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Michael R Boyd - One of the best experts on this subject based on the ideXlab platform.
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structures of the complexes of a potent anti hiv protein cyanovirin n and high mannose oligosaccharides
Journal of Biological Chemistry, 2002Co-Authors: Istvan Botos, Laura K Cartner, Daniel M. Ratner, Michael R Boyd, Peter H. Seeberger, Shilpa R Shenoy, Barry R Okeefe, Alexander WlodawerAbstract:Abstract The development of anti-human immunodeficiency virus (HIV) microbicides for either topical or ex vivo use is of considerable interest, mainly due to the difficulties in creating a vaccine that would be active against multiple clades of HIV. Cyanovirin-N (CV-N), an 11-kDa protein from the cyanobacterium (blue-green algae) Nostoc Ellipsosporum with potent virucidal activity, was identified in the search for such antiviral agents. The binding of CV-N to the heavily glycosylated HIV envelope protein gp120 is carbohydrate-dependent. Since previous CV-N-dimannose structures could not fully explain CV-N-oligomannose binding, we determined the crystal structures of recombinant CV-N complexed to Man-9 and a synthetic hexamannoside, at 2.5- and 2.4-A resolution, respectively. CV-N is a three-dimensional domain-swapped dimer in the crystal structures with two primary sites near the hinge region and two secondary sites on the opposite ends of the dimer. The binding interface is constituted of three stacked α1→2-linked mannose rings for Man-9 and two stacked mannose rings for hexamannoside with the rest of the saccharide molecules pointing to the solution. These structures show unequivocally the binding geometry of high mannose sugars to CV-N, permitting a better understanding of carbohydrate binding to this potential new lead for the design of drugs against AIDS.
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Cyanovirin-N, a Potent Human Immunodeficiency Virus-Inactivating Protein, Blocks both CD4-Dependent and CD4-Independent Binding of Soluble gp120 (sgp120) to Target Cells, Inhibits sCD4-Induced Binding of sgp120 to Cell-Associated CXCR4, and Dissociat
Antimicrobial agents and chemotherapy, 2001Co-Authors: Toshiyuki Mori, Michael R BoydAbstract:Cyanovirin-N (CV-N), an 11-kDa protein originally isolated from the cyanobacterium Nostoc Ellipsosporum, potently inactivates diverse strains of human immunodeficiency virus type 1 (HIV-1), HIV-2, simian immunodeficiency virus, and feline immunodeficiency virus. It has been well established that the HIV surface envelope glycoprotein gp120 is a molecular target of CV-N. We recently reported that CV-N impaired the binding of virion-associated gp120 to cell-associated CD4 and that CV-N preferentially inhibited binding of the glycosylation-dependent neutralizing monoclonal antibody 2G12 to gp120. However, CV-N did not interfere with the interactions of soluble CD4 (sCD4) with either soluble gp120 (sgp120) or virion-associated gp120. In the present study, we have evaluated the effects of CV-N on the binding of sgp120 to cell-associated CD4 to clarify the experimental basis of the previous binding results, and we further address the detailed mechanism of action of CV-N. Here we present evidence that (i) CV-N impairs both CD4-dependent and CD4-independent binding of sgp120 to the target cells, (ii) CV-N blocks the sCD4-induced binding of sgp120 with cell-associated coreceptor CXCR4, and (iii) CV-N dissociates bound sgp120 from target cells. The results illustrate that the measured effects of CV-N on gp120-CD4 binding interactions depend upon the type of CD4 (soluble or cell associated), but not upon the type of gp120 (soluble or virion associated), employed in the experimental protocