we focus on induction of protective antibodies, both broadly neutralizing and non-neutralizing, with the viral envelope being the key target antigen. Prime/boost approaches are complicated by the diversity of autologous and heterologous priming vectors, and by various forms of envelope booster immunogens, many still in development as structural studies aim to design stable constructs with exposure of critical epitopes for protective antibody elicitation. This review discusses individual vaccine components, reviews recent prime/boost strategies and their outcomes, and highlights complicating factors arising as greater knowledge concerning induction of adaptive, protective immunity is acquired. PMID:26910195

  8. The deletion of residues 268-292 of E1 impairs the ability of HCV envelope proteins to induce pore formation.

    PubMed

    Lombana, Laura; Ortega-Atienza, Sara; Gómez-Gutiérrez, Julián; Yélamos, Belén; Peterson, Darrell L; Gavilanes, Francisco

    2016-06-01

    We have obtained a chimeric protein containing the ectodomains of hepatitis C virus (HCV) envelope proteins but lacking the region 268-292 of E1. All its structural properties are coincident with those of the corresponding full length chimera. The deleted and entire chimeras were compared in terms of their membrane destabilizing properties. No differences were found in their ability to induce vesicle aggregation and lipid mixing but the deleted chimera showed a reduced capacity to promote leakage. The role of the deletion was also studied by obtaining HCV pseudoparticles (HCVpp). Both E1 and E2, and also the E1 deleted mutant, were incorporated into HCVpp to a similar level. However, HCVpp containing the E1 deleted protein are almost unable to infect Huh7 cells. These results point to the involvement of the region 268-292 in the formation of pores in the membrane necessary for the complete fusion of the membranes. PMID:26945847

  9. 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

  10. A dengue-2 Envelope fragment inserted within the structure of the P64k meningococcal protein carrier enables a functional immune response against the virus in mice.

    PubMed

    Hermida, Lisset; Rodríguez, Rayner; Lazo, Laura; Silva, Ricardo; Zulueta, Aída; Chinea, Glay; López, Carlos; Guzmán, María G; Guillén, Gerardo

    2004-01-01

    A gene fragment encoding for the amino acids (aa) 286-426 from the dengue Envelope (E) protein was expressed in Escherichia coli as two forms of fusion proteins. In one case, the E fragment was fused to the first 45 aa of the P64k protein from Neisseria meningitidis (PD2) while, in the other, it was inserted within the lipoil-binding domain of the aforementioned bacterial protein (PD3). PD2 was obtained as insoluble form within the cytoplasm of the bacteria while PD3 was distributed equally as soluble and insoluble forms. The insoluble forms of each protein as well as the soluble fraction of PD3 were semipurified to test the antigenicity and the immunogenicity in mice. The forms containing the entire P64k protein exhibited the highest recognition with different polyclonal and monoclonal antibodies. Consequently, the neutralizing antibodies elicited by the recombinant proteins were higher in the case of PD3 forms than with PD2, independently of the solubility status. In addition, mice inoculated with the semipurified insoluble form of PD3 were partially protected against lethal challenge with dengue-2 virus, administered by intracerebral inoculation. The results suggested the folding and carrier capacity of the P64k protein over the E fragment, converting PD3 as an attractive vaccine candidate against dengue-2 virus. PMID:14656459

  11. 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.

  12. DNA vaccines expressing soluble CD4-envelope proteins fused to C3d elicit cross-reactive neutralizing antibodies to HIV-1

    SciTech Connect

    Bower, Joseph F.; Green, Thomas D.; Ross, Ted M. . E-mail: tmr15@pitt.edu

    2004-10-25

    DNA vaccines expressing the envelope (Env) of the human immunodeficiency virus type 1 (HIV-1) have been relatively ineffective at generating high-titer, long-lasting, neutralizing antibodies in a variety of animal models. In this study, DNA vaccines were constructed to express a fusion protein of the soluble human CD4 (sCD4) and the gp120 subunit of the HIV-1 envelope. To enhance the immunogenicity of the expressed fusion protein, three copies of the murine C3d (mC3d{sub 3}) were added to the carboxyl terminus of the complex. Monoclonal antibodies that recognize CD4-induced epitopes on gp120 efficiently bound to sCD4-gp120 or sCD4-gp120-mC3d{sub 3}. In addition, both sCD4-gp120 and sCD4-gp120-mC3d{sub 3} bound to cells expressing appropriate coreceptors in the absence of cell surface hCD4. Mice (BALB/c) vaccinated with DNA vaccines expressing either gp120-mC3d{sub 3} or sCD4-gp120-mC3d{sub 3} elicited antibodies that neutralized homologous virus infection. However, the use of sCD4-gp120-mC3d{sub 3}-DNA elicited the highest titers of neutralizing antibodies that persisted after depletion of anti-hCD4 antibodies. Interestingly, only mice vaccinated with DNA expressing sCD4-gp120-mC3d{sub 3} had antibodies that elicited cross-protective neutralizing antibodies. The fusion of sCD4 to the HIV-1 envelope exposes neutralizing epitopes that elicit broad protective immunity when the fusion complex is coupled with the molecular adjuvant, C3d.

  13. Iron-regulated envelope proteins of mycobacteria grown in vitro and their occurrence in Mycobacterium avium and Mycobacterium leprae grown in vivo.

    PubMed

    Sritharan, M; Ratledge, C

    1990-01-01

    Several iron-regulated envelope proteins (IREPs), 11-180 kDa, have been detected in preparations of walls and membranes of Mycobacterium smegmatis, in an armadillo-derived mycobacterium (ADM) and in M. avium. The same sized proteins from M. vacae appeared under both iron-deficient and iron-sufficient growth conditions. Two larger proteins, of 240 and 250 kDa, appeared in the membranes of M. smegmatis and M. avium only when grown iron-sufficiently but were constitutively present in both ADM and M. vaccae. The IREPs from M. smegmatis were not induced under zinc-deficient growth conditions. Three of the four IREPs (14, 21 and 29 kDa) recognized in M. avium grown in vitro were also recovered from membrane fractions of the same strain grown in mice. In addition, these membranes contained both the high-molecular-mass proteins associated with iron-sufficient growth conditions. Membranes of M. leprae, recovered from infected armadillos, showed the faint presence of a possible IREP at 29 kDa and wall preparations showed the presence of a 21-kDa protein. Membranes also contained the two larger proteins at 240 and 250 kDa. An explanation for the simultaneous occurrence of both low-iron-regulated and high-iron-regulated proteins is offered. PMID:2202378

  14. The Cpx Envelope Stress Response Modifies Peptidoglycan Cross-Linking via the l,d-Transpeptidase LdtD and the Novel Protein YgaU

    PubMed Central

    Bernal-Cabas, Margarita; Ayala, Juan Alfonso

    2014-01-01

    The Cpx envelope stress response mediates a complex adaptation to conditions that cause protein misfolding in the periplasm. A recent microarray study demonstrated that Cpx response activation led to changes in the expression of genes known, or predicted, to be involved in cell wall remodeling. We sought to characterize the changes that the cell wall undergoes during activation of the Cpx pathway in Escherichia coli. Luminescent reporters of gene expression confirmed that LdtD, a putative l,d-transpeptidase; YgaU, a protein of unknown function; and Slt, a lytic transglycosylase, are upregulated in response to Cpx-inducing conditions. Phosphorylated CpxR binds to the upstream regions of these genes, which contain putative CpxR binding sites, suggesting that regulation is direct. We show that the activation of the Cpx response causes an increase in the abundance of diaminopimelic acid (DAP)-DAP cross-links that involves LdtD and YgaU. Altogether, our data indicate that changes in peptidoglycan structure are part of the Cpx-mediated adaptation to envelope stress and indicate a role for the uncharacterized gene ygaU in regulating cross-linking. PMID:25422305

  15. Evolutionary Dynamics of a Highly Pathogenic Type 2 Porcine Reproductive and Respiratory Syndrome Virus: Analyses of Envelope Protein-Coding Genes.

    PubMed

    Nguyen, V G; Kim, H K; Moon, H J; Park, S J; Chung, H C; Choi, M K; Park, B K

    2015-08-01

    Porcine reproductive and respiratory syndrome virus (PRRSV) has long been an economically devastating swine viral disease. The recent emergence of a highly pathogenic type 2 PRRSV with high mobility and mortality in China, spreading in Vietnam, Laos, and Thailand has placed neighbouring countries at risk. This study applied a codon-based extension of the Bayesian relaxed clock model and the fixed effects maximum-likelihood method to investigate and compare the evolutionary dynamics of type 2 PRRSV for all of known structural envelope protein-coding genes. By comparing the highly pathogenic type 2 PRRSV clade against the typical type 2 PRRSV clade, this study demonstrated that the highly pathogenic clade evolved at high rates in all of the known structural genes but did not display rapid evolutionary dynamics compared with typical type 2 PRRSV. In contrast, the ORF3, ORF5 and ORF6 genes of the highly pathogenic clade evolved in a qualitatively different manner from the genes of the typical clade. At the population level, several codons of the sequence elements that were involved in viral neutralization, as well as codons that were associated with in vitro attenuation/over-attenuation, were predicted to be selected differentially between the typical clade and the highly pathogenic clade. The results of this study suggest that the multigenic factors of the envelope protein-coding genes contribute to diversifying the biological properties (virulence, antigenicity, etc.) of the highly pathogenic clade compared with the typical clade of type 2 PRRSV. PMID:23981823

  16. Expression profile of key immune-related genes in Penaeus monodon juveniles after oral administration of recombinant envelope protein VP28 of white spot syndrome virus.

    PubMed

    Thomas, Ancy; Sudheer, Naduvilamuriparampu Saidumuhammed; Kiron, Viswanath; Bright Singh, Issac S; Narayanan, Rangarajan Badri

    2016-07-01

    White spot syndrome virus (WSSV) is the most catastrophic pathogen the shrimp industry has ever encountered. VP28, the abundant envelope protein of WSSV was expressed in bacteria, the purified protein administered orally to Penaeus monodon juveniles and its immune modulatory effects examined. The results indicated significant up-regulation of caspase, penaeidin, crustin, astakine, syntenin, PmRACK, Rab7, STAT and C-type lectin in animals orally administered with this antigen. This revealed the immune modulations in shrimps followed by oral administration of rVP28P which resulted in the reduced transcription of viral gene vp28 and delay in mortality after WSSV challenge. The study suggests the potential of rVP28P to elicit a non-specific immune stimulation in shrimps. PMID:27154537

  17. Multiple Envelope Stress Response Pathways Are Activated in an Escherichia coli Strain with Mutations in Two Members of the DedA Membrane Protein Family

    PubMed Central

    Sikdar, Rakesh; Simmons, Angelica R.

    2013-01-01

    We have reported that simultaneous deletion of two Escherichia coli genes, yqjA and yghB, encoding related and conserved inner membrane proteins belonging to the DedA protein family results in a number of intriguing phenotypes, including temperature sensitivity at 42°C, altered membrane lipid composition, and cell division defects. We sought to characterize these and other phenotypes in an effort to establish a function for this protein family in E. coli. Here, using reporter assays, we show that the major envelope stress response pathways Cpx, Psp, Bae, and Rcs are activated in strain BC202 (W3110; ΔyqjA ΔyghB) at the permissive growth temperature of 30°C. We previously demonstrated that 10 mM Mg2+, 400 mM NaCl, and overexpression of tatABC are capable of restoring normal growth to BC202 at elevated growth temperatures. Deletion of the cpxR gene from BC202 results in the loss of the ability of these supplements to restore growth at 42°C. Additionally, we report that the membrane potential of BC202 is significantly reduced and that cell division and growth can be restored either by expression of the multidrug transporter MdfA from a multicopy plasmid or by growth at pH 6.0. Together, these results suggest that the DedA family proteins YqjA and YghB are required for general envelope maintenance and homeostasis of the proton motive force under a variety of growth conditions. PMID:23042993

  18. Nuclear envelope-localized EGF family protein amphiregulin activates breast cancer cell migration in an EGF-like domain independent manner

    SciTech Connect

    Tanaka, Hisae; Nishioka, Yu; Yokoyama, Yuhki; Higashiyama, Shigeki; Matsuura, Nariaki; Matsuura, Shuji; Hieda, Miki

    2012-04-20

    Highlights: Black-Right-Pointing-Pointer Nuclear envelope-localized proAREG activates cancer cell migration via its cytoplasmic domain. Black-Right-Pointing-Pointer The induction of cell migration does not require the EGF-like domain or EGR function. Black-Right-Pointing-Pointer Nuclear envelope-localized proAREG suppresses breast cancer cell growth without EGFR function. Black-Right-Pointing-Pointer This study revealed a novel function mediated by the intracellular domain of proAREG. -- Abstract: Amphiregulin (AREG), an EGF family protein, is synthesized as a type I transmembrane precursor (proAREG) and expressed on the cell surface with an extracellular EGF-like domain and an intracellular short cytoplasmic tail. The ectodomain shedding yields a soluble EGF receptor ligand (soluble AREG) which binds to EGF receptor (EGFR) and concomitantly induces migration of unshed proAREG from the plasma membrane to the nuclear envelope (NE). AREG is known to play a potential role in breast cancer and has been intensively investigated as an EGF receptor ligand, while the function of the NE-localized proAREG remains unknown. In this study we used a truncated mutant that mimics NE-localized proAREG without shedding stimuli to discriminate between the functions of NE-localized and plasma membrane-localized proAREG and demonstrate that NE-localized proAREG activates breast cancer cell migration, but suppresses cell growth. Moreover, the present study shows that induction of cell migration by NE-localized proAREG does not require the extracellular growth factor domain or EGF receptor function. Collectively these data demonstrate a novel function mediated by the intracellular domain of proAREG and suggest a significant role for NE-localized proAREG in driving human breast cancer progression.

  19. Enhanced immune responses against Japanese encephalitis virus using recombinant adenoviruses coexpressing Japanese encephalitis virus envelope and porcine interleukin-6 proteins in mice.

    PubMed

    Liu, Hanyang; Wu, Rui; Liu, Kai; Yuan, Lei; Huang, Xiaobo; Wen, Yiping; Ma, Xiaoping; Yan, Qigui; Zhao, Qin; Wen, Xintian; Cao, Sanjie

    2016-08-15

    Japanese encephalitis is a reproductive disorder caused by Japanese encephalitis virus (JEV) in swine. Previous studies have demonstrated that recombinant adenovirus serotype 5 (Ad5) may be a potential vaccine candidate because it can express JEV envelope epitopes and induce immune responses against JEV. Still, it will be necessary to develop an adjuvant that can enhance both humoral and cellular immune responses to the recombinant antigen delivered by non-replicating Ad5. In this study, we investigated the systemic immune responses of BALB/c mice immunized with recombinant adenovirus expressing JEV envelope epitopes in combination with porcine interleukin-6 (rAdE-IL-6).The rAdE-IL-6 immunized group had the highest titers of anti-JEV antibody as detected by an enzyme-linked immunosorbent assay (ELISA), as well as the highest levels of neutralizing antibody (1:75) as detected by a serum neutralization test. Similarly, higher concentrations of interferon-gamma (834.7pg/ml) and interleukin-6 (IL-6) (229.7pg/ml) were detected in the rAdE-IL-6 group using an ELISA assay. These data indicate that immunized BALB/c induce a strong cellular response against rAdE-IL-6. Furthermore, after challenge with the virulent JEV SCYA201201 strain, the rAdE-IL-6 group generated an immune protective response 70% greater than that of the control group, indicating that rAdE-IL-6 induced a protective immune response against JEV challenge in mice. The results from this study demonstrated that IL-6 is a strong adjuvant that can enhance both humoral and cellular immune responses in mice. Furthermore, a recombinant adenovirus coexpressing JEV envelope epitopes and porcine IL-6 protein may be an effective vaccine in animals. PMID:27235810

  20. OlpB, a new outer layer protein of Clostridium thermocellum, and binding of its S-layer-like domains to components of the cell envelope.

    PubMed Central

    Lemaire, M; Ohayon, H; Gounon, P; Fujino, T; Béguin, P

    1995-01-01

    Several proteins of Clostridium thermocellum possess a C-terminal triplicated sequence related to bacterial cell surface proteins. This sequence was named the SLH domain (for S-layer homology), and it was proposed that it might serve to anchor proteins to the cell surface (A. Lupas, H. Engelhardt, J. Peters, U. Santarius, S. Volker, and W. Baumeister, J. Bacteriol. 176:1224-1233, 1994). This hypothesis was investigated by using the SLH-containing protein ORF1p from C. thermocellum as a model. Subcellular fractionation, immunoblotting, and electron microscopy of immunocytochemically labeled cells indicated that ORF1p was located on the surface of C. thermocellum. To detect C. thermocellum components interacting with the SLH domains of ORF1p, a probe was constructed by grafting these domains on the C terminus of the MalE protein of Escherichia coli. The SLH domains conferred on the chimeric protein (MalE-ORF1p-C) the ability to bind noncovalently to the peptidoglycan of C. thermocellum. In addition, 125I-labeled MalE-ORF1p-C was shown to bind to SLH-bearing proteins transferred onto nitrocellulose, and to a 26- to 28-kDa component of the cell envelope. These results agree with the hypothesis that SLH domains contribute to the binding of exocellular proteins to the cell surface of bacteria. The gene carrying ORF1 and its product, ORF1p, are renamed olpB and OlpB (for outer layer protein B), respectively. PMID:7730277

  1. Development of electron spin echo envelope modulation spectroscopy to probe the secondary structure of recombinant membrane proteins in a lipid bilayer.

    PubMed

    Zhang, Rongfu; Sahu, Indra D; Gibson, Kaylee R; Muhammad, Nefertiti B; Bali, Avnika P; Comer, Raven G; Liu, Lishan; Craig, Andrew F; Mccarrick, Robert M; Dabney-Smith, Carole; Sanders, Charles R; Lorigan, Gary A

    2015-11-01

    Membrane proteins conduct many important biological functions essential to the survival of organisms. However, due to their inherent hydrophobic nature, it is very difficult to obtain structural information on membrane-bound proteins using traditional biophysical techniques. We are developing a new approach to probe the secondary structure of membrane proteins using the pulsed EPR technique of Electron Spin Echo Envelope Modulation (ESEEM) Spectroscopy. This method has been successfully applied to model peptides made synthetically. However, in order for this ESEEM technique to be widely applicable to larger membrane protein systems with no size limitations, protein samples with deuterated residues need to be prepared via protein expression methods. For the first time, this study shows that the ESEEM approach can be used to probe the local secondary structure of a (2) H-labeled d8 -Val overexpressed membrane protein in a membrane mimetic environment. The membrane-bound human KCNE1 protein was used with a known solution NMR structure to demonstrate the applicability of this methodology. Three different α-helical regions of KCNE1 were probed: the extracellular domain (Val21), transmembrane domain (Val50), and cytoplasmic domain (Val95). These results indicated α-helical structures in all three segments, consistent with the micelle structure of KCNE1. Furthermore, KCNE1 was incorporated into a lipid bilayer and the secondary structure of the transmembrane domain (Val50) was shown to be α-helical in a more native-like environment. This study extends the application of this ESEEM approach to much larger membrane protein systems that are difficult to study with X-ray crystallography and/or NMR spectroscopy. PMID:26355804

  2. HIV-1 viral envelope protein gp41: An NMR investigation of dodecyl phosphocholine embedded gp41 reveals a dynamic pre-fusion intermediate conformation

    PubMed Central

    Lakomek, Nils-Alexander; Kaufman, Joshua D.; Stahl, Stephen J.; Wingfield, Paul T.

    2014-01-01

    Summary Human immunodeficiency viral (HIV-1) fusion is mediated by the viral envelope gp120/gp41 complex (ENVelope glycoprotein). After gp120 shedding, gp41 is exposed and elicits membrane fusion via a cascade of conformational changes. In contrast to pre-fusion and post-fusion conformation, little is known about any intermediate conformation. We report on a solution NMR investigation of homotrimeric HIV-1 gp4127–194, comprising the transmembrane region and reconstituted in dodecyl phosphocholine (DPC) micelles. The protein is mainly α-helical but experiences internal dynamics on the nanosecond and micro-to millisecond time scale and transient α-helical behavior for certain residues in the N-terminal heptad repeat (NHR). Strong lipid interactions are observed, in particular for C-terminal residues of the NHR and imunodominant loop region connecting NHR and C-terminal heptad repeat (CHR). Our data indicate an extended conformation with features anticipated for a pre-fusion intermediate, presumably in exchange with a lowly populated post-fusion six-helical bundle conformation. PMID:25132083

  3. Use of the quartz crystal microbalance to monitor ligand-induced conformational rearrangements in HIV-1 envelope protein gp120

    PubMed Central

    Lee, Hyun-Su; Contarino, Mark; Umashankara, M.; Schön, Arne; Freire, Ernesto; Smith, Amos B.; Chaiken, Irwin M.

    2010-01-01

    We evaluated the potential of a quartz crystal microbalance with dissipation monitoring (QCM-D) to provide a sensitive, label-free method for detecting the conformational rearrangement of glycoprotein gp120 upon binding to different ligands. This glycoprotein is normally found on the envelope of the HIV-1 virus and is involved in viral entry into host cells. It was immobilized on the surface of the sensing element of the QCM-D and was exposed to individual solutions of several different small-molecule inhibitors as well as to a solution of soluble form of the host cell receptor to which gp120 binds. Instrument responses to ligand-triggered changes were in qualitative agreement with conformational changes suggested by other biophysical methods. PMID:20016882

  4. Dengue virus envelope protein domain I/II hinge determines long-lived serotype-specific dengue immunity.

    PubMed

    Messer, William B; de Alwis, Ruklanthi; Yount, Boyd L; Royal, Scott R; Huynh, Jeremy P; Smith, Scott A; Crowe, James E; Doranz, Benjamin J; Kahle, Kristen M; Pfaff, Jennifer M; White, Laura J; Sariol, Carlos A; de Silva, Aravinda M; Baric, Ralph S

    2014-02-01

    The four dengue virus (DENV) serotypes, DENV-1, -2, -3, and -4, are endemic throughout tropical and subtropical regions of the world, with an estimated 390 million acute infections annually. Infection confers long-term protective immunity against the infecting serotype, but secondary infection with a different serotype carries a greater risk of potentially fatal severe dengue disease, including dengue hemorrhagic fever and dengue shock syndrome. The single most effective measure to control this threat to global health is a tetravalent DENV vaccine. To date, attempts to develop a protective vaccine have progressed slowly, partly because the targets of type-specific human neutralizing antibodies (NAbs), which are critical for long-term protection, remain poorly defined, impeding our understanding of natural immunity and hindering effective vaccine development. Here, we show that the envelope glycoprotein domain I/II hinge of DENV-3 and DENV-4 is the primary target of the long-term type-specific NAb response in humans. Transplantation of a DENV-4 hinge into a recombinant DENV-3 virus showed that the hinge determines the serotype-specific neutralizing potency of primary human and nonhuman primate DENV immune sera and that the hinge region both induces NAbs and is targeted by protective NAbs in rhesus macaques. These results suggest that the success of live dengue vaccines may depend on their ability to stimulate NAbs that target the envelope glycoprotein domain I/II hinge region. More broadly, this study shows that complex conformational antibody epitopes can be transplanted between live viruses, opening up similar possibilities for improving the breadth and specificity of vaccines for influenza, HIV, hepatitis C virus, and other clinically important viral pathogens. PMID:24385585

  5. Nuclear envelope: positioning nuclei and organizing synapses

    PubMed Central

    Razafsky, David; Hodzic, Didier

    2015-01-01

    The nuclear envelope plays an essential role in nuclear positioning within cells and tissues. This review highlights advances in understanding the mechanisms of nuclear positioning during skeletal muscle and central nervous system development. New findings, particularly about Atype lamins and Nesprin1, may link nuclear envelope integrity to synaptic integrity. Thus synaptic defects, rather than nuclear mispositioning, may underlie human pathologies associated with mutations of nuclear envelope proteins. PMID:26079712

  6. Quick and low cost immobilization of proteinases on polyesters: Comparison of lactobacilli cell-envelope proteinase and trypsin for protein degradation.

    PubMed

    Agyei, Dominic; Tambimuttu, Shaun; Kasargod, Bhuvana; Gao, Yuan; He, Lizhong

    2014-10-20

    Cell-envelope proteinases (CEPs) are a class of proteolytic enzymes produced by lactic acid bacteria and have several industrially relevant applications. However, soluble CEPs are economically unfavorable for such applications due to their poor stability and lack of reusability. In a quest to prepare stable biocatalysts with improved performance, CEP from Lactobacillus delbrueckii subsp. lactis 313 and trypsin (as a model enzyme) were immobilized onto nonwoven polyester fabrics in a three-step protocol including ethylenediamine activation and glutaraldehyde crosslinking. Immobilization gave protein loading yields of 21.9% (CEP) and 67.7% (trypsin) while residual activity yields were 85.6% (CEP) and 4.1% (trypsin). The activity of the immobilized enzymes was dependent on pH, but was retained at elevated temperatures (40-70 °C). An increase in Km values was observed for both enzymes after immobilization. After 70 days of storage, the immobilized CEP retained ca. 62% and 96% of initial activity when the samples were stored in a lyophilized form at -20 °C or in a buffer at 4 °C, respectively. Both immobilized CEP and trypsin were able to hydrolyze proteins such as casein, skimmed milk proteins and bovine serum albumin. This immobilization protocol can be used to prepare immobilized biocatalyst for various protein degradation processes. PMID:25128611

  7. Clupeiformes' Egg Envelope Proteins characterization: The case of Engraulis encrasicolus as a proxy for stock assessment through a novel molecular tool.

    PubMed

    Miccoli, Andrea; Leonori, Iole; Estonba, Andone; De Felice, Andrea; Piccinetti, Chiara Carla; Carnevali, Oliana

    2016-07-01

    Zona radiata proteins are essential for ensuring bactericidal resistance, oocyte nutrients uptake and functional buoyancy, sperm binding and guidance to the micropyle, and protection to the growing oocyte or embryo from the physical environment. Such glycoproteins have been characterized in terms of molecular structure, protein composition and phylogenetics in several chordate models. Nevertheless, research on teleost has not been extensive. In Clupeiformes, one of the most biologically relevant and commercially important order which accounts for over 400 species and totally contributes to more than a quarter of the world fish catch, Egg Envelope Protein (EEP) information exist only for the Clupea pallasii and Engraulis japonicus species. The European anchovy, Engraulis encrasicolus, the target of a well-consolidated fishery in the Mediterranean Sea, has been ignored until now and the interest on the Otocephala superorder has been fragmentally limited to some Cypriniformes and Gonorynchiformes, as well. The aim of the present study was to fill the ZP protein-wise gap of knowledge afflicting the understanding of the European anchovy's reproductive process and to expand the background on Clupeiformes. We cloned the five Engraulis encrasicolus' zp genes and deduced their products, determined their tissue distribution, quantified their mRNA expression throughout the reproductive cycle and provided an insight into their evolution through phylogenetic tools. Furthermore, we proposed a multivariate statistics-based method to objectively infer and/or confirm the classification of Engraulis encrasicolus' sexual maturity stages by analyzing data of zp mRNAs' relative abundance. PMID:27060425

  8. Structural analysis and immunogenicity of recombinant major envelope protein (rA27L) of buffalopox virus, a zoonotic Indian vaccinia-like virus.

    PubMed

    Kumar, Amit; Yogisharadhya, Revanaiah; Bhanuprakash, Veerakyathappa; Venkatesan, Gnanavel; Shivachandra, Sathish Bhadravati

    2015-10-01

    Buffalopox virus (BPXV), an Indian variant of vaccinia virus (VACV), is a zoonotic agent and affects buffaloes, cattle and humans. A27L is one of the conserved major immuno-dominant envelope proteins of orthopox viruses (OPVs) involved in viral entry/maturation and elicits neutralizing antibodies. In this study, the A27L gene of BPXV-Vij/96 strain encoding recombinant mature A27L (21S to E110) and C-terminal truncated A27L-LZD (21S to N84aa) proteins were cloned and over-expressed in Escherichia coli as fusion proteins. Structurally, A27L of BPXV was similar to that of VACV and found to contain four regions including a potential coiled-coil motif (CCM) in the centre (43 to 84aa). Oligomerization of recombinant A27L fusion protein (∼30 kDa) leads to the formation of dimer/trimers/tetramers under non-reducing conditions. Further, the purified rA27L protein was used for active immunization of rabbit (250 μg/rabbit) and adult mice (10 μg and 50 μg/mice) with or without adjuvants (FCA, alum and CpG). Immune response measured by using indirect-ELISA and SNT revealed a gradual increase in antigen specific serum IgG as well as neutralization antibody titers. Upon challenge with virulent BPXV strain, a protection of 60% was observed in suckling mice passively administered with anti-rA27L sera. No cross-reactivity of rA27L protein with hyperimmune sera against ORFV, GTPV, SPPV, PPRV, FMDV and BTV was noticed in indirect-ELISA. The study indicated that the rA27L protein is a safe and potential prophylactic as well as diagnostic antigen for buffalopox. PMID:26319070

  9. Humoral immune responses in humanized BLT mice immunized with West Nile virus and HIV-1 envelope proteins are largely mediated via human CD5+ B cells.

    PubMed

    Biswas, Subhabrata; Chang, Hong; Sarkis, Phuong T N; Fikrig, Erol; Zhu, Quan; Marasco, Wayne A

    2011-12-01

    BLT mice, constructed by surgical implantation of human fetal thymus-liver tissues and intravenous delivery of autologous CD34+ haematopoietic stem cells into adult non-obese diabetic/severe combined immunodeficiency mice, were evaluated for vaccine-induced humoral immune responses. Following engraftment, these mice developed a human lymphoid system; however, the majority of the peripheral human B lymphocytes displayed an immature phenotype as evidenced by surface CD10 expression. Over 50% of the human B cells in the periphery but not in the bone marrow also expressed the CD5 antigen, which is found only infrequently on mature follicular B cells in humans. A single intramuscular immunization with recombinant viral envelope antigens, e.g., HIVgp140 and West Nile Virus envelope proteins, together with the immune stimulatory KLK/ODN1a composition) [corrected] adjuvant resulted in seroconversion characterized by antigen-specific human antibodies predominantly of the IgM isotype. However, repeated booster immunizations did not induce secondary immune responses as evidenced by the lack of class switching and specific IgM levels remaining relatively unchanged. Interestingly, the peripheral CD19+  CD5+ but not the CD19+  CD5- human B lymphocytes displayed a late developing CD27+  IgM+ memory phenotype, suggesting that the CD5+ B-cell subset, previously implicated in 'natural antibody' production, may play a role in the vaccine-induced antibody response. Furthermore, human T lymphocytes from these mice demonstrated suboptimal proliferative responses and loss of co-stimulatory surface proteins ex vivo that could be partially reversed with human interleukin-2 and interleukin-7. Therefore, vaccine-induced immune responses in BLT mice resemble a T-cell-independent pathway that can potentially be modulated in vivo by the exogenous delivery of human cytokines/growth factors. PMID:22044090

  10. Mutation of the dengue virus type 2 envelope protein heparan sulfate binding sites or the domain III lateral ridge blocks replication in Vero cells prior to membrane fusion

    SciTech Connect

    Roehrig, John T.; Butrapet, Siritorn; Liss, Nathan M.; Bennett, Susan L.; Luy, Betty E.; Childers, Thomas; Boroughs, Karen L.; Stovall, Janae L.; Calvert, Amanda E.; Blair, Carol D.; Huang, Claire Y.-H.

    2013-07-05

    Using an infectious cDNA clone we engineered seven mutations in the putative heparan sulfate- and receptor-binding motifs of the envelope protein of dengue virus serotype 2, strain 16681. Four mutant viruses, KK122/123EE, E202K, G304K, and KKK305/307/310EEE, were recovered following transfection of C6/36 cells. A fifth mutant, KK291/295EE, was recovered from C6/36 cells with a compensatory E295V mutation. All mutants grew in and mediated fusion of virus-infected C6/36 cells, but three of the mutants, KK122/123EE, E202K, G304K, did not grow in Vero cells without further modification. Two Vero cell lethal mutants, KK291/295EV and KKK307/307/310EEE, failed to replicate in DC-SIGN-transformed Raji cells and did not react with monoclonal antibodies known to block DENV attachment to Vero cells. Additionally, both mutants were unable to initiate negative-strand vRNA synthesis in Vero cells by 72 h post-infection, suggesting that the replication block occurred prior to virus-mediated membrane fusion. - Highlights: • Heparan sulfate- and receptor-binding motifs of DENV2 envelope protein were mutated. • Four mutant viruses were isolated—all could fuse C6/36 cells. • Two of these mutants were lethal in Vero cells without further modification. • Lethal mutations were KK291/295EV and KKK305/307/310EEE. • Cell attachment was implicated as the replication block for both mutants.

  11. Identification and Characterization of a Major Cell Wall-Associated Iron-Regulated Envelope Protein (Irep-28) in Mycobacterium tuberculosis

    PubMed Central

    Yeruva, Veena C.; Duggirala, Sridevi; Lakshmi, V.; Kolarich, Daniel; Altmann, Friedrich; Sritharan, Manjula

    2006-01-01

    Iron limitation and the expression of mycobactin and carboxymycobactin by Mycobacterium tuberculosis are known. Here, we report how iron regulated the coordinate expression of these two siderophores and a 28-kDa cell wall-associated iron-regulated protein (Irep-28). Irep-28 is identified as the DNA-binding HU homologue HupB protein (hupB [Rv2986c]). Antibodies to this protein were detected in sera from tuberculosis patients. The location of the protein in the cell wall makes it a potential drug target. PMID:17028216

  12. Evaluation of protection induced by a dengue virus serotype 2 envelope domain III protein scaffold/DNA vaccine in non-human primates.

    PubMed

    McBurney, Sean P; Sunshine, Justine E; Gabriel, Sarah; Huynh, Jeremy P; Sutton, William F; Fuller, Deborah H; Haigwood, Nancy L; Messer, William B

    2016-06-24

    We describe the preclinical development of a dengue virus vaccine targeting the dengue virus serotype 2 (DENV2) envelope domain III (EDIII). This study provides proof-of-principle that a dengue EDIII protein scaffold/DNA vaccine can protect against dengue challenge. The dengue vaccine (EDIII-E2) is composed of both a protein particle and a DNA expression plasmid delivered simultaneously via intramuscular injection (protein) and gene gun (DNA) into rhesus macaques. The protein component can contain a maximum of 60 copies of EDIII presented on a multimeric scaffold of Geobacillus stearothermophilus E2 proteins. The DNA component is composed of the EDIII portion of the envelope gene cloned into an expression plasmid. The EDIII-E2 vaccine elicited robust antibody responses to DENV2, with neutralizing antibody responses detectable following the first boost and reaching titers of greater than 1:100,000 following the second and final boost. Vaccinated and naïve groups of macaques were challenged with DENV2. All vaccinated macaques were protected from detectable viremia by infectious assay, while naïve animals had detectable viremia for 2-7 days post-challenge. All naïve macaques had detectable viral RNA from day 2-10 post-challenge. In the EDIII-E2 group, three macaques were negative for viral RNA and three were found to have detectable viral RNA post challenge. Viremia onset was delayed and the duration was shortened relative to naïve controls. The presence of viral RNA post-challenge corresponded to a 10-30-fold boost in neutralization titers 28 days post challenge, whereas no boost was observed in the fully protected animals. Based on these results, we determine that pre-challenge 50% neutralization titers of >1:6000 correlated with sterilizing protection against DENV2 challenge in EDIII-E2 vaccinated macaques. Identification of the critical correlate of protection for the EDIII-E2 platform in the robust non-human primate model lays the groundwork for further

  13. The hemagglutinin envelope protein of canine distemper virus (CDV) confers cell tropism as illustrated by CDV and measles virus complementation analysis.

    PubMed Central

    Stern, L B; Greenberg, M; Gershoni, J M; Rozenblatt, S

    1995-01-01

    Measles virus (MV) and canine distemper virus (CDV) are morbilliviruses that cause acute illnesses and several persistent central nervous system infections in humans and in dogs, respectively. Characteristically, the cytopathic effect of these viruses is the formation of syncytia in permissive cells. In this study, a vaccinia virus expression system was used to express MV and CDV hemagglutinin (HA) and fusion (F) envelope proteins. We found that cotransfecting F and HA genes of MV or F and HA genes of CDV resulted in extensive syncytium formation in permissive cells while transfecting either F or HA alone did not. Similar experiments with heterologous pairs of proteins, CDV-F with MV-HA or MV-F with CDV-HA, caused significant cell fusion in both cases. These results indicate that in this expression system, cell fusion requires both F and HA; however, the functions of these proteins are interchangeable between the two types of morbilliviruses. Human-mouse somatic hybrids were used to determine the human chromosome conferring susceptibility to either MV and CDV. Of the 12 hybrids screened, none were sensitive to MV. Two of the hybrids containing human chromosome 19 formed syncytia following CDV infection. In addition, these two hybrids underwent cell fusion when cotransfected with CDV-F and CDV-HA (but not MV-F and MV-HA) glycoproteins by using the vaccinia virus expression system. To discover the viral component responsible for cell specificity, complementation experiments coexpressing CDV-HA with MV-F or CDV-F with MV-HA in the CDV-sensitive hybrids were performed. We found that syncytia were formed only in the presence of CDV-HA. These results support the idea that the HA protein is responsible for cell tropism. Furthermore, while the F protein is necessary for the fusion process, it is interchangeable with the F protein from other morbilliviruses. PMID:7853502

  14. Epitope map of human immunodeficiency virus type 1 gp41 derived from 47 monoclonal antibodies produced by immunization with oligomeric envelope protein.

    PubMed Central

    Earl, P L; Broder, C C; Doms, R W; Moss, B

    1997-01-01

    The biologically relevant form of the human immunodeficiency virus type 1 (HIV-1) envelope (Env) glycoprotein is oligomeric, with the major points of contact between oligomeric partners located in the ectodomain of gp41. To identify and map conserved epitopes and regions in gp41 where structure is influenced by quaternary interactions, we used a panel of 38 conformation-dependent and 9 conformation-independent anti-gp41 monoclonal antibodies (MAbs) produced by immunization of mice with oligomeric Env protein. By cross-competition experiments using these MAbs and several others previously described, six distinct antigenic determinants were identified and mapped. Three of these determinants are conformational in nature and dependent in part on Env oligomeric structure. MAbs to two of these determinants were broadly cross-reactive with Env proteins derived from primary virus strains. The prevalence of antibodies in HIV-1-positive human sera to the antigenic determinants was determined by the ability of such sera to block binding of MAbs to Env protein. Strong blocking activity that correlated with cross-reactivity was found. PMID:9060620

  15. The autographa californica multiple nucleopolyhedrovirus ODV-E56 envelope protein is required for oral infectivity and can be functionally substituted by rachiplusia ou multiple nucleopolyhedrovirus ODV-E56

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The Autographa californica multiple nucleopolyhedrovirus (AcMNPV) odv-e56 gene encodes an occlusion-derived virus (ODV)-specific envelope protein, ODV-E56. In a previous analysis, the odv-e56 gene was found to be under positive selection pressure, suggesting that it may be a determinant of viral ho...

  16. The Immunodominance Change and Protection of CD4+ T-Cell Responses Elicited by an Envelope Protein Domain III-Based Tetravalent Dengue Vaccine in Mice

    PubMed Central

    Wu, Szu-Hsien; Chiang, Chen-Yi; Hsiao, Yu-Ju; Wu, Chia-Kai; Hsieh, Chun-Hsiang; Chung, Han-Hsuan; Chong, Pele; Leng, Chih-Hsiang; Pan, Chien-Hsiung

    2015-01-01

    Dengue is the leading cause of mosquito-borne viral infections and no vaccine is available now. Envelope protein domain III (ED3) is the major target for the binding of dengue virus neutralizing antibodies; however, the ED3-specifc T-cell response is less well understood. To investigate the T-cell responses to four serotypes of dengue virus (DENV-1 to 4), we immunized mice using either a tetravalent ED3-based DNA or protein vaccine, or combined both as a DNA prime-protein boost strategy (prime-boost). A significant serotype-dependent IFN-γ or IL-4 response was observed in mice immunized with either the DNA or protein vaccine. The IFN-γ response was dominant to DENV-1 to 3, whereas the IL-4 response was dominant to DENV-4. Although the similar IgG titers for the four serotypes were observed in mice immunized with the tetravalent vaccines, the neutralizing antibody titers varied and followed the order of 2 = 3>1>4. Interestingly, the lower IFN-γ response to DENV-4 is attributable to the immunodominance change between two CD4+ T-cell epitopes; one T-cell epitope located at E349-363 of DENV-1 to 3 was more immunogenic than the DENV-4 epitope E313-327. Despite DENV-4 specific IFN-γ responses were suppressed by immunodominance change, either DENV-4-specific IFN-γ or neutralizing antibody responses were still recalled after DENV-4 challenge and contributed to virus clearance. Immunization with the prime-boost elicited both IFN-γ and neutralizing antibody responses and provided better protection than either DNA or protein immunization. Our findings shed light on how ED3-based tetravalent dengue vaccines sharpen host CD4 T-cell responses and contribute to protection against dengue virus. PMID:26714037

  17. Crystal Structure of Dengue Type 1 Envelope Protein in the Postfusion Conformation and its Implication for Receptor Binding, Membrane Fusion and Antibody Recognition

    SciTech Connect

    Nayak, V.; Dessau, M; Kucera, K; Anthony, K; Ledizet, M; Modis, Y

    2009-01-01

    Dengue virus relies on a conformational change in its envelope protein, E, to fuse the viral lipid membrane with the endosomal membrane and thereby deliver the viral genome into the cytosol. We have determined the crystal structure of a soluble fragment E (sE) of dengue virus type 1 (DEN-1). The protein is in the postfusion conformation even though it was not exposed to a lipid membrane or detergent. At the domain I-domain III interface, 4 polar residues form a tight cluster that is absent in other flaviviral postfusion structures. Two of these residues, His-282 and His-317, are conserved in flaviviruses and are part of the pH sensor that triggers the fusogenic conformational change in E, at the reduced pH of the endosome. In the fusion loop, Phe-108 adopts a distinct conformation, forming additional trimer contacts and filling the bowl-shaped concavity observed at the tip of the DEN-2 sE trimer.

  18. Sulfated Escherichia coli K5 Polysaccharide Derivatives Inhibit Dengue Virus Infection of Human Microvascular Endothelial Cells by Interacting with the Viral Envelope Protein E Domain III

    PubMed Central

    Vervaeke, Peter; Alen, Marijke; Noppen, Sam; Schols, Dominique; Oreste, Pasqua; Liekens, Sandra

    2013-01-01

    Dengue virus (DENV) is an emerging mosquito-borne pathogen that causes cytokine-mediated alterations in the barrier function of the microvascular endothelium, leading to dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). We observed that DENV (serotype 2) productively infects primary (HMVEC-d) and immortalized (HMEC-1) human dermal microvascular endothelial cells, despite the absence of well-described DENV receptors, such as dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) or the mannose receptor on the cell surface. However, heparan sulfate proteoglycans (HSPGs) were highly expressed on these cells and pre-treatment of HMEC-1 cells with heparinase II or with glycosaminoglycans reduced DENV infectivity up to 90%, suggesting that DENV uses HSPGs as attachment receptor on microvascular endothelial cells. Sulfated Escherichia coli K5 derivatives, which are structurally similar to heparin/heparan sulfate but lack anticoagulant activity, were able to block DENV infection of HMEC-1 and HMVEC-d cells in the nanomolar range. The highly sulfated K5-OS(H) and K5-N,OS(H) inhibited virus attachment and subsequent entry into microvascular endothelial cells by interacting with the viral envelope (E) protein, as shown by surface plasmon resonance (SPR) analysis using the receptor-binding domain III of the E protein. PMID:24015314

  19. Induction of antigen-specific immune responses in mice by recombinant baculovirus expressing premembrane and envelope proteins of West Nile virus

    PubMed Central

    2012-01-01

    Background West Nile Virus (WNV) is an emerging arthropod-born flavivirus with increasing distribution worldwide that is responsible for a large proportion of viral encephalitis in humans and horses. Given that there are no effective antiviral drugs available for treatment of the disease, efforts have been directed to develop vaccines to prevent WNV infection. Recently baculovirus has emerged as a novel and attractive gene delivery vehicle for mammalian cells. Results In the present study, recombinant baculoviruses expressing WNV premembrane (prM) and envelope (E) proteins under the cytomegalovirus (CMV) promoter with or without vesicular stomatitis virus glycoprotein (VSV/G) were constructed. The recombinant baculoviruses designated Bac-G-prM/E and Bac-prM/E, efficiently express E protein in mammalian cells. Intramuscular injection of the two recombinant baculoviruses (at doses of 108 or 109 PFU/mouse) induced the production of WNV-specific antibodies, neutralizing antibodies as well as gamma interferon (IFN-γ) in a dose-dependent pattern. Interestingly, the recombinant baculovirus Bac-G-prM/E was found to be a more efficient immunogen than Bac-prM/E to elicit a robust immune response upon intramuscular injection. In addition, inoculation of baculovirus resulted in the secretion of inflammatory cytokines, such as TNF-α, IL-2 and IL-6. Conclusions These recombinant baculoviruses are capable of eliciting robust humoral and cellular immune responses in mice, and may be considered as novel vaccine candidates for West Nile Virus. PMID:22799608

  20. Analysis of Rab GTPase-Activating Proteins Indicates that Rab1a/b and Rab43 Are Important for Herpes Simplex Virus 1 Secondary Envelopment

    PubMed Central

    Zenner, Helen L.; Yoshimura, Shin-ichiro; Barr, Francis A.; Crump, Colin M.

    2011-01-01

    Assembly of herpes simplex virus 1 (HSV-1) occurs in the cytoplasm, where the capsid and tegument bud into host cell membranes. It is at this point that the viral glycoproteins are incorporated into the virion, as they are located at the assembly site. We investigated the role of the Rab GTPases in coordinating the assembly process by overexpressing 37 human Rab GTPase-activating proteins (GAPs) and assessing infectious titers. Rab GTPases are key cellular regulators of membrane trafficking events that, by their membrane association and binding of effector proteins, ensure the appropriate fusion of membranes. We identified that TBC1D20 and RN-tre and their partner Rabs, Rab1a/b and Rab43, respectively, are important for virion assembly. In the absence of Rab1a/b, the viral glycoproteins are unable to traffic from the endoplasmic reticulum to the assembly compartment, and thus unenveloped particles build up in the cytoplasm. The defect resulting from Rab43 depletion is somewhat more complex, but it appears that the fragmentation and dispersal of the trans-Golgi network and associated membranes render these compartments unable to support secondary envelopment. PMID:21680502

  1. Identification of Estrogen-responsive Vitelline Envelope Protein Fragments from Rainbow Trout (Oncorhynchus mykiss) Plasma Using Mass Spectrometry

    EPA Science Inventory

    Plasma protein biomarkers associated with exposure of rainbow trout (Oncorhynchus mykiss) to 17β-estradiol were isolated and identified using novel sample preparation techniques and state-of-the-art mass spectrometry and bioinformatics approaches. Juvenile male and female trout ...

  2. 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.

  3. A fragment of the envelope protein from dengue-1 virus, fused in two different sites of the meningococcal P64k protein carrier, induces a functional immune response in mice.

    PubMed

    Hermida, Lisset; Rodríguez, Rayner; Lazo, Laura; Bernardo, Lídice; Silva, Ricardo; Zulueta, Aída; López, Carlos; Martín, Jorge; Valdés, Iris; del Rosario, Delfina; Guillén, Gerardo; Guzmán, María G

    2004-02-01

    Previously we have reported the capacity of the fusion protein PD3, composed of the P64k protein and the envelope (E) fragment from amino acids (aa) 286-426 of dengue-2 virus (DEN-2), to induce a functional immune response in mice against the homologous virus. In that case, the E fragment was inserted within the lipoyl-binding domain of the meningococcal P64k protein. In the present study, to test the functionality of the same E region from dengue-1 (DEN-1), a similar construct was made. Furthermore, another alternative of fusion protein was also constructed where the same E fragment from DEN-1 was fused to the C-terminus of the P64k protein. The recombinant proteins obtained (PD11 and PD10) were semi-purified and analysed for their antigenicity, immunogenicity and the ability to protect mice against lethal challenge. Both molecules exhibited the same recognition patterns against anti-DEN-1 polyclonal antibodies. In addition, when administered to mice, they elicited high levels of neutralizing antibodies and induced significant protection against lethal challenge with DEN-1 after intracerebral inoculation. These results reveal the availability of two sites within the P64k for the further insertion of DEN fragments, enabling a construct carrying two fragments from heterologous serotypes within the same molecule of this protein carrier. PMID:12887334

  4. An interaction site of the envelope proteins of Semliki Forest virus that is preserved after proteolytic activation

    SciTech Connect

    Zhang Xinyong; Kielian, Margaret . E-mail: kielian@aecom.yu.edu

    2005-07-05

    Semliki Forest virus (SFV) membrane fusion is mediated by the viral E1 protein at acidic pH and regulated by the dimeric interaction of E1 with the E2 membrane protein. During low pH-triggered fusion, the E2/E1 heterodimer dissociates, freeing E1 to drive membrane fusion. E2 is synthesized as a precursor, p62, which is processed to mature E2 by the cellular protease furin. Both the dissociation of the p62/E1 dimer and the fusion reaction of p62 virus have a more acidic pH threshold than that of the mature E2 virus. We have previously isolated SFV mutations that allow virus growth in furin-deficient cells. Here we have used such pci mutations to compare the interactions of the p62/E1 and E2/E1 dimers. Our data suggest that there is an important p62/E1 dimer interaction site identified by an E2 R250G mutation and that this interaction is maintained after processing to the mature E2 protein.

  5. MEPPitope: spatial, electrostatic and secondary structure perturbations in the post-fusion Dengue virus envelope protein highlights known epitopes and conserved residues in the Zika virus.

    PubMed

    Chakraborty, Sandeep

    2016-01-01

    The dramatic transformation of the Zika virus (ZIKV) from a relatively unknown virus to a pathogen generating global-wide panic has exposed the dearth of detailed knowledge about this virus. Decades of research in the related Dengue virus (DENV), finally culminating in a vaccine registered for use in endemic regions (CYD-TDV), provides key insights in developing strategies for tackling ZIKV. The previously established MEPP methodology compares two conformations of the same protein and identifies residues with significant spatial and electrostatic perturbations. In the current work, MEPP analyzed the pre-and post-fusion DENV type 2 envelope (E) protein, and identified several known epitopes (His317, Tyr299, Glu26, Arg188, etc.) (MEPPitope). These residues are overwhelmingly conserved in ZIKV and all DENV serotypes. Characterization of α-helices in E-proteins show that α1 is not conserved in the sequence space of ZIKV and DENV. Furthermore, perturbation of α1 in the post-fusion DENV structure includes a known epitope Asp215, a residue absent in the pre-fusion α1. A cationic β-sheet in the GAG-binding domain that is stereochemically equivalent in ZIKV and all DENV serotypes is also highlighted due to a residue pair (Arg286-Arg288) that has a significant electrostatic polarity reversal upon fusion. Finally, two highly conserved residues (Thr32 and Thr40), with little emphasis in existing literature, are found to have significant electrostatic perturbation. Thus, a combination of different computational methods enable the rapid and rational detection of critical residues that can be made the target of small drugs, or as epitopes in the search for an elusive therapy or vaccine that neutralizes multiple members of the Flaviviridae family. PMID:27540468

  6. Development and evaluation of a DAS-ELISA for rapid detection of Tembusu virus using monoclonal antibodies against the envelope protein.

    PubMed

    Chen, Hao; Ou, Quanbin; Tang, Yi; Gao, Xuhui; Wu, Lili; Xue, Cong; Yu, Chunmei; Cui, Jingteng; Diao, Youxiang

    2014-01-01

    Since April 2010, Tembusu virus (TMUV) which is a contagious pathogen of waterfowls, causing symptoms of high fever, loss of appetite and fall in egg production, has been reported in east of China. A double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) which detects for TMUV was developed, using two monoclonal antibodies (mAbs) against the TMUV envelope (E) protein. BALB/c mice were immunized with purified recombinant E protein expressed in E. coli. Three hybridoma cell lines designated as 12B1, 10C6 and 2D2, were screened by cell fusion and indirect ELISA for their ability to recognize different linear epitopes on the E protein, and were characterized subsequently. High-affinity mAbs 12B1 and 2D2 were used as capture and detection antibodies, respectively. The reaction conditions for the DAS-ELISA were optimized for TMUV detection. The cross-reactivity of the DAS-ELISA was determined using TMUV, duck plague virus, avian influenza virus subtype H9, Newcastle disease virus, duck hepatitis A virus type 1 and duck reovirus samples. A total of 191 homogenized tissues of field samples were simultaneously detected by DAS-ELISA and by RT-PCR. The former was found to have a high specificity of 99.1% and a sensitivity of 93.1%. These results reveal a positive coincidence between DAS-ELISA and RT-PCR at a coincidence rate of 95.8%. The method developed in this study can be used for the diagnosis of TMUV infection of duck origin. PMID:24797141

  7. Development and Evaluation of a DAS-ELISA for Rapid Detection of Tembusu Virus Using Monoclonal Antibodies against the Envelope Protein

    PubMed Central

    Chen, Hao; Ou, Quanbin; Tang, Yi; Gao, Xuhui; Wu, Lili; Xue, Cong; Yu, Chunmei; Cui, Jingteng; Diao, Youxiang

    2014-01-01

    Since April 2010, Tembusu virus (TMUV) which is a contagious pathogen of waterfowls, causing symptoms of high fever, loss of appetite and fall in egg production, has been reported in east of China. A double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) which detects for TMUV was developed, using two monoclonal antibodies (mAbs) against the TMUV envelope (E) protein. BALB/c mice were immunized with purified recombinant E protein expressed in E. coli. Three hybridoma cell lines designated as 12B1, 10C6 and 2D2, were screened by cell fusion and indirect ELISA for their ability to recognize different linear epitopes on the E protein, and were characterized subsequently. High-affinity mAbs 12B1 and 2D2 were used as capture and detection antibodies, respectively. The reaction conditions for the DAS-ELISA were optimized for TMUV detection. The cross-reactivity of the DAS-ELISA was determined using TMUV, duck plague virus, avian influenza virus subtype H9, Newcastle disease virus, duck hepatitis A virus type 1 and duck reovirus samples. A total of 191 homogenized tissues of field samples were simultaneously detected by DAS-ELISA and by RT-PCR. The former was found to have a high specificity of 99.1% and a sensitivity of 93.1%. These results reveal a positive coincidence between DAS-ELISA and RT-PCR at a coincidence rate of 95.8%. The method developed in this study can be used for the diagnosis of TMUV infection of duck origin. PMID:24797141

  8. Characterization of an antigenic site that contains a dominant, type-specific neutralization determinant on the envelope protein domain III (ED3) of dengue 2 virus

    SciTech Connect

    Gromowski, Gregory D.; Barrett, Alan D.T.

    2007-09-30

    The surface of the mature dengue virus (DENV) particle consists of 90 envelope (E) protein dimers that mediate both receptor binding and fusion. The E protein ectodomain can be divided into three structural domains designated ED1, ED2, and ED3, of which ED3 contains the critical and dominant virus-specific neutralization sites. In this study the ED3 epitopes recognized by seven, murine, IgG1 DENV-2 type-specific, monoclonal antibodies (MAbs) were determined using site-directed mutagenesis of a recombinant DENV-2 ED3 (rED3) protein. A total of 41 single amino acid substitutions were introduced into the rED3 at 30 different surface accessible residues. The affinity of each MAb with the mutant rED3s was assessed by indirect ELISA and the results indicate that all seven MAbs recognize overlapping epitopes with residues K305 and P384 critical for binding. These residues are conserved among DENV-2 strains and cluster together on the upper lateral face of ED3. A linear relationship was observed between relative occupancy of ED3 on the virion by MAb and neutralization of the majority of virus infectivity ({approx} 90%) for all seven MAbs. Depending on the MAb, it is predicted that between 10% and 50% relative occupancy of ED3 on the virion is necessary for virus neutralization and for all seven MAbs occupancy levels approaching saturation were required for 100% neutralization of virus infectivity. Overall, the conserved antigenic site recognized by all seven MAbs is likely to be a dominant DENV-2 type-specific, neutralization determinant.

  9. MEPPitope: spatial, electrostatic and secondary structure perturbations in the post-fusion Dengue virus envelope protein highlights known epitopes and conserved residues in the Zika virus

    PubMed Central

    Chakraborty, Sandeep

    2016-01-01

    The dramatic transformation of the Zika virus (ZIKV) from a relatively unknown virus to a pathogen generating global-wide panic has exposed the dearth of detailed knowledge about this virus. Decades of research in the related Dengue virus (DENV), finally culminating in a vaccine registered for use in endemic regions (CYD-TDV), provides key insights in developing strategies for tackling ZIKV. The previously established MEPP methodology compares two conformations of the same protein and identifies residues with significant spatial and electrostatic perturbations. In the current work, MEPP analyzed the pre-and post-fusion DENV type 2 envelope (E) protein, and identified several known epitopes (His317, Tyr299, Glu26, Arg188, etc.) (MEPPitope). These residues are overwhelmingly conserved in ZIKV and all DENV serotypes. Characterization of α-helices in E-proteins show that α1 is not conserved in the sequence space of ZIKV and DENV. Furthermore, perturbation of α1 in the post-fusion DENV structure includes a known epitope Asp215, a residue absent in the pre-fusion α1. A cationic β-sheet in the GAG-binding domain that is stereochemically equivalent in ZIKV and all DENV serotypes is also highlighted due to a residue pair (Arg286-Arg288) that has a significant electrostatic polarity reversal upon fusion. Finally, two highly conserved residues (Thr32 and Thr40), with little emphasis in existing literature, are found to have significant electrostatic perturbation. Thus, a combination of different computational methods enable the rapid and rational detection of critical residues that can be made the target of small drugs, or as epitopes in the search for an elusive therapy or vaccine that neutralizes multiple members of the Flaviviridae family. PMID:27540468

  10. Formalin Inactivation of Japanese Encephalitis Virus Vaccine Alters the Antigenicity and Immunogenicity of a Neutralization Epitope in Envelope Protein Domain III

    PubMed Central

    Fan, Yi-Chin; Chiu, Hsien-Chung; Chen, Li-Kuang; Chang, Gwong-Jen J.; Chiou, Shyan-Song

    2015-01-01

    Formalin-inactivated Japanese encephalitis virus (JEV) vaccines are widely available, but the effects of formalin inactivation on the antigenic structure of JEV and the profile of antibodies elicited after vaccination are not well understood. We used a panel of monoclonal antibodies (MAbs) to map the antigenic structure of live JEV virus, untreated control virus (UCV), formalin-inactivated commercial vaccine (FICV), and formalin-inactivated virus (FIV). The binding activity of T16 MAb against Nakayama-derived FICV and several strains of FIV was significantly lower compared to live virus and UCV. T16 MAb, a weakly neutralizing JEV serocomplex antibody, was found to inhibit JEV infection at the post-attachment step. The T16 epitope was mapped to amino acids 329, 331, and 389 within domain III (EDIII) of the envelope (E) glycoprotein. When we explored the effect of formalin inactivation on the immunogenicity of JEV, we found that Nakayama-derived FICV, FIV, and UCV all exhibited similar immunogenicity in a mouse model, inducing anti-JEV and anti-EDII 101/106/107 epitope-specific antibodies. However, the EDIII 329/331/389 epitope-specific IgG antibody and neutralizing antibody titers were significantly lower for FICV-immunized and FIV-immunized mouse serum than for UCV-immunized. Formalin inactivation seems to alter the antigenic structure of the E protein, which may reduce the potency of commercially available JEV vaccines. Virus inactivation by H2O2, but not by UV or by short-duration and higher temperature formalin treatment, is able to maintain the antigenic structure of the JEV E protein. Thus, an alternative inactivation method, such as H2O2, which is able to maintain the integrity of the E protein may be essential to improving the potency of inactivated JEV vaccines. PMID:26495991

  11. Identification of a cell envelope protein (MtrF) involved in hydrophobic antimicrobial resistance in Neisseria gonorrhoeae.

    PubMed

    Veal, Wendy L; Shafer, William M

    2003-01-01

    The mtrCDE-encoded efflux pump of Neisseria gonorrhoeae provides gonococci with a mechanism to resist structurally diverse antimicrobial hydrophobic agents (HAs). Strains of N. gonorrhoeae that display hypersusceptibility to HAs often contain mutations in the efflux pump genes, mtrCDE. Such strains frequently contain a phenotypically suppressed mutation in mtrR, a gene that encodes a repressor (MtrR) of mtrCDE gene expression, and one that would normally result in HA resistance. We have recently examined HA-hypersusceptible clinical isolates of gonococci that contain such phenotypically suppressed mtrR mutations, in order to determine whether genes other than mtrCDE are involved in HA resistance. These studies led to the discovery of a gene that we have designated mtrF, located downstream of the mtrR gene, that is predicted to encode a 56.1 kDa cytoplasmic membrane protein containing 12 transmembrane domains. Expression of mtrF was enhanced in a strain deficient in MtrR production, indicating that this gene, together with the closely linked mtrCDE operon, is subject to MtrR-dependent transcriptional control. Orthologues of mtrF were identified in a number of diverse bacteria. Except for the AbgT protein of Escherichia coli, their products have been identified as hypothetical proteins with unknown function(s). Genetic evidence is presented that MtrF is important in the expression of high-level detergent resistance by gonococci. We propose that MtrF acts in conjunction with the MtrC-MtrD-MtrE efflux pump, to confer on gonococci high-level resistance to certain HAs. PMID:12493784

  12. Tissue specificity in the nuclear envelope supports its functional complexity

    PubMed Central

    de las Heras, Jose I; Meinke, Peter; Batrakou, Dzmitry G; Srsen, Vlastimil; Zuleger, Nikolaj; Kerr, Alastair RW; Schirmer, Eric C

    2013-01-01

    Nuclear envelope links to inherited disease gave the conundrum of how mutations in near-ubiquitous proteins can yield many distinct pathologies, each focused in different tissues. One conundrum-resolving hypothesis is that tissue-specific partner proteins mediate these pathologies. Such partner proteins may have now been identified with recent proteome studies determining nuclear envelope composition in different tissues. These studies revealed that the majority of the total nuclear envelope proteins are tissue restricted in their expression. Moreover, functions have been found for a number these tissue-restricted nuclear envelope proteins that fit with mechanisms proposed to explain how the nuclear envelope could mediate disease, including defects in mechanical stability, cell cycle regulation, signaling, genome organization, gene expression, nucleocytoplasmic transport, and differentiation. The wide range of functions to which these proteins contribute is consistent with not only their involvement in tissue-specific nuclear envelope disease pathologies, but also tissue evolution. PMID:24213376

  13. The cell envelope proteome of Aggregatibacter actinomycetemcomitans

    PubMed Central

    Smith, Kenneth P.; Fields, Julia G.; Voogt, Richard D.; Deng, Bin; Lam, Ying-Wai; Mintz, Keith P.

    2014-01-01

    Summary The cell envelope of Gram-negative bacteria serves a critical role in maintenance of cellular homeostasis, resistance to external stress, and host-pathogen interactions. Envelope protein composition is influenced by the physiological and environmental demands placed on the bacterium. In this study, we report a comprehensive compilation of cell envelope proteins from the periodontal and systemic pathogen Aggregatibacter actinomycetemcomitans VT1169, an afimbriated serotype b strain. The urea-extracted membrane proteins were identified by mass spectrometry-based shotgun proteomics. The membrane proteome, isolated from actively growing bacteria under normal laboratory conditions, included 648 proteins representing 28% of the predicted ORFs in the genome. Bioinformatic analyses were used to annotate and predict the cellular location and function of the proteins. Surface adhesins, porins, lipoproteins, numerous influx and efflux pumps, multiple sugar, amino acid and iron transporters, and components of the type I, II and V secretion systems were identified. Periplasmic space and cytoplasmic proteins with chaperone function were also identified. 107 proteins with unknown function were associated with the cell envelope. Orthologs of a subset of these uncharacterized proteins are present in other bacterial genomes, while others are found exclusively in A. actinomycetemcomitans. This knowledge will contribute to elucidating the role of cell envelope proteins in bacterial growth and survival in the oral cavity. PMID:25055881

  14. Sequences in Gibbon Ape Leukemia Virus Envelope That Confer Sensitivity to HIV-1 Accessory Protein Vpu▿†

    PubMed Central

    Janaka, Sanath Kumar; Lucas, Tiffany M.; Johnson, Marc C.

    2011-01-01

    HIV-1 efficiently forms pseudotyped particles with many gammaretrovirus glycoproteins, such as Friend murine leukemia virus (F-MLV) Env, but not with the related gibbon ape leukemia virus (GaLV) Env or with a chimeric F-MLV Env with a GaLV cytoplasmic tail domain (CTD). This incompatibility is modulated by the HIV-1 accessory protein Vpu. Because the GaLV Env CTD does not resemble tetherin or CD4, the well-studied targets of Vpu, we sought to characterize the modular sequence in the GaLV Env CTD required for this restriction in the presence of Vpu. Using a systematic mutagenesis scan, we determined that the motif that makes GaLV Env sensitive to Vpu is INxxIxxVKxxVxRxK. This region in the CTD of GaLV Env is predicted to form a helix. Mutations in the CTD that would break this helix abolish sensitivity to Vpu. Although many of these positions can be replaced with amino acids with similar biophysical properties without disrupting the Vpu sensitivity, the final lysine residue is required. This Vpu sensitivity sequence appears to be modular, as the unrelated Rous sarcoma virus (RSV) Env can be made Vpu sensitive by replacing its CTD with the GaLV Env CTD. In addition, F-MLV Env can be made Vpu sensitive by mutating two amino acids in its cytoplasmic tail to make it resemble more closely the Vpu sensitivity motif. Surprisingly, the core components of this Vpu sensitivity sequence are also present in the host surface protein CD4, which is also targeted by Vpu through its CTD. PMID:21917962

  15. 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

  16. Structural and Biochemical Characterization of the Vaccinia Virus Envelope Protein D8 and Its Recognition by the Antibody LA5

    PubMed Central

    Matho, Michael H.; Maybeno, Matt; Benhnia, Mohammed Rafii-El-Idrissi; Becker, Danielle; Meng, Xiangzhi; Xiang, Yan; Crotty, Shane; Peters, Bjoern

    2012-01-01

    Smallpox vaccine is considered a gold standard of vaccines, as it is the only one that has led to the complete eradication of an infectious disease from the human population. B cell responses are critical for the protective immunity induced by the vaccine, yet their targeted epitopes recognized in humans remain poorly described. Here we describe the biochemical and structural characterization of one of the immunodominant vaccinia virus (VACV) antigens, D8, and its binding to the monoclonal antibody LA5, which is capable of neutralizing VACV in the presence of complement. The full-length D8 ectodomain was found to form a tetramer. We determined the crystal structure of the LA5 Fab-monomeric D8 complex at a resolution of 2.1 Å, as well as the unliganded structures of D8 and LA5-Fab at resolutions of 1.42 Å and 1.6 Å, respectively. D8 features a carbonic anhydrase (CAH) fold that has evolved to bind to the glycosaminoglycan (GAG) chondroitin sulfate (CS) on host cells. The central positively charged crevice of D8 was predicted to be the CS binding site by automated docking experiments. Furthermore, sequence alignment of various poxvirus D8 orthologs revealed that this crevice is structurally conserved. The D8 epitope is formed by 23 discontinuous residues that are spread across 80% of the D8 protein sequence. Interestingly, LA5 binds with a high-affinity lock-and-key mechanism above this crevice with an unusually large antibody-antigen interface, burying 2,434 Å2 of protein surface. PMID:22623786

  17. Highly Efficient Transfer of Chromosomes to a Broad Range of Target Cells Using Chinese Hamster Ovary Cells Expressing Murine Leukemia Virus-Derived Envelope Proteins.

    PubMed

    Suzuki, Teruhiko; Kazuki, Yasuhiro; Oshimura, Mitsuo; Hara, Takahiko

    2016-01-01

    Microcell-mediated chromosome transfer (MMCT) is an essential step for introducing chromosomes from donor cells to recipient cells. MMCT allows not only for genetic/epigenetic analysis of specific chromosomes, but also for utilization of human and mouse artificial chromosomes (HACs/MACs) as gene delivery vectors. Although the scientific demand for genome scale analyses is increasing, the poor transfer efficiency of the current method has hampered the application of chromosome engineering technology. Here, we developed a highly efficient chromosome transfer method, called retro-MMCT, which is based on Chinese hamster ovary cells expressing envelope proteins derived from ecotropic or amphotropic murine leukemia viruses. Using this method, we transferred MACs to NIH3T3 cells with 26.5 times greater efficiency than that obtained using the conventional MMCT method. Retro-MMCT was applicable to a variety of recipient cells, including embryonic stem cells. Moreover, retro-MMCT enabled efficient transfer of MAC to recipient cells derived from humans, monkeys, mice, rats, and rabbits. These results demonstrate the utility of retro-MMCT for the efficient transfer of chromosomes to various types of target cell. PMID:27271046

  18. The HIV-1 envelope protein gp120 is captured and displayed for B cell recognition by SIGN-R1(+) lymph node macrophages.

    PubMed

    Park, Chung; Arthos, James; Cicala, Claudia; Kehrl, John H

    2015-01-01

    The HIV-1 envelope protein gp120 is both the target of neutralizing antibodies and a major focus of vaccine efforts; however how it is delivered to B cells to elicit an antibody response is unknown. Here, we show that following local gp120 injection lymph node (LN) SIGN-R1(+) sinus macrophages located in interfollicular pockets and underlying SIGN-R1(+) macrophages form a cellular network that rapidly captures gp120 from the afferent lymph. In contrast, two other antigens, phycoerythrin and hen egg lysozyme, were not captured by these cells. Intravital imaging of mouse LNs revealed persistent, but transient interactions between gp120 bearing interfollicular network cells and both trafficking and LN follicle resident gp120 specific B cells. The gp120 specific, but not the control B cells repetitively extracted gp120 from the network cells. Our findings reveal a specialized LN antigen delivery system poised to deliver gp120 and likely other pathogen derived glycoproteins to B cells. PMID:26258881

  19. The HIV-1 envelope protein gp120 is captured and displayed for B cell recognition by SIGN-R1+ lymph node macrophages

    PubMed Central

    Park, Chung; Arthos, James; Cicala, Claudia; Kehrl, John H

    2015-01-01

    The HIV-1 envelope protein gp120 is both the target of neutralizing antibodies and a major focus of vaccine efforts; however how it is delivered to B cells to elicit an antibody response is unknown. Here, we show that following local gp120 injection lymph node (LN) SIGN-R1+ sinus macrophages located in interfollicular pockets and underlying SIGN-R1+ macrophages form a cellular network that rapidly captures gp120 from the afferent lymph. In contrast, two other antigens, phycoerythrin and hen egg lysozyme, were not captured by these cells. Intravital imaging of mouse LNs revealed persistent, but transient interactions between gp120 bearing interfollicular network cells and both trafficking and LN follicle resident gp120 specific B cells. The gp120 specific, but not the control B cells repetitively extracted gp120 from the network cells. Our findings reveal a specialized LN antigen delivery system poised to deliver gp120 and likely other pathogen derived glycoproteins to B cells. DOI: http://dx.doi.org/10.7554/eLife.06467.001 PMID:26258881

  20. 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

  1. Highly Efficient Transfer of Chromosomes to a Broad Range of Target Cells Using Chinese Hamster Ovary Cells Expressing Murine Leukemia Virus-Derived Envelope Proteins

    PubMed Central

    Kazuki, Yasuhiro; Oshimura, Mitsuo; Hara, Takahiko

    2016-01-01

    Microcell-mediated chromosome transfer (MMCT) is an essential step for introducing chromosomes from donor cells to recipient cells. MMCT allows not only for genetic/epigenetic analysis of specific chromosomes, but also for utilization of human and mouse artificial chromosomes (HACs/MACs) as gene delivery vectors. Although the scientific demand for genome scale analyses is increasing, the poor transfer efficiency of the current method has hampered the application of chromosome engineering technology. Here, we developed a highly efficient chromosome transfer method, called retro-MMCT, which is based on Chinese hamster ovary cells expressing envelope proteins derived from ecotropic or amphotropic murine leukemia viruses. Using this method, we transferred MACs to NIH3T3 cells with 26.5 times greater efficiency than that obtained using the conventional MMCT method. Retro-MMCT was applicable to a variety of recipient cells, including embryonic stem cells. Moreover, retro-MMCT enabled efficient transfer of MAC to recipient cells derived from humans, monkeys, mice, rats, and rabbits. These results demonstrate the utility of retro-MMCT for the efficient transfer of chromosomes to various types of target cell. PMID:27271046

  2. Dengue virus serotyping based on envelope and membrane and nonstructural protein NS1 serotype-specific capture immunoglobulin M enzyme-linked immunosorbent assays.

    PubMed

    Shu, Pei-Yun; Chen, Li-Kuang; Chang, Shu-Fen; Su, Chien-Ling; Chien, Li-Jung; Chin, Chuan; Lin, Ting-Hsiang; Huang, Jyh-Hsiung

    2004-06-01

    Envelope and membrane (E/M) and nonstructural protein NS1 serotype-specific capture Immunoglobulin M (IgM) enzyme-linked immunosorbent assays (ELISAs) were developed to differentiate four dengue virus serotypes. A total of 93 anti-dengue virus IgM-positive serum samples collected between days 5 and 45 of illness from 59 confirmed dengue patients were analyzed. The results showed that positive serotype specificity could be identified for 86.1 and 47.6% of serum samples tested for E/M-specific IgM antibodies versus 83.3 and 42.9% of serum samples tested for NS1-specific IgM antibodies from patients with primary and secondary dengue virus infections, respectively. Dual analyses with both E/M and NS1 serotype-specific capture IgM ELISAs showed that positive serotype specificity could be correctly identified for 98.6 and 61.9% of all of the primary and secondary serum samples tested, respectively. These findings suggested that E/M and NS1 serotype-specific capture IgM ELISAs have the potential to be of use in dengue virus serotyping. PMID:15184425

  3. Molecular epidemiology of dengue virus serotype 2 in the Taiwan 2002 outbreak with envelope gene and nonstructural protein 1 gene analysis.

    PubMed

    Tung, Yi-Ching; Lin, Kuei-Hsiang; Chiang, Hung-Che; Ke, Liang-Yin; Chen, Yen-Hsu; Ke, Guan-Ming; Chen, Tun-Chieh; Chou, Lee-Chiu; Lu, Po-Liang

    2008-08-01

    The genetic relationships among dengue virus serotype 2 (DEN-2) isolates from the Taiwan 2002 epidemic were studied by sequence analysis of the envelope (E) and nonstructural protein 1 (NS1) genes. A 0-0.4% divergence among 10 isolates revealed an epidemic strain in the outbreak. Phylogenetic study demonstrated that the 2002 Taiwan isolates were of the Cosmopolitan genotype, which is different from the Asian 1 and Asian 2 genotypes of Taiwan DEN-2 isolates from 1981 to 1998 and the American/Asian genotype of 2005 Taiwan isolates. Although grouping results from both E and NS1 gene sequence analyses were the same, the usage of the NS1 gene as a sequence analysis target has not been validated for the lower bootstrap support values of branches in the phylogenetic tree. Our result showing the same genotype changes in Taiwan and Philippines isolates suggests strain transfer of DEN-2 to nearby countries resulting in the same trend of genotype change. PMID:18926953

  4. Secretion of dengue virus envelope protein ectodomain from mammalian cells is dependent on domain II serotype and affects the immune response upon DNA vaccination.

    PubMed

    Slon Campos, J L; Poggianella, M; Marchese, S; Bestagno, M; Burrone, O R

    2015-11-01

    Dengue virus (DENV) is currently among the most important human pathogens and affects millions of people throughout the tropical and subtropical regions of the world. Although it has been a World Health Organization priority for several years, there is still no efficient vaccine available to prevent infection. The envelope glycoprotein (E), exposed on the surface on infective viral particles, is the main target of neutralizing antibodies. For this reason it has been used as the antigen of choice for vaccine development efforts. Here we show a detailed analysis of factors involved in the expression, secretion and folding of E ectodomain from all four DENV serotypes in mammalian cells, and how this affects their ability to induce neutralizing antibody responses in DNA-vaccinated mice. Proper folding of E domain II (DII) is essential for efficient E ectodomain secretion, with DIII playing a significant role in stabilizing soluble dimers. We also show that the level of protein secreted from transfected cells determines the strength and efficiency of antibody responses in the context of DNA vaccination and should be considered a pivotal feature for the development of E-based DNA vaccines against DENV. PMID:26358704

  5. trans-dominant interference with virus infection at two different stages by a mutant envelope protein of Friend murine leukemia virus.

    PubMed Central

    Matano, T; Odawara, T; Ohshima, M; Yoshikura, H; Iwamoto, A

    1993-01-01

    A dominant negative mutant Friend murine leukemia virus (FMLV) env gene was cloned from an immunoselected Friend erythroleukemia cell. The mutant env had a point mutation which resulted in a Cys-to-Arg substitution at the 361st amino acid in the FMLV envelope protein (Env). The mutant Env was retained in the endoplasmic reticulum (ER) and accumulated because of its slow degradation. The NIH 3T3 cells expressing the mutant env were resistant to ecotropic Moloney MLV (MoMLV) penetration, suggesting that the mutant Env traps the ecotropic MLV receptors in the ER. When the mutant env gene was transfected into and expressed in the cells persistently infected with MoMLV, the wild-type Env was trapped in the ER, and the MoMLV production was suppressed. Thus, the mutant Env accumulating in the ER trans-dominantly and efficiently interfered with the ecotropic MLV infection at both the early and the late stages. Images PMID:8445721

  6. 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

  7. Nuclear envelope precursor vesicle targeting to chromatin is stimulated by protein phosphatase 1 in Xenopus egg extracts

    SciTech Connect

    Ito, Hiromi; Koyama, Yuhei; Takano, Makoto; Ishii, Kohei; Maeno, Mitsugu; Furukawa, Kazuhiro; Horigome, Tsuneyoshi . E-mail: thori@chem.sc.niigata-u.ac.jp

    2007-05-15

    The mechanism underlying targeting of the nuclear membrane to chromatin at the end of mitosis was studied using an in vitro cell-free system comprising Xenopus egg membrane and cytosol fractions, and sperm chromatin. The mitotic phase membrane, which was separated from a mitotic phase extract of Xenopus eggs and could not bind to chromatin, became able to bind to chromatin on pretreatment with a synthetic phase cytosol fraction of Xenopus eggs. When the cytosol fraction was depleted of protein phosphatase 1 (PP1) with anti-Xenopus PP1{gamma}1 antibodies, this ability was lost. The addition of recombinant xPP1{gamma}1 to the PP1-depleted cytosol fraction restored the ability. These and other results suggested that dephosphorylation of mitotic phosphorylation sites on membranes by PP1 in the synthetic phase cytosol fraction promoted targeting of the membranes to chromatin. On the other hand, a fragment containing the chromatin-binding domain of lamin B receptor (LBR) but not emerin inhibited targeting of membrane vesicles. It was also shown that PP1 dephosphorylates a phosphate group(s) responsible for regulation of the binding of LBR to chromatin. A possible mechanism involving PP1 and LBR for the regulation of nuclear membrane targeting to chromatin was discussed.

  8. Nuclear envelope breakdown may deliver an inhibitor of protein phosphatase 1 which triggers cyclin B translation in starfish oocytes.

    PubMed

    Lapasset, Laure; Pradet-Balade, Bérengère; Lozano, Jean-Claude; Peaucellier, Gérard; Picard, André

    2005-09-01

    In vertebrates, enhanced translation of mRNAs in oocytes and early embryos entering M-phase is thought to occur through polyadenylation, involving binding, hyperphosphorylation and proteolytic degradation of Aurora-activated CPEB. In starfish, an unknown component of the oocyte nucleus is required for cyclin B synthesis following the release of G2/prophase block by hormonal stimulation. We have found that CPEB cannot be hyperphosphorylated following hormonal stimulation in starfish oocytes from which the nucleus has been removed. Activation of Aurora kinase, known to interact with protein phosphatase 1 and its specific inhibitor Inh-2, is also prevented. The microinjection of Inh-2 restores Aurora activation, CPEB hyperphosphorylation and cyclin B translation in enucleated oocytes. Nevertheless, we provide evidence that CPEB is in fact hyperphosphorylated by cdc2, without apparent involvement of Aurora or MAP kinase, and that cyclin B synthesis can be stimulated without previous degradation of phosphorylated CPEB. Thus, the regulation of cyclin B synthesis necessary for progression through meiosis can be explained by an equilibrium between CPEB phosphorylation and dephosphorylation, and both aspects of this control may rely on the sole activation of Cdc2 and subsequent nuclear breakdown. PMID:16081061

  9. Identification of immunodominant regions and linear B cell epitopes of the gE envelope protein of varicella-zoster virus.

    PubMed

    Fowler, W J; Garcia-Valcarcel, M; Hill-Perkins, M S; Murphy, G; Harper, D R; Jeffries, D J; Burns, N R; Adams, S E; Kingsman, A J; Layton, G T

    1995-12-20

    The envelope proteins of varicella-zoster virus (VZV) are highly immunogenic and one of the most abundant is glycoprotein E (gE). However, its immunodominant regions and epitopes have not been identified. In this study, using human sera from individuals with recent varicella or zoster infections, we have localized antigenic sequences of gE using recombinant hybrid Ty-virus-like particles (VLPs) carrying overlapping fragments of the gE protein. gE(1-134)-VLPs (particles carrying amino acids 1-134 of gE) and, to a lesser extent, gE(101-161)-VLPs were found to be the most antigenic when tested by Western blotting and ELISA. Other fragments of gE (spanning residues 161-623) showed weak or no antigenicity. Pepscan analysis of human sera on overlapping synthetic peptides representing residues 1-135 of gE revealed that the most antigenic region was between residues 50 and 135. Three immunodominant sequences (residues 86-105, 116-135, and, to a lesser extent, 56-75) were detected using sera from both varicella and zoster patients. All sera from varicella, but not zoster, patients reacted strongly with an epitope in peptide 66-85. Other epitopes were recognized weakly by some varicella or zoster sera. More sera need to be tested to assess the potential disease specificity of these epitopes. The neutralizing monoclonal antibody (MAb) IF-B9 reacted with residues 71-90; however, another neutralizing MAb, SG1A, which bound to both gE(1-134)-VLPs and gE(101-161)-VLPs did not bind to any peptide. The identification of immunodominant sequences of gE will help toward the development of a subunit VZV vaccine. PMID:8553555

  10. A fusion DNA vaccine encoding middle version of HBV envelope protein fused to interleukin-21 did not enhance HBV-specific immune response in mice.

    PubMed

    Zhang, Ye; Su, Wen-Jing; Wang, Jue; Bai, Xue-Fan; Huang, Chang-Xing; Lian, Jian-Qi

    2014-11-01

    DNA vaccination can generate both humoral and cellular immunity, resulting in potential prophylactic and therapeutic vaccines in variety of conditions, including hepatitis B virus (HBV) infection. Fusion of cytokine gene is one of the ways to increase the immunogenicity of DNA vaccine. Interleukin (IL)-21 has been demonstrated to play an immunomodulatory role in HBV infection. Thus, we aimed to investigate the ability of IL-21 in the regulation of middle version of HBV envelop protein (MS) DNA vaccine. Fusion plasmid encoding IL-21 linked with MS was constructed. Normal and HBV transgenic mice were immunized by plasmid. pcDNA-IL-21/S2S induced a comparable level of anti-HBs antibody and HBsAg-specific CD8+ T-cell response with pcDNA-S2S. Furthermore, the level of circulating HBsAg was decreased by induction of anti-HBs antibody and HBsAg-specific CD8+ T-cell response to both pcDNA-IL-21/S2S and pcDNA-S2S vaccination in HBV transgenic mice. Thus, immunization with DNA vaccine encoding HBV MS protein induced both T- and B-cell response by targeting the specific antigen. Furthermore, it was also revealed that MS DNA vaccination could break immune tolerance in HBV transgenic mice. But IL-21 did not strengthen immune response induced by HBV DNA immunization. Our study suggested that MS-expressing plasmid may be useful for both preventive and therapeutic methods in HBV infection. However, IL-21 does not improve the immunogenicity and efficacy of MS DNA vaccination, and thus may not be used as a therapeutic marker for chronic hepatitis B. PMID:25211639

  11. Total and Envelope Protein-Specific Antibody-Secreting Cell Response in Pediatric Dengue Is Highly Modulated by Age and Subsequent Infections

    PubMed Central

    Toro, Jessica F.; Salgado, Doris M.; Vega, Rocío; Rodríguez, Jairo A.; Rodríguez, Luz-Stella; Angel, Juana; Franco, Manuel A.; Greenberg, Harry B.; Narváez, Carlos F.

    2016-01-01

    The response of antibody-secreting cells (ASC) induced by dengue has only recently started to be characterized. We propose that young age and previous infections could be simple factors that affect this response. Here, we evaluated the primary and secondary responses of circulating ASC in infants (6–12 months old) and children (1–14 years old) infected with dengue showing different degrees of clinical severity. The ASC response was delayed and of lower magnitude in infants, compared with older children. In primary infection (PI), the total and envelope (E) protein-specific IgM ASC were dominant in infants but not in children, and a negative correlation was found between age and the number of IgM ASC (rho = −0.59, P = 0.03). However, infants with plasma dengue-specific IgG detectable in the acute phase developed an intense ASC response largely dominated by IgG and comparable to that of children with secondary infection (SI). IgM and IgG produced by ASC circulating in PI or SI were highly cross-reactive among the four serotypes. Dengue infection caused the disturbance of B cell subsets, particularly a decrease in the relative frequency of naïve B cells. Higher frequencies of total and E protein-specific IgM ASC in the infants and IgG in the children were associated with clinically severe forms of infection. Therefore, the ASC response induced by dengue is highly influenced by the age at which infection occurs and previous immune status, and its magnitude is a relevant element in the clinical outcome. These results are important in the search for correlates of protection and for determining the ideal age for vaccinating against dengue. PMID:27560782

  12. Total and Envelope Protein-Specific Antibody-Secreting Cell Response in Pediatric Dengue Is Highly Modulated by Age and Subsequent Infections.

    PubMed

    Toro, Jessica F; Salgado, Doris M; Vega, Rocío; Rodríguez, Jairo A; Rodríguez, Luz-Stella; Angel, Juana; Franco, Manuel A; Greenberg, Harry B; Narváez, Carlos F

    2016-01-01

    The response of antibody-secreting cells (ASC) induced by dengue has only recently started to be characterized. We propose that young age and previous infections could be simple factors that affect this response. Here, we evaluated the primary and secondary responses of circulating ASC in infants (6-12 months old) and children (1-14 years old) infected with dengue showing different degrees of clinical severity. The ASC response was delayed and of lower magnitude in infants, compared with older children. In primary infection (PI), the total and envelope (E) protein-specific IgM ASC were dominant in infants but not in children, and a negative correlation was found between age and the number of IgM ASC (rho = -0.59, P = 0.03). However, infants with plasma dengue-specific IgG detectable in the acute phase developed an intense ASC response largely dominated by IgG and comparable to that of children with secondary infection (SI). IgM and IgG produced by ASC circulating in PI or SI were highly cross-reactive among the four serotypes. Dengue infection caused the disturbance of B cell subsets, particularly a decrease in the relative frequency of naïve B cells. Higher frequencies of total and E protein-specific IgM ASC in the infants and IgG in the children were associated with clinically severe forms of infection. Therefore, the ASC response induced by dengue is highly influenced by the age at which infection occurs and previous immune status, and its magnitude is a relevant element in the clinical outcome. These results are important in the search for correlates of protection and for determining the ideal age for vaccinating against dengue. PMID:27560782

  13. Analysis of Epitopes on Dengue Virus Envelope Protein Recognized by Monoclonal Antibodies and Polyclonal Human Sera by a High Throughput Assay

    PubMed Central

    Lin, Hong-En; Tsai, Wen-Yang; Liu, I-Ju; Li, Pi-Chun; Liao, Mei-Ying; Tsai, Jih-Jin; Wu, Yi-Chieh; Lai, Chih-Yun; Lu, Chih-Hsuan; Huang, Jyh-Hsiung; Chang, Gwong-Jen; Wu, Han-Chung; Wang, Wei-Kung

    2012-01-01

    Background The envelope (E) protein of dengue virus (DENV) is the major target of neutralizing antibodies and vaccine development. While previous studies on domain III or domain I/II alone have reported several epitopes of monoclonal antibodies (mAbs) against DENV E protein, the possibility of interdomain epitopes and the relationship between epitopes and neutralizing potency remain largely unexplored. Methodology/Principal Findings We developed a dot blot assay by using 67 alanine mutants of predicted surface-exposed E residues as a systematic approach to identify epitopes recognized by mAbs and polyclonal sera, and confirmed our findings using a capture-ELISA assay. Of the 12 mouse mAbs tested, three recognized a novel epitope involving residues (Q211, D215, P217) at the central interface of domain II, and three recognized residues at both domain III and the lateral ridge of domain II, suggesting a more frequent presence of interdomain epitopes than previously appreciated. Compared with mAbs generated by traditional protocols, the potent neutralizing mAbs generated by a new protocol recognized multiple residues in A strand or residues in C strand/CC′ loop of DENV2 and DENV1, and multiple residues in BC loop and residues in DE loop, EF loop/F strand or G strand of DENV1. The predominant epitopes of anti-E antibodies in polyclonal sera were found to include both fusion loop and non-fusion residues in the same or adjacent monomer. Conclusions/Significance Our analyses have implications for epitope-specific diagnostics and epitope-based dengue vaccines. This high throughput method has tremendous application for mapping both intra and interdomain epitopes recognized by human mAbs and polyclonal sera, which would further our understanding of humoral immune responses to DENV at the epitope level. PMID:22235356

  14. Enhanced Immune Responses to HIV-1 Envelope Elicited by a Vaccine Regimen Consisting of Priming with Newcastle Disease Virus Expressing HIV gp160 and Boosting with gp120 and SOSIP gp140 Proteins.

    PubMed

    Khattar, Sunil K; DeVico, Anthony L; LaBranche, Celia C; Panda, Aruna; Montefiori, David C; Samal, Siba K

    2016-02-01

    Newcastle disease virus (NDV) expressing HIV-1 BaL gp160 was evaluated either alone or with monomeric BaL gp120 and BaL SOSIP gp140 protein in a prime-boost combination in guinea pigs to enhance envelope (Env)-specific humoral and mucosal immune responses. We showed that a regimen consisting of an NDV prime followed by a protein boost elicited stronger serum and mucosal Th-1-biased IgG responses and neutralizing antibody responses than NDV-only immunizations. Additionally, these responses were higher after the gp120 than after the SOSIP gp140 protein boost. PMID:26581986

  15. Cell Envelope Protein PPE68 Contributes to Mycobacterium tuberculosis RD1 Immunogenicity Independently of a 10-Kilodalton Culture Filtrate Protein and ESAT-6

    PubMed Central

    Demangel, Caroline; Brodin, Priscille; Cockle, Paul J.; Brosch, Roland; Majlessi, Laleh; Leclerc, Claude; Cole, Stewart T.

    2004-01-01

    The protective efficacy of Mycobacterium bovis BCG can be markedly augmented by stable integration of Mycobacterium tuberculosis genomic region RD1. BCG complemented with RD1 (BCG::RD1) encodes nine additional proteins. Among them, 10-kDa culture filtrate protein (CFP-10) and ESAT-6 (6-kDa early secreted antigenic target) are low-molecular-weight proteins that induce potent Th1 responses. Using pools of synthetic peptides, we have examined the potential immunogenicity of four other RD1 products (PE35, PPE68, Rv3878, and Rv3879c). PPE68, the protein encoded by rv3873, was the only one to elicit gamma interferon (IFN-γ)-producing cells in C57BL/6 mice infected with M. tuberculosis. Anti-PPE68 T cells were predominantly raised against an epitope mapped in the N-terminal end of the protein. Importantly, inactivation of rv3873 in BCG::RD1 did not modify CFP-10 and ESAT-6 secretion. Moreover, the generation of IFN-γ responses to these antigens following immunization with BCG::RD1 was independent of PPE68 expression. Taken together, these results show that PPE68 is an immunogenic product of the RD1 region, which does not interfere with the secretion and immunogenicity of CFP-10 and ESAT-6. PMID:15039340

  16. Molecular determinants of dengue virus 2 envelope protein important for virus entry in FcγRIIA-mediated antibody-dependent enhancement of infection

    SciTech Connect

    Chotiwan, Nunya; Roehrig, John T.; Schlesinger, Jacob J.; Blair, Carol D.; Huang, Claire Y.-H.

    2014-05-15

    Antibody-dependent enhancement (ADE) of infection may cause severe illness in patients suffering a secondary infection by a heterologous dengue virus (DENV) serotype. During ADE of infection, cross-reactive non- or poorly-neutralizing antibodies form infectious virus-Ab complexes with the newly infecting serotype and enhance virus infection by binding to the Fcγ receptors (FcγR) on FcγR-bearing cells. In this study, we determined that molecular determinants of DENV2 envelope protein critical for virus entry during non-ADE infection are also required for ADE infection mediated by FcγRIIA, and binding of virus-Ab complexes with FcγRIIA alone is not sufficient for ADE of infection. The FcγRIIA mainly plays an auxiliary role in concentrating the virus–Ab complex to the cell surface, and other primary cellular receptors are required for virus entry. Understanding the viral entry pathway in ADE of DENV infection will greatly facilitate rational designs of anti-viral therapeutics against severe dengue disease associated with ADE. - Highlights: • KKK305/307/310 in DENV2 E-DIII is critical for virus attachment in ADE and non-ADE infection. • Binding of DENV2–Ab complex with FcγRII alone is not sufficient for virus entry in ADE infection. • Other primary receptors were required for DENV2 internalization during FcγRII–mediated ADE. • G104 and L135 of DENV2 E are critical for virus-mediated membrane fusion. • DENV2 virus-mediated membrane fusion is required for both ADE and non-ADE infection.

  17. The nuclear pore complex protein ALADIN is anchored via NDC1 but not via POM121 and GP210 in the nuclear envelope

    SciTech Connect

    Kind, Barbara; Koehler, Katrin; Lorenz, Mike; Huebner, Angela

    2009-12-11

    The nuclear pore complex (NPC) consists of {approx}30 different proteins and provides the only sites for macromolecular transport between cytoplasm and nucleus. ALADIN was discovered as a new member of the NPC. Mutations in ALADIN are known to cause triple A syndrome, a rare autosomal recessive disorder characterized by adrenal insufficiency, alacrima, and achalasia. The function and exact location of the nucleoporin ALADIN within the NPC multiprotein complex is still unclear. Using a siRNA-based approach we downregulated the three known membrane integrated nucleoporins NDC1, GP210, and POM121 in stably expressing GFP-ALADIN HeLa cells. We identified NDC1 but not GP210 and POM121 as the main anchor of ALADIN within the NPC. Solely the depletion of NDC1 caused mislocalization of ALADIN. Vice versa, the depletion of ALADIN led also to disappearance of NDC1 at the NPC. However, the downregulation of two further membrane-integral nucleoporins GP210 and POM121 had no effect on ALADIN localization. Furthermore, we could show a direct association of NDC1 and ALADIN in NPCs by fluorescence resonance energy transfer (FRET) measurements. Based on our findings we conclude that ALADIN is anchored in the nuclear envelope via NDC1 and that this interaction gets lost, if ALADIN is mutated. The loss of integration of ALADIN in the NPC is a main pathogenetic aspect for the development of the triple A syndrome and suggests that the interaction between ALADIN and NDC1 may be involved in the pathogenesis of the disease.

  18. Lung cancer induced in mice by the envelope protein of jaagsiekte sheep retrovirus (JSRV) closely resembles lung cancer in sheep infected with JSRV

    PubMed Central

    Wootton, Sarah K; Metzger, Michael J; Hudkins, Kelly L; Alpers, Charles E; York, Denis; DeMartini, James C; Miller, A Dusty

    2006-01-01

    Background Jaagsiekte sheep retrovirus (JSRV) causes a lethal lung cancer in sheep and goats. Expression of the JSRV envelope (Env) protein in mouse lung, by using a replication-defective adeno-associated virus type 6 (AAV6) vector, induces tumors resembling those seen in sheep. However, the mouse and sheep tumors have not been carefully compared to determine if Env expression alone in mice can account for the disease features observed in sheep, or whether additional aspects of virus replication in sheep are important, such as oncogene activation following retrovirus integration into the host cell genome. Results We have generated mouse monoclonal antibodies (Mab) against JSRV Env and have used these to study mouse and sheep lung tumor histology. These Mab detect Env expression in tumors in sheep infected with JSRV from around the world with high sensitivity and specificity. Mouse and sheep tumors consisted mainly of well-differentiated adenomatous foci with little histological evidence of anaplasia, but at long times after vector exposure some mouse tumors did have a more malignant appearance typical of adenocarcinoma. In addition to epithelial cell tumors, lungs of three of 29 sheep examined contained fibroblastic cell masses that expressed Env and appeared to be separate neoplasms. The Mab also stained nasal adenocarcinoma tissue from one United States sheep, which we show was due to expression of Env from ovine enzootic nasal tumor virus (ENTV), a virus closely related to JSRV. Systemic administration of the AAV6 vector encoding JSRV Env to mice produced numerous hepatocellular tumors, and some hemangiomas and hemangiosarcomas, showing that the Env protein can induce tumors in multiple cell types. Conclusion Lung cancers induced by JSRV infection in sheep and by JSRV Env expression in mice have similar histologic features and are primarily characterized by adenomatous proliferation of peripheral lung epithelial cells. Thus it is unnecessary to invoke a role for

  19. Spectrin repeat containing nuclear envelope 1 and forkhead box protein E1 are promising markers for the detection of colorectal cancer in blood.

    PubMed

    Melotte, Veerle; Yi, Joo Mi; Lentjes, Marjolein H F M; Smits, Kim M; Van Neste, Leander; Niessen, Hanneke E C; Wouters, Kim A D; Louwagie, Joost; Schuebel, Kornel E; Herman, James G; Baylin, Stephen B; van Criekinge, Wim; Meijer, Gerrit A; Ahuja, Nita; van Engeland, Manon

    2015-02-01

    Identifying biomarkers in body fluids may improve the noninvasive detection of colorectal cancer. Previously, we identified N-Myc downstream-regulated gene 4 (NDRG4) and GATA binding protein 5 (GATA5) methylation as promising biomarkers for colorectal cancer in stool DNA. Here, we examined the utility of NDRG4, GATA5, and two additional markers [Forkhead box protein E1 (FOXE1) and spectrin repeat containing nuclear envelope 1 (SYNE1)] promoter methylation as biomarkers in plasma DNA. Quantitative methylation-specific PCR was performed on plasma DNA from 220 patients with colorectal cancer and 684 noncancer controls, divided in a training set and a test set. Receiver operating characteristic analysis was performed to measure the area under the curve of GATA5, NDRG4, SYNE1, and FOXE1 methylation. Functional assays were performed in SYNE1 and FOXE1 stably transfected cell lines. The sensitivity of NDRG4, GATA5, FOXE1, and SYNE1 methylation in all stages of colorectal cancer (154 cases, 444 controls) was 27% [95% confidence interval (CI), 20%-34%), 18% (95% CI, 12%-24%), 46% (95% CI, 38%-54%), and 47% (95% CI, 39%-55%), with a specificity of 95% (95% CI, 93%-97%), 99% (95% CI, 98%-100%), 93% (95% CI, 91%-95%), and 96% (95% CI, 94%-98%), respectively. Combining SYNE1 and FOXE1, increased the sensitivity to 56% (95% CI, 48%-64%), while the specificity decreased to 90% (95% CI, 87%-93%) in the training set and to 58% sensitivity (95% CI, 46%-70%) and 91% specificity (95% CI, 80%-100%) in a test set (66 cases, 240 controls). SYNE1 overexpression showed no major differences in cell proliferation, migration, and invasion compared with controls. Overexpression of FOXE1 significantly decreased the number of colonies in SW480 and HCT116 cell lines. Overall, our data suggest that SYNE1 and FOXE1 are promising markers for colorectal cancer detection. PMID:25538088

  20. Broadly neutralizing HIV antibodies define a glycan-dependent epitope on the pre-fusion conformation of the gp41 protein on cleaved Envelope trimers

    PubMed Central

    Falkowska, Emilia; Le, Khoa M.; Ramos, Alejandra; Doores, Katie J.; Lee, Jeong Hyun; Blattner, Claudia; Ramirez, Alejandro; Derking, Ronald; van Gils, Marit J.; Liang, Chi-Hui; Mcbride, Ryan; von Bredow, Benjamin; Shivatare, Sachin S.; Wu, Chung-Yi; Chan-Hui, Po-Ying; Liu, Yan; Feizi, Ten; Zwick, Michael B.; Koff, Wayne C.; Seaman, Michael S.; Swiderek, Kristine; Moore, John P.; Evans, David; Paulson, James C.; Wong, Chi-Huey; Ward, Andrew B.; Wilson, Ian A.; Sanders, Rogier W.; Poignard, Pascal; Burton, Dennis R.

    2014-01-01

    SUMMARY Broadly neutralizing antibodies to HIV are much sought-after (a) to guide vaccine design, both as templates and to inform on the authenticity of vaccine candidates, (b) to assist in structural studies and (c) as potential therapeutics. However, the number of targets on the viral envelope spike for such antibodies is limited. Here, we describe a set of human monoclonal antibodies that define a previously undefined target on HIV Env. The antibodies recognize a glycan-dependent epitope on the prefusion conformation of gp41 and unambiguously distinguish cleaved from uncleaved Env trimers, an important property given increasing evidence that cleavage is required for vaccine candidates that seek to mimic the functional HIV envelope spike. The availability of this set of antibodies expands the number of vaccine targets on HIV and provides reagents to characterize the native envelope spike. PMID:24768347

  1. Defining the Core Proteome of the Chloroplast Envelope Membranes

    PubMed Central

    Simm, Stefan; Papasotiriou, Dimitrios G.; Ibrahim, Mohamed; Leisegang, Matthias S.; Müller, Bernd; Schorge, Tobias; Karas, Michael; Mirus, Oliver; Sommer, Maik S.; Schleiff, Enrico

    2013-01-01

    High-throughput protein localization studies require multiple strategies. Mass spectrometric analysis of defined cellular fractions is one of the complementary approaches to a diverse array of cell biological methods. In recent years, the protein content of different cellular (sub-)compartments was approached. Despite of all the efforts made, the analysis of membrane fractions remains difficult, in that the dissection of the proteomes of the envelope membranes of chloroplasts or mitochondria is often not reliable because sample purity is not always warranted. Moreover, proteomic studies are often restricted to single (model) species, and therefore limited in respect to differential individual evolution. In this study we analyzed the chloroplast envelope proteomes of different plant species, namely, the individual proteomes of inner and outer envelope (OE) membrane of Pisum sativum and the mixed envelope proteomes of Arabidopsis thaliana and Medicago sativa. The analysis of all three species yielded 341 identified proteins in total, 247 of them being unique. 39 proteins were genuine envelope proteins found in at least two species. Based on this and previous envelope studies we defined the core envelope proteome of chloroplasts. Comparing the general overlap of the available six independent studies (including ours) revealed only a number of 27 envelope proteins. Depending on the stringency of applied selection criteria we found 231 envelope proteins, while less stringent criteria increases this number to 649 putative envelope proteins. Based on the latter we provide a map of the outer and inner envelope core proteome, which includes many yet uncharacterized proteins predicted to be involved in transport, signaling, and response. Furthermore, a foundation for the functional characterization of yet unidentified functions of the inner and OE for further analyses is provided. PMID:23390424

  2. 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

  3. Genetic diversity of koala retroviral envelopes.

    PubMed

    Xu, Wenqin; Gorman, Kristen; Santiago, Jan Clement; Kluska, Kristen; Eiden, Maribeth V

    2015-03-01

    Genetic diversity, attributable to the low fidelity of reverse transcription, recombination and mutation, is an important feature of infectious retroviruses. Under selective pressure, such as that imposed by superinfection interference, gammaretroviruses commonly adapt their envelope proteins to use alternative receptors to overcome this entry block. The first characterized koala retroviruses KoRV subgroup A (KoRV-A) were remarkable in their absence of envelope genetic variability. Once it was determined that KoRV-A was present in all koalas in US zoos, regardless of their disease status, we sought to isolate a KoRV variant whose presence correlated with neoplastic malignancies. More than a decade after the identification of KoRV-A, we isolated a second subgroup of KoRV, KoRV-B from koalas with lymphomas. The envelope proteins of KoRV-A and KoRV-B are sufficiently divergent to confer the ability to bind and employ distinct receptors for infection. We have now obtained a number of additional KoRV envelope variants. In the present studies we report these variants, and show that they differ from KoRV-A and KoRV-B envelopes in their host range and superinfection interference properties. Thus, there appears to be considerable variation among KoRVs envelope genes suggesting genetic diversity is a factor following the KoRV-A infection process. PMID:25789509

  4. 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.

  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. Comparison of Immunogenicity in Rhesus Macaques of Transmitted-Founder, HIV-1 Group M Consensus, and Trivalent Mosaic Envelope Vaccines Formulated as a DNA Prime, NYVAC, and Envelope Protein Boost

    PubMed Central

    Hulot, Sandrine L.; Korber, Bette; Giorgi, Elena E.; Vandergrift, Nathan; Saunders, Kevin O.; Balachandran, Harikrishnan; Mach, Linh V.; Lifton, Michelle A.; Pantaleo, Giuseppe; Tartaglia, Jim; Phogat, Sanjay; Jacobs, Bertram; Kibler, Karen; Perdiguero, Beatriz; Gomez, Carmen E.; Esteban, Mariano; Rosati, Margherita; Felber, Barbara K.; Pavlakis, George N.; Parks, Robert; Lloyd, Krissey; Sutherland, Laura; Scearce, Richard; Letvin, Norman L.; Seaman, Michael S.; Alam, S. Munir; Montefiori, David; Liao, Hua-Xin; Haynes, Barton F.

    2015-01-01

    ABSTRACT An effective human immunodeficiency virus type 1 (HIV-1) vaccine must induce protective antibody responses, as well as CD4+ and CD8+ T cell responses, that can be effective despite extraordinary diversity of HIV-1. The consensus and mosaic immunogens are complete but artificial proteins, computationally designed to elicit immune responses with improved cross-reactive breadth, to attempt to overcome the challenge of global HIV diversity. In this study, we have compared the immunogenicity of a transmitted-founder (T/F) B clade Env (B.1059), a global group M consensus Env (Con-S), and a global trivalent mosaic Env protein in rhesus macaques. These antigens were delivered using a DNA prime-recombinant NYVAC (rNYVAC) vector and Env protein boost vaccination strategy. While Con-S Env was a single sequence, mosaic immunogens were a set of three Envs optimized to include the most common forms of potential T cell epitopes. Both Con-S and mosaic sequences retained common amino acids encompassed by both antibody and T cell epitopes and were central to globally circulating strains. Mosaics and Con-S Envs expressed as full-length proteins bound well to a number of neutralizing antibodies with discontinuous epitopes. Also, both consensus and mosaic immunogens induced significantly higher gamma interferon (IFN-γ) enzyme-linked immunosorbent spot assay (ELISpot) responses than B.1059 immunogen. Immunization with these proteins, particularly Con-S, also induced significantly higher neutralizing antibodies to viruses than B.1059 Env, primarily to tier 1 viruses. Both Con-S and mosaics stimulated more potent CD8-T cell responses against heterologous Envs than did B.1059. Both antibody and cellular data from this study strengthen the concept of using in silico-designed centralized immunogens for global HIV-1 vaccine development strategies. IMPORTANCE There is an increasing appreciation for the importance of vaccine-induced anti-Env antibody responses for preventing HIV-1

  7. DNA vaccines encoding the envelope protein of West Nile virus lineages 1 or 2 administered intramuscularly, via electroporation and with recombinant virus protein induce partial protection in large falcons (Falco spp.).

    PubMed

    Fischer, Dominik; Angenvoort, Joke; Ziegler, Ute; Fast, Christine; Maier, Kristina; Chabierski, Stefan; Eiden, Martin; Ulbert, Sebastian; Groschup, Martin H; Lierz, Michael

    2015-01-01

    As West Nile virus (WNV) can cause lethal diseases in raptors, a vaccination prophylaxis of free-living and captive populations is desirable. In the absence of vaccines approved for birds, equine vaccines have been used in falcons, but full protection against WNV infection was not achieved. Therefore, two DNA vaccines encoding the ectodomain of the envelope protein of WNV lineages 1 and 2, respectively, were evaluated in 28 large falcons. Four different vaccination protocols were used, including electroporation and booster-injections of recombinant WNV domain III protein, before challenge with the live WNV lineage 1 strain NY99. Drug safety, plasmid shedding and antibody production were monitored during the vaccination period. Serological, virological, histological, immunohistochemical and molecular biological investigations were performed during the challenge trials. Antibody response following vaccination was low overall and lasted for a maximum of three weeks. Plasmid shedding was not detected at any time. Viremia, mortality and levels, but not duration, of oral virus shedding were reduced in all of the groups during the challenge trial compared to the non-vaccinated control group. Likewise, clinical scoring, levels of cloacal virus shedding and viral load in organs were significantly reduced in three vaccination groups. Histopathological findings associated with WNV infections (meningo-encephalitis, myocarditis, and arteritis) were present in all groups, but immunohistochemical detection of the viral antigen was reduced. In conclusion, the vaccines can be used safely in falcons to reduce mortality and clinical signs and to lower the risk of virus transmission due to decreased levels of virus shedding and viremia, but full protection was not achieved in all groups. PMID:26282836

  8. 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.

  9. 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

  10. 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.

  11. 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.

  12. An unresolved LINC in the nuclear envelope

    PubMed Central

    Torbati, Mehdi; Lele, Tanmay P; Agrawal, Ashutosh

    2016-01-01

    The nuclear envelope segregates the nucleoplasm from the cytoplasm and is a key feature of eukaryotic cells. Nuclear envelope architecture is comprised of two concentric membrane shells which fuse at multiple sites and yet maintain a uniform separation of 30–50 nm over the rest of the membrane. Studies have revealed the roles for numerous nuclear proteins in forming and maintaining the architecture of the nuclear envelope. However, there is a lack of consensus on the fundamental forces and physical mechanisms that establish the geometry. The objective of this review is to discuss recent findings in the context of membrane mechanics in an effort to define open questions and possible answers. PMID:27330571

  13. Drug design from the cryptic inhibitor envelope

    PubMed Central

    Lee, Chul-Jin; Liang, Xiaofei; Wu, Qinglin; Najeeb, Javaria; Zhao, Jinshi; Gopalaswamy, Ramesh; Titecat, Marie; Sebbane, Florent; Lemaitre, Nadine; Toone, Eric J.; Zhou, Pei

    2016-01-01

    Conformational dynamics plays an important role in enzyme catalysis, allosteric regulation of protein functions and assembly of macromolecular complexes. Despite these well-established roles, such information has yet to be exploited for drug design. Here we show by nuclear magnetic resonance spectroscopy that inhibitors of LpxC—an essential enzyme of the lipid A biosynthetic pathway in Gram-negative bacteria and a validated novel antibiotic target—access alternative, minor population states in solution in addition to the ligand conformation observed in crystal structures. These conformations collectively delineate an inhibitor envelope that is invisible to crystallography, but is dynamically accessible by small molecules in solution. Drug design exploiting such a hidden inhibitor envelope has led to the development of potent antibiotics with inhibition constants in the single-digit picomolar range. The principle of the cryptic inhibitor envelope approach may be broadly applicable to other lead optimization campaigns to yield improved therapeutics. PMID:26912110

  14. 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'.

  15. The Arabidopsis Nuclear Pore and Nuclear Envelope

    PubMed Central

    Meier, Iris; Brkljacic, Jelena

    2010-01-01

    The nuclear envelope is a double membrane structure that separates the eukaryotic cytoplasm from the nucleoplasm. The nuclear pores embedded in the nuclear envelope are the sole gateways for macromolecular trafficking in and out of the nucleus. The nuclear pore complexes assembled at the nuclear pores are large protein conglomerates composed of multiple units of about 30 different nucleoporins. Proteins and RNAs traffic through the nuclear pore complexes, enabled by the interacting activities of nuclear transport receptors, nucleoporins, and elements of the Ran GTPase cycle. In addition to directional and possibly selective protein and RNA nuclear import and export, the nuclear pore gains increasing prominence as a spatial organizer of cellular processes, such as sumoylation and desumoylation. Individual nucleoporins and whole nuclear pore subcomplexes traffic to specific mitotic locations and have mitotic functions, for example at the kinetochores, in spindle assembly, and in conjunction with the checkpoints. Mutants of nucleoporin genes and genes of nuclear transport components lead to a wide array of defects from human diseases to compromised plant defense responses. The nuclear envelope acts as a repository of calcium, and its inner membrane is populated by functionally unique proteins connected to both chromatin and—through the nuclear envelope lumen—the cytoplasmic cytoskeleton. Plant nuclear pore and nuclear envelope research—predominantly focusing on Arabidopsis as a model—is discovering both similarities and surprisingly unique aspects compared to the more mature model systems. This chapter gives an overview of our current knowledge in the field and of exciting areas awaiting further exploration. PMID:22303264

  16. Identification of the gC1qR sites for the HIV-1 viral envelope protein gp41 and the HCV core protein: Implications in viral-specific pathogenesis and therapy

    PubMed Central

    Pednekar, Lina; Valentino, Alisa; Ji, Yan; Tumma, Nithin; Valentino, Christopher; Kadoor, Adarsh; Hosszu, Kinga K.; Ramadass, Mahalakshmi; Kew, Richard R.; Kishore, Uday; Peerschke, Ellinor I.B.; Ghebrehiwet, Berhane

    2016-01-01

    A substantial body of evidence accumulated over the past 20 years supports the concept that gC1qR is a major pathogen-associated pattern recognition receptor (PRR). This conclusion is based on the fact that, a wide range of bacterial and viral ligands are able to exploit gC1qR to either suppress the host’s immune response and thus enhance their survival, or to gain access into cells to initiate disease. Of the extensive array of viral ligands that have affinity for gC1qR, the HIV-1 envelope glycoprotein gp41, and the core protein of hepatitis C virus (HCV) are of major interest as they are known to contribute to the high morbidity and mortality caused by these pathogens. While the HCV core protein binds gC1qR and suppresses T cell proliferation resulting in a significantly diminished immune response, the gp41 employs gC1qR to induce the surface expression of the NK cell ligand, NKp44L, on uninfected CD4+ T cells, thereby rendering them susceptible to autologous destruction by NKp44 receptor expressing NK cells. Because of the potential for the design of peptide-based or antibody-based therapeutic options, the present studies were undertaken to define the gC1qR interaction sites for these pathogen-associated molecular ligands. Employing a solid phase microplate-binding assay, we examined the binding of each viral ligand to wild type gC1qR and 11 gC1qR deletion mutants. The results obtained from these studies have identified two major HCV core protein sites on a domain of gC1qR comprising of residues 144–148 and 196–202. Domain 196–202 in turn, is located in the last half of the larger gC1qR segment encoded by exons IV–VI (residues 159–282), which was proposed previously to contain the site for HCV core protein. The major gC1qR site for gp41 on the other hand, was found to be in a highly conserved region encoded by exon IV and comprises of residues 174–180. Interestingly, gC1qR residues 174–180 also constitute the cell surface-binding site for soluble

  17. Identification of the gC1qR sites for the HIV-1 viral envelope protein gp41 and the HCV core protein: Implications in viral-specific pathogenesis and therapy.

    PubMed

    Pednekar, Lina; Valentino, Alisa; Ji, Yan; Tumma, Nithin; Valentino, Christopher; Kadoor, Adarsh; Hosszu, Kinga K; Ramadass, Mahalakshmi; Kew, Richard R; Kishore, Uday; Peerschke, Ellinor I B; Ghebrehiwet, Berhane

    2016-06-01

    A substantial body of evidence accumulated over the past 20 years supports the concept that gC1qR is a major pathogen-associated pattern recognition receptor (PRR). This conclusion is based on the fact that, a wide range of bacterial and viral ligands are able to exploit gC1qR to either suppress the host's immune response and thus enhance their survival, or to gain access into cells to initiate disease. Of the extensive array of viral ligands that have affinity for gC1qR, the HIV-1 envelope glycoprotein gp41, and the core protein of hepatitis C virus (HCV) are of major interest as they are known to contribute to the high morbidity and mortality caused by these pathogens. While the HCV core protein binds gC1qR and suppresses T cell proliferation resulting in a significantly diminished immune response, the gp41 employs gC1qR to induce the surface expression of the NK cell ligand, NKp44L, on uninfected CD4(+) T cells, thereby rendering them susceptible to autologous destruction by NKp44 receptor expressing NK cells. Because of the potential for the design of peptide-based or antibody-based therapeutic options, the present studies were undertaken to define the gC1qR interaction sites for these pathogen-associated molecular ligands. Employing a solid phase microplate-binding assay, we examined the binding of each viral ligand to wild type gC1qR and 11 gC1qR deletion mutants. The results obtained from these studies have identified two major HCV core protein sites on a domain of gC1qR comprising of residues 144-148 and 196-202. Domain 196-202 in turn, is located in the last half of the larger gC1qR segment encoded by exons IV-VI (residues 159-282), which was proposed previously to contain the site for HCV core protein. The major gC1qR site for gp41 on the other hand, was found to be in a highly conserved region encoded by exon IV and comprises of residues 174-180. Interestingly, gC1qR residues 174-180 also constitute the cell surface-binding site for soluble gC1qR (sgC1q

  18. Ultrastructural Localization of the Herpes Simplex Virus Type 1 UL31, UL34, and US3 Proteins Suggests Specific Roles in Primary Envelopment and Egress of Nucleocapsids

    PubMed Central

    Reynolds, Ashley E.; Wills, Elizabeth G.; Roller, Richard J.; Ryckman, Brent J.; Baines, Joel D.

    2002-01-01

    The wild-type UL31, UL34, and US3 proteins localized on nuclear membranes and perinuclear virions; the US3 protein was also on cytoplasmic membranes and extranuclear virions. The UL31 and UL34 proteins were not detected in extracellular virions. US3 deletion caused (i) virion accumulation in nuclear membrane invaginations, (ii) delayed virus production onset, and (iii) reduced peak virus titers. These data support the herpes simplex virus type 1 deenvelopment-reenvelopment model of virion egress and suggest that the US3 protein plays an important, but nonessential, role in the egress pathway. PMID:12163613

  19. Fusion of Enveloped Viruses in Endosomes.

    PubMed

    White, Judith M; Whittaker, Gary R

    2016-06-01

    Ari Helenius launched the field of enveloped virus fusion in endosomes with a seminal paper in the Journal of Cell Biology in 1980. In the intervening years, a great deal has been learned about the structures and mechanisms of viral membrane fusion proteins as well as about the endosomes in which different enveloped viruses fuse and the endosomal cues that trigger fusion. We now recognize three classes of viral membrane fusion proteins based on structural criteria and four mechanisms of fusion triggering. After reviewing general features of viral membrane fusion proteins and viral fusion in endosomes, we delve into three characterized mechanisms for viral fusion triggering in endosomes: by low pH, by receptor binding plus low pH and by receptor binding plus the action of a protease. We end with a discussion of viruses that may employ novel endosomal fusion-triggering mechanisms. A key take-home message is that enveloped viruses that enter cells by fusing in endosomes traverse the endocytic pathway until they reach an endosome that has all of the environmental conditions (pH, proteases, ions, intracellular receptors and lipid composition) to (if needed) prime and (in all cases) trigger the fusion protein and to support membrane fusion. PMID:26935856

  20. Model scattering envelopes of young stellar objects. II - Infalling envelopes

    NASA Technical Reports Server (NTRS)

    Whitney, Barbara A.; Hartmann, Lee

    1993-01-01

    We present scattered light images for models of young stellar objects surrounded by dusty envelopes. The envelopes are assumed to have finite angular momentum and are falling in steady flow onto a disk. The model envelopes include holes, such as might be created by energetic bipolar flows. We calculate images using the Monte Carlo method to follow the light scattered in the dusty envelope and circumstellar disk, assuming that the photons originate from the central source. Adopting typical interstellar medium dust opacities and expected mass infall rates for protostars of about 10 exp -6 solar mass/yr, we find that detectable amounts of optical radiation can escape from envelopes falling into a disk as small as about 10-100 AU, depending upon the viewing angle and the size of the bipolar flow cavity. We suggest that the extended optical and near-IR light observed around several young stars is scattered by dusty infalling envelopes rather than disks.

  1. Fluorescence molecular painting of enveloped viruses.

    PubMed

    Metzner, Christoph; Kochan, Feliks; Dangerfield, John A

    2013-01-01

    In this study, we describe a versatile, flexible, and quick method to label different families of enveloped viruses with glycosylphosphatidylinositol-modified green fluorescent protein, termed fluorescence molecular painting (FMP). As an example for a potential application, we investigated virus attachment by means of flow cytometry to determine if viral binding behavior may be analyzed after FMP of enveloped viruses. Virus attachment was inhibited by using either dextran sulfate or by blocking attachment sites with virus pre-treatment. Results from the FMP-flow cytometry approach were verified by immunoblotting and enzyme-linked immunosorbent assay. Since the modification strategy is applicable to a broad range of proteins and viruses, variations of this method may be useful in a range of research and applied applications from bio-distribution studies to vaccine development and targeted infection for gene delivery. PMID:23104232

  2. Refrigerated cryogenic envelope

    DOEpatents

    Loudon, John D.

    1976-11-16

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

  3. Identification of two Isoforms of Vitelline Envelope Protein as Complementary Biomarkers to Vitellogenin in the Plasma of Rainbow Trout Exposed to 17beta-estradiol

    EPA Science Inventory

    In the present study, protein markers of estrogenic exposure in rainbow trout (Oncorhynchus mykiss) were isolated and identified using innovative sample preparation techniques followed by advanced MS and bioinformatics approaches. Juvenile trout were administered 17ß-estradiol t...

  4. Light-induced Changes of the Carotenoid Levels in Chloroplast Envelopes 1

    PubMed Central

    Siefermann-Harms, Dorothea; Joyard, Jacques; Douce, Roland

    1978-01-01

    The carotenoid content of thylakoids and envelopes isolated from dark-or light-treated spinach (Spinacia oleracea L.) chloroplasts was compared. In thylakoids, light induced a decrease of violaxanthin parallel with a stoichiometric increase of zeaxanthin due to violaxanthin deepoxidation. In envelopes, violaxanthin was also decreased and the relative decrease was similar to thylakoids, but zeaxanthin increase was small resulting in an over-all decrease of the amount of envelope carotenoids. When violaxanthin deepoxidation in thylakoids was partly inhibited by 10 nm nigericin, violaxanthin decrease in the envelope was inhibited to a similar degree. These observations together with the absence of deepoxidase activity in isolated envelopes plus added stroma proteins suggest that light-induced violaxanthin decrease in the envelope is not caused by an envelope or stroma deepoxidase but results from violaxanthin exchange between envelope and thylakoids. PMID:16660330

  5. Replicating Adenovirus-Simian Immunodeficiency Virus (SIV) Recombinant Priming and Envelope Protein Boosting Elicits Localized, Mucosal IgA Immunity in Rhesus Macaques Correlated with Delayed Acquisition following a Repeated Low-Dose Rectal SIVmac251 Challenge

    PubMed Central

    Xiao, Peng; Patterson, L. Jean; Kuate, Seraphin; Brocca-Cofano, Egidio; Thomas, Michael A.; Venzon, David; Zhao, Jun; DiPasquale, Janet; Fenizia, Claudio; Lee, Eun Mi; Kalisz, Irene; Kalyanaraman, Vaniambadi S.; Pal, Ranajit; Montefiori, David; Keele, Brandon F.

    2012-01-01

    We have shown that sequential replicating adenovirus type 5 host range mutant human immunodeficiency virus/simian immunodeficiency virus (HIV/SIV) recombinant priming delivered first intranasally (i.n.) plus orally and then intratracheally (i.t.), followed by envelope protein boosting, elicits broad cellular immunity and functional, envelope-specific serum and mucosal antibodies that correlate with protection from high-dose SIV and simian/human immunodeficiency virus (SHIV) challenges in rhesus macaques. Here we extended these studies to compare the standard i.n./i.t. regimen with additional mucosal administration routes, including sublingual, rectal, and vaginal routes. Similar systemic cellular and humoral immunity was elicited by all immunization routes. Central and effector memory T cell responses were also elicited by the four immunization routes in bronchoalveolar lavage fluid and jejunal, rectal, and vaginal tissue samples. Cellular responses in vaginal tissue were more compartmentalized, being induced primarily by intravaginal administration. In contrast, all immunization routes elicited secretory IgA (sIgA) responses at multiple mucosal sites. Following a repeated low-dose intrarectal (i.r.) challenge with SIVmac251 at a dose transmitting one or two variants, protection against acquisition was not achieved except in one macaque in the i.r. immunized group. All immunized macaques exhibited reduced peak viremia compared to that of controls, correlated inversely with prechallenge serum antienvelope avidity, antibody-dependent cellular cytotoxicity (ADCC) titers, and percent antibody-dependent cell-mediated viral inhibition. Both antibody avidity and ADCC titers were correlated with the number of exposures required for infection. Notably, we show for the first time a significant correlation of vaccine-induced sIgA titers in rectal secretions with delayed acquisition. Further investigation of the characteristics and properties of the sIgA should elucidate the

  6. Effects of Deletion and Overexpression of the Autographa californica Nuclear Polyhedrosis Virus FP25K Gene on Synthesis of Two Occlusion-Derived Virus Envelope Proteins and Their Transport into Virus-Induced Intranuclear Membranes

    PubMed Central

    Rosas-Acosta, Germán; Braunagel, Sharon C.; Summers, Max D.

    2001-01-01

    Partial deletions within Autographa californica open reading frame 61 (FP25K) alter the expression and accumulation profile of several viral proteins and the transport of occlusion-derived virus (ODV)-E66 to intranuclear membranes during infection (S. C. Braunagel et al., J. Virol. 73:8559–8570, 1999). Here we show the effects of a full deletion and overexpression of FP25K on the transport and expression of two ODV envelope proteins, ODV-E66 (E66) and ODV-E25 (E25). Deletion and overexpression of FP25K substantially altered the levels of expression of E66 during infection. Compared with cells infected with wild-type (wt) virus, the levels of E66 were reduced fivefold in cells infected with a viral mutant lacking FP25K (ΔFP25K) and were slightly increased in cells infected with a viral mutant overexpressing FP25K (FP25Kpolh). In contrast, no significant changes were observed in the levels of E25 among wt-, ΔFP25K-, and FP25Kpolh-infected cells. The changes observed in the levels of E66 among the different viral mutants were not accompanied by changes in either the time of synthesis, membrane association, protein turnover, or steady-state transcript abundance. Deletion of FP25K also substantially altered the transport and localization of E66 during infection. In cells infected with the ΔFP25K mutant virus, E66 accumulated in localized regions at the nuclear periphery and the outer nuclear membrane and did not traffic to intranuclear membranes. In contrast, in cells infected with the FP25Kpolh mutant virus E66 trafficked to intranuclear membranes. For comparison, E25 was normally transported to intranuclear membranes in both ΔFP25K- and FP25Kpolh-infected cells. Altogether these studies suggest that FP25K affects the synthesis of E66 at a posttranscriptional level, probably by altering the translation of E66; additionally, the block in transport of E66 at the nuclear envelope in ΔFP25K-infected cells suggests that the pathway of E66 trafficking to the inner

  7. Solubilization and reconstitution of vesicular stomatitis virus envelope using octylglucoside.

    PubMed Central

    Paternostre, M; Viard, M; Meyer, O; Ghanam, M; Ollivon, M; Blumenthal, R

    1997-01-01

    Reconstituted vesicular stomatitis virus envelopes or virosomes are formed by detergent removal from solubilized intact virus. We have monitored the solubilization process of the intact vesicular stomatitis virus by the nonionic surfactant octylglucoside at various initial virus concentrations by employing turbidity measurements. This allowed us to determine the phase boundaries between the membrane and the mixed micelles domains. We have also characterized the lipid and protein content of the solubilized material and of the reconstituted envelope. Both G and M proteins and all of the lipids of the envelope were extracted by octylglucoside and recovered in the reconstituted envelope. Fusion activity of the virosomes tested either on Vero cells or on liposomes showed kinetics and pH dependence similar to those of the intact virus. Images FIGURE 4 PMID:9083672

  8. Border Safety: Quality Control at the Nuclear Envelope.

    PubMed

    Webster, Brant M; Lusk, C Patrick

    2016-01-01

    The unique biochemical identity of the nuclear envelope confers its capacity to establish a barrier that protects the nuclear compartment and directly contributes to nuclear function. Recent work uncovered quality control mechanisms employing the endosomal sorting complexes required for transport (ESCRT) machinery and a new arm of endoplasmic reticulum-associated protein degradation (ERAD) to counteract the unfolding, damage, or misassembly of nuclear envelope proteins and ensure the integrity of the nuclear envelope membranes. Moreover, cells have the capacity to recognize and triage defective nuclear pore complexes to prevent their inheritance and preserve the longevity of progeny. These mechanisms serve to highlight the diverse strategies used by cells to maintain nuclear compartmentalization; we suggest they mitigate the progression and severity of diseases associated with nuclear envelope malfunction such as the laminopathies. PMID:26437591

  9. The neurovirulence and neuroinvasiveness of chimeric tick-borne encephalitis/dengue virus can be attenuated by introducing defined mutations into the envelope and NS5 protein genes and the 3' non-coding region of the genome

    SciTech Connect

    Engel, Amber R.; Rumyantsev, Alexander A.; Maximova, Olga A.; Speicher, James M.; Heiss, Brian; Murphy, Brian R.; Pletnev, Alexander G.

    2010-09-15

    Tick-borne encephalitis (TBE) is a severe disease affecting thousands of people throughout Eurasia. Despite the use of formalin-inactivated vaccines in endemic areas, an increasing incidence of TBE emphasizes the need for an alternative vaccine that will induce a more durable immunity against TBE virus (TBEV). The chimeric attenuated virus vaccine candidate containing the structural protein genes of TBEV on a dengue virus genetic background (TBEV/DEN4) retains a high level of neurovirulence in both mice and monkeys. Therefore, attenuating mutations were introduced into the envelope (E{sub 315}) and NS5 (NS5{sub 654,655}) proteins, and into the 3' non-coding region ({Delta}30) of TBEV/DEN4. The variant that contained all three mutations (v{Delta}30/E{sub 315}/NS5{sub 654,655}) was significantly attenuated for neuroinvasiveness and neurovirulence and displayed a reduced level of replication and virus-induced histopathology in the brains of mice. The high level of safety in the central nervous system indicates that v{Delta}30/E{sub 315}/NS5{sub 654,655} should be further evaluated as a TBEV vaccine.

  10. MEASUREMENT OF ESTROGEN-INDUCED VITELLOGENIN AND VITELLINE ENVELOPE PROTEIN MRNA IN SHEEPSHEAD MINNOW (CYPRINODON VARIEGATUS) USING QUANTITATIVE REAL-TIME PCR TECHNOLOGY

    EPA Science Inventory

    Many environmentally persistent chemicals found in both European and U.S. waterways can act as estrogens by binding to estrogen receptors and modifying the expression of genes regulated by endogenous estrogens. Synthesis of female-specific proteins (Vitellogenin [VTG], vitelline ...

  11. The Lack of the Essential LptC Protein in the Trans-Envelope Lipopolysaccharide Transport Machine Is Circumvented by Suppressor Mutations in LptF, an Inner Membrane Component of the Escherichia coli Transporter

    PubMed Central

    Benedet, Mattia; Falchi, Federica A.; Puccio, Simone; Di Benedetto, Cristiano; Peano, Clelia; Polissi, Alessandra

    2016-01-01

    The lipopolysaccharide (LPS) transport (Lpt) system is responsible for transferring LPS from the periplasmic surface of the inner membrane (IM) to the outer leaflet of the outer membrane (OM), where it plays a crucial role in OM selective permeability. In E. coli seven essential proteins are assembled in an Lpt trans-envelope complex, which is conserved in γ-Proteobacteria. LptBFG constitute the IM ABC transporter, LptDE form the OM translocon for final LPS delivery, whereas LptC, an IM-anchored protein with a periplasmic domain, interacts with the IM ABC transporter, the periplasmic protein LptA, and LPS. Although essential, LptC can tolerate several mutations and its role in LPS transport is unclear. To get insights into the functional role of LptC in the Lpt machine we searched for viable mutants lacking LptC by applying a strong double selection for lptC deletion mutants. Genome sequencing of viable ΔlptC mutants revealed single amino acid substitutions at a unique position in the predicted large periplasmic domain of the IM component LptF (LptFSupC). In complementation tests, lptFSupC mutants suppress lethality of both ΔlptC and lptC conditional expression mutants. Our data show that mutations in a specific residue of the predicted LptF periplasmic domain can compensate the lack of the essential protein LptC, implicate such LptF domain in the formation of the periplasmic bridge between the IM and OM complexes, and suggest that LptC may have evolved to improve the performance of an ancestral six-component Lpt machine. PMID:27529623

  12. Anisotropic charged core envelope star

    NASA Astrophysics Data System (ADS)

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

    2016-08-01

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

  13. The Cholera Toxin B Subunit (CTB) Fused to the Porcine Arterivirus Matrix M and GP5 Envelope Proteins Fails to Enhance the GP5-Specific Antibody Response in Pigs Immunized with Adenovectors.

    PubMed

    Roques, Elodie; Lessard, Martin; Archambault, Denis

    2015-08-01

    The porcine reproductive and respiratory syndrome virus (PRRSV) is an arterivirus of the Arteriviridae family. As the current commercial vaccines are incompletely protective effective against PRRSV infection, we developed a vaccine strategy using replicating but non-disseminating adenovectors (rAdVs) expressing the PRRSV M matrix protein in fusion with the neutralizing major epitope-carrying GP5 envelope protein (Roques et al. in Vet Res 44:17, 2013). Although production of GP5-specific antibodies (Abs) was observed, no PRRSV-specific neutralizing Abs (NAbs) were induced in pigs given the rAdVs expressing M-GP5 or M-GP5m (GP5m being a mutant form of GP5). Nevertheless, partial protection was observed in the M-GP5m-rAdV-inoculated pigs experimentally infected with PRRSV. Here, we determined the impact of the cholera toxin B subunit (CTB, known for its adjuvant effect) in fusion with the C-terminus of M-GP5m on the Ab response to PRRSV. Three-week-old pigs were immunized twice both intramuscularly and intranasally at 3-week intervals with rAdV-expressing the green fluorescent protein (rAdV-GFP), rAdV-M-GP5m, or rAdV-M-GP5m-CTB. Pigs immunized with rAdV-M-GP5m showed a high level of serum GP5-specific Abs (as determined by an indirect ELISA). In contrast, CTB in fusion with M-GP5m had an unexpected severe negative impact on GP5-specific Ab production. PRRSV-specific NAbs could not be detected in any pigs of all groups. PMID:25801418

  14. DltABCD- and MprF-Mediated Cell Envelope Modifications of Staphylococcus aureus Confer Resistance to Platelet Microbicidal Proteins and Contribute to Virulence in a Rabbit Endocarditis Model

    PubMed Central

    Weidenmaier, Christopher; Peschel, Andreas; Kempf, Volkhard A. J.; Lucindo, Natalie; Yeaman, Michael R.; Bayer, Arnold S.

    2005-01-01

    The DltABCD and MprF proteins contribute a net positive charge to the Staphylococcus aureus surface envelope by alanylating and lysinylating teichoic acids and membrane phosphatidylglycerol, respectively. These surface charge modifications are associated with increased in vitro resistance profiles of S. aureus to a number of endogenous cationic antimicrobial peptides (CAPs), such as α-defensins. The current study investigated the effects of dltA and mprF mutations on the following host factors relevant to endovascular infections: (i) in vitro susceptibility to the CAP thrombin-induced platelet microbicidal protein 1 (tPMP-1), (ii) in vitro adherence to endothelial cells (EC) and matrix proteins, and (iii) in vivo virulence in an endovascular infection model (rabbit endocarditis) in which tPMP-1 is felt to play a role in limiting S. aureus pathogenesis. Both mutations resulted in substantial increases in the in vitro susceptibility to tPMP-1 compared to that of the parental strain. The dltA (but not the mprF) mutation resulted in a significantly reduced capacity to bind to EC in vitro, while neither mutation adversely impacted in vitro binding to fibronectin, fibrinogen, or platelets. In vivo, both mutations significantly attenuated virulence in terms of early colonization of sterile vegetations and subsequent proliferation at this site (versus the parental strain). However, only the dltA mutation significantly reduced metastatic infections in kidneys and spleens compared to those in animals infected with the parental strain. These data underscore the importance of resistance to distinct CAPs and of teichoic acid-dependent EC interactions in the context of endovascular infection pathogenesis. PMID:16299297

  15. Genomic Methylation Inhibits Expression of Hepatitis B Virus Envelope Protein in Transgenic Mice: A Non-Infectious Mouse Model to Study Silencing of HBV Surface Antigen Genes

    PubMed Central

    Graumann, Franziska; Churin, Yuri; Tschuschner, Annette; Reifenberg, Kurt; Glebe, Dieter; Roderfeld, Martin; Roeb, Elke

    2015-01-01

    Objective The Hepatitis B virus genome persists in the nucleus of virus infected hepatocytes where it serves as template for viral mRNA synthesis. Epigenetic modifications, including methylation of the CpG islands contribute to the regulation of viral gene expression. The present study investigates the effects of spontaneous age dependent loss of hepatitis B surface protein- (HBs) expression due to HBV-genome specific methylation as well as its proximate positive effects in HBs transgenic mice. Methods Liver and serum of HBs transgenic mice aged 5–33 weeks were analyzed by Western blot, immunohistochemistry, serum analysis, PCR, and qRT-PCR. Results From the third month of age hepatic loss of HBs was observed in 20% of transgenic mice. The size of HBs-free area and the relative number of animals with these effects increased with age and struck about 55% of animals aged 33 weeks. Loss of HBs-expression was strongly correlated with amelioration of serum parameters ALT and AST. In addition lower HBs-expression went on with decreased ER-stress. The loss of surface protein expression started on transcriptional level and appeared to be regulated epigenetically by DNA methylation. The amount of the HBs-expression correlated negatively with methylation of HBV DNA in the mouse genome. Conclusions Our data suggest that methylation of specific CpG sites controls gene expression even in HBs-transgenic mice with truncated HBV genome. More important, the loss of HBs expression and intracellular aggregation ameliorated cell stress and liver integrity. Thus, targeted modulation of HBs expression may offer new therapeutic approaches. Furthermore, HBs-transgenic mice depict a non-infectious mouse model to study one possible mechanism of HBs gene silencing by hypermethylation. PMID:26717563

  16. Antiretrovirals, Methamphetamine, and HIV-1 Envelope Protein gp120 Compromise Neuronal Energy Homeostasis in Association with Various Degrees of Synaptic and Neuritic Damage

    PubMed Central

    Sanchez, Ana B.; Varano, Giuseppe P.; de Rozieres, Cyrus M.; Maung, Ricky; Catalan, Irene C.; Dowling, Cari C.; Sejbuk, Natalia E.; Hoefer, Melanie M.

    2015-01-01

    HIV-1 infection frequently causes HIV-associated neurocognitive disorders (HAND) despite combination antiretroviral therapy (cART). Evidence is accumulating that components of cART can themselves be neurotoxic upon long-term exposure. In addition, abuse of psychostimulants, such as methamphetamine, seems to aggravate HAND and compromise antiretroviral therapy. However, the combined effect of virus and recreational and therapeutic drugs on the brain is poorly understood. Therefore, we exposed mixed neuronal-glial cerebrocortical cells to antiretrovirals (ARVs) (zidovudine [AZT], nevirapine [NVP], saquinavir [SQV], and 118-D-24) of four different pharmacological categories and to methamphetamine and, in some experiments, the HIV-1 gp120 protein for 24 h and 7 days. Subsequently, we assessed neuronal injury by fluorescence microscopy, using specific markers for neuronal dendrites and presynaptic terminals. We also analyzed the disturbance of neuronal ATP levels and assessed the involvement of autophagy by using immunofluorescence and Western blotting. ARVs caused alterations of neurites and presynaptic terminals primarily during the 7-day incubation and depending on the specific compounds and their combinations with and without methamphetamine. Similarly, the loss of neuronal ATP was context specific for each of the drugs or combinations thereof, with and without methamphetamine or viral gp120. Loss of ATP was associated with activation of AMP-activated protein kinase (AMPK) and autophagy, which, however, failed to restore normal levels of neuronal ATP. In contrast, boosting autophagy with rapamycin prevented the long-term drop of ATP during exposure to cART in combination with methamphetamine or gp120. Our findings indicate that the overall positive effect of cART on HIV infection is accompanied by detectable neurotoxicity, which in turn may be aggravated by methamphetamine. PMID:26482305

  17. Multifamily Envelope Leakage Model

    SciTech Connect

    Faakye, Omari; Griffiths, Dianne

    2015-05-08

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

  18. A New Model for Nuclear Envelope BreakdownV⃞

    PubMed Central

    Terasaki, Mark; Campagnola, Paul; Rolls, Melissa M.; Stein, Pascal A.; Ellenberg, Jan; Hinkle, Beth; Slepchenko, Boris

    2001-01-01

    Nuclear envelope breakdown was investigated during meiotic maturation of starfish oocytes. Fluorescent 70-kDa dextran entry, as monitored by confocal microscopy, consists of two phases, a slow uniform increase and then a massive wave. From quantitative analysis of the first phase of dextran entry, and from imaging of green fluorescent protein chimeras, we conclude that nuclear pore disassembly begins several minutes before nuclear envelope breakdown. The best fit for the second phase of entry is with a spreading disruption of the membrane permeability barrier determined by three-dimensional computer simulations of diffusion. We propose a new model for the mechanism of nuclear envelope breakdown in which disassembly of the nuclear pores leads to a fenestration of the nuclear envelope double membrane. PMID:11179431

  19. Reducing systems protecting the bacterial cell envelope from oxidative damage.

    PubMed

    Arts, Isabelle S; Gennaris, Alexandra; Collet, Jean-François

    2015-06-22

    Exposure of cells to elevated levels of reactive oxygen species (ROS) damages DNA, membrane lipids and proteins, which can potentially lead to cell death. In proteins, the sulfur-containing residues cysteine and methionine are particularly sensitive to oxidation, forming sulfenic acids and methionine sulfoxides, respectively. The presence of protection mechanisms to scavenge ROS and repair damaged cellular components is therefore essential for cell survival. The bacterial cell envelope, which constitutes the first protection barrier from the extracellular environment, is particularly exposed to the oxidizing molecules generated by the host cells to kill invading microorganisms. Therefore, the presence of oxidative stress defense mechanisms in that compartment is crucial for cell survival. Here, we review recent findings that led to the identification of several reducing pathways protecting the cell envelope from oxidative damage. We focus in particular on the mechanisms that repair envelope proteins with oxidized cysteine and methionine residues and we discuss the major questions that remain to be solved. PMID:25957772

  20. Immunization against HTLV-I with chitosan and tri-methylchitosan nanoparticles loaded with recombinant env23 and env13 antigens of envelope protein gp46.

    PubMed

    Amirnasr, Maryam; Fallah Tafti, Tannan; Sankian, Mojtaba; Rezaei, Abdorrahim; Tafaghodi, Mohsen

    2016-08-01

    To prevent the spread of HTLV-I (Human T-lymphotropic virus type 1), a safe and effective vaccine is required. To increase immune responses against the peptide antigens can be potentiated with polymer-based nanoparticles, like chitosan (CHT) and trimethylchitosan (TMC), as delivery system/adjuvant. CHT and TMC nanoparticles loaded with recombinant proteins (env23 & env13) of gp46 were prepared by direct coating of antigens with positively charged polymers. The size of CHT and TMC nanoparticles (NPs) loaded with each antigen was about 400 nm. The physical stability of NPs was followed for 4 weeks. Both formulations showed to be stable for about 15 days. The immunogenicity of NPs loaded with antigens was studied after nasal and subcutaneous immunization in mice. Three immunizations (7.5 μg antigen) were performed with 2 weeks intervals. Two weeks after the last booster dose, sera IgG subtypes were measured. After subcutaneous administration, for both nanoparticulate antigens, serum IgG1 and IgGtotal levels were higher than antigen solution (P < 0.001). After nasal administration, for env23, IgG2a levels and IgG2a/IgG1 ratio was significantly higher than groups with subcutaneous administration (P < 0.001). Both nanoparticles showed good immunoadjuvant potential. Env23 antigen was a better candidate for vaccination against HTLV-I, as it induced higher cellular immune responses, compared with env13. PMID:27235335

  1. Genetic Diversity and Positive Selection Analysis of Classical Swine Fever Virus Envelope Protein Gene E2 in East China under C-Strain Vaccination

    PubMed Central

    Hu, Dongfang; Lv, Lin; Gu, Jinyuan; Chen, Tongyu; Xiao, Yihong; Liu, Sidang

    2016-01-01

    Classical swine fever virus (CSFV) causes an economically important and highly contagious disease of pigs worldwide. C-strain vaccination is one of the most effective ways to contain this disease. Since 2014, sporadic CSF outbreaks have been occurring in some C-strain vaccinated provinces of China. To decipher the disease etiology, 25 CSFV E2 genes from 169 clinical samples were cloned and sequenced. Phylogenetic analyses revealed that all 25 isolates belonged to subgenotype 2.1. Twenty-three of the 25 isolates were clustered in a newly defined subgenotype, 2.1d, and shared some consistent molecular characteristics. To determine whether the complete E2 gene was under positive selection pressure, we used a site-by-site analysis to identify specific codons that underwent evolutionary selection, and seven positively selected codons were found. Three positively selected sites (amino acids 17, 34, and 72) were identified in antigenicity-relevant domains B/C of the amino-terminal half of the E2 protein. In addition, another positively selected site (amino acid 200) exhibited a polarity change from hydrophilic to hydrophobic, which may change the antigenicity and virulence of CSFV. The results indicate that the circulating CSFV strains in Shandong province were mostly clustered in subgenotype 2.1d. Moreover, the identification of these positively selected sites could help to reveal molecular determinants of virulence or pathogenesis, and to clarify the driving force of CSFV evolution in East China. PMID:26903966

  2. Sequence comparison of JSRV with endogenous proviruses: envelope genotypes and a novel ORF with similarity to a G-protein-coupled receptor.

    PubMed

    Bai, J; Bishop, J V; Carlson, J O; DeMartini, J C

    1999-06-01

    Ovine pulmonary carcinoma, a contagious lung cancer of sheep, is caused by the oncogenic jaagsiekte sheep retrovirus (JSRV) that is closely related to a family of endogenous sheep retroviral sequences (ESRVs). By using exogenous virus-specific U3 oligonucleotide primers, the entire JSRV proviral genome or its 3' part was amplified from tumor DNA. Analysis of these proviral sequences revealed a novel open reading frame (ORF) within the pol coding region, designated ORF X, which was well conserved in ESRV and JSRV sequences. Deduced amino acids of ORF X showed similarity to a portion of the mammalian adenosine receptor subtype 3, a member of the G-protein-coupled receptor family. Comparison of deduced env amino acids of six JSRV strains from three continents identified 15 residues that defined two distinct genotypes of JSRVs. Sequence analysis identified two highly variable regions between JSRV and ESRV in the transmembrane domain of env (TM) and the 3' unique sequence (U3) of the long terminal repeat, from which JSRV-specific DNA probes were derived. By using these DNA probes in Southern hybridization, for the first time we successfully identified JSRV proviral sequences in tumor genomic DNA in the presence of multiple ESRV loci, validating the use of exogenous virus-specific DNA probes in the analysis of oncogenic proviral integration sites and identification of integrated exogenous proviral sequences. PMID:10366570

  3. 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

  4. Genetic Diversity and Positive Selection Analysis of Classical Swine Fever Virus Envelope Protein Gene E2 in East China under C-Strain Vaccination.

    PubMed

    Hu, Dongfang; Lv, Lin; Gu, Jinyuan; Chen, Tongyu; Xiao, Yihong; Liu, Sidang

    2016-01-01

    Classical swine fever virus (CSFV) causes an economically important and highly contagious disease of pigs worldwide. C-strain vaccination is one of the most effective ways to contain this disease. Since 2014, sporadic CSF outbreaks have been occurring in some C-strain vaccinated provinces of China. To decipher the disease etiology, 25 CSFV E2 genes from 169 clinical samples were cloned and sequenced. Phylogenetic analyses revealed that all 25 isolates belonged to subgenotype 2.1. Twenty-three of the 25 isolates were clustered in a newly defined subgenotype, 2.1d, and shared some consistent molecular characteristics. To determine whether the complete E2 gene was under positive selection pressure, we used a site-by-site analysis to identify specific codons that underwent evolutionary selection, and seven positively selected codons were found. Three positively selected sites (amino acids 17, 34, and 72) were identified in antigenicity-relevant domains B/C of the amino-terminal half of the E2 protein. In addition, another positively selected site (amino acid 200) exhibited a polarity change from hydrophilic to hydrophobic, which may change the antigenicity and virulence of CSFV. The results indicate that the circulating CSFV strains in Shandong province were mostly clustered in subgenotype 2.1d. Moreover, the identification of these positively selected sites could help to reveal molecular determinants of virulence or pathogenesis, and to clarify the driving force of CSFV evolution in East China. PMID:26903966

  5. Comparative genomics in Chlamydomonas and Plasmodium identifies an ancient nuclear envelope protein family essential for sexual reproduction in protists, fungi, plants, and vertebrates

    PubMed Central

    Ning, Jue; Otto, Thomas D.; Pfander, Claudia; Schwach, Frank; Brochet, Mathieu; Bushell, Ellen; Goulding, David; Sanders, Mandy; Lefebvre, Paul A.; Pei, Jimin; Grishin, Nick V.; Vanderlaan, Gary; Billker, Oliver; Snell, William J.

    2013-01-01

    Fertilization is a crucial yet poorly characterized event in eukaryotes. Our previous discovery that the broadly conserved protein HAP2 (GCS1) functioned in gamete membrane fusion in the unicellular green alga Chlamydomonas and the malaria pathogen Plasmodium led us to exploit the rare biological phenomenon of isogamy in Chlamydomonas in a comparative transcriptomics strategy to uncover additional conserved sexual reproduction genes. All previously identified Chlamydomonas fertilization-essential genes fell into related clusters based on their expression patterns. Out of several conserved genes in a minus gamete cluster, we focused on Cre06.g280600, an ortholog of the fertilization-related Arabidopsis GEX1. Gene disruption, cell biological, and immunolocalization studies show that CrGEX1 functions in nuclear fusion in Chlamydomonas. Moreover, CrGEX1 and its Plasmodium ortholog, PBANKA_113980, are essential for production of viable meiotic progeny in both organisms and thus for mosquito transmission of malaria. Remarkably, we discovered that the genes are members of a large, previously unrecognized family whose first-characterized member, KAR5, is essential for nuclear fusion during yeast sexual reproduction. Our comparative transcriptomics approach provides a new resource for studying sexual development and demonstrates that exploiting the data can lead to the discovery of novel biology that is conserved across distant taxa. PMID:23699412

  6. Serial femtosecond X-ray diffraction of enveloped virus microcrystals

    DOE PAGESBeta

    Lawrence, Robert M.; Conrad, Chelsie E.; Zatsepin, Nadia A.; Grant, Thomas D.; Liu, Haiguang; James, Daniel; Nelson, Garrett; Subramanian, Ganesh; Aquila, Andrew; Hunter, Mark S.; et al

    2015-08-20

    Serial femtosecond crystallography (SFX) using X-ray free-electron lasers has produced high-resolution, room temperature, time-resolved protein structures. We report preliminary SFX of Sindbis virus, an enveloped icosahedral RNA virus with ~700 Å diameter. Microcrystals delivered in viscous agarose medium diffracted to ~40 Å resolution. Small-angle diffuse X-ray scattering overlaid Bragg peaks and analysis suggests this results from molecular transforms of individual particles. Viral proteins undergo structural changes during entry and infection, which could, in principle, be studied with SFX. This is a pertinent step toward determining room temperature structures from virus microcrystals that may enable time-resolved studies of enveloped viruses.

  7. Closing a gap in the nuclear envelope.

    PubMed

    Vietri, Marina; Stenmark, Harald; Campsteijn, Coen

    2016-06-01

    The nuclear envelope (NE) ensures nucleo-cytoplasmic compartmentalization, with trafficking of macromolecules across this double membrane controlled by embedded nuclear pore complexes (NPCs). The NE and associated proteins are dismantled during open mitosis and reestablishment of this barrier during mitotic exit requires dynamic remodeling of endoplasmic reticulum (ER) membranes and coordination with NPC reformation, with NPC deposition continuing during subsequent interphase. In this review, we discuss recent progress in our understanding of NE reformation and nuclear pore complex generation, with special focus on work implicating the endosomal sorting complex required for transport (ESCRT) membrane remodeling machinery in these events. PMID:27016712

  8. Characterization of a chloroplast inner envelope K+ channel.

    PubMed Central

    Mi, F; Peters, J S; Berkowitz, G A

    1994-01-01

    A K(+)-conducting protein of the chloroplast inner envelope was characterized as a K+ channel. Studies of this transport protein in the native membrane documented its sensitivity to K+ channel blockers. Further studies of native membranes demonstrated a sensitivity of K+ conductance to divalent cations such as Mg2+, which modulate ion conduction through interaction with negative surface charges on the inner-envelope membrane. Purified chloroplast inner-envelope vesicles were fused into an artificial planar lipid bilayer to facilitate recording of single-channel K+ currents. These single-channel K+ currents had a slope conductance of 160 picosiemens. Antibodies generated against the conserved amino acid sequence that serves as a selectivity filter in the pore of K+ channels immunoreacted with a 62-kD polypeptide derived from the chloroplast inner envelope. This polypeptide was fractionated using density gradient centrifugation. Comigration of this immunoreactive polypeptide and K+ channel activity in sucrose density gradients further suggested that this polypeptide is the protein facilitating K+ conductance across the chloroplast inner envelope. PMID:8058841

  9. LINCing complex functions at the nuclear envelope

    PubMed Central

    Rothballer, Andrea; Schwartz, Thomas U.; Kutay, Ulrike

    2013-01-01

    Linker of nucleoskeleton and cytoskeleton (LINC) complexes span the double membrane of the nuclear envelope (NE) and physically connect nuclear structures to cytoskeletal elements. LINC complexes are envisioned as force transducers in the NE, which facilitate processes like nuclear anchorage and migration, or chromosome movements. The complexes are built from members of two evolutionary conserved families of transmembrane (TM) proteins, the SUN (Sad1/UNC-84) domain proteins in the inner nuclear membrane (INM) and the KASH (Klarsicht/ANC-1/SYNE homology) domain proteins in the outer nuclear membrane (ONM). In the lumen of the NE, the SUN and KASH domains engage in an intimate assembly to jointly form a NE bridge. Detailed insights into the molecular architecture and atomic structure of LINC complexes have recently revealed the molecular basis of nucleo-cytoskeletal coupling. They bear important implications for LINC complex function and suggest new potential and as yet unexplored roles, which the complexes may play in the cell. PMID:23324460

  10. Envelope gene of the Friend spleen focus-forming virus: deletion and insertions in 3' gp70/p15E-encoding region have resulted in unique features in the primary structure of its protein product.

    PubMed Central

    Wolff, L; Scolnick, E; Ruscetti, S

    1983-01-01

    A nucleotide sequence was determined for the envelope (env) gene of the polycythemia-inducing strain of the acute leukemia-inducing Friend spleen focus-forming virus (SFFV) and from this the amino acid sequence of its gene product, gp52, was deduced. All major elements of the gene were found to be related to genes of other retroviruses that code for functional glycoproteins. Although the carboxyl terminus of gp52 is encoded by sequences highly related to sequences in its putative parent, ecotropic Friend murine leukemia virus, the majority of the protein (69%), including the amino terminus, is encoded by dualtropic virus-like sequences. Nucleotide sequence comparisons suggest that the nonecotropic region may be more closely related to the 5' substitution in dualtropic mink cell focus-inducing viruses that it is to the 5' end of xenotropic virus env genes. A large deletion and two unique insertions have been located in the env gene of polycythemia-inducing SFFV and may account for some of the unusual structural characteristics, aberrant processing, and pathogenic properties of gp52. As a consequence of the deletion, amino-terminal gp70 and carboxyl-terminal p15E-encoding sequences are juxtaposed and it appears that translation from the p15E region, 3' to the deletion, continues in the standard reading frame used by other retroviruses. Insertions of six base pairs and one base pair at the very 3' end of the gp52-encoding region results in a SFFV-unique amino acid sequence and a premature termination codon. PMID:6308646

  11. Structure of Phage P22 Cell Envelope-Penetrating Needle

    SciTech Connect

    Olia,A.; Casjens, S.; Cingolani, G.

    2007-01-01

    Bacteriophage P22 infects Salmonella enterica by injecting its genetic material through the cell envelope. During infection, a specialized tail needle, gp26, is injected into the host, likely piercing a hole in the host cell envelope. The 2.1-Angstroms crystal structure of gp26 reveals a 240-Angstroms elongated protein fiber formed by two trimeric coiled-coil domains interrupted by a triple beta-helix. The N terminus of gp26 plugs the portal protein channel, retaining the genetic material inside the virion. The C-terminal tip of the fiber exposes beta-hairpins with hydrophobic tips similar to those seen in class II fusion peptides. The alpha-helical core connecting these two functionally polarized tips presents four trimerization octads with consensus sequence IXXLXXXV. The slender conformation of the gp26 fiber minimizes the surface exposed to solvent, which is consistent with the idea that gp26 traverses the cell envelope lipid bilayers.

  12. Structure of Phage P22 Cell Envelope-Penetrating Needle

    SciTech Connect

    Olia, A.S.; Casjens, S.; Cingolani, G.

    2009-06-02

    Bacteriophage P22 infects Salmonella enterica by injecting its genetic material through the cell envelope. During infection, a specialized tail needle, gp26, is injected into the host, likely piercing a hole in the host cell envelope. The 2.1-{angstrom} crystal structure of gp26 reveals a 240-{angstrom} elongated protein fiber formed by two trimeric coiled-coil domains interrupted by a triple {beta}-helix. The N terminus of gp26 plugs the portal protein channel, retaining the genetic material inside the virion. The C-terminal tip of the fiber exposes {beta}-hairpins with hydrophobic tips similar to those seen in class II fusion peptides. The {alpha}-helical core connecting these two functionally polarized tips presents four trimerization octads with consensus sequence IXXLXXXV. The slender conformation of the gp26 fiber minimizes the surface exposed to solvent, which is consistent with the idea that gp26 traverses the cell envelope lipid bilayers.

  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. Heat Recovery in Building Envelopes

    SciTech Connect

    Sherman, Max H.; Walker, Iain S.

    2001-01-01

    Infiltration has traditionally been assumed to contribute to the energy load of a building by an amount equal to the product of the infiltration flow rate and the enthalpy difference between inside and outside. Application of such a simple formula may produce an unreasonably high contribution because of heat recovery within the building envelope. Previous laboratory and simulation research has indicated that such heat transfer between the infiltrating air and walls may be substantial. In this study, Computational Fluid Dynamics was used to simulate sensible heat transfer in typical envelope constructions. The results show that the traditional method may over-predict the infiltration energy load by up to 95 percent at low leakage rates. A simplified physical model has been developed and used to predict the infiltration heat recovery based on the Peclet number of the flow and the fraction of the building envelope active in infiltration heat recovery.

  16. Envelope Inflation or Stellar Wind?

    NASA Astrophysics Data System (ADS)

    Ro, S.; Matzner, C. D.

    We an optically-thick, transonic, steady wind model for a H-free Wolf-Rayet star. A bifurcation is found across a critical mass loss rate Mb. Slower winds M < Mb extend by several hydrostatic stellar radii, reproduce features of envelope in ation from Petrovic et al. (2006) and Gräfener et al. (2012), and are energetically unbound. This work is of particular interest for extended envelopes and winds, radiative hydrodynamic instabilities (eg. wind stagnation, clumping, etc.), and NLTE atmospheric models.

  17. Carbon chemistry of circumstellar envelopes

    NASA Technical Reports Server (NTRS)

    Bieging, John H.

    1990-01-01

    The chemical composition of envelopes surrounding cool evolved stars, as determined from microwave spectroscopic observations, is reviewed. Emphasis is placed on recent observations with the new large mm-wavelength telescopes and interferometer arrays, and on new theoretical work, especially concerning ion-molecule chemistry of carbon-bearing in these envelopes. Thermal (as opposed to maser) emission lines are discussed. Much progress has been made in the past few years in the theoretical understanding of these objects. It is already clear, however, that observations with the new generation of mm-telescopes will require substantial improvements in the theoretical models to achieve a thorough understanding of the data now becoming available.

  18. Diversity in the fertilization envelopes of echinoderms

    PubMed Central

    Oulhen, Nathalie; Reich, Adrian; Wong, Julian L.; Wessel, Gary M.

    2013-01-01

    Cell surface changes in an egg at fertilization are essential to begin development and for protecting the zygote. Most fertilized eggs construct a barrier around themselves by modifying their original extracellular matrix. This construction usually results from calcium induced exocytosis of cortical granules, the contents of which in sea urchins function to form the fertilization envelope (FE), an extracellular matrix of cortical granule contents built upon a vitelline layer scaffold. Here we examined the molecular mechanism of this process in sea stars, a close relative of the sea urchins, and analyze the evolutionary changes that likely occurred in the functionality of this structure between these two organisms. We find that the FE of sea stars is more permeable than in sea urchins, allowing diffusion of molecules in excess of 2 megadaltons. Through a proteomic and transcriptomic approach, we find that most, but not all of the proteins present in the sea urchin envelope are present in sea stars, including SFE9, proteoliaisin, rendezvin, and ovoperoxidase. The mRNAs encoding these FE proteins accumulated most densely in early oocytes, and then beginning with vitellogenesis, these mRNAs deceased in abundance to levels nearly undetectable in eggs. Antibodies to the SFE9 protein of sea stars showed that the cortical granules in sea star also accumulated most significantly in early oocytes, and different from sea urchins, they translocated to the cortex of the oocytes well before meiotic initiation. These results suggest that the preparation of the cell surface changes in sea urchins has been shifted to later in oogenesis and perhaps reflects the meiotic differences among the species–sea star oocytes are stored in prophase of meiosis and fertilized during the meiotic divisions, as in most animals, whereas sea urchins are one of the few taxa in which eggs have completed meiosis prior to fertilization. PMID:23331915

  19. 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

  20. Safeguards Envelope Progress FY08

    SciTech Connect

    Robert Bean; Richard Metcalf; Aaron Bevill

    2008-09-01

    The Safeguards Envelope Project met its milestones by creating a rudimentary safeguards envelope, proving the value of the approach on a small scale, and determining the most appropriate path forward. The Idaho Chemical Processing Plant’s large cache of reprocessing process monitoring data, dubbed UBER Data, was recovered and used in the analysis. A probabilistic Z test was used on a Markov Monte Carlo simulation of expected diversion data when compared with normal operating data. The data regarding a fully transient event in a tank was used to create a simple requirement, representative of a safeguards envelope, whose impact was a decrease in operating efficiency by 1.3% but an increase in material balance period of 26%. This approach is operator, state, and international safeguards friendly and should be applied to future reprocessing plants. Future requirements include tank-to-tank correlations in reprocessing facilities, detailed operations impact studies, simulation inclusion, automated optimization, advanced statistics analysis, and multi-attribute utility analysis.

  1. 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

  2. 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

  3. Monoclonal antibody to spleen focus-forming virus-encoded gp52 provides a probe for the amino-terminal region of retroviral envelope proteins that confers dual tropism and xenotropism.

    PubMed Central

    Wolff, L; Koller, R; Ruscetti, S

    1982-01-01

    Monoclonal antibodies which recognize a region common to Friend spleen focus-forming virus encoded gp52 and Friend mink cell focus-inducing viral gp70 were isolated. One such antibody from hybridoma 7C10 was tested extensively in immune precipitation and was found to react with a determinant on envelope gp70s of all mink cell focus-inducing, xenotropic, and amphotropic mouse retroviruses tested, but not with envelope gp70s of ecotropic viruses, including Friend, Moloney, and AKR murine leukemia viruses. Monoclonal antibody from hybridoma 7C10 precipitated a 23,000-molecular-weight fragment, derived by V8 protease digestion of Friend mink cell focus-inducing gp70. This 23,000-molecular-weight peptide was determined to derive from the amino terminus of the molecule. These results correlate well with other genetic data which indicate that endogenously acquired sequences of mink cell focus-inducing viruses are found at the 5' end of the envelope gene. Images PMID:6180179

  4. The structure of common-envelope remnants

    NASA Astrophysics Data System (ADS)

    Hall, Philip D.

    2015-05-01

    We investigate the structure and evolution of the remnants of common-envelope evolution in binary star systems. In a common-envelope phase, two stars become engulfed in a gaseous envelope and, under the influence of drag forces, spiral to smaller separations. They may merge to form a single star or the envelope may be ejected to leave the stars in a shorter period orbit. This process explains the short orbital periods of many observed binary systems, such as cataclysmic variables and low-mass X-ray binary systems. Despite the importance of these systems, and of common-envelope evolution to their formation, it remains poorly understood. Specifically, we are unable to confidently predict the outcome of a common-envelope phase from the properties at its onset. After presenting a review of work on stellar evolution, binary systems, common-envelope evolution and the computer programs used, we describe the results of three computational projects on common-envelope evolution. Our work specifically relates to the methods and prescriptions which are used for predicting the outcome. We use the Cambridge stellar-evolution code STARS to produce detailed models of the structure and evolution of remnants of common-envelope evolution. We compare different assumptions about the uncertain end-of-common envelope structure and envelope mass of remnants which successfully eject their common envelopes. In the first project, we use detailed remnant models to investigate whether planetary nebulae are predicted after common-envelope phases initiated by low-mass red giants. We focus on the requirement that a remnant evolves rapidly enough to photoionize the nebula and compare the predictions for different ideas about the structure at the end of a common-envelope phase. We find that planetary nebulae are possible for some prescriptions for the end-of-common envelope structure. In our second contribution, we compute a large set of single-star models and fit new formulae to the core radii of

  5. ESCRT III repairs nuclear envelope ruptures during cell migration to limit DNA damage and cell death.

    PubMed

    Raab, M; Gentili, M; de Belly, H; Thiam, H R; Vargas, P; Jimenez, A J; Lautenschlaeger, F; Voituriez, Raphaël; Lennon-Duménil, A M; Manel, N; Piel, M

    2016-04-15

    In eukaryotic cells, the nuclear envelope separates the genomic DNA from the cytoplasmic space and regulates protein trafficking between the two compartments. This barrier is only transiently dissolved during mitosis. Here, we found that it also opened at high frequency in migrating mammalian cells during interphase, which allowed nuclear proteins to leak out and cytoplasmic proteins to leak in. This transient opening was caused by nuclear deformation and was rapidly repaired in an ESCRT (endosomal sorting complexes required for transport)-dependent manner. DNA double-strand breaks coincided with nuclear envelope opening events. As a consequence, survival of cells migrating through confining environments depended on efficient nuclear envelope and DNA repair machineries. Nuclear envelope opening in migrating leukocytes could have potentially important consequences for normal and pathological immune responses. PMID:27013426

  6. 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

  7. 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

  8. Isolating The Building Thermal Envelope

    NASA Astrophysics Data System (ADS)

    Harrje, D. T.; Dutt, G. S.; Gadsby, K. J.

    1981-01-01

    The evaluation of the thermal integrity of building envelopes by infrared scanning tech-niques is often hampered in mild weather because temperature differentials across the envelope are small. Combining the infrared scanning with positive or negative building pressures, induced by a "blower door" or the building ventilation system, considerably extends the periods during which meaningful diagnostics can be conducted. Although missing or poorly installed insulation may lead to a substantial energy penalty, it is the search for air leakage sites that often has the largest potential for energy savings. Infrared inspection of the attic floor with air forced from the occupied space through ceiling by-passes, and inspecting the interior of the building when outside air is being sucked through the envelope reveals unexpected leakage sites. Portability of the diagnostic equipment is essential in these surveys which may include access into some tight spaces. A catalog of bypass heat losses that have been detected in residential housing using the combined infrared pressure differential technique is included to point out the wide variety of leakage sites which may compromise the benefits of thermal insulation and allow excessive air infiltration. Detection and suppression of such leaks should be key items in any building energy audit program. Where a calibrated blower door is used to pressurize or evacuate the house, the leakage rate can be quantified and an excessively tight house recognized. Houses that are too tight may be improved with a minimal energy penalty by forced ventilation,preferably with a heat recuperator and/or by providing combustion air directly to the furnace.

  9. Serial femtosecond X-ray diffraction of enveloped virus microcrystals

    PubMed Central

    Lawrence, Robert M.; Conrad, Chelsie E.; Zatsepin, Nadia A.; Grant, Thomas D.; Liu, Haiguang; James, Daniel; Nelson, Garrett; Subramanian, Ganesh; Aquila, Andrew; Hunter, Mark S.; Liang, Mengning; Boutet, Sébastien; Coe, Jesse; Spence, John C. H.; Weierstall, Uwe; Liu, Wei; Fromme, Petra; Cherezov, Vadim; Hogue, Brenda G.

    2015-01-01

    Serial femtosecond crystallography (SFX) using X-ray free-electron lasers has produced high-resolution, room temperature, time-resolved protein structures. We report preliminary SFX of Sindbis virus, an enveloped icosahedral RNA virus with ∼700 Å diameter. Microcrystals delivered in viscous agarose medium diffracted to ∼40 Å resolution. Small-angle diffuse X-ray scattering overlaid Bragg peaks and analysis suggests this results from molecular transforms of individual particles. Viral proteins undergo structural changes during entry and infection, which could, in principle, be studied with SFX. This is an important step toward determining room temperature structures from virus microcrystals that may enable time-resolved studies of enveloped viruses. PMID:26798819

  10. Flexible Envelope Request Notation (FERN)

    NASA Technical Reports Server (NTRS)

    Zoch, David R.; Lavallee, David; Weinstein, Stuart

    1991-01-01

    The following topics are presented in view graph form and include the following: scheduling application; the motivation for the Flexible Envelope Request Notation (FERN); characteristics of FERN; types of information needed in requests; where information is stored in requests; FERN structures; generic requests; resource availability for pooled resources; expressive notation; temporal constraints; time formats; changes to FERN; sample FERN requests; the temporal relationship between two steps; maximum activity length to limit step delays; alternative requests; the temporal relationship between two activities; and idle resource usage between steps.

  11. The Metabolite Transporters of the Plastid Envelope: An Update

    PubMed Central

    Facchinelli, Fabio; Weber, Andreas P. M.

    2011-01-01

    The engulfment of a photoautotrophic cyanobacterium by a primitive mitochondria-bearing eukaryote traces back to more than 1.2 billion years ago. This single endosymbiotic event not only provided the early petroalgae with the metabolic capacity to perform oxygenic photosynthesis, but also introduced a plethora of other metabolic routes ranging from fatty acids and amino acids biosynthesis, nitrogen and sulfur assimilation to secondary compounds synthesis. This implicated the integration and coordination of the newly acquired metabolic entity with the host metabolism. The interface between the host cytosol and the plastidic stroma became of crucial importance in sorting precursors and products between the plastid and other cellular compartments. The plastid envelope membranes fulfill different tasks: they perform important metabolic functions, as they are involved in the synthesis of carotenoids, chlorophylls, and galactolipids. In addition, since most genes of cyanobacterial origin have been transferred to the nucleus, plastidial proteins encoded by nuclear genes are post-translationally transported across the envelopes through the TIC–TOC import machinery. Most importantly, chloroplasts supply the photoautotrophic cell with photosynthates in form of reduced carbon. The innermost bilayer of the plastidic envelope represents the permeability barrier for the metabolites involved in the carbon cycle and is literally stuffed with transporter proteins facilitating their transfer. The intracellular metabolite transporters consist of polytopic proteins containing membrane spans usually in the number of four or more α-helices. Phylogenetic analyses revealed that connecting the plastid with the host metabolism was mainly a process driven by the host cell. In Arabidopsis, 58% of the metabolite transporters are of host origin, whereas only 12% are attributable to the cyanobacterial endosymbiont. This review focuses on the metabolite transporters of the inner envelope

  12. Enveloped virus flocculation and removal in osmolyte solutions.

    PubMed

    Gencoglu, Maria F; Heldt, Caryn L

    2015-07-20

    Our ability to reduce infectious disease burden throughout the world has been greatly improved by the creation of vaccines. However, worldwide immunization rates are low. The two most likely reasons are the lack of sufficient distribution in underdeveloped countries and the high cost of vaccine products. The high costs are due to the difficulties of manufacturing individual vaccine products with specialized purification trains. In this study, we propose to use virus flocculation in osmolytes, followed by microfiltration, as an alternative vaccine purification operation. In our previous work, we demonstrated that osmolytes preferentially flocculate a non-enveloped virus, porcine parvovirus (PPV). In this work we show that osmolytes flocculate the enveloped virus, Sindbis virus heat resistant strain (SVHR), and demonstrate a >80% removal with a 0.2 μm microfilter membrane while leaving proteins in solution. The best osmolytes were tested for their ability to flocculate SVHR at different concentrations, pH and ionic strengths. Our best removal was 98% of SVHR in 0.3M mannitol at a pH of 5. We propose that osmolytes are able to flocculate hydrophobic non-enveloped and enveloped virus particles by the reduction of the hydration layer around the particles, which stimulates virus aggregation. Now that we have demonstrated that protecting osmolytes flocculate viruses, this method has the potential to be a future platform purification process for vaccines. PMID:25865274

  13. Circumplanetary disc or circumplanetary envelope?

    NASA Astrophysics Data System (ADS)

    Szulágyi, J.; Masset, F.; Lega, E.; Crida, A.; Morbidelli, A.; Guillot, T.

    2016-08-01

    We present three-dimensional simulations with nested meshes of the dynamics of the gas around a Jupiter mass planet with the JUPITER and FARGOCA codes. We implemented a radiative transfer module into the JUPITER code to account for realistic heating and cooling of the gas. We focus on the circumplanetary gas flow, determining its characteristics at very high resolution (80 per cent of Jupiter's diameter). In our nominal simulation where the temperature evolves freely by the radiative module and reaches 13000 K at the planet, a circumplanetary envelope was formed filling the entire Roche lobe. Because of our equation of state is simplified and probably overestimates the temperature, we also performed simulations with limited maximal temperatures in the planet region (1000, 1500, and 2000 K). In these fixed temperature cases circumplanetary discs (CPDs) were formed. This suggests that the capability to form a CPD is not simply linked to the mass of the planet and its ability to open a gap. Instead, the gas temperature at the planet's location, which depends on its accretion history, plays also fundamental role. The CPDs in the simulations are hot and cooling very slowly, they have very steep temperature and density profiles, and are strongly sub-Keplerian. Moreover, the CPDs are fed by a strong vertical influx, which shocks on the CPD surfaces creating a hot and luminous shock-front. In contrast, the pressure supported circumplanetary envelope is characterized by internal convection and almost stalled rotation.

  14. Safeguards Envelope Progress FY10

    SciTech Connect

    Richard Metcalf

    2010-10-01

    The Safeguards Envelope is a strategy to determine a set of specific operating parameters within which nuclear facilities may operate to maximize safeguards effectiveness without sacrificing safety or plant efficiency. This paper details the additions to the advanced operating techniques that will be applied to real plant process monitoring (PM) data from the Idaho Chemical Processing Plant (ICPP). Research this year focused on combining disparate pieces of data together to maximize operating time with minimal downtime due to safeguards. A Chi-Square and Croiser's cumulative sum were both included as part of the new analysis. Because of a major issue with the original data, the implementation of the two new tests did not add to the existing set of tests, though limited one-variable optimization made a small increase in detection probability. Additional analysis was performed to determine if prior analysis would have caused a major security or safety operating envelope issue. It was determined that a safety issue would have resulted from the prior research, but that the security may have been increased under certain conditions.

  15. Pushing the Envelope: Dengue Viral Membrane Coaxed into Shape by Molecular Simulations.

    PubMed

    Marzinek, Jan K; Holdbrook, Daniel A; Huber, Roland G; Verma, Chandra; Bond, Peter J

    2016-08-01

    Dengue virus is a flavivirus responsible for millions of infections per year. Its surface contains a phospholipid bilayer, within which are embedded the envelope (E) and membrane (M) proteins, arranged with icosahedral geometry. Exposure to low pH triggers the E proteins to undergo conformational changes, which precede fusion with the host cell membrane and release of the viral genome. The flavivirus membrane exhibits significant local curvature and deformation, as revealed by cryoelectron microscopy (cryo-EM), but its precise structure and interactions with envelope components remain unclear. We now report simulations of the dengue viral particle that refine its envelope structure in unprecedented detail. Our final models are morphologically consistent with cryo-EM data, and reveal the structural basis for membrane curvature. Electrostatic interactions increased envelope complex stability; this coupling has potential functional significance in the context of the viral fusion mechanism and infective states. PMID:27396828

  16. The Role of the Membrane in the Structure and Biophysical Robustness of the Dengue Virion Envelope

    PubMed Central

    Reddy, Tyler; Sansom, Mark S.P.

    2016-01-01

    Summary The dengue virion is surrounded by an envelope of membrane proteins surrounding a lipid bilayer. We have combined the cryoelectron microscopy structures of the membrane proteins (PDB: 3J27) with a lipid bilayer whose composition is based on lipidomics data for insect cell membranes, to obtain a near-atomic resolution computational model of the envelope of the dengue virion. A coarse-grained molecular dynamics simulation on the microsecond timescale enables analysis of key biophysical properties of the dengue outer envelope. Properties analyzed include area per lipid values (for a spherical virion with a mixed lipid composition), bilayer thickness, and lipid diffusion coefficients. Despite the absence of cholesterol from the lipid bilayer, the virion exhibits biophysical robustness (slow lipid diffusion alongside stable bilayer thickness, virion diameter, and shape) that matches the cholesterol-rich membrane of influenza A, with similarly anomalous diffusion of lipids. Biophysical robustness of the envelope may confer resilience to environmental perturbations. PMID:26833387

  17. Characterization of sciellin, a precursor to the cornified envelope of human keratinocytes.

    PubMed

    Kvedar, J C; Manabe, M; Phillips, S B; Ross, B S; Baden, H P

    1992-04-01

    The cornified envelope, located beneath the plasma membrane of terminally differentiated keratinocytes, is formed as protein precursors are cross-linked by a membrane associated transglutaminase. This report characterizes a new precursor to the cornified envelope. A monoclonal antibody derived from mice immunized with cornified envelopes of human cultured keratinocytes stained the periphery of more differentiated cells in epidermis and other stratified squamous epithelia including hair and nails. The epitope was widely conserved among mammals as determined by immunohistochemical and Western analysis. Immunoelectron microscopy localized the epitope to the cell periphery in the upper stratum spinosum and granulosum of epidermis. In the hair follicle, the epitope was present in the internal root sheath and in the infundibulum, the innermost aspect of the external root sheath. The antibody recognized a protein of relative mobility (M(r)) 82,000, pI 7.8. The protein was a transglutaminase substrate as shown by a dansylcadaverine incorporation assay. Purified cornified envelopes absorbed the reactivity of the antibody to the partially purified protein and cleavage of envelopes by cyanogen bromide resulted in release of immunoreactive fragments. The protein was soluble only in denaturing buffers such as 8 M urea or 2% sodium dodecyl-sulfate (SDS). Partial solubility could be achieved in 50 mM TRIS pH 8.3 plus 0.3 M NaCl (high salt buffer); the presence of a reducing agent did not affect solubility. Extraction of cultured keratinocytes in 8 M urea and subsequent dialysis against 50 mM TRIS pH 8.3 buffer resulted in precipitation of the protein with the keratin filaments. Dialysis against high salt buffer prevented precipitation of the protein. The unique solubility properties of this protein suggest that it aggregates with itself and/or with keratin filaments. The possible role of the protein in cornified envelope assembly is discussed. We have named this protein Sciellin

  18. 14 CFR 23.333 - Flight envelope.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 14 Aeronautics and Space 1 2010-01-01 2010-01-01 false Flight envelope. 23.333 Section 23.333... STANDARDS: NORMAL, UTILITY, ACROBATIC, AND COMMUTER CATEGORY AIRPLANES Structure Flight Loads § 23.333 Flight envelope. (a) General. Compliance with the strength requirements of this subpart must be shown...

  19. 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

  20. An Accurate In Vitro Model of the E. coli Envelope

    PubMed Central

    Clifton, Luke A; Holt, Stephen A; Hughes, Arwel V; Daulton, Emma L; Arunmanee, Wanatchaporn; Heinrich, Frank; Khalid, Syma; Jefferies, Damien; Charlton, Timothy R; Webster, John R P; Kinane, Christian J; Lakey, Jeremy H

    2015-01-01

    Gram-negative bacteria are an increasingly serious source of antibiotic-resistant infections, partly owing to their characteristic protective envelope. This complex, 20 nm thick barrier includes a highly impermeable, asymmetric bilayer outer membrane (OM), which plays a pivotal role in resisting antibacterial chemotherapy. Nevertheless, the OM molecular structure and its dynamics are poorly understood because the structure is difficult to recreate or study in vitro. The successful formation and characterization of a fully asymmetric model envelope using Langmuir–Blodgett and Langmuir–Schaefer methods is now reported. Neutron reflectivity and isotopic labeling confirmed the expected structure and asymmetry and showed that experiments with antibacterial proteins reproduced published in vivo behavior. By closely recreating natural OM behavior, this model provides a much needed robust system for antibiotic development. PMID:26331292

  1. Structural basis for membrane anchoring of HIV-1 envelope spike.

    PubMed

    Dev, Jyoti; Park, Donghyun; Fu, Qingshan; Chen, Jia; Ha, Heather Jiwon; Ghantous, Fadi; Herrmann, Tobias; Chang, Weiting; Liu, Zhijun; Frey, Gary; Seaman, Michael S; Chen, Bing; Chou, James J

    2016-07-01

    HIV-1 envelope spike (Env) is a type I membrane protein that mediates viral entry. We used nuclear magnetic resonance to determine an atomic structure of the transmembrane (TM) domain of HIV-1 Env reconstituted in bicelles that mimic a lipid bilayer. The TM forms a well-ordered trimer that protects a conserved membrane-embedded arginine. An amino-terminal coiled-coil and a carboxyl-terminal hydrophilic core stabilize the trimer. Individual mutations of conserved residues did not disrupt the TM trimer and minimally affected membrane fusion and infectivity. Major changes in the hydrophilic core, however, altered the antibody sensitivity of Env. These results show how a TM domain anchors, stabilizes, and modulates a viral envelope spike and suggest that its influence on Env conformation is an important consideration for HIV-1 immunogen design. PMID:27338706

  2. Role of HIV-2 envelope in Lv2-mediated restriction

    SciTech Connect

    Reuter, Sandra; Kaumanns, Patrick; Buschhorn, Sabine B.; Dittmar, Matthias T. . E-mail: Matthias_Dittmar@med.uni-heidelberg.de

    2005-02-05

    We have characterized envelope protein pseudotyped HIV-2 particles derived from two HIV-2 isolates termed prCBL23 and CBL23 in order to define the role of the envelope protein for the Lv2-mediated restriction to infection. Previously, it has been described that the primary isolate prCBL23 is restricted to infection of several human cell types, whereas the T cell line adapted isolate CBL23 is not restricted in these cell types. Molecular cloning of the two isolates revealed that the env and the gag gene are responsible for the observed phenotype and that this restriction is mediated by Lv2, which is distinct from Ref1/Lv1 (Schmitz, C., Marchant, D., Neil, S.J., Aubin, K., Reuter, S., Dittmar, M.T., McKnight, A., Kizhatil, K., Albritton, L.M., 2004. Lv2, a novel postentry restriction, is mediated by both capsid and envelope. J. Virol. 78 (4), 2006-2016). We generated pseudotyped viruses consisting of HIV-2 (ROD-A{delta}env-GFP, ROD-A{delta}env-RFP, or ROD-A{delta}env-REN) and the prCBL23 or CBL23 envelope proteins as well as chimeric proteins between these envelopes. We demonstrate that a single amino acid exchange at position 74 in the surface unit of CBL23-Env confers restriction to infection. This single point mutation causes tighter CD4 binding, resulting in a less efficient fusion into the cytosol of the restricted cell line. Prevention of endosome formation and prevention of endosome acidification enhance infectivity of the restricted particles for GHOST/X4 cells indicating a degradative lysosomal pathway as a cause for the reduced cytosolic entry. The described restriction to infection of the primary isolate prCBL23 is therefore largely caused by an entry defect. A remaining restriction to infection (19-fold) is preserved when endosomal acidification is prevented. This restriction to infection is also dependent on the presence of the point mutation at position 74 (G74E)

  3. Seasonal benefits of a natural propolis envelope to honey bee immunity and colony health.

    PubMed

    Borba, Renata S; Klyczek, Karen K; Mogen, Kim L; Spivak, Marla

    2015-11-01

    Honey bees, as social insects, rely on collective behavioral defenses that produce a colony-level immune phenotype, or social immunity, which in turn impacts the immune response of individuals. One behavioral defense is the collection and deposition of antimicrobial plant resins, or propolis, in the nest. We tested the effect of a naturally constructed propolis envelope within standard beekeeping equipment on the pathogen and parasite load of large field colonies, and on immune system activity, virus and storage protein levels of individual bees over the course of a year. The main effect of the propolis envelope was a decreased and more uniform baseline expression of immune genes in bees during summer and autumn months each year, compared with the immune activity in bees with no propolis envelope in the colony. The most important function of the propolis envelope may be to modulate costly immune system activity. As no differences were found in levels of bacteria, pathogens and parasites between the treatment groups, the propolis envelope may act directly on the immune system, reducing the bees' need to activate the physiologically costly production of humoral immune responses. Colonies with a natural propolis envelope had increased colony strength and vitellogenin levels after surviving the winter in one of the two years of the study, despite the fact that the biological activity of the propolis diminished over the winter. A natural propolis envelope acts as an important antimicrobial layer enshrouding the colony, benefiting individual immunity and ultimately colony health. PMID:26449975

  4. The theoretical polarization of pure scattering axisymmetric circumstellar envelopes

    NASA Technical Reports Server (NTRS)

    Fox, G. K.

    1994-01-01

    The Sobolev approach to the scattering of starlight through a pure scattering circumstellar envelope is developed. The theoretical polarization due to electron scattering in Be star envelopes is calculated for two geometries (an equatorially enhanced envelope and a spheroidal envelope). Only the disk-type envelope is found to yield a maximum polarization consistent with the observed range for Be stars. A lower limit, analytical approximation to the theoretical polarization from a pure scattering envelope is obtained.

  5. Personnel occupied woven envelope robot

    NASA Technical Reports Server (NTRS)

    Wessling, Francis; Teoh, William; Ziemke, M. Carl

    1988-01-01

    The Personnel Occupied Woven Envelope Robot (POWER) provides an alternative to extravehicular activity (EVA) of space suited astronauts and/or use of long slender manipulator arms such as are used in the Shuttle Remote Manipulator System. POWER provides the capability for a shirt sleeved astronaut to perform such work by entering a control pod through air locks at both ends of an inflated flexible bellows (access tunnel). The exoskeleton of the tunnel is a series of six degrees of freedom (Six-DOF) articulated links compressible to 1/6 of their fully extended length. The operator can maneuver the control pod to almost any location within about 50 m of the base attachment to the space station. POWER can be envisioned as a series of hollow Six-DOF manipulator segments or arms wherein each arm grasps the shoulder of the next arm. Inside the hollow arms ia a bellow-type access tunnel. The control pod is the fist of the series of linked hollow arms. The fingers of the fist are conventional manipulator arms under direct visual control of the nearby operator in the pod. The applications and progress to date of the POWER system is given.

  6. Safeguards Envelope Progress FY09

    SciTech Connect

    Richard Metcalf; Robert Bean

    2009-09-01

    The Safeguards Envelope is a strategy to determine a set of specific operating parameters which nuclear facilities may operate within to maximize safeguards effectiveness without sacrificing safety or plant efficiency. This paper details advanced statistical techniques will be applied to real plant process monitoring (PM) data from the Idaho Chemical Processing Plant (ICPP). As a result of the U.S. having no operating nuclear chemical reprocessing plants, there has been a strong interest in obtaining process monitoring data from the ICPP. The ICPP was shut down in 1996 and a recent effort has been made to retrieve the PM data from storage in a data mining effort. In a simulation based on this data, multi-tank and multi-attribute correlations were tested against synthetic diversion scenarios. Kernel regression smoothing was used to fit a curve to the historical data, and multivariable, residual analysis and cumulative sum techniques set parameters for operating conditions. Diversion scenarios were created and tested, showing improved results when compared with a previous study utilizing only one-variable Z- testing7.

  7. Retroviral retargeting by envelopes expressing an N-terminal binding domain.

    PubMed Central

    Cosset, F L; Morling, F J; Takeuchi, Y; Weiss, R A; Collins, M K; Russell, S J

    1995-01-01

    We have engineered ecotropic Moloney murine leukemia virus-derived envelopes targeted to cell surface molecules expressed on human cells by the N-terminal insertion of polypeptides able to bind either Ram-1 phosphate transporter (the first 208 amino acids of amphotropic murine leukemia virus surface protein) or epidermal growth factor receptor (EGFR) (the 53 amino acids of EGF). Both envelopes were correctly processed and incorporated into viral particles. Virions carrying these envelopes could specifically bind the new cell surface receptors. Virions targeted to Ram-1 could infect human cells, although the efficiency was reduced compared with that of virions carrying wild-type amphotropic murine leukemia virus envelopes. The infectivity of virions targeted to EGFR was blocked at a postbinding step, and our results suggest that EGFR-bound virions were rapidly trafficked to lysosomes. These data suggest that retroviruses require specific properties of cell surface molecules to allow the release of viral cores into the correct cell compartment. PMID:7666532

  8. Isolation, Proteomic Analysis, and Microscopy Confirmation of the Liver Nuclear Envelope Proteome.

    PubMed

    Korfali, Nadia; Florens, Laurence; Schirmer, Eric C

    2016-01-01

    Nuclei can be relatively easily extracted from homogenized liver due to the softness of the tissue and crudely separated from other cellular organelles by low-speed centrifugation due to the comparatively large size of nuclei. However, further purification is complicated by nuclear envelope continuity with the endoplasmic reticulum, invaginations containing mitochondria, and connections to the cytoskeleton. Subsequent purification to nuclear envelopes is additionally confounded by connections of inner nuclear membrane proteins to chromatin. For these reasons, it is necessary to confirm proteomic identification of nuclear envelope proteins by testing targeting of individual proteins. The proteomic identification of nuclear envelope fractions is affected by the tendencies of transmembrane proteins to have extreme isoelectric points, strongly hydrophobic peptides, posttranslational modifications, and a propensity to aggregate, thus making proteolysis inefficient. To circumvent these problems, we have developed a MudPIT approach that uses multiple extractions and sequential proteolysis to increase identifications. Here we describe methods for isolating nuclear envelopes, determining their proteome by MudPIT, and confirming their targeting to the nuclear periphery by microscopy. PMID:27147032

  9. Resource envelope concepts for mission planning

    NASA Technical Reports Server (NTRS)

    Ibrahim, K. Y.; Weiler, J. D.; Tokaz, J. C.

    1991-01-01

    Seven proposed methods for creating resource envelopes for Space Station Freedom mission planning are detailed. Four reference science activity models are used to illustrate the effect of adding operational flexibility to mission timelines. For each method, a brief explanation is given along with graphs to illustrate the application of the envelopes to the power and crew resources. The benefits and costs of each method are analyzed in terms of resource utilization. In addition to the effect on individual activities, resource envelopes are analyzed at the experiment level.

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

    PubMed Central

    Moller-Tank, Sven; Maury, Wendy

    2014-01-01

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

  11. Thioridazine Alters the Cell-Envelope Permeability of Mycobacterium tuberculosis.

    PubMed

    de Keijzer, Jeroen; Mulder, Arnout; de Haas, Petra E W; de Ru, Arnoud H; Heerkens, Evy M; Amaral, Leonard; van Soolingen, Dick; van Veelen, Peter A

    2016-06-01

    The increasing occurrence of multidrug resistant tuberculosis exerts a major burden on treatment of this infectious disease. Thioridazine, previously used as a neuroleptic, is active against extensively drug resistant tuberculosis when added to other second- and third-line antibiotics. By quantitatively studying the proteome of thioridazine-treated Mycobacterium tuberculosis, we discovered the differential abundance of several proteins that are involved in the maintenance of the cell-envelope permeability barrier. By assessing the accumulation of fluorescent dyes in mycobacterial cells over time, we demonstrate that long-term drug exposure of M. tuberculosis indeed increased the cell-envelope permeability. The results of the current study demonstrate that thioridazine induced an increase in cell-envelope permeability and thereby the enhanced uptake of compounds. These results serve as a novel explanation to the previously reported synergistic effects between thioridazine and other antituberculosis drugs. This new insight in the working mechanism of this antituberculosis compound could open novel perspectives of future drug-administration regimens in combinational therapy. PMID:27068340

  12. African Swine Fever Virus Undergoes Outer Envelope Disruption, Capsid Disassembly and Inner Envelope Fusion before Core Release from Multivesicular Endosomes.

    PubMed

    Hernáez, Bruno; Guerra, Milagros; Salas, María L; Andrés, Germán

    2016-04-01

    African swine fever virus (ASFV) is a nucleocytoplasmic large DNA virus (NCLDV) that causes a highly lethal disease in domestic pigs. As other NCLDVs, the extracellular form of ASFV possesses a multilayered structure consisting of a genome-containing nucleoid successively wrapped by a thick protein core shell, an inner lipid membrane, an icosahedral protein capsid and an outer lipid envelope. This structural complexity suggests an intricate mechanism of internalization in order to deliver the virus genome into the cytoplasm. By using flow cytometry in combination with pharmacological entry inhibitors, as well as fluorescence and electron microscopy approaches, we have dissected the entry and uncoating pathway used by ASFV to infect the macrophage, its natural host cell. We found that purified extracellular ASFV is internalized by both constitutive macropinocytosis and clathrin-mediated endocytosis. Once inside the cell, ASFV particles move from early endosomes or macropinosomes to late, multivesicular endosomes where they become uncoated. Virus uncoating requires acidic pH and involves the disruption of the outer membrane as well as of the protein capsid. As a consequence, the inner viral membrane becomes exposed and fuses with the limiting endosomal membrane to release the viral core into the cytosol. Interestingly, virus fusion is dependent on virus protein pE248R, a transmembrane polypeptide of the inner envelope that shares sequence similarity with some members of the poxviral entry/fusion complex. Collective evidence supports an entry model for ASFV that might also explain the uncoating of other multienveloped icosahedral NCLDVs. PMID:27110717

  13. African Swine Fever Virus Undergoes Outer Envelope Disruption, Capsid Disassembly and Inner Envelope Fusion before Core Release from Multivesicular Endosomes

    PubMed Central

    Hernáez, Bruno; Guerra, Milagros; Salas, María L.

    2016-01-01

    African swine fever virus (ASFV) is a nucleocytoplasmic large DNA virus (NCLDV) that causes a highly lethal disease in domestic pigs. As other NCLDVs, the extracellular form of ASFV possesses a multilayered structure consisting of a genome-containing nucleoid successively wrapped by a thick protein core shell, an inner lipid membrane, an icosahedral protein capsid and an outer lipid envelope. This structural complexity suggests an intricate mechanism of internalization in order to deliver the virus genome into the cytoplasm. By using flow cytometry in combination with pharmacological entry inhibitors, as well as fluorescence and electron microscopy approaches, we have dissected the entry and uncoating pathway used by ASFV to infect the macrophage, its natural host cell. We found that purified extracellular ASFV is internalized by both constitutive macropinocytosis and clathrin-mediated endocytosis. Once inside the cell, ASFV particles move from early endosomes or macropinosomes to late, multivesicular endosomes where they become uncoated. Virus uncoating requires acidic pH and involves the disruption of the outer membrane as well as of the protein capsid. As a consequence, the inner viral membrane becomes exposed and fuses with the limiting endosomal membrane to release the viral core into the cytosol. Interestingly, virus fusion is dependent on virus protein pE248R, a transmembrane polypeptide of the inner envelope that shares sequence similarity with some members of the poxviral entry/fusion complex. Collective evidence supports an entry model for ASFV that might also explain the uncoating of other multienveloped icosahedral NCLDVs. PMID:27110717

  14. Solar envelope concepts: moderate density building applications

    NASA Astrophysics Data System (ADS)

    Knowles, R. L.; Berry, R. D.

    1980-04-01

    The public policy mechanism for guaranteeing solar access is conceptualized as a solar zoning envelope that allows the largest possible building bulk on a land parcel without shadowing neighboring properties during specified times. Step-by-step methods for generating solar envelopes are described with extensive drawings, showing a variety of urban platting and lot configurations. Development and design possibilities are examined on a selected set of Los Angeles sites with typically diverse urban characteristics. Envelope attributes suitable for encouraging moderate-density commercial and residential building are examined in the context of two hypothetical but realistic development programs: one for speculative office buildings and one for condominium housing. Numerous illustrations of envelope forms and prototypical building designs are provided.

  15. Personnel occupied woven envelope robot power

    NASA Technical Reports Server (NTRS)

    Wessling, F. C.

    1988-01-01

    The Personnel Occupied Woven Envelope Robot (POWER) concept has evolved over the course of the study. The goal of the project was the development of methods and algorithms for solid modeling for the flexible robot arm.

  16. Survival of an Enveloped Virus on Toys.

    PubMed

    Bearden, Richard L; Casanova, Lisa M

    2016-08-01

    Children's toys may carry respiratory viruses. Inactivation of a lipid-enveloped bacteriophage, Φ6, was measured on a nonporous toy at indoor temperature and relative humidity (RH). Inactivation was approximately 2log10 after 24 hours at 60% RH and 6.8log10 at 10 hours at 40% RH. Enveloped viruses can potentially survive on toys long enough to result in exposures. PMID:27144972

  17. Creating a Lunar EVA Work Envelope

    NASA Technical Reports Server (NTRS)

    Griffin, Brand N.; Howard, Robert; Rajulu, Sudhakar; Smitherman, David

    2009-01-01

    A work envelope has been defined for weightless Extravehicular Activity (EVA) based on the Space Shuttle Extravehicular Mobility Unit (EMU), but there is no equivalent for planetary operations. The weightless work envelope is essential for planning all EVA tasks because it determines the location of removable parts, making sure they are within reach and visibility of the suited crew member. In addition, using the envelope positions the structural hard points for foot restraints that allow placing both hands on the job and provides a load path for reacting forces. EVA operations are always constrained by time. Tasks are carefully planned to ensure the crew has enough breathing oxygen, cooling water, and battery power. Planning first involves computers using a virtual work envelope to model tasks, next suited crew members in a simulated environment refine the tasks. For weightless operations, this process is well developed, but planetary EVA is different and no work envelope has been defined. The primary difference between weightless and planetary work envelopes is gravity. It influences anthropometry, horizontal and vertical mobility, and reaction load paths and introduces effort into doing "overhead" work. Additionally, the use of spacesuits other than the EMU, and their impacts on range of motion, must be taken into account. This paper presents the analysis leading to a concept for a planetary EVA work envelope with emphasis on lunar operations. There is some urgency in creating this concept because NASA has begun building and testing development hardware for the lunar surface, including rovers, habitats and cargo off-loading equipment. Just as with microgravity operations, a lunar EVA work envelope is needed to guide designers in the formative stages of the program with the objective of avoiding difficult and costly rework.

  18. Passage of heme-iron across the envelope of Staphylococcus aureus.

    PubMed

    Mazmanian, Sarkis K; Skaar, Eric P; Gaspar, Andrew H; Humayun, Munir; Gornicki, Piotr; Jelenska, Joanna; Joachmiak, Andrzej; Missiakas, Dominique M; Schneewind, Olaf

    2003-02-01

    The cell wall envelope of Gram-positive pathogens functions as a scaffold for the attachment of virulence factors and as a sieve that prevents diffusion of molecules. Here the isd genes (iron-regulated surface determinant) of Staphylococcus aureus were found to encode factors responsible for hemoglobin binding and passage of heme-iron to the cytoplasm, where it acts as an essential nutrient. Heme-iron passage required two sortases that tether Isd proteins to unique locations within the cell wall. Thus, Isd appears to act as an import apparatus that uses cell wall-anchored proteins to relay heme-iron across the bacterial envelope. PMID:12574635

  19. Human inositol 1,4,5-trisphosphate 3-kinase isoform B (IP3KB) is a nucleocytoplasmic shuttling protein specifically enriched at cortical actin filaments and at invaginations of the nuclear envelope.

    PubMed

    Nalaskowski, Marcus M; Fliegert, Ralf; Ernst, Olga; Brehm, Maria A; Fanick, Werner; Windhorst, Sabine; Lin, Hongying; Giehler, Susanne; Hein, Jamin; Lin, Yuan-Na; Mayr, Georg W

    2011-02-11

    Recent studies have shown that inositol 1,4,5-trisphosphate 3-kinase isoform B (IP3KB) possesses important roles in the development of immune cells. IP3KB can be targeted to multiple cellular compartments, among them nuclear localization and binding in close proximity to the plasma membrane. The B isoform is the only IP3K that is almost ubiquitously expressed in mammalian cells. Detailed mechanisms of its targeting regulation will be important in understanding the role of Ins(1,4,5)P(3) phosphorylation on subcellular calcium signaling and compartment-specific initiation of pathways leading to regulatory active higher phosphorylated inositol phosphates. Here, we identified an exportin 1-dependent nuclear export signal ((134)LQRELQNVQV) and characterized the amino acids responsible for nuclear localization of IP3KB ((129)RKLR). These two targeting domains regulate the amount of nuclear IP3KB in cells. We also demonstrated that the localization of IP3KB at the plasma membrane is due to its binding to cortical actin structures. Intriguingly, all three of these targeting activities reside in one small polypeptide segment (amino acids 104-165), which acts as a multitargeting domain (MTD). Finally, a hitherto unknown subnuclear localization of IP3KB could be demonstrated in rapidly growing H1299 cells. IP3KB is specifically enriched at nuclear invaginations extending perpendicular between the apical and basal surface of the nucleus of these flat cells. Such nuclear invaginations are known to be involved in Ins(1,4,5)P(3)-mediated Ca(2+) signaling of the nucleus. Our findings indicate that IP3KB not only regulates cytoplasmic Ca(2+) signals by phosphorylation of subplasmalemmal and cytoplasmic Ins(1,4,5)P(3) but may also be involved in modulating nuclear Ca(2+) signals generated from these nuclear envelope invaginations. PMID:21148483

  20. Cooling of neutron stars with diffusive envelopes

    NASA Astrophysics Data System (ADS)

    Beznogov, M. V.; Fortin, M.; Haensel, P.; Yakovlev, D. G.; Zdunik, J. L.

    2016-08-01

    We study the effects of heat blanketing envelopes of neutron stars on their cooling. To this aim, we perform cooling simulations using newly constructed models of the envelopes composed of binary ion mixtures (H-He, He-C, C-Fe) varying the mass of lighter ions (H, He or C) in the envelope. The results are compared with those calculated using the standard models of the envelopes which contain the layers of lighter (accreted) elements (H, He and C) on top of the Fe layer, varying the mass of accreted elements. The main effect is that the chemical composition of the envelopes influences their thermal conductivity and, hence, thermal insulation of the star. For illustration, we apply these results to estimate the internal temperature of the Vela pulsar and to study the cooling of neutron stars of ages of 105 - 106 yr at the photon cooling stage. The uncertainties of the cooling models associated with our poor knowledge of chemical composition of the heat insulating envelopes strongly complicate theoretical reconstruction of the internal structure of cooling neutron stars from observations of their thermal surface emission.

  1. The joke envelope: a neglected precursor of the psychic envelope concept in Freud's writing.

    PubMed

    Spero, Moshe Halevi

    2009-01-01

    The concepts of the primeval skin ego, psychic envelope, and related pre-ego containing and wrapping functions elaborated respectively by Esther Bick, Didier Anzieu, and Francis Tustin occupy an important position in contemporary psychoanalytic theory and clinical practice. The psychic envelope begins as a virtual mental protostructure ("proto" because it is not yet based on fully symbolized representations) that holds the budding mind together pending further developments. With maturity, the enveloping functions adopt symbolized, metaphoric form (for example, the aesthetic use of cloth, the analytic framework), but can regress to more concrete and pathological forms. The aforementioned authors based their ideas on a cluster of specific allusions to the idea of a psychic covering, barrier, or envelope in Freud's work. Yet they neglected one reference, hidden in Freud's analysis of the structure ofjokes and humor: the 'joke envelope"--die witzige Einkleidung. The present essay explores Freud's use of the term Einkleidung, including his intriguing idea that a joke requires three people whereas a dream does not and the fact that Freud nowhere speaks of a "dream envelope. "I take the "joke envelope" beyond its original context and posit a relationship between laughter and the early, normative traumas of breathing, crying, and loss, and the dawn of rhythmic envelopes that enable mentalization. Jokes and joking symbolically repeat the early rupture and rapture of breathing and self-other differentiation and the internalization of maternal containing and envelopment. PMID:20578439

  2. Isolation of the envelope of vesicular stomatitis virus.

    PubMed Central

    Taube, S E; Rothfield, L I

    1978-01-01

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

  3. 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

  4. Targeted Entry of Enveloped Viruses: Measles and Herpes Simplex Virus I

    PubMed Central

    Navaratnarajah, Chanakha K.; Miest, Tanner S.; Carfi, Andrea; Cattaneo, Roberto

    2011-01-01

    We compare the receptor-based mechanisms that a small RNA virus and a larger DNA virus have evolved to drive the fusion of viral and cellular membranes. Both systems rely on tight control over triggering the concerted refolding of a trimeric fusion protein. While measles virus entry depends on a receptor-binding protein and a fusion protein only, the herpes simplex virus is more complex and requires four viral proteins. Nevertheless, in both viruses a receptor-binding protein is required for triggering the membrane fusion process. Moreover, specificity domains can be appended to these receptor-binding proteins to target virus entry to cells expressing a designated receptor. We discuss how principles established with measles and herpes simplex virus can be applied to targeting other enveloped viruses, and alternatively how retargeted envelopes can be fitted on foreign capsids. PMID:22440965

  5. Comparative analysis of envelope proteomes in Escherichia coli B and K-12 strains.

    PubMed

    Han, Mee-Jung; Lee, Sang Yup; Hong, Soon Ho

    2012-04-01

    Recent genome comparisons of E. coli B and K-12 strains have indicated that the makeup of the cell envelopes in these two strains is quite different. Therefore, we analyzed and compared the envelope proteomes of E. coli BL21(DE3) and MG1655. A total of 165 protein spots, including 62 nonredundant proteins, were unambiguously identified by two-dimensional gel electrophoresis and mass spectrometry. Of these, 43 proteins were conserved between the two strains, whereas 4 and 16 strain-specific proteins were identified only in E. coli BL21(DE3) and MG1655, respectively. Additionally, 24 proteins showed more than 2-fold differences in intensities between the B and K-12 strains. The reference envelope proteome maps showed that E. coli envelope mainly contained channel proteins and lipoproteins. Interesting proteomic observations between the two strains were as follows: (i) B produced more OmpF porin with a larger pore size than K-12, indicating an increase in the membrane permeability; (ii) B produced higher amounts of lipoproteins, which facilitates the assembly of outer membrane beta-barrel proteins; and (iii) motility- (FliC) and chemotaxis-related proteins (CheA and CheW) were detected only in K-12, which showed that E. coli B is restricted with regard to migration under unfavorable conditions. These differences may influence the permeability and integrity of the cell envelope, showing that E. coli B may be more susceptible than K-12 to certain stress conditions. Thus, these findings suggest that E. coli K-12 and its derivatives will be more favorable strains in certain biotechnological applications, such as cell surface display or membrane engineering studies. PMID:22534293

  6. Simulating Convection in Stellar Envelopes

    NASA Astrophysics Data System (ADS)

    Tanner, Joel

    Understanding convection in stellar envelopes, and providing a mathematical description of it, would represent a substantial advance in stellar astrophysics. As one of the largest sources of uncertainty in stellar models, existing treatments of convection fail to account for many of the dynamical effects of convection, such as turbulent pressure and asymmetry in the velocity field. To better understand stellar convection, we must be able to study and examine it in detail, and one of the best tools for doing so is numerical simulation. Near the stellar surface, both convective and radiative process play a critical role in determining the structure and gas dynamics. By following these processes from first principles, convection can be simulated self-consistently and accurately, even in regions of inefficient energy transport where existing descriptions of convection fail. Our simulation code includes two radiative transfer solvers that are based on different assumptions and approximations. By comparing simulations that differ only in their respective radiative transfer methods, we are able to isolate the effect that radiative efficiency has on the structure of the superadiabatic layer. We find the simulations to be in good general agreement, but they show distinct differences in the thermal structure in the superadiabatic layer and atmosphere. Using the code to construct a grid of three-dimensional radiation hydrodynamic simulations, we investigate the link between convection and various chemical compositions. The stellar parameters correspond to main-sequence stars at several surface gravities, and span a range in effective temperatures (4500 < Teff < 6400). Different chemical compositions include four metallicities (Z = 0.040, 0.020, 0.010, 0.001), three helium abundances (Y = 0.1, 0.2, 0.3) and several levels of alpha-element enhancement. Our grid of simulations shows that various convective properties, such as velocity and the degree of superadiabaticity, are

  7. Detection of an Immunogenic HERV-E Envelope with Selective Expression in Clear Cell Kidney Cancer.

    PubMed

    Cherkasova, Elena; Scrivani, Claire; Doh, Susan; Weisman, Quinn; Takahashi, Yoshiyuki; Harashima, Nanae; Yokoyama, Hisayuki; Srinivasan, Ramaprasad; Linehan, W Marston; Lerman, Michael I; Childs, Richard W

    2016-04-15

    VHL-deficient clear cell renal cell carcinomas (ccRCC), the most common form of kidney cancer, express transcripts derived from the novel human endogenous retrovirus HERV-E (named CT-RCC HERV-E). In this study, we define a transcript encoding the entire envelope gene of HERV-E as expressed selectively in ccRCC tumors, as distinct from normal kidney tissues or other tumor types. Sequence analysis of this envelope transcript revealed long open reading frames encoding putative surface and transmembrane envelope proteins. Retroviral envelopes are known to be capable of eliciting immunity in humans. Accordingly, we found that HLA-A*0201-restricted peptides predicted to be products of the CT-RCC HERV-E envelope transcript-stimulated CD8(+) T cells, which could recognize HLA-A*0201-positive HERV-E-expressing kidney tumor cells. Overall, our results offer evidence of unique HERV-E envelope peptides presented on the surface of ccRCC cells, offering potentially useful tumor-restricted targets for T-cell-based immunotherapy of kidney cancer. Cancer Res; 76(8); 2177-85. ©2016 AACR. PMID:26862115

  8. Envelope-receptor interactions in Nipah virus pathobiology.

    PubMed

    Lee, Benhur

    2007-04-01

    Nipah (NiV) and Hendra (HeV) viruses are members of the newly defined Henipavirus genus of the Paramyxoviridae. Nipah virus (NiV) is an emergent paramyxovirus that causes fatal encephalitis in up to 70% of infected patients, and there is increasing evidence of human-to-human transmission. NiV is designated a priority pathogen in the NIAID Biodefense Research Agenda, and could be a devastating agent of agrobioterrorism if used against the pig farming industry. Endothelial syncytium is a pathognomonic feature of NiV infections, and is mediated by the fusion (F) and attachment (G) envelope glycoproteins. This review summarizes what is known about the pathophysiology of NiV infections, and documents the identification of the NiV receptor. EphrinB2, the NiV and HeV receptor, is expressed on endothelial cells and neurons, consistent with the known cellular tropism for NiV. We discuss how the identification of the henipahvirus receptor sheds light on the pathobiology of NiV infection, and how it will spur the rational development of effective therapeutics. In addition, ephrinB3, a related protein, can serve as an alternative receptor, and we suggest that differential usage of ephrinB2 versus B3 may explain the variant pathogenic profiles observed between NiV and HeV. Thus, identifying the NiV receptors opens the door for a more comprehensive analysis of the envelope-receptor interactions in NiV pathobiology. Finally, we also describe how galectin-1 (an innate immune defense lectin) can interact with specific N-glycans on the Nipah envelope fusion protein, underscoring the potential role that innate immune defense mechanisms may play against emerging pathogens. PMID:17470911

  9. Featured Image: Orbiting Stars Share an Envelope

    NASA Astrophysics Data System (ADS)

    Kohler, Susanna

    2016-03-01

    This beautiful series of snapshots from a simulation (click for a better look!) shows what happens when two stars in a binary system become enclosed in the same stellar envelope. In this binary system, one of the stars has exhausted its hydrogen fuel and become a red giant, complete with an expanding stellar envelope composed of hydrogen and helium. Eventually, the envelope expands so much that the companion star falls into it, where it releases gravitational potential energy into the common envelope. A team led by Sebastian Ohlmann (Heidelberg Institute for Theoretical Studies and University of Wrzburg) recently performed hydrodynamic simulations of this process. Ohlmann and collaborators discovered that the energy release eventually triggers large-scale flow instabilities, which leads to turbulence within the envelope. This process has important consequences for how these systems next evolve (for instance, determining whether or not a supernova occurs!). You can check out the authors video of their simulated stellar inspiral below, or see their paper for more images and results from their study.CitationSebastian T. Ohlmann et al 2016 ApJ 816 L9. doi:10.3847/2041-8205/816/1/L9

  10. Envelope Solitons in Acoustically Dispersive Vitreous Silica

    NASA Technical Reports Server (NTRS)

    Cantrell, John H.; Yost, William T.

    2012-01-01

    Acoustic radiation-induced static strains, displacements, and stresses are manifested as rectified or dc waveforms linked to the energy density of an acoustic wave or vibrational mode via the mode nonlinearity parameter of the material. An analytical model is developed for acoustically dispersive media that predicts the evolution of the energy density of an initial waveform into a series of energy solitons that generates a corresponding series of radiation-induced static strains (envelope solitons). The evolutionary characteristics of the envelope solitons are confirmed experimentally in Suprasil W1 vitreous silica. The value (-11.9 plus or minus 1.43) for the nonlinearity parameter, determined from displacement measurements of the envelope solitons via a capacitive transducer, is in good agreement with the value (-11.6 plus or minus 1.16) obtained independently from acoustic harmonic generation measurements. The agreement provides strong, quantitative evidence for the validity of the model.

  11. Common Envelope and the Binding Energy Consideration

    NASA Astrophysics Data System (ADS)

    Irawati, P.; Mahasena, P.

    2014-08-01

    We report the results of our study on the common-envelope phase of the cataclysmic variables. We are investigating the role of additional energies, such as recombination energy and internal energy, in expelling the envelope of the primary star. In this work, we use the TWIN stellar evolution code which can evolve both stars in binary simultaneously. We analysed the energies involved by considering the binding energy of the core at the onset of the common envelope phase. The core of the primary is calculated using the hydrogen-exhausted layer with 10% hydrogen fraction. Our preliminary result shows that the internal energy plays a significant role while the recombination energy has only a small contribution to the energy budget of the cataclysmic variable evolution.

  12. Computational and experimental approaches to chart the Escherichia coli cell-envelope-associated proteome and interactome.

    PubMed

    Díaz-Mejía, Juan Javier; Babu, Mohan; Emili, Andrew

    2009-01-01

    The bacterial cell-envelope consists of a complex arrangement of lipids, proteins and carbohydrates that serves as the interface between a microorganism and its environment or, with pathogens, a human host. Escherichia coli has long been investigated as a leading model system to elucidate the fundamental mechanisms underlying microbial cell-envelope biology. This includes extensive descriptions of the molecular identities, biochemical activities and evolutionary trajectories of integral transmembrane proteins, many of which play critical roles in infectious disease and antibiotic resistance. Strikingly, however, only half of the c. 1200 putative cell-envelope-related proteins of E. coli currently have experimentally attributed functions, indicating an opportunity for discovery. In this review, we summarize the state of the art of computational and proteomic approaches for determining the components of the E. coli cell-envelope proteome, as well as exploring the physical and functional interactions that underlie its biogenesis and functionality. We also provide a comprehensive comparative benchmarking analysis on the performance of different bioinformatic and proteomic methods commonly used to determine the subcellular localization of bacterial proteins. PMID:19054114

  13. Computational and experimental approaches to chart the Escherichia coli cell-envelope-associated proteome and interactome

    PubMed Central

    Díaz-Mejía, Juan Javier; Babu, Mohan; Emili, Andrew

    2009-01-01

    The bacterial cell-envelope consists of a complex arrangement of lipids, proteins and carbohydrates that serves as the interface between a microorganism and its environment or, with pathogens, a human host. Escherichia coli has long been investigated as a leading model system to elucidate the fundamental mechanisms underlying microbial cell-envelope biology. This includes extensive descriptions of the molecular identities, biochemical activities and evolutionary trajectories of integral transmembrane proteins, many of which play critical roles in infectious disease and antibiotic resistance. Strikingly, however, only half of the c. 1200 putative cell-envelope-related proteins of E. coli currently have experimentally attributed functions, indicating an opportunity for discovery. In this review, we summarize the state of the art of computational and proteomic approaches for determining the components of the E. coli cell-envelope proteome, as well as exploring the physical and functional interactions that underlie its biogenesis and functionality. We also provide a comprehensive comparative benchmarking analysis on the performance of different bioinformatic and proteomic methods commonly used to determine the subcellular localization of bacterial proteins. PMID:19054114

  14. Perception and coding of envelopes in weakly electric fishes.

    PubMed

    Stamper, Sarah A; Fortune, Eric S; Chacron, Maurice J

    2013-07-01

    Natural sensory stimuli have a rich spatiotemporal structure and can often be characterized as a high frequency signal that is independently modulated at lower frequencies. This lower frequency modulation is known as the envelope. Envelopes are commonly found in a variety of sensory signals, such as contrast modulations of visual stimuli and amplitude modulations of auditory stimuli. While psychophysical studies have shown that envelopes can carry information that is essential for perception, how envelope information is processed in the brain is poorly understood. Here we review the behavioral salience and neural mechanisms for the processing of envelopes in the electrosensory system of wave-type gymnotiform weakly electric fishes. These fish can generate envelope signals through movement, interactions of their electric fields in social groups or communication signals. The envelopes that result from the first two behavioral contexts differ in their frequency content, with movement envelopes typically being of lower frequency. Recent behavioral evidence has shown that weakly electric fish respond in robust and stereotypical ways to social envelopes to increase the envelope frequency. Finally, neurophysiological results show how envelopes are processed by peripheral and central electrosensory neurons. Peripheral electrosensory neurons respond to both stimulus and envelope signals. Neurons in the primary hindbrain recipient of these afferents, the electrosensory lateral line lobe (ELL), exhibit heterogeneities in their responses to stimulus and envelope signals. Complete segregation of stimulus and envelope information is achieved in neurons in the target of ELL efferents, the midbrain torus semicircularis (Ts). PMID:23761464

  15. 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.

  16. 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

  17. Fumarate or a fumarate metabolite restores switching ability to rotating flagella of bacterial envelopes.

    PubMed Central

    Barak, R; Eisenbach, M

    1992-01-01

    Flagella of cytoplasm-free envelopes of Escherichia coli or Salmonella typhimurium can rotate in either the counterclockwise or clockwise direction, but they never switch from one direction of rotation to another. Exogenous fumarate, in the intracellular presence of the chemotaxis protein CheY, restored switching ability to envelopes, with a concomitant increase in clockwise rotation. An increase in clockwise rotation was also observed after fumarate was added to partially lysed cells of E. coli, but the proportion of switching cells remained unchanged. Images PMID:1729255

  18. Functional Analysis of the Hsp93/ClpC Chaperone at the Chloroplast Envelope1[OPEN

    PubMed Central

    Tanabe, Noriaki; Clarke, Adrian K.

    2016-01-01

    The Hsp100-type chaperone Hsp93/ClpC has crucial roles in chloroplast biogenesis. In addition to its role in proteolysis in the stroma, biochemical and genetic evidence led to the hypothesis that this chaperone collaborates with the inner envelope TIC complex to power preprotein import. Recently, it was suggested that Hsp93, working together with the Clp proteolytic core, can confer a protein quality control mechanism at the envelope. Thus, the role of envelope-localized Hsp93, and the mechanism by which it participates in protein import, remain unclear. To analyze the function of Hsp93 in protein import independently of its ClpP association, we created a mutant of Hsp93 affecting its ClpP-binding motif (PBM) (Hsp93[P-]), which is essential for the chaperone’s interaction with the Clp proteolytic core. The Hsp93[P-] construct was ineffective at complementing the pale-yellow phenotype of hsp93 Arabidopsis (Arabidopsis thaliana) mutants, indicating that the PBM is essential for Hsp93 function. As expected, the PBM mutation negatively affected the degradation activity of the stromal Clp protease. The mutation also disrupted association of Hsp93 with the Clp proteolytic core at the envelope, without affecting the envelope localization of Hsp93 itself or its association with the TIC machinery, which we demonstrate to be mediated by a direct interaction with Tic110. Nonetheless, Hsp93[P-] expression did not detectably improve the protein import efficiency of hsp93 mutant chloroplasts. Thus, our results do not support the proposed function of Hsp93 in protein import propulsion, but are more consistent with the notion of Hsp93 performing a quality control role at the point of import. PMID:26586836

  19. The Methodology of Data Envelopment Analysis.

    ERIC Educational Resources Information Center

    Sexton, Thomas R.

    1986-01-01

    The methodology of data envelopment analysis, (DEA) a linear programming-based method, is described. Other procedures often used for measuring relative productive efficiency are discussed in relation to DEA, including ratio analysis and multiple regression analysis. The DEA technique is graphically illustrated for only two inputs and one output.…

  20. Diffusive heat blanketing envelopes of neutron stars

    NASA Astrophysics Data System (ADS)

    Beznogov, M. V.; Potekhin, A. Y.; Yakovlev, D. G.

    2016-06-01

    We construct new models of outer heat blanketing envelopes of neutron stars composed of binary ion mixtures (H-He, He-C, C-Fe) in and out of diffusive equilibrium. To this aim, we generalize our previous work on diffusion of ions in isothermal gaseous or Coulomb liquid plasmas to handle non-isothermal systems. We calculate the relations between the effective surface temperature Ts and the temperature Tb at the bottom of heat blanketing envelopes (at a density ρb ˜ 108 - 1010 g cm-3) for diffusively equilibrated and non-equilibrated distributions of ion species at different masses ΔM of lighter ions in the envelope. Our principal result is that the Ts-Tb relations are fairly insensitive to detailed distribution of ion fractions over the envelope (diffusively equilibrated or not) and depend almost solely on ΔM. The obtained relations are approximated by analytic expressions which are convenient for modelling the evolution of neutron stars.

  1. Thermal Damage to Chloroplast Envelope Membranes 1

    PubMed Central

    McCain, Douglas C.; Croxdale, Judith; Markley, John L.

    1989-01-01

    Nuclear magnetic resonance was used to detect thermal injury to chloroplasts in vivo. A lesion occurs in the chloroplast envelope membrane at temperatures between 53°C and 57°C, depending on species, leaf condition, and heating rate. The injury is associated with a sudden loss of water from the chloroplast. PMID:16666815

  2. Discriminating Dysarthria Type from Envelope Modulation Spectra

    ERIC Educational Resources Information Center

    Liss, Julie M.; LeGendre, Sue; Lotto, Andrew J.

    2010-01-01

    Purpose: Previous research demonstrated the ability of temporally based rhythm metrics to distinguish among dysarthrias with different prosodic deficit profiles (J. M. Liss et al., 2009). The authors examined whether comparable results could be obtained by an automated analysis of speech envelope modulation spectra (EMS), which quantifies the…

  3. SAFEGUARDS ENVELOPE: PREVIOUS WORK AND EXAMPLES

    SciTech Connect

    Richard Metcalf; Aaron Bevill; William Charlton; Robert Bean

    2008-07-01

    The future expansion of nuclear power will require not just electricity production but fuel cycle facilities such as fuel fabrication and reprocessing plants. As large reprocessing facilities are built in various states, they must be built and operated in a manner to minimize the risk of nuclear proliferation. Process monitoring has returned to the spotlight as an added measure that can increase confidence in the safeguards of special nuclear material (SNM). Process monitoring can be demonstrated to lengthen the allowable inventory period by reducing accountancy requirements, and to reduce the false positive indications. The next logical step is the creation of a Safeguards Envelope, a set of operational parameters and models to maximize anomaly detection and inventory period by process monitoring while minimizing operator impact and false positive rates. A brief example of a rudimentary Safeguards Envelope is presented, and shown to detect synthetic diversions overlaying a measured processing plant data set. This demonstration Safeguards Envelope is shown to increase the confidence that no SNM has been diverted with minimal operator impact, even though it is based on an information sparse environment. While the foundation on which a full Safeguards Envelope can be built has been presented in historical demonstrations of process monitoring, several requirements remain yet unfulfilled. Future work will require reprocessing plant transient models, inclusion of “non-traditional” operating data, and exploration of new methods of identifying subtle events in transient processes.

  4. Trumpet synthesis using context-dependent envelopes

    NASA Astrophysics Data System (ADS)

    Dannenberg, Roger B.

    2002-05-01

    Synthesizing trumpet music in a natural-sounding way requires careful control. Even when synthesis is achieved by splicing together actual recorded trumpet tones, the result can sound artificial and unnatural. This is because natural notes are not played in isolation and are therefore influenced by neighboring notes and the musical context. In fact, a succession of notes played on the trumpet is likely to be a continuous sound with no separating silences. Improved synthesis can be obtained by calculating amplitude and frequency control envelopes that take context into consideration. In the combined spectral interpolation synthesis (CSIS) method, the spectrum is controlled by instantaneous frequency and rms amplitude. These, in turn, are controlled by envelopes computed by a rule-based system. To reduce the high dimensionality of envelopes (typically a vector of 200 samples per second), envelopes are specified by about ten parameters. This reduced set of parameters is computed from note attributes, most importantly, the duration and pitches of the current and preceding notes, and whether or not the notes are tongued or slurred. This procedure is described in detail, and synthesis results will be demonstrated.

  5. Ozone Reductions Using Residential Building Envelopes

    SciTech Connect

    Walker, Iain S.; Sherman, Max; Nazaroff, William W.

    2009-02-01

    Ozone is an air pollutant with that can have significant health effects and a significant source of ozone in some regions of California is outdoor air. Because people spend the vast majority of their time indoors, reduction in indoor levels of ozone could lead to improved health for many California residents. Ozone is removed from indoor air by surface reactions and can also be filtered by building envelopes. The magnitude of the envelope impact depends on the specific building materials that the air flows over and the geometry of the air flow paths through the envelope that can be changes by mechanical ventilation operation. The 2008 Residential Building Standards in California include minimum requirements for mechanical ventilation by referencing ASHRAE Standard 62.2. This study examines the changes in indoor ozone depending on the mechanical ventilation system selected to meet these requirements. This study used detailed simulations of ventilation in a house to examine the impacts of different ventilation systems on indoor ozone concentrations. The simulation results showed that staying indoors reduces exposure to ozone by 80percent to 90percent, that exhaust ventilation systems lead to lower indoor ozone concentrations, that opening of windows should be avoided at times of high outdoor ozone, and that changing the time at which mechanical ventilation occurs has the ability to halve exposure to ozone. Future work should focus on the products of ozone reactions in the building envelope and the fate of these products with respect to indoor exposures.

  6. 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

  7. The preprotein conducting channel at the inner envelope membrane of plastids

    PubMed Central

    Heins, Lisa; Mehrle, Alexander; Hemmler, Roland; Wagner, Richard; Küchler, Michael; Hörmann, Friederike; Sveshnikov, Dmitry; Soll, Jürgen

    2002-01-01

    The preprotein translocation at the inner envelope membrane of chloroplasts so far involves five proteins: Tic110, Tic55, Tic40, Tic22 and Tic20. The molecular function of these proteins has not yet been established. Here, we demonstrate that Tic110 constitutes a central part of the preprotein translocation pore. Dependent on the presence of intact Tic110, radiolabelled preprotein specifically interacts with isolated inner envelope vesicles as well as with purified, recombinant Tic110 reconstituted into liposomes. Circular dichroism analysis reveals that Tic110 consists mainly of β-sheets, a structure typically found in pore proteins. In planar lipid bilayers, recombinant Tic110 forms a cation-selective high-conductance channel with a calculated inner pore opening of 1.7 nm. Purified transit peptide causes strong flickering and a voltage-dependent block of the channel. Moreover, at the inner envelope membrane, a peptide-sensitive channel is described that shows properties basically identical to the channel formed by recombinant Tic110. We conclude that Tic110 has a distinct preprotein binding site and functions as a preprotein translocation pore at the inner envelope membrane. PMID:12032074

  8. Octaarginine-modified multifunctional envelope-type nanoparticles for gene delivery

    PubMed Central

    Khalil, IA; Kogure, K; Futaki, S; Hama, S; Akita, H; Ueno, M; Kishida, H; Kudoh, M; Mishina, Y; Kataoka, K; Yamada, M; Harashima, H

    2007-01-01

    This study describes a multifunctional envelope-type nano device (MEND) that mimics an envelope-type virus based on a novel packaging strategy. MEND particles contain a DNA core packaged into a lipid envelope modified with an octaarginine peptide. The peptide mediates internalization via macropinocytosis, which avoids lysosomal degradation. MEND-mediated transfection of a luciferase expression plasmid achieved comparable efficiency to adenovirus-mediated transfection, with lower associated cytotoxicity. Furthermore, topical application of MEND particles containing constitutively active bone morphogenetic protein (BMP) type IA receptor (caBmpr1a) gene had a significant impact on hair growth in vivo. These data demonstrate that MEND is a promising non-viral gene delivery system that may provide superior results to existing non-viral gene delivery technologies. PMID:17268535

  9. Identification of Peptide Inhibitors of Enveloped Viruses Using Support Vector Machine

    PubMed Central

    Xu, Yongtao; Yu, Shui; Zou, Jian-Wei; Hu, Guixiang; Rahman, Noorsaadah A. B. D.; Othman, Rozana Binti; Tao, Xia; Huang, Meilan

    2015-01-01

    The peptides derived from envelope proteins have been shown to inhibit the protein-protein interactions in the virus membrane fusion process and thus have a great potential to be developed into effective antiviral therapies. There are three types of envelope proteins each exhibiting distinct structure folds. Although the exact fusion mechanism remains elusive, it was suggested that the three classes of viral fusion proteins share a similar mechanism of membrane fusion. The common mechanism of action makes it possible to correlate the properties of self-derived peptide inhibitors with their activities. Here we developed a support vector machine model using sequence-based statistical scores of self-derived peptide inhibitors as input features to correlate with their activities. The model displayed 92% prediction accuracy with the Matthew’s correlation coefficient of 0.84, obviously superior to those using physicochemical properties and amino acid decomposition as input. The predictive support vector machine model for self- derived peptides of envelope proteins would be useful in development of antiviral peptide inhibitors targeting the virus fusion process. PMID:26636321

  10. 48 CFR 14.202-3 - Bid envelopes.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... AND CONTRACT TYPES SEALED BIDDING Solicitation of Bids 14.202-3 Bid envelopes. (a) Postage or envelopes bearing Postage and Fees Paid indicia shall not be distributed with the invitation for bids...

  11. 48 CFR 14.202-3 - Bid envelopes.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... AND CONTRACT TYPES SEALED BIDDING Solicitation of Bids 14.202-3 Bid envelopes. (a) Postage or envelopes bearing Postage and Fees Paid indicia shall not be distributed with the invitation for bids...

  12. Focal Targeting of the Bacterial Envelope by Antimicrobial Peptides.

    PubMed

    Rashid, Rafi; Veleba, Mark; Kline, Kimberly A

    2016-01-01

    Antimicrobial peptides (AMPs) are utilized by both eukaryotic and prokaryotic organisms. AMPs such as the human beta defensins, human neutrophil peptides, human cathelicidin, and many bacterial bacteriocins are cationic and capable of binding to anionic regions of the bacterial surface. Cationic AMPs (CAMPs) target anionic lipids [e.g., phosphatidylglycerol (PG) and cardiolipins (CL)] in the cell membrane and anionic components [e.g., lipopolysaccharide (LPS) and lipoteichoic acid (LTA)] of the cell envelope. Bacteria have evolved mechanisms to modify these same targets in order to resist CAMP killing, e.g., lysinylation of PG to yield cationic lysyl-PG and alanylation of LTA. Since CAMPs offer a promising therapeutic alternative to conventional antibiotics, which are becoming less effective due to rapidly emerging antibiotic resistance, there is a strong need to improve our understanding about the AMP mechanism of action. Recent literature suggests that AMPs often interact with the bacterial cell envelope at discrete foci. Here we review recent AMP literature, with an emphasis on focal interactions with bacteria, including (1) CAMP disruption mechanisms, (2) delocalization of membrane proteins and lipids by CAMPs, and (3) CAMP sensing systems and resistance mechanisms. We conclude with new approaches for studying the bacterial membrane, e.g., lipidomics, high resolution imaging, and non-detergent-based membrane domain extraction. PMID:27376064

  13. Focal Targeting of the Bacterial Envelope by Antimicrobial Peptides

    PubMed Central

    Rashid, Rafi; Veleba, Mark; Kline, Kimberly A.

    2016-01-01

    Antimicrobial peptides (AMPs) are utilized by both eukaryotic and prokaryotic organisms. AMPs such as the human beta defensins, human neutrophil peptides, human cathelicidin, and many bacterial bacteriocins are cationic and capable of binding to anionic regions of the bacterial surface. Cationic AMPs (CAMPs) target anionic lipids [e.g., phosphatidylglycerol (PG) and cardiolipins (CL)] in the cell membrane and anionic components [e.g., lipopolysaccharide (LPS) and lipoteichoic acid (LTA)] of the cell envelope. Bacteria have evolved mechanisms to modify these same targets in order to resist CAMP killing, e.g., lysinylation of PG to yield cationic lysyl-PG and alanylation of LTA. Since CAMPs offer a promising therapeutic alternative to conventional antibiotics, which are becoming less effective due to rapidly emerging antibiotic resistance, there is a strong need to improve our understanding about the AMP mechanism of action. Recent literature suggests that AMPs often interact with the bacterial cell envelope at discrete foci. Here we review recent AMP literature, with an emphasis on focal interactions with bacteria, including (1) CAMP disruption mechanisms, (2) delocalization of membrane proteins and lipids by CAMPs, and (3) CAMP sensing systems and resistance mechanisms. We conclude with new approaches for studying the bacterial membrane, e.g., lipidomics, high resolution imaging, and non-detergent-based membrane domain extraction. PMID:27376064

  14. A New Family of Membrane Electron Transporters and Its Substrates, Including a New Cell Envelope Peroxiredoxin, Reveal a Broadened Reductive Capacity of the Oxidative Bacterial Cell Envelope

    PubMed Central

    Cho, Seung-Hyun; Parsonage, Derek; Thurston, Casey; Dutton, Rachel J.; Poole, Leslie B.; Collet, Jean-Francois; Beckwith, Jon

    2012-01-01

    ABSTRACT The Escherichia coli membrane protein DsbD functions as an electron hub that dispatches electrons received from the cytoplasmic thioredoxin system to periplasmic oxidoreductases involved in protein disulfide isomerization, cytochrome c biogenesis, and sulfenic acid reduction. Here, we describe a new class of DsbD proteins, named ScsB, whose members are found in proteobacteria and Chlamydia. ScsB has a domain organization similar to that of DsbD, but its amino-terminal domain differs significantly. In DsbD, this domain directly interacts with substrates to reduce them, which suggests that ScsB acts on a different array of substrates. Using Caulobacter crescentus as a model organism, we searched for the substrates of ScsB. We discovered that ScsB provides electrons to the first peroxide reduction pathway identified in the bacterial cell envelope. The reduction pathway comprises a thioredoxin-like protein, TlpA, and a peroxiredoxin, PprX. We show that PprX is a thiol-dependent peroxidase that efficiently reduces both hydrogen peroxide and organic peroxides. Moreover, we identified two additional proteins that depend on ScsB for reduction, a peroxiredoxin-like protein, PrxL, and a novel protein disulfide isomerase, ScsC. Altogether, our results reveal that the array of proteins involved in reductive pathways in the oxidative cell envelope is significantly broader than was previously thought. Moreover, the identification of a new periplasmic peroxiredoxin indicates that in some bacteria, it is important to directly scavenge peroxides in the cell envelope even before they reach the cytoplasm. PMID:22493033

  15. Single Mutation in the Flavivirus Envelope Protein Hinge Region Increases Neurovirulence for Mice and Monkeys but Decreases Viscerotropism for Monkeys: Relevance to Development and Safety Testing of Live, Attenuated Vaccines

    PubMed Central

    Monath, Thomas P.; Arroyo, Juan; Levenbook, Inessa; Zhang, Zhen-Xi; Catalan, John; Draper, Ken; Guirakhoo, Farshad

    2002-01-01

    A chimeric yellow fever (YF) virus/Japanese encephalitis (JE) virus vaccine (ChimeriVax-JE) was constructed by insertion of the prM-E genes from the attenuated JE virus SA14-14-2 vaccine strain into a full-length cDNA clone of YF 17D virus. Passage in fetal rhesus lung (FRhL) cells led to the emergence of a small-plaque virus containing a single Met→Lys amino acid mutation at E279, reverting this residue from the SA14-14-2 to the wild-type amino acid. A similar virus was also constructed by site-directed mutagenesis (J. Arroyo, F. Guirakhoo, S. Fenner, Z.-X. Zhang, T. P. Monath, and T. J. Chambers, J. Virol. 75:934-942, 2001). The E279 mutation is located in a beta-sheet in the hinge region of the E protein that is responsible for a pH-dependent conformational change during virus penetration from the endosome into the cytoplasm of the infected cell. In independent transfection-passage studies with FRhL or Vero cells, mutations appeared most frequently in hinge 4 (bounded by amino acids E266 to E284), reflecting genomic instability in this functionally important region. The E279 reversion caused a significant increase in neurovirulence as determined by the 50% lethal dose and survival distribution in suckling mice and by histopathology in rhesus monkeys. Based on sensitivity and comparability of results with those for monkeys, the suckling mouse is an appropriate host for safety testing of flavivirus vaccine candidates for neurotropism. After intracerebral inoculation, the E279 Lys virus was restricted with respect to extraneural replication in monkeys, as viremia and antibody levels (markers of viscerotropism) were significantly reduced compared to those for the E279 Met virus. These results are consistent with the observation that empirically derived vaccines developed by mouse brain passage of dengue and YF viruses have increased neurovirulence for mice but reduced viscerotropism for humans. PMID:11799188

  16. Solar Effective Envelope Design Advisor (SEEDA)

    NASA Astrophysics Data System (ADS)

    Mahaek, Ekkachai

    The lack of effort by mainstream architects in integrating energy-efficient strategies in architectural designing is due to the complexity in a building's energy conscious concepts and theories, the difficulties to visualize and quantify energy consumption, and the late implementing of energy consumption analysis in the conventional design process. This task would be accomplishing by a building system's engineer where results might be determined only after the basic architectural design has been completed. An effective simple tool and method should then be available to assist architects in building's energy-efficient designing at the beginning of the design. The building's energy consumption is directly and mainly influenced by the relationship of the sun, site, and its building configuration. The solar radiations will first impact on the building's envelope, which will have a direct effect on the amount of energy a building will consume. If an architect can define or map the intensity of solar energy on the site's buildable volume, and use this information to determine the levels of solar insolation, a more energy efficient building form can be proposed. This research hypothesis has shared the fundamental techniques of the Solar Envelope projection by Professor Ralph Knowles [Knowles, 1981] of the University of Southern California. However a different approach is taken by including the influence of regional restrictions and the surrounding buildings' shadows when projecting of solar volumes and solar envelope. The research methodology will discuss the development of a computer-based approach to develop a three-dimensional architectural form based on an insolation map related to the design site. The prototype computer program is referred as the Solar Effective Envelope Design Advisor (SEEDA). The solar insolation volume of the site is determined by integrating three types of computer-generated models include the Buildable Volume model based on design constraints

  17. Application of the Envelope Difference Index to Spectrally Sparse Speech

    ERIC Educational Resources Information Center

    Souza, Pamela; Hoover, Eric; Gallun, Frederick

    2012-01-01

    Purpose: Amplitude compression is a common hearing aid processing strategy that can improve speech audibility and loudness comfort but also has the potential to alter important cues carried by the speech envelope. In previous work, a measure of envelope change, the Envelope Difference Index (EDI; Fortune, Woodruff, & Preves, 1994), was moderately…

  18. Analysis of Building Envelope Construction in 2003 CBECS

    SciTech Connect

    Winiarski, David W.; Halverson, Mark A.; Jiang, Wei

    2007-06-01

    The purpose of this analysis is to determine "typical" building envelope characteristics for buildings built after 1980. We address three envelope components in this paper - roofs, walls, and window area. These typical building envelope characteristics were used in the development of DOE’s Reference Buildings .

  19. 200 Area Deactivation Project Facilities Authorization Envelope Document

    SciTech Connect

    DODD, E.N.

    2000-03-28

    Project facilities as required by HNF-PRO-2701, Authorization Envelope and Authorization Agreement. The Authorization Agreements (AA's) do not identify the specific set of environmental safety and health requirements that are applicable to the facility. Therefore, the facility Authorization Envelopes are defined here to identify the applicable requirements. This document identifies the authorization envelopes for the 200 Area Deactivation.

  20. Quantitative proteomics of the Neisseria gonorrhoeae cell envelope and membrane vesicles for the discovery of potential therapeutic targets.

    PubMed

    Zielke, Ryszard A; Wierzbicki, Igor H; Weber, Jacob V; Gafken, Philip R; Sikora, Aleksandra E

    2014-05-01

    Neisseria gonorrhoeae (GC) is a human-specific pathogen, and the agent of a sexually transmitted disease, gonorrhea. There is a critical need for new approaches to study and treat GC infections because of the growing threat of multidrug-resistant isolates and the lack of a vaccine. Despite the implied role of the GC cell envelope and membrane vesicles in colonization and infection of human tissues and cell lines, comprehensive studies have not been undertaken to elucidate their constituents. Accordingly, in pursuit of novel molecular therapeutic targets, we have applied isobaric tagging for absolute quantification coupled with liquid chromatography and mass spectrometry for proteome quantitative analyses. Mining the proteome of cell envelopes and native membrane vesicles revealed 533 and 168 common proteins, respectively, in analyzed GC strains FA1090, F62, MS11, and 1291. A total of 22 differentially abundant proteins were discovered including previously unknown proteins. Among those proteins that displayed similar abundance in four GC strains, 34 were found in both cell envelopes and membrane vesicles fractions. Focusing on one of them, a homolog of an outer membrane protein LptD, we demonstrated that its depletion caused loss of GC viability. In addition, we selected for initial characterization six predicted outer membrane proteins with unknown function, which were identified as ubiquitous in the cell envelopes derived from examined GC isolates. These studies entitled a construction of deletion mutants and analyses of their resistance to different chemical probes. Loss of NGO1985, in particular, resulted in dramatically decreased GC viability upon treatment with detergents, polymyxin B, and chloramphenicol, suggesting that this protein functions in the maintenance of the cell envelope permeability barrier. Together, these findings underscore the concept that the cell envelope and membrane vesicles contain crucial, yet under-explored determinants of GC

  1. Quantitative Proteomics of the Neisseria Gonorrhoeae Cell Envelope and Membrane Vesicles for the Discovery of Potential Therapeutic Targets*

    PubMed Central

    Zielke, Ryszard A.; Wierzbicki, Igor H.; Weber, Jacob V.; Gafken, Philip R.; Sikora, Aleksandra E.

    2014-01-01

    Neisseria gonorrhoeae (GC) is a human-specific pathogen, and the agent of a sexually transmitted disease, gonorrhea. There is a critical need for new approaches to study and treat GC infections because of the growing threat of multidrug-resistant isolates and the lack of a vaccine. Despite the implied role of the GC cell envelope and membrane vesicles in colonization and infection of human tissues and cell lines, comprehensive studies have not been undertaken to elucidate their constituents. Accordingly, in pursuit of novel molecular therapeutic targets, we have applied isobaric tagging for absolute quantification coupled with liquid chromatography and mass spectrometry for proteome quantitative analyses. Mining the proteome of cell envelopes and native membrane vesicles revealed 533 and 168 common proteins, respectively, in analyzed GC strains FA1090, F62, MS11, and 1291. A total of 22 differentially abundant proteins were discovered including previously unknown proteins. Among those proteins that displayed similar abundance in four GC strains, 34 were found in both cell envelopes and membrane vesicles fractions. Focusing on one of them, a homolog of an outer membrane protein LptD, we demonstrated that its depletion caused loss of GC viability. In addition, we selected for initial characterization six predicted outer membrane proteins with unknown function, which were identified as ubiquitous in the cell envelopes derived from examined GC isolates. These studies entitled a construction of deletion mutants and analyses of their resistance to different chemical probes. Loss of NGO1985, in particular, resulted in dramatically decreased GC viability upon treatment with detergents, polymyxin B, and chloramphenicol, suggesting that this protein functions in the maintenance of the cell envelope permeability barrier. Together, these findings underscore the concept that the cell envelope and membrane vesicles contain crucial, yet under-explored determinants of GC

  2. PRA1 co-localizes with envelope but does not influence primate lentivirus production, infectivity or envelope incorporation.

    PubMed

    Blancou, Philippe; Evans, David T; Desrosiers, Ronald C

    2005-06-01

    The results of yeast and mammalian two-hybrid assays previously indicated complex formation between prenylated Rab acceptor 1 (PRA1) and the cytoplasmic domain of gp41 (gp41CD) for both the human and simian immunodeficiency viruses [Evans, D. T., Tilman, K. C. & Desrosiers, R. C. (2002). J Virol 76, 327-337]. The assembly and release of infectious virus particles was studied under conditions of PRA1 overexpression in a transient transfection assay or suppression by RNA interference. Although a clear pattern of co-localization of PRA1 and gp41 was observed, no changes in virion release, infectivity or envelope content were observed as a result of either PRA1 suppression or overexpression. These data show that PRA1 co-localizes with gp41 inside cells and they are consistent with a direct or indirect interaction between these proteins. However, variation in the levels of PRA1 expression did not influence virion production, infectivity or envelope incorporation under the conditions of these assays. PMID:15914857

  3. Formaldehyde in envelopes of interstellar dark clouds

    NASA Technical Reports Server (NTRS)

    Federman, S. R.; Allen, M.

    1991-01-01

    Observed formaldehyde column densities of 1 x 10 to the 12th - 3 x 10 to the 13th/sq cm in cloud envelopes along lines of sight with A(V) = 1-4 mag can not be explained with the current understanding of interstellar gas phase chemistry. However, these column densities can be reproduced by a simple time-dependent model in which H2CO is supplied to the gas phase by the erosion of icy grain mantles. The release of H2CO from the grain mantles must occur on time scales comparable to the time scales for mixing from the cloud interior to the cloud envelope. Thus, in low-density regions of clouds, it appears that formaldehyde is the second molecule whose gas phase source is primarily ejection from grains. This simple model suggests understanding gas phase steady state in clouds on macroscopic, rather than microscopic, spatial scales.

  4. Discontinuous envelope function in semiconductor heterostructures

    NASA Astrophysics Data System (ADS)

    Drouhin, Henri-Jean; Bottegoni, Federico; Nguyen, T. L. Hoai; Wegrowe, Jean-Eric; Fishman, Guy

    2013-09-01

    Based on a proper definition of the current operators for non-quadratic Hamiltonians, we derive the expression for the transport current which involves the derivative of the imaginary part of the free-electron current, highlighting peculiarities of the extra terms. The expression of the probability current, when Spin-Orbit Interaction (SOI) is taken into account, requires a reformulation of the boudary conditions. This is especially important for tunnel heterojunctions made of non-centrosymmetric semiconductors. Therefore, we consider a model case: tunneling of conduction electrons through a [110]-oriented GaAs barrier. The new boundary conditions are reduced to two set of equations: the first one expresses the discontinuity of the envelope function at the interface while the other one expresses the discontinuity of the derivative of the envelope function.

  5. Development of High Specific Strength Envelope Materials

    NASA Astrophysics Data System (ADS)

    Komatsu, Keiji; Sano, Masa-Aki; Kakuta, Yoshiaki

    Progress in materials technology has produced a much more durable synthetic fabric envelope for the non-rigid airship. Flexible materials are required to form airship envelopes, ballonets, load curtains, gas bags and covering rigid structures. Polybenzoxazole fiber (Zylon) and polyalirate fiber (Vectran) show high specific tensile strength, so that we developed membrane using these high specific tensile strength fibers as a load carrier. The main material developed is a Zylon or Vectran load carrier sealed internally with a polyurethane bonded inner gas retention film (EVOH). The external surface provides weather protecting with, for instance, a titanium oxide integrated polyurethane or Tedlar film. The mechanical test results show that tensile strength 1,000 N/cm is attained with weight less than 230g/m2. In addition to the mechanical properties, temperature dependence of the joint strength and solar absorptivity and emissivity of the surface are measured. 

  6. Digital image envelope: method and evaluation

    NASA Astrophysics Data System (ADS)

    Huang, H. K.; Cao, Fei; Zhou, Michael Z.; Mogel, Greg T.; Liu, Brent J.; Zhou, Xiaoqiang

    2003-05-01

    Health data security, characterized in terms of data privacy, authenticity, and integrity, is a vital issue when digital images and other patient information are transmitted through public networks in telehealth applications such as teleradiology. Mandates for ensuring health data security have been extensively discussed (for example The Health Insurance Portability and Accountability Act, HIPAA) and health informatics guidelines (such as the DICOM standard) are beginning to focus on issues of data continue to be published by organizing bodies in healthcare; however, there has not been a systematic method developed to ensure data security in medical imaging Because data privacy and authenticity are often managed primarily with firewall and password protection, we have focused our research and development on data integrity. We have developed a systematic method of ensuring medical image data integrity across public networks using the concept of the digital envelope. When a medical image is generated regardless of the modality, three processes are performed: the image signature is obtained, the DICOM image header is encrypted, and a digital envelope is formed by combining the signature and the encrypted header. The envelope is encrypted and embedded in the original image. This assures the security of both the image and the patient ID. The embedded image is encrypted again and transmitted across the network. The reverse process is performed at the receiving site. The result is two digital signatures, one from the original image before transmission, and second from the image after transmission. If the signatures are identical, there has been no alteration of the image. This paper concentrates in the method and evaluation of the digital image envelope.

  7. Nuclear envelope and genome interactions in cell fate

    PubMed Central

    Talamas, Jessica A.; Capelson, Maya

    2015-01-01

    The eukaryotic cell nucleus houses an organism’s genome and is the location within the cell where all signaling induced and development-driven gene expression programs are ultimately specified. The genome is enclosed and separated from the cytoplasm by the nuclear envelope (NE), a double-lipid membrane bilayer, which contains a large variety of trans-membrane and associated protein complexes. In recent years, research regarding multiple aspects of the cell nucleus points to a highly dynamic and coordinated concert of efforts between chromatin and the NE in regulation of gene expression. Details of how this concert is orchestrated and how it directs cell differentiation and disease are coming to light at a rapid pace. Here we review existing and emerging concepts of how interactions between the genome and the NE may contribute to tissue specific gene expression programs to determine cell fate. PMID:25852741

  8. Cell Envelope of Corynebacteria: Structure and Influence on Pathogenicity

    PubMed Central

    Burkovski, Andreas

    2013-01-01

    To date the genus Corynebacterium comprises 88 species. More than half of these are connected to human and animal infections, with the most prominent member of the pathogenic species being Corynebacterium diphtheriae, which is also the type species of the genus. Corynebacterium species are characterized by a complex cell wall architecture: the plasma membrane of these bacteria is followed by a peptidoglycan layer, which itself is covalently linked to a polymer of arabinogalactan. Bound to this, an outer layer of mycolic acids is found which is functionally equivalent to the outer membrane of Gram-negative bacteria. As final layer, free polysaccharides, glycolipids, and proteins are found. The composition of the different substructures of the corynebacterial cell envelope and their influence on pathogenicity are discussed in this paper. PMID:23724339

  9. 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

  10. The cell envelope glycoconjugates of Mycobacterium tuberculosis

    PubMed Central

    Angala, Shiva Kumar; Belardinelli, Juan Manuel; Huc-Claustre, Emilie; Wheat, William H.; Jackson, Mary

    2015-01-01

    Tuberculosis (TB) remains the second most common cause of death due to a single infectious agent. The cell envelope of Mycobacterium tuberculosis (Mtb), the causative agent of the disease in humans, is a source of unique glycoconjugates and the most distinctive feature of the biology of this organism. It is the basis of much of Mtb pathogenesis and one of the major causes of its intrinsic resistance to chemotherapeutic agents. At the same time, the unique structures of Mtb cell envelope glycoconjugates, their antigenicity and essentiality for mycobacterial growth provide opportunities for drug, vaccine, diagnostic and biomarker development, as clearly illustrated by recent advances in all of these translational aspects. This review focuses on our current understanding of the structure and biogenesis of Mtb glycoconjugates with particular emphasis on one of most intriguing and least understood aspect of the physiology of mycobacteria: the translocation of these complex macromolecules across the different layers of the cell envelope. It further reviews the rather impressive progress made in the last ten years in the discovery and development of novel inhibitors targeting their biogenesis. PMID:24915502

  11. On the hybrid localization/envelopment problem

    SciTech Connect

    Chu, Y.X.; Gou, J.B.; Li, Z.X.

    1999-05-01

    The problem of aligning the CAD model of a workpiece such that all points measured on the finished surfaces of the workpiece match closely to corresponding surfaces on the model while all unmachined surfaces lie outside the model is referred to as the hybrid localization/envelopment problem. The hybrid problem has important applications in setting up for machining of partially finished workpieces. This paper gives a formulation of the hybrid localization/envelopment problem, and presents a simple algorithm for computing its solutions. First, the authors show that when the finished surfaces of a workpiece are inadequate to fully constrain the rigid motions of the workpiece, then the set of free motions remaining must form a subgroup G{sub 0} of the Euclidean group SE(3). This allows the authors to decompose the hybrid problem into a (symmetric) localization problem on G{sub 0}. While the symmetric localization problem is solved using the fast symmetric localization (FSL) algorithm developed in one of the earlier papers, the envelopment problem is solved by computing the solutions of a sequence of linear programming (LP) problems. The authors derive explicitly the LP problems, and apply standard linear programming techniques to solve the LP problems. They present simulation results to demonstrate the effectiveness of the method for the hybrid problem.

  12. Spectral envelope sensitivity of musical instrument sounds.

    PubMed

    Gunawan, David; Sen, D

    2008-01-01

    It is well known that the spectral envelope is a perceptually salient attribute in musical instrument timbre perception. While a number of studies have explored discrimination thresholds for changes to the spectral envelope, the question of how sensitivity varies as a function of center frequency and bandwidth for musical instruments has yet to be addressed. In this paper a two-alternative forced-choice experiment was conducted to observe perceptual sensitivity to modifications made on trumpet, clarinet and viola sounds. The experiment involved attenuating 14 frequency bands for each instrument in order to determine discrimination thresholds as a function of center frequency and bandwidth. The results indicate that perceptual sensitivity is governed by the first few harmonics and sensitivity does not improve when extending the bandwidth any higher. However, sensitivity was found to decrease if changes were made only to the higher frequencies and continued to decrease as the distorted bandwidth was widened. The results are analyzed and discussed with respect to two other spectral envelope discrimination studies in the literature as well as what is predicted from a psychoacoustic model. PMID:18177177

  13. NET23/STING Promotes Chromatin Compaction from the Nuclear Envelope

    PubMed Central

    de las Heras, Jose I.; Saiz-Ros, Natalia; Makarov, Alexandr A.; Lazou, Vassiliki; Meinke, Peter; Waterfall, Martin; Kelly, David A.; Schirmer, Eric C.

    2014-01-01

    Changes in the peripheral distribution and amount of condensed chromatin are observed in a number of diseases linked to mutations in the lamin A protein of the nuclear envelope. We postulated that lamin A interactions with nuclear envelope transmembrane proteins (NETs) that affect chromatin structure might be altered in these diseases and so screened thirty-one NETs for those that promote chromatin compaction as determined by an increase in the number of chromatin clusters of high pixel intensity. One of these, NET23 (also called STING, MITA, MPYS, ERIS, Tmem173), strongly promoted chromatin compaction. A correlation between chromatin compaction and endogenous levels of NET23/STING was observed for a number of human cell lines, suggesting that NET23/STING may contribute generally to chromatin condensation. NET23/STING has separately been found to be involved in innate immune response signaling. Upon infection cells make a choice to either apoptose or to alter chromatin architecture to support focused expression of interferon genes and other response factors. We postulate that the chromatin compaction induced by NET23/STING may contribute to this choice because the cells expressing NET23/STING eventually apoptose, but the chromatin compaction effect is separate from this as the condensation was still observed when cells were treated with Z-VAD to block apoptosis. NET23/STING-induced compacted chromatin revealed changes in epigenetic marks including changes in histone methylation and acetylation. This indicates a previously uncharacterized nuclear role for NET23/STING potentially in both innate immune signaling and general chromatin architecture. PMID:25386906

  14. NET23/STING promotes chromatin compaction from the nuclear envelope.

    PubMed

    Malik, Poonam; Zuleger, Nikolaj; de las Heras, Jose I; Saiz-Ros, Natalia; Makarov, Alexandr A; Lazou, Vassiliki; Meinke, Peter; Waterfall, Martin; Kelly, David A; Schirmer, Eric C

    2014-01-01

    Changes in the peripheral distribution and amount of condensed chromatin are observed in a number of diseases linked to mutations in the lamin A protein of the nuclear envelope. We postulated that lamin A interactions with nuclear envelope transmembrane proteins (NETs) that affect chromatin structure might be altered in these diseases and so screened thirty-one NETs for those that promote chromatin compaction as determined by an increase in the number of chromatin clusters of high pixel intensity. One of these, NET23 (also called STING, MITA, MPYS, ERIS, Tmem173), strongly promoted chromatin compaction. A correlation between chromatin compaction and endogenous levels of NET23/STING was observed for a number of human cell lines, suggesting that NET23/STING may contribute generally to chromatin condensation. NET23/STING has separately been found to be involved in innate immune response signaling. Upon infection cells make a choice to either apoptose or to alter chromatin architecture to support focused expression of interferon genes and other response factors. We postulate that the chromatin compaction induced by NET23/STING may contribute to this choice because the cells expressing NET23/STING eventually apoptose, but the chromatin compaction effect is separate from this as the condensation was still observed when cells were treated with Z-VAD to block apoptosis. NET23/STING-induced compacted chromatin revealed changes in epigenetic marks including changes in histone methylation and acetylation. This indicates a previously uncharacterized nuclear role for NET23/STING potentially in both innate immune signaling and general chromatin architecture. PMID:25386906

  15. Multiscale envelope manifold for enhanced fault diagnosis of rotating machines

    NASA Astrophysics Data System (ADS)

    Wang, Jun; He, Qingbo; Kong, Fanrang

    2015-02-01

    The wavelet transform has been widely used in the field of machinery fault diagnosis for its good property of band-pass filtering. However, the filtered signal still faces the contamination of in-band noise. This paper focuses on wavelet enveloping, and proposes a new method, called multiscale envelope manifold (MEM), to extract the envelope information of fault impacts with in-band noise suppression. The MEM addresses manifold learning on the wavelet envelopes at multiple scales. Specifically, the proposed method is conducted by three following steps. First, the continuous wavelet transform (CWT) with complex Morlet wavelet base is introduced to obtain the wavelet envelopes at all scales. Second, the wavelet envelopes are restricted in one or more narrow scale bands to simply include the envelope information of fault impacts. The scale band is determined through a smoothness index-based (SI-based) selection method by considering the impulsiveness inside the power spectrum. Third, the manifold learning algorithm is conducted on the wavelet envelopes at selected scales to extract the intrinsic envelope manifold of fault-related impulses. The MEM combines the envelope information at multiple scales in a nonlinear approach, and may thus preserve the factual envelope structure of machinery fault. Simulation studies and experimental verifications confirm that the new method is effective for enhanced fault diagnosis of rotating machines.

  16. Antiviral Activity of Graphene–Silver Nanocomposites against Non-Enveloped and Enveloped Viruses

    PubMed Central

    Chen, Yi-Ning; Hsueh, Yi-Huang; Hsieh, Chien-Te; Tzou, Dong-Ying; Chang, Pai-Ling

    2016-01-01

    The discovery of novel antiviral materials is important because many infectious diseases are caused by viruses. Silver nanoparticles have demonstrated strong antiviral activity, and graphene is a potential antimicrobial material due to its large surface area, high carrier mobility, and biocompatibility. No studies on the antiviral activity of nanomaterials on non-enveloped viruses have been reported. To investigate the antiviral activity of graphene oxide (GO) sheets and GO sheets with silver particles (GO-Ag) against enveloped and non-enveloped viruses, feline coronavirus (FCoV) with an envelope and infectious bursal disease virus (IBDV) without an envelope were chosen. The morphology and sizes of GO and GO-Ag were characterized by transmission, scanning electron microscopy, and X-ray diffraction. A virus inhibition assay was used to identify the antiviral activity of GO and GO-Ag. Go-Ag inhibited 25% of infection by FCoV and 23% by IBDV, whereas GO only inhibited 16% of infection by FCoV but showed no antiviral activity against the infection by IBDV. Further application of GO and GO-Ag can be considered for personal protection equipment to decrease the transmission of viruses. PMID:27104546

  17. Antiviral Activity of Graphene-Silver Nanocomposites against Non-Enveloped and Enveloped Viruses.

    PubMed

    Chen, Yi-Ning; Hsueh, Yi-Huang; Hsieh, Chien-Te; Tzou, Dong-Ying; Chang, Pai-Ling

    2016-01-01

    The discovery of novel antiviral materials is important because many infectious diseases are caused by viruses. Silver nanoparticles have demonstrated strong antiviral activity, and graphene is a potential antimicrobial material due to its large surface area, high carrier mobility, and biocompatibility. No studies on the antiviral activity of nanomaterials on non-enveloped viruses have been reported. To investigate the antiviral activity of graphene oxide (GO) sheets and GO sheets with silver particles (GO-Ag) against enveloped and non-enveloped viruses, feline coronavirus (FCoV) with an envelope and infectious bursal disease virus (IBDV) without an envelope were chosen. The morphology and sizes of GO and GO-Ag were characterized by transmission, scanning electron microscopy, and X-ray diffraction. A virus inhibition assay was used to identify the antiviral activity of GO and GO-Ag. Go-Ag inhibited 25% of infection by FCoV and 23% by IBDV, whereas GO only inhibited 16% of infection by FCoV but showed no antiviral activity against the infection by IBDV. Further application of GO and GO-Ag can be considered for personal protection equipment to decrease the transmission of viruses. PMID:27104546

  18. Masses and Envelope Binding Energies of Primary Stars at the Onset of a Common Envelope

    NASA Astrophysics Data System (ADS)

    van der Sluys, Marc; Politano, Michael; Taam, Ronald E.

    2010-12-01

    We present basic properties of primary stars that initiate a common envelope (CE) in a binary, while on the giant branch. We use the population-synthesis code described in Politano et al. [1] and follow the evolution of a population of binary stars up to the point where the primary fills its Roche lobe and initiates a CE. We then collect the properties of each system, in particular the donor mass and the binding energy of the donor's envelope, which are important for the treatment of a CE. We find that for most CEs, the donor mass is sufficiently low to define the core-envelope boundary reasonably well. We compute the envelope-structure parameter λenv from the binding energy and compare its distribution to typical assumptions that are made in population-synthesis codes. We conclude that λenv varies appreciably and that the assumption of a constant value for this parameter results in typical errors of 20-50%. In addition, such an assumption may well result in the implicit assumption of unintended and/or unphysical values for the CE parameter αCE. Finally, we discuss accurate existing analytic fits for the envelope binding energy, which make these oversimplified assumptions for λenv, and the use of λenv in general, unnecessary.

  19. Envelope Variants Circulating as Initial Neutralization Breadth Developed in Two HIV-Infected Subjects Stimulate Multiclade Neutralizing Antibodies in Rabbits

    PubMed Central

    Malherbe, Delphine C.; Pissani, Franco; Sather, D. Noah; Guo, Biwei; Pandey, Shilpi; Sutton, William F.; Stuart, Andrew B.; Robins, Harlan; Park, Byung; Krebs, Shelly J.; Schuman, Jason T.; Kalams, Spyros; Hessell, Ann J.

    2014-01-01

    ABSTRACT Identifying characteristics of the human immunodeficiency virus type 1 (HIV-1) envelope that are effective in generating broad, protective antibodies remains a hurdle to HIV vaccine design. Emerging evidence of the development of broad and potent neutralizing antibodies in HIV-infected subjects suggests that founder and subsequent progeny viruses may express unique antigenic motifs that contribute to this developmental pathway. We hypothesize that over the course of natural infection, B cells are programmed to develop broad antibodies by exposure to select populations of emerging envelope quasispecies variants. To test this hypothesis, we identified two unrelated subjects whose antibodies demonstrated increasing neutralization breadth against a panel of HIV-1 isolates over time. Full-length functional env genes were cloned longitudinally from these subjects from months after infection through 2.6 to 5.8 years of infection. Motifs associated with the development of breadth in published, cross-sectional studies were found in both subjects. We compared the immunogenicity of envelope vaccines derived from time points obtained during and after broadening of neutralization activity within these subjects. Rabbits were coimmunized four times with selected multiple gp160 DNAs and gp140-trimeric envelope proteins. The affinity of the polyclonal response increased as a function of boosting. The most rapid and persistent neutralization of multiclade tier 1 viruses was elicited by envelopes that were circulating in plasma at time points prior to the development of 50% neutralization breadth in both human subjects. The breadth elicited in rabbits was not improved by exposure to later envelope variants. These data have implications for vaccine development in describing a target time point to identify optimal envelope immunogens. IMPORTANCE Vaccine protection against viral infections correlates with the presence of neutralizing antibodies; thus, vaccine components capable

  20. Visualizing the Spatial Relationship of the Genome with the Nuclear Envelope Using Fluorescence In Situ Hybridization.

    PubMed

    Clements, Craig S; Bikkul, Ural; Ahmed, Mai Hassan; Foster, Helen A; Godwin, Lauren S; Bridger, Joanna M

    2016-01-01

    The genome has a special relationship with the nuclear envelope in cells. Much of the genome is anchored at the nuclear periphery, tethered by chromatin binding proteins such nuclear lamins and other integral membrane proteins. Even though there are global assays such as DAM-ID or ChIP to assess what parts of the genome are associated with the nuclear envelope, it is also essential to be able to visualize regions of the genome in order to reveal their individual relationships with nuclear structures in single cells. This is executed by fluorescence in situ hybridization (FISH) in 2-dimensional flattened nuclei (2D-FISH) or 3-dimensionally preserved cells (3D-FISH) in combination with indirect immunofluorescence to reveal structural proteins. This chapter explains the protocols for 2D- and 3D-FISH in combination with indirect immunofluorescence and discusses options for image capture and analysis. Due to the nuclear envelope proteins being part of the non-extractable nucleoskeleton, we also describe how to prepare DNA halos through salt extraction and how they can be used to study genome behavior and association when combined with 2D-FISH. PMID:27147055

  1. Essential role of VIPP1 in chloroplast envelope maintenance in Arabidopsis.

    PubMed

    Zhang, Lingang; Kato, Yusuke; Otters, Stephanie; Vothknecht, Ute C; Sakamoto, Wataru

    2012-09-01

    VESICLE-INDUCING PROTEIN IN PLASTIDS1 (VIPP1), proposed to play a role in thylakoid biogenesis, is conserved in photosynthetic organisms and is closely related to Phage Shock Protein A (PspA), which is involved in plasma membrane integrity in Escherichia coli. This study showed that chloroplasts/plastids in Arabidopsis thaliana vipp1 knockdown and knockout mutants exhibit a unique morphology, forming balloon-like structures. This altered morphology, as well as lethality of vipp1, was complemented by expression of VIPP1 fused to green fluorescent protein (VIPP1-GFP). Several lines of evidence show that the balloon chloroplasts result from chloroplast swelling related to osmotic stress, implicating VIPP1 in the maintenance of plastid envelopes. In support of this, Arabidopsis VIPP1 rescued defective proton leakage in an E. coli pspA mutant. Microscopy observation of VIPP1-GFP in transgenic Arabidopsis revealed that VIPP1 forms large macrostructures that are integrated into various morphologies along the envelopes. Furthermore, live imaging revealed that VIPP1-GFP is highly mobile when chloroplasts are subjected to osmotic stress. VIPP1-GFP showed dynamic movement in the transparent area of spherical chloroplasts, as the fluorescent molecules formed filament-like structures likely derived from disassembly of the large VIPP1 complex. Collectively, our data demonstrate that VIPP1 is a multifunctional protein in chloroplasts that is critically important for envelope maintenance. PMID:23001039

  2. In Vivo Dynamics of Drosophila Nuclear Envelope Components

    PubMed Central

    Katsani, Katerina R.; Karess, Roger E.; Dostatni, Nathalie

    2008-01-01

    Nuclear pore complexes (NPCs) are multisubunit protein entities embedded into the nuclear envelope (NE). Here, we examine the in vivo dynamics of the essential Drosophila nucleoporin Nup107 and several other NE-associated proteins during NE and NPCs disassembly and reassembly that take place within each mitosis. During both the rapid mitosis of syncytial embryos and the more conventional mitosis of larval neuroblasts, Nup107 is gradually released from the NE, but it remains partially confined to the nuclear (spindle) region up to late prometaphase, in contrast to nucleoporins detected by wheat germ agglutinin and lamins. We provide evidence that in all Drosophila cells, a structure derived from the NE persists throughout metaphase and early anaphase. Finally, we examined the dynamics of the spindle checkpoint proteins Mad2 and Mad1. During mitotic exit, Mad2 and Mad1 are actively imported back from the cytoplasm into the nucleus after the NE and NPCs have reformed, but they reassociate with the NE only later in G1, concomitantly with the recruitment of the basket nucleoporin Mtor (the Drosophila orthologue of vertebrate Tpr). Surprisingly, Drosophila Nup107 shows no evidence of localization to kinetochores, despite the demonstrated importance of this association in mammalian cells. PMID:18562695

  3. Envelope tracking CMOS power amplifier with high-speed CMOS envelope amplifier for mobile handsets

    NASA Astrophysics Data System (ADS)

    Yoshida, Eiji; Sakai, Yasufumi; Oishi, Kazuaki; Yamazaki, Hiroshi; Mori, Toshihiko; Yamaura, Shinji; Suto, Kazuo; Tanaka, Tetsu

    2014-01-01

    A high-efficiency CMOS power amplifier (PA) based on envelope tracking (ET) has been reported for a wideband code division multiple access (W-CDMA) and long term evolution (LTE) application. By adopting a high-speed CMOS envelope amplifier with current direction sensing, a 5% improvement in total power-added efficiency (PAE) and a 11 dB decrease in adjacent channel leakage ratio (ACLR) are achieved with a W-CDMA signal. Moreover, the proposed PA achieves a PAE of 25.4% for a 10 MHz LTE signal at an output power (Pout) of 25.6 dBm and a gain of 24 dB.

  4. Ultrastructural Study of Salmonella typhimurium Treated with Membrane-Active Agents: Specific Reaction of Dansylchloride with Cell Envelope Components

    PubMed Central

    Schindler, Peter R. G.; Teuber, Michael

    1978-01-01

    Amino groups of cell envelope proteins, lipids, and lipopolysaccharides cannot be labeled in intact cells of Salmonella typhimurium G 30 by using 5-dimethylaminonaphthalene-1-sulfonylchloride incorporated in lecithin-cholesterol vesicles. However, application of membrane-interacting agents like tris(hydroxymethyl)aminomethane (Tris)-hydrochloride, ethylenediaminetetraacetate (Na salt) (EDTA), divalent cations, and sublethal doses of the cationic antibacterial agents polymyxin B and chlorhexidine induced specific fluorescent labeling of envelope proteins and lipids but not of cytoplasmic compounds, with the exception of a soluble protein with a molecular weight of 46,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Treatment with Tris-hydrochloride buffer produced labeling of the heat-modifiable protein B/B+ and of proteins with molecular weights of 26,000, 22,000, and below 17,000. A combination of Tris-hydrochloride and EDTA induced additional dansylation of the major protein A and of proteins of molecular weights 80,000, 60,000, and 44,000. Polymyxin B and chlorhexidine caused similar labeling patterns. In every case, except with divalent cation treatment, protein B/B+ was the most prominently labeled species. Phosphatidylethanolamine was dansylated up to 30%. Lipopolysaccharide was not reactive under any condition or treatment. In addition, the peptidoglycan-bound lipoprotein did not react with dansylchloride in either intact or Tris-hydrochloride-treated cells. The results are discussed with regard to a possible localization of labeled and unlabeled compounds of the cell envelope on the basis of a model placing cell envelope amino groups into ion-ion interactions with anionic components of other envelope compounds like phosphate and carboxyl groups. Images PMID:97268

  5. Modeling pollutant penetration across building envelopes

    SciTech Connect

    Liu, De-Ling; Nazaroff, William W.

    2001-04-01

    As air infiltrates through unintentional openings in building envelopes, pollutants may interact with adjacent surfaces. Such interactions can alter human exposure to air pollutants of outdoor origin. We present modeling explorations of the proportion of particles and reactive gases (e.g., ozone) that penetrate building envelopes as air enters through cracks and wall cavities. Calculations were performed for idealized rectangular cracks, assuming regular geometry, smooth inner crack surface and steady airflow. Particles of 0.1-1.0 {micro}m diameter are predicted to have the highest penetration efficiency, nearly unity for crack heights of 0.25 mm or larger, assuming a pressure difference of 4 Pa or greater and a flow path length of 3 cm or less. Supermicron and ultrafine particles are significantly removed by means of gravitational settling and Brownian diffusion, respectively. In addition to crack geometry, ozone penetration depends on its reactivity with crack surfaces, as parameterized by the reaction probability. For reaction probabilities less than {approx}10{sup -5}, penetration is complete for cracks heights greater than 1 mm. However, penetration through mm scale cracks is small if the reaction probability is {approx}10{sup -4} or greater. For wall cavities, fiberglass insulation is an efficient particle filter, but particles would penetrate efficiently through uninsulated wall cavities or through insulated cavities with significant airflow bypass. The ozone reaction probability on fiberglass fibers was measured to be 10{sup -7} for fibers previously exposed to high ozone levels and 6 x 10{sup -6} for unexposed fibers. Over this range, ozone penetration through fiberglass insulation would vary from >90% to {approx}10-40%. Thus, under many conditions penetration is high; however, there are realistic circumstances in which building envelopes can provide substantial pollutant removal. Not enough is yet known about the detailed nature of pollutant penetration

  6. Antireflection pyrex envelopes for parabolic solar collectors

    SciTech Connect

    McCollister, H.L.; Pettit, R.B.

    1983-01-01

    Parabolic trough solar collectors utilize glass envelopes around the receiver tube in order to reduce thermal losses. Antireflective (AR) coatings applied to the envelope can potentially increase the solar transmittance by 0.07. An excellent AR surface can be formed on Pyrex (Corning Code 7740 glass) by first heat treating the glass to cause a compositional phase separation. After heat treating, a surface layer is removed using a pre-etch solution of aqueous ammonium bifluoride. Finally the AR layer is formed by etching in a solution containing hydrofluorosilic and ammonium bifluoride acid. Processing parameters studied included the phase separation temperature and heat treatment time, the pre-etch time, and the etching bath temperature and time. AR-coated samples with solar transmittance values >0.97, as compared to a value of 0.91 in untreated samples, were obtained for a range of heat treatment temperatures from 560 to 630/sup 0/C. The phase separation time and temperature interact so that at 630/sup 0/C short times are required (3 hours) while at 560/sup 0/C longer times are necessary (24 hours). Optimum values for the other processing parameters are 12 to 18 minutes in the pre-etching bath, and 5 to 10 minutes in the film forming bath when maintained between 35 and 45/sup 0/C. Application of this process to full scale 3 m long x 6 cm diameter Pyrex envelopes was successful in producing solar transmittance values greater than or equal to 0.97.

  7. Antireflection Pyrex envelopes for parabolic solar collectors

    SciTech Connect

    McCollister, H.L.; Pettit, R.B.

    1983-11-01

    Parabolic trough solar collectors utilize glass envelopes around the receiver tube in order to reduce thermal losses. Antireflective (AR) coatings applied to the envelope can potentially increase the solar transmittance by 7 percent. An excellent AR surface can be formed on Pyrex (Corning Code 7740 glass) by first heat treating the glass to cause a compositional phase separation. After heat treating, a surface layer is removed using a pre-etch solution of aqueous ammonium bifluoride. Finally, the AR layer is formed by etching in a solution containing hydrofluorosilic and ammonium bifluoride acid. Processing parameters studied included the phase separation temperature and heat treatment time, the pre-etch time, and the etching bath temperature and time. AR-coated samples with solar transmittance values > 0.97, as compared to a value of 0.91 in untreated samples, were obtained for a range of heat treatment temperatures from 560-630/sup 0/C. The phase separation time and temperature interact so that at 630/sup 0/C short times are required (3 hrs) while at 560/sup 0/C longer times are necessary (24 hrs). Optimum values for the other processing parameters are 12-18 min in the pre-etching bath, and 5-10 min in the film forming bath when maintained between 35-45/sup 0/C. Application of this process to full scale 3-m-long X 6-cm dia Pyrex envelopes was successful in producing solar transmittance values greater than or equal to 0.97.

  8. Lentivirus infection in the brain induces matrix metalloproteinase expression: role of envelope diversity.

    PubMed

    Johnston, J B; Jiang, Y; van Marle, G; Mayne, M B; Ni, W; Holden, J; McArthur, J C; Power, C

    2000-08-01

    Infection of the brain by lentiviruses, including human immunodeficiency virus (HIV) and feline immunodeficiency virus (FIV), causes inflammation and results in neurodegeneration. Molecular diversity within the lentivirus envelope gene has been implicated in the regulation of cell tropism and the host response to infection. Here, we examine the hypothesis that envelope sequence diversity modulates the expression of host molecules implicated in lentivirus-induced brain disease, including matrix metalloproteinases (MMP) and related transcription factors. Infection of primary macrophages by chimeric HIV clones containing brain-derived envelope fragments from patients with HIV-associated dementia (HAD) or nondemented AIDS patients (HIV-ND) showed that MMP-2 and -9 levels in conditioned media were significantly higher for the HAD clones. Similarly, STAT-1 and JAK-1 levels were higher in macrophages infected by HAD clones. Infections of primary feline macrophages by the neurovirulent FIV strain (V(1)CSF), the less neurovirulent strain (Petaluma), and a chimera containing the V(1)CSF envelope in a Petaluma background (FIV-Ch) revealed that MMP-2 and -9 levels were significantly higher in conditioned media from V(1)CSF- and FIV-Ch-infected macrophages, which was associated with increased intracellular STAT-1 and JAK-1 levels. The STAT-1 inhibitor fludarabine significantly reduced MMP-2 expression, but not MMP-9 expression, in FIV-infected macrophages. Analysis of MMP mRNA and protein levels in brain samples from HIV-infected persons or FIV-infected cats showed that MMP-2 and -9 levels were significantly increased in lentivirus-infected brains compared to those of uninfected controls. Elevated MMP expression was accompanied by significant increases in STAT-1 and JAK-1 mRNA and protein levels in the same brain samples. The present findings indicate that two lentiviruses, HIV and FIV, have common mechanisms of MMP-2 and -9 induction, which is modulated in part by envelope

  9. Cryo Electron Tomography of Herpes Simplex Virus during Axonal Transport and Secondary Envelopment in Primary Neurons

    PubMed Central

    Ibiricu, Iosune; Huiskonen, Juha T.; Döhner, Katinka; Bradke, Frank; Sodeik, Beate; Grünewald, Kay

    2011-01-01

    During herpes simplex virus 1 (HSV1) egress in neurons, viral particles travel from the neuronal cell body along the axon towards the synapse. Whether HSV1 particles are transported as enveloped virions as proposed by the ‘married’ model or as non-enveloped capsids suggested by the ‘separate’ model is controversial. Specific viral proteins may form a recruitment platform for microtubule motors that catalyze such transport. However, their subviral location has remained elusive. Here we established a system to analyze herpesvirus egress by cryo electron tomography. At 16 h post infection, we observed intra-axonal transport of progeny HSV1 viral particles in dissociated hippocampal neurons by live-cell fluorescence microscopy. Cryo electron tomography of frozen-hydrated neurons revealed that most egressing capsids were transported independently of the viral envelope. Unexpectedly, we found not only DNA-containing capsids (cytosolic C-capsids), but also capsids lacking DNA (cytosolic A-/B-capsids) in mid-axon regions. Subvolume averaging revealed lower amounts of tegument on cytosolic A-/B-capsids than on C-capsids. Nevertheless, all capsid types underwent active axonal transport. Therefore, even few tegument proteins on the capsid vertices seemed to suffice for transport. Secondary envelopment of capsids was observed at axon terminals. On their luminal face, the enveloping vesicles were studded with typical glycoprotein-like spikes. Furthermore, we noted an accretion of tegument density at the concave cytosolic face of the vesicle membrane in close proximity to the capsids. Three-dimensional analysis revealed that these assembly sites lacked cytoskeletal elements, but that filamentous actin surrounded them and formed an assembly compartment. Our data support the ‘separate model’ for HSV1 egress, i.e. progeny herpes viruses being transported along axons as subassemblies and not as complete virions within transport vesicles. PMID:22194682

  10. 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.

  11. Low heat-leak cryogenic envelope

    DOEpatents

    DeHaan, James R.

    1976-10-19

    A plurality of cryogenic envelope sections are joined together to form a power transmission line. Each of the sections is comprised of inner and outer tubes having multilayer metalized plastic spirally wrapped within a vacuum chamber formed between the inner and outer tubes. A refrigeration tube traverses the vacuum chamber, but exits one section and enters another through thermal standoffs for reducing heat-leak from the outer tube to the refrigeration tube. The refrigeration tube passes through a spirally wrapped shield within each section's vacuum chamber in a manner so that the refrigeration tube is in close thermal contact with the shield, but is nevertheless slideable with respect thereto.

  12. Snell Envelope with Small Probability Criteria

    SciTech Connect

    Del Moral, Pierre Hu, Peng; Oudjane, Nadia

    2012-12-15

    We present a new algorithm to compute the Snell envelope in the specific case where the criteria to optimize is associated with a small probability or a rare event. This new approach combines the Stochastic Mesh approach of Broadie and Glasserman with a particle approximation scheme based on a specific change of measure designed to concentrate the computational effort in regions pointed out by the criteria. The theoretical analysis of this new algorithm provides non asymptotic convergence estimates. Finally, the numerical tests confirm the practical interest of this approach.

  13. Surface area coefficients for airship envelopes

    NASA Technical Reports Server (NTRS)

    Diehl, W S

    1922-01-01

    In naval architecture, it is customary to determine the wetted surface of a ship by means of some formula which involves the principal dimensions of the design and suitable constants. These formulas of naval architecture may be extended and applied to the calculation of the surface area of airship envelopes by the use of new values of the constants determined for this purpose. Surface area coefficients were calculated from the actual dimensions, surfaces, and volumes of 52 streamline bodies, which form a series covering the entire range of shapes used in the present aeronautical practice.

  14. 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

  15. Protein

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Proteins are the major structural and functional components of all cells in the body. They are macromolecules that comprise 1 or more chains of amino acids that vary in their sequence and length and are folded into specific 3-dimensional structures. The sizes and conformations of proteins, therefor...

  16. Proteins.

    ERIC Educational Resources Information Center

    Doolittle, Russell F.

    1985-01-01

    Examines proteins which give rise to structure and, by virtue of selective binding to other molecules, make genes. Binding sites, amino acids, protein evolution, and molecular paleontology are discussed. Work with encoding segments of deoxyribonucleic acid (exons) and noncoding stretches (introns) provides new information for hypotheses. (DH)

  17. Models of Structure of the Envelope of Influenza Virus*†

    PubMed Central

    Tiffany, John M.; Blough, Herbert A.

    1970-01-01

    Possible models of the structure of the influenza virus envelope are considered in terms of the known chemical composition. Models incorporating lipid in the form of a bimolecular leaflet are shown to be unlikely on geometrical grounds. A model having „inverted toadstool” protein units separated by spherical lipid micelles is favored, and is capable of explaining the appearance of the virus in the electron microscope and differences between normal and incomplete (von Magnus) forms of the virus. Images PMID:5266149

  18. Envelope as Climate Negotiator: Evaluating adaptive building envelope's capacity to moderate indoor climate and energy

    NASA Astrophysics Data System (ADS)

    Erickson, James

    Through manipulation of adaptable opportunities available within a given environment, individuals become active participants in managing personal comfort requirements, by exercising control over their comfort without the assistance of mechanical heating and cooling systems. Similarly, continuous manipulation of a building skin's form, insulation, porosity, and transmissivity qualities exerts control over the energy exchanged between indoor and outdoor environments. This research uses four adaptive response variables in a modified software algorithm to explore an adaptive building skin's potential in reacting to environmental stimuli with the purpose of minimizing energy use without sacrificing occupant comfort. Results illustrate that significant energy savings can be realized with adaptive envelopes over static building envelopes even under extreme summer and winter climate conditions; that the magnitude of these savings are dependent on climate and orientation; and that occupant thermal comfort can be improved consistently over comfort levels achieved by optimized static building envelopes. The resulting adaptive envelope's unique climate-specific behavior could inform designers in creating an intelligent kinetic aesthetic that helps facilitate adaptability and resiliency in architecture.

  19. Envelope Membranes from Spinach Chloroplasts Are a Site of Metabolism of Fatty Acid Hydroperoxides.

    PubMed Central

    Blee, E.; Joyard, J.

    1996-01-01

    Enzymes in envelope membranes from spinach (Spinacia oleracea L.) chloroplasts were found to catalyze the rapid breakdown of fatty acid hydroperoxides. In contrast, no such activities were detected in the stroma or in thylakoids. In preparations of envelope membranes, 9S-hydroperoxy-10(E),12(Z)-octadecadienoic acid, 13S-hydroperoxy-9(Z),11(E)-octadecadienoic acid, or 13S-hydroperoxy-9(Z),11(E),15(Z)-octadecatrienoic acid were transformed at almost the same rates (1-2 [mu]mol min-1 mg-1 protein). The products formed were separated by reversed-phase high-pressure liquid chromatography and further characterized by gas chromatography-mass spectrometry. Fatty acid hydroperoxides were cleaved (a) into aldehydes and oxoacid fragments, corresponding to the functioning of a hydroperoxide lyase, (b) into ketols that were spontaneously formed from allene oxide synthesized by a hydroperoxide dehydratase, (c) into hydroxy compounds synthesized enzymatically by a system that has not yet been characterized, and (d) into oxoenes resulting from the hydroperoxidase activity of a lipoxygenase. Chloroplast envelope membranes therefore contain a whole set of enzymes that catalyze the synthesis of a variety of fatty acid derivatives, some of which may act as regulatory molecules. The results presented demonstrate a new role for the plastid envelope within the plant cell. PMID:12226196

  20. Non-Enveloped Virus Entry: Structural Determinants and Mechanism of Functioning of a Viral Lytic Peptide.

    PubMed

    Bajaj, Saumya; Dey, Debajit; Bhukar, Rohan; Kumar, Mohit; Banerjee, Manidipa

    2016-08-28

    In the absence of lipid envelopes and associated fusion proteins, non-enveloped viruses employ membrane lytic peptides to breach the limiting membranes of host cells. Although several of these lytic peptides have been identified and characterized, their manner of deployment and interaction with host membranes remains unclear in most cases. We are using the gamma peptide of Flock House Virus (FHV), a model non-enveloped virus, to understand the mechanistic details of non-enveloped virus interaction with host cell membranes. We utilized a combination of biophysical assays, molecular dynamics simulation studies, and single-particle cryo-electron microscopy to elucidate the functional and structural determinants for membrane penetration by gamma in context of the FHV capsid. Although the amphipathic, helical N-terminal region of gamma (γ1) was previously thought to be the membrane-penetrating module, with the C-terminal region having a supporting role in correct structural positioning of γ1, we demonstrate that the C terminus of gamma directly participates in membrane penetration. Our studies suggest that full-length gamma, including the hydrophobic C terminus, forms an alpha-helical hairpin motif, and any disruption in this motif drastically reduces its functionality, in spite of the correct positioning of amphipathic γ1 in the virus capsid. Taken together, our data suggest that the most effective module for membrane disruption is a pentameric unit of full-length gamma, released from the virus, which associates with membranes via both N- and C-terminal ends. PMID:27320388

  1. Dynamic properties of meiosis-specific lamin C2 and its impact on nuclear envelope integrity

    PubMed Central

    Jahn, Daniel; Schramm, Sabine; Benavente, Ricardo

    2010-01-01

    A hallmark of meiosis is the precise pairing and the stable physical connection (synapsis) of the homologous chromosomes. These processes are essential prerequisite for their proper segregation. Pairing of the homologs during meiotic prophase I critically depends on characteristic movements of chromosomes. These movements, in turn, require attachment of meiotic telomeres to the nuclear envelope and their subsequent dynamic repositioning. Dynamic repositioning of meiotic telomeres goes along with profound structural reorganization of the nuclear envelope. The short A-type lamin C2 is thought to play a critical role in this process due to its specific expression during meiotic prophase I and the unique localization surrounding telomere attachments. Consistent with this notion, here we provide compelling evidence that meiosis-specific lamin C2 features a significantly increased mobility compared to somatic lamins as revealed by photobleaching techniques. We show that this property can be clearly ascribed to the lack of the N-terminal head and the significantly shorter α-helical coil domain. Moreover, expression of lamin C2 in somatic cells induces nuclear deformations and alters the distribution of the endogenous nuclear envelope proteins lamin B1, LAP2, SUN1 and SUN2. Together, our data define lamin C2 as a “natural lamin deletion mutant” that confers unique properties to the nuclear envelope which would be essential for dynamic telomere repositioning during meiotic prophase I. PMID:21327075

  2. On-Line Safe Flight Envelope Determination for Impaired Aircraft

    NASA Technical Reports Server (NTRS)

    Lombaerts, Thomas; Schuet, Stefan; Acosta, Diana; Kaneshige, John

    2015-01-01

    The design and simulation of an on-line algorithm which estimates the safe maneuvering envelope of aircraft is discussed in this paper. The trim envelope is estimated using probabilistic methods and efficient high-fidelity model based computations of attainable equilibrium sets. From this trim envelope, a robust reachability analysis provides the maneuverability limitations of the aircraft through an optimal control formulation. Both envelope limits are presented to the flight crew on the primary flight display. In the results section, scenarios are considered where this adaptive algorithm is capable of computing online changes to the maneuvering envelope due to impairment. Furthermore, corresponding updates to display features on the primary flight display are provided to potentially inform the flight crew of safety critical envelope alterations caused by the impairment.

  3. Precision envelope detector and linear rectifier circuitry

    DOEpatents

    Davis, Thomas J.

    1980-01-01

    Disclosed is a method and apparatus for the precise linear rectification and envelope detection of oscillatory signals. The signal is applied to a voltage-to-current converter which supplies current to a constant current sink. The connection between the converter and the sink is also applied through a diode and an output load resistor to a ground connection. The connection is also connected to ground through a second diode of opposite polarity from the diode in series with the load resistor. Very small amplitude voltage signals applied to the converter will cause a small change in the output current of the converter, and the difference between the output current and the constant current sink will be applied either directly to ground through the single diode, or across the output load resistor, dependent upon the polarity. Disclosed also is a full-wave rectifier utilizing constant current sinks and voltage-to-current converters. Additionally, disclosed is a combination of the voltage-to-current converters with differential integrated circuit preamplifiers to boost the initial signal amplitude, and with low pass filtering applied so as to obtain a video or signal envelope output.

  4. Glycolate transporter of the pea chloroplast envelope

    SciTech Connect

    Howitz, K.T.

    1985-01-01

    The discovery of a glycolate transporter in the pea (Pisum sativum) chloroplast envelope is described. Several novel silicone oil centrifugation methods were developed to resolve the initial rate kinetics of (/sup 14/C)glycolate transport by isolated, intact pea chloroplasts. Chloroplast glycolate transport was found to be carrier mediated. Transport rates saturated with increasing glycolate concentration. N-Ethylmaleimide (NEM) pretreatment of chloroplasts inhibited transport, an inhibition prevented by glycolate. Glycolate distributed across the envelope in a way which equalized stromal and medium glycolic acid concentrations, limiting possible transport mechanisms to facilitated glycolic acid diffusion, proton symport or hydroxyl antiport. The effects of stomal and medium pH's on the K/sub m/ and V/sub max/ fit the predictions of mobile carrier kinetic models of hydroxyl antiport or proton symport (H/sup +/ binds first). The carrier mediated transport was fast enough to be consistent with in vivo rates of photorespiration. The 2-hydroxymonocarboxylates, glycerate, lactate and glyoxylate are competitive inhibitors of chloroplast glycolate uptake. Glyoxylate, D-lactate and D-glycerate cause glycolate counterflow, indicating that they are also substrates of the glycolate carrier. This finding was confirmed for D-glycerate by studies on glycolate effects on (1-/sup 14/C)D-glycerate transport.

  5. Transparent Helium in Stripped Envelope Supernovae

    NASA Astrophysics Data System (ADS)

    Piro, Anthony L.; Morozova, Viktoriya S.

    2014-09-01

    Using simple arguments based on photometric light curves and velocity evolution, we propose that some stripped envelope supernovae (SNe) show signs that a significant fraction of their helium is effectively transparent. The main pieces of evidence are the relatively low velocities with little velocity evolution, as are expected deep inside an exploding star, along with temperatures that are too low to ionize helium. This means that the helium should not contribute to the shaping of the main SN light curve, and thus the total helium mass may be difficult to measure from simple light curve modeling. Conversely, such modeling may be more useful for constraining the mass of the carbon/oxygen core of the SN progenitor. Other stripped envelope SNe show higher velocities and larger velocity gradients, which require an additional opacity source (perhaps the mixing of heavier elements or radioactive nickel) to prevent the helium from being transparent. We discuss ways in which similar analysis can provide insights into the differences and similarities between SNe Ib and Ic, which will lead to a better understanding of their respective formation mechanisms.

  6. Sensitivity to changes in amplitude envelope

    NASA Astrophysics Data System (ADS)

    Gallun, Erick; Hafter, Ervin R.; Bonnel, Anne-Marie

    2002-05-01

    Detection of a brief increment in a tonal pedestal is less well predicted by energy-detection (e.g., Macmillan, 1973; Bonnel and Hafter, 1997) than by sensitivity to changes in the stimulus envelope. As this implies a mechanism similar to an envelope extractor (Viemeister, 1979), sinusoidal amplitude modulation was used to mask a single ramped increment (10, 45, or 70 ms) added to a 1000-ms pedestal with carrier frequency (cf)=477 Hz. As in informational masking (Neff, 1994) and ``modulation-detection interference'' (Yost and Sheft, 1989), interference occurred with masker cfs of 477 and 2013 Hz. While slight masking was found with modulation frequencies (mfs) from 16 to 96 Hz, masking grew inversely with still lower mfs, being greatest for mf=4 Hz. This division is reminiscent of that said to separate sensations of ``roughness'' and ``beats,'' respectively (Terhardt, 1974), with the latter also being related to durations associated with auditory groupings in music and speech. Importantly, this result held for all of the signal durations and onset-offset ramps tested, suggesting that an increment on a pedestal is treated as a single auditory object whose detection is most difficult in the presence of other objects (in this case, ``beats'').

  7. Solution of K-V envelope equations

    SciTech Connect

    Anderson, O.A.

    1995-04-01

    The envelope equations for a KV beam with space charge have been analyzed systematically by an e expansion followed by integrations. The focusing profile as a function of axial length is assumed to be symmetric but otherwise arbitrary. Given the bean current, emittance, and peak focusing field, we find the envelopes a(s) and b(s) and obtain , a{sub max}, {sigma}, and {sigma}{sub 0}. Explicit results are presented for various truncations of the expansion. The zeroth order results correspond to those from the well-known smooth approximation; the same convenient format is retained for the higher order cases. The first order results, involving single correction terms, give 3--10 times better accuracy and are good to {approximately}1% at {sigma}{sub 0} = 70{degree}. Third order gives a factor of 10--30 improvement over the smooth approximation and derived quantities accurate to {approximately}1% at {sigma}{sub 0} = 112 {degree}. The first order expressions are convenient design tools. They lend themselves to variable energy problems and have been applied to the design, construction, and testing of ESQ accelerators at LBL.

  8. TRANSPARENT HELIUM IN STRIPPED ENVELOPE SUPERNOVAE

    SciTech Connect

    Piro, Anthony L.; Morozova, Viktoriya S.

    2014-09-01

    Using simple arguments based on photometric light curves and velocity evolution, we propose that some stripped envelope supernovae (SNe) show signs that a significant fraction of their helium is effectively transparent. The main pieces of evidence are the relatively low velocities with little velocity evolution, as are expected deep inside an exploding star, along with temperatures that are too low to ionize helium. This means that the helium should not contribute to the shaping of the main SN light curve, and thus the total helium mass may be difficult to measure from simple light curve modeling. Conversely, such modeling may be more useful for constraining the mass of the carbon/oxygen core of the SN progenitor. Other stripped envelope SNe show higher velocities and larger velocity gradients, which require an additional opacity source (perhaps the mixing of heavier elements or radioactive nickel) to prevent the helium from being transparent. We discuss ways in which similar analysis can provide insights into the differences and similarities between SNe Ib and Ic, which will lead to a better understanding of their respective formation mechanisms.

  9. Groupwise Dimension Reduction via Envelope Method

    PubMed Central

    Guo, Zifang; Li, Lexin; Lu, Wenbin; Li, Bing

    2016-01-01

    The family of sufficient dimension reduction (SDR) methods that produce informative combinations of predictors, or indices, are particularly useful for high dimensional regression analysis. In many such analyses, it becomes increasingly common that there is available a priori subject knowledge of the predictors; e.g., they belong to different groups. While many recent SDR proposals have greatly expanded the scope of the methods’ applicability, how to effectively incorporate the prior predictor structure information remains a challenge. In this article, we aim at dimension reduction that recovers full regression information while preserving the predictor group structure. Built upon a new concept of the direct sum envelope, we introduce a systematic way to incorporate the group information in most existing SDR estimators. As a result, the reduction outcomes are much easier to interpret. Moreover, the envelope method provides a principled way to build a variety of prior structures into dimension reduction analysis. Both simulations and real data analysis demonstrate the competent numerical performance of the new method. PMID:26973362

  10. Cellular phosphoinositides and the maturation of bluetongue virus, a non-enveloped capsid virus

    PubMed Central

    2013-01-01

    Background Bluetongue virus (BTV), a member of Orbivirus genus in the Reoviridae family is a double capsid virus enclosing a genome of 10 double-stranded RNA segments. A non-structural protein of BTV, NS3, which is associated with cellular membranes and interacts with outer capsid proteins, has been shown to be involved in virus morphogenesis in infected cells. In addition, studies have also shown that during the later stages of virus infection NS3 behaves similarly to HIV protein Gag, an enveloped viral protein. Since Gag protein is known to interact with membrane lipid phosphatidylinositol (4,5) bisphosphate [PI(4,5)P2] and one of the known binding partners of NS3, cellular protein p11 also interacts with annexin a PI(4,5)P2 interacting protein, this study was designed to understand the role of this negatively charged membrane lipid in BTV assembly and maturation. Methods Over expression of cellular enzymes that either depleted cells of PI(4,5)P2 or altered the distribution of PI(4,5)P2, were used to analyze the effect of the lipid on BTV maturation at different times post-infection. The production of mature virus particles was monitored by plaque assay. Microscopic techniques such as confocal microscopy and electron microscopy (EM) were also undertaken to study localization of virus proteins and virus particles in cells, respectively. Results Initially, confocal microscopic analysis demonstrated that PI(4,5)P2 not only co-localized with NS3, but it also co-localized with VP5, one of the outer capsid proteins of BTV. Subsequently, experiments involving depletion of cellular PI(4,5)P2 or its relocation demonstrated an inhibitory effect on normal BTV maturation and it also led to a redistribution of BTV proteins within the cell. The data was supported further by EM visualization showing that modulation of PI(4,5)P2 in cells indeed resulted in less particle production. Conclusion This study to our knowledge, is the first report demonstrating involvement of PI(4,5)P2

  11. The Escherichia coli Cpx Envelope Stress Response Regulates Genes of Diverse Function That Impact Antibiotic Resistance and Membrane Integrity

    PubMed Central

    Leblanc, Shannon K. D.; Price, Nancy L.

    2013-01-01

    The Cpx envelope stress response mediates adaptation to stresses that cause envelope protein misfolding. Adaptation is partly conferred through increased expression of protein folding and degradation factors. The Cpx response also plays a conserved role in the regulation of virulence determinant expression and impacts antibiotic resistance. We sought to identify adaptive mechanisms that may be involved in these important functions by characterizing changes in the transcriptome of two different Escherichia coli strains when the Cpx response is induced. We show that, while there is considerable strain- and condition-specific variability in the Cpx response, the regulon is enriched for proteins and functions that are inner membrane associated under all conditions. Genes that were changed by Cpx pathway induction under all conditions were involved in a number of cellular functions and included several intergenic regions, suggesting that posttranscriptional regulation is important during Cpx-mediated adaptation. Some Cpx-regulated genes are centrally involved in energetics and play a role in antibiotic resistance. We show that a number of small, uncharacterized envelope proteins are Cpx regulated and at least two of these affect phenotypes associated with membrane integrity. Altogether, our work suggests new mechanisms of Cpx-mediated envelope stress adaptation and antibiotic resistance. PMID:23564175

  12. The Cpx envelope stress response both facilitates and inhibits elaboration of the enteropathogenic Escherichia coli bundle-forming pilus

    PubMed Central

    Vogt, Stefanie L; Nevesinjac, Anna Z; Humphries, Romney M; Donnenberg, Michael S; Armstrong, Glen D; Raivio, Tracy L

    2010-01-01

    The Cpx envelope stress response is induced by the misfolding of periplasmic proteins and restores envelope homeostasis by upregulating several periplasmic protein folding and degrading factors. The Cpx response also regulates the expression of a variety of envelope-spanning protein complexes, including flagella, secretion systems and pili, which play an important role in pathogenesis. In a previous study, we inactivated the Cpx response in enteropathogenic Escherichia coli (EPEC), a causative agent of infant diarrhoea, and observed decreased expression of its major adhesin, the bundle-forming pilus (BFP). Here, we examined the mechanism underlying this BFP expression defect, and found that this phenotype can be attributed to insufficient expression of periplasmic folding factors, such as DsbA, DegP and CpxP. Hence, a low level of Cpx pathway activity promotes BFP synthesis by upregulating factors important for folding of BFP component proteins. Conversely, we found that full induction of the Cpx response inhibits BFP expression, mainly by repressing transcription of the bfp gene cluster. In combination with a previous report examining EPEC type III secretion, our results demonstrate that the Cpx response co-ordinates the repression of cell-surface structures during periods of envelope stress. PMID:20444097

  13. Remodeling of the Nuclear Envelope and Lamina during Bovine Preimplantation Development and Its Functional Implications.

    PubMed

    Popken, Jens; Graf, Alexander; Krebs, Stefan; Blum, Helmut; Schmid, Volker J; Strauss, Axel; Guengoer, Tuna; Zakhartchenko, Valeri; Wolf, Eckhard; Cremer, Thomas

    2015-01-01

    The present study demonstrates a major remodeling of the nuclear envelope and its underlying lamina during bovine preimplantation development. Up to the onset of major embryonic genome activation (MGA) at the 8-cell stage nuclei showed a non-uniform distribution of nuclear pore complexes (NPCs). NPCs were exclusively present at sites where DNA contacted the nuclear lamina. Extended regions of the lamina, which were not contacted by DNA, lacked NPCs. In post-MGA nuclei the whole lamina was contacted rather uniformly by DNA. Accordingly, NPCs became uniformly distributed throughout the entire nuclear envelope. These findings shed new light on the conditions which control the integration of NPCs into the nuclear envelope. The switch from maternal to embryonic production of mRNAs was accompanied by multiple invaginations covered with NPCs, which may serve the increased demands of mRNA export and protein import. Other invaginations, as well as interior nuclear segments and vesicles without contact to the nuclear envelope, were exclusively positive for lamin B. Since the abundance of these invaginations and vesicles increased in concert with a massive nuclear volume reduction, we suggest that they reflect a mechanism for fitting the nuclear envelope and its lamina to a shrinking nuclear size during bovine preimplantation development. In addition, a deposit of extranuclear clusters of NUP153 (a marker for NPCs) without associated lamin B was frequently observed from the zygote stage up to MGA. Corresponding RNA-Seq data revealed deposits of spliced, maternally provided NUP153 mRNA and little unspliced, newly synthesized RNA prior to MGA, which increased strongly at the initiation of embryonic expression of NUP153 at MGA. PMID:25932910

  14. Remodeling of the Nuclear Envelope and Lamina during Bovine Preimplantation Development and Its Functional Implications

    PubMed Central

    Popken, Jens; Graf, Alexander; Krebs, Stefan; Blum, Helmut; Schmid, Volker J.; Strauss, Axel; Guengoer, Tuna; Zakhartchenko, Valeri; Wolf, Eckhard; Cremer, Thomas

    2015-01-01

    The present study demonstrates a major remodeling of the nuclear envelope and its underlying lamina during bovine preimplantation development. Up to the onset of major embryonic genome activation (MGA) at the 8-cell stage nuclei showed a non-uniform distribution of nuclear pore complexes (NPCs). NPCs were exclusively present at sites where DNA contacted the nuclear lamina. Extended regions of the lamina, which were not contacted by DNA, lacked NPCs. In post-MGA nuclei the whole lamina was contacted rather uniformly by DNA. Accordingly, NPCs became uniformly distributed throughout the entire nuclear envelope. These findings shed new light on the conditions which control the integration of NPCs into the nuclear envelope. The switch from maternal to embryonic production of mRNAs was accompanied by multiple invaginations covered with NPCs, which may serve the increased demands of mRNA export and protein import. Other invaginations, as well as interior nuclear segments and vesicles without contact to the nuclear envelope, were exclusively positive for lamin B. Since the abundance of these invaginations and vesicles increased in concert with a massive nuclear volume reduction, we suggest that they reflect a mechanism for fitting the nuclear envelope and its lamina to a shrinking nuclear size during bovine preimplantation development. In addition, a deposit of extranuclear clusters of NUP153 (a marker for NPCs) without associated lamin B was frequently observed from the zygote stage up to MGA. Corresponding RNA-Seq data revealed deposits of spliced, maternally provided NUP153 mRNA and little unspliced, newly synthesized RNA prior to MGA, which increased strongly at the initiation of embryonic expression of NUP153 at MGA. PMID:25932910

  15. Pushing the Envelope of Extreme Space Weather

    NASA Astrophysics Data System (ADS)

    Pesnell, W. D.

    2014-12-01

    Extreme Space Weather events are large solar flares or geomagnetic storms, which can cost billions of dollars to recover from. We have few examples of such events; the Carrington Event (the solar superstorm) is one of the few that had superlatives in three categories: size of solar flare, drop in Dst, and amplitude of aa. Kepler observations show that stars similar to the Sun can have flares releasing millions of times more energy than an X-class flare. These flares and the accompanying coronal mass ejections could strongly affect the atmosphere surrounding a planet. What level of solar activity would be necessary to strongly affect the atmosphere of the Earth? Can we map out the envelope of space weather along the evolution of the Sun? What would space weather look like if the Sun stopped producing a magnetic field? To what extreme should Space Weather go? These are the extremes of Space Weather explored in this talk.

  16. Cricket team selection using data envelopment analysis.

    PubMed

    Amin, Gholam R; Sharma, Sujeet Kumar

    2014-01-01

    This paper suggests a new method for cricket team selection using data envelopment analysis (DEA). We propose a DEA formulation for evaluation of cricket players in different capabilities using multiple outputs. This evaluation determines efficient and inefficient cricket players and ranks them on the basis of DEA scores. The ranking can be used to choose the required number of players for a cricket team in each cricketing capability. A real dataset, Indian Premier League 4 (IPL 2011), cricket players having various capabilities is used to choose the best cricket team. The proposed method has the advantage of considering multiple factors related to the performance of players in multiple capabilities collected from IPL 4 and aggregates their scores using a linear programming DEA model. This DEA Aggregation gives the scores of players objectively instead of using subjective computations. The proposed DEA method can be used to form a national cricket team from several clubs or a team of top cricketers. PMID:24444231

  17. Fullerenes and fulleranes in circumstellar envelopes

    NASA Astrophysics Data System (ADS)

    Zhang, Yong; Kwok, Sun; Sadjadi, SeyedAbdolreza

    2016-07-01

    Three decades of search have recently led to convincing discoveries of cosmic fullerenes. The presence of C60 and C+ 60 in both circumstellar and interstellar environments suggests that these molecules and their derivatives can be efficiently formed in circumstellar envelopes and survive in harsh conditions. Detailed analysis of the infrared bands from fullerenes and their connections with the local properties can provide valuable information on the physical conditions and chemical processes that occurred in the late stages of stellar evolution. The identification of C+ 60 as the carrier of four diffuse interstellar bands (DIBs) suggests that fullerene- related compounds are abundant in interstellar space and are essential for resolving the DIB mystery. Experiments have revealed a high hydrogenation rate when C60 is exposed to atomic hydrogen, motivating the attempt to search for cosmic fulleranes. In this paper, we present a short review of current knowledge of cosmic fullerenes and fulleranes and briefly discuss the implications on circumstellar chemistry.

  18. Antireflection Pyrex envelopes for parabolic solar collectors

    NASA Astrophysics Data System (ADS)

    McCollister, H. L.; Pettit, R. B.

    1983-11-01

    Antireflective (AR) coatings, applied to the glass envelopes used in parabolic trough solar collectors around the receiver tube in order to reduce thermal losses, can increase solar transmittance by 7 percent. An AR surface has been formed on Pyrex by first heat treating the glass to cause a compositional phase separation, removing a surface layer after heat treatment through the use of a preetching solution, and finally etching in a solution that contains hydrofluorosilic and ammonium bifluoride acids. AR-coated samples with solar transmittance values of more than 0.97, by comparison to an untreated sample value of 0.91, have been obtained for the 560-630 C range of heat treatment temperatures. Optimum values have also been determined for the other processing parameters.

  19. SO2 and SO in circumstellar envelopes

    NASA Astrophysics Data System (ADS)

    Guilloteau, S.; Lucas, R.; Omont, A.; Nguyen-Q-Rieu

    1986-09-01

    After its first detection in circumstellar envelopes (Lucas et al. 1986) SO2 has been systematically searched for with the IRAM 30-m telescope. It has been found in 3 new stars, with very strong lines in OH 231.8+4.2 (TA* ≈ 0.7 - 1.4K, Trot ≈ 25K, Δv ≈ 80 km s-1, TA*(SO2) > TA*(CO) ) and relatively strong ones in OH 26.5+0.6. SO has been detected for the first time in a circumstellar shell, in OH 231.8+4.2. H13CN has been observed in the same star, suggesting a very large abundance of 13C.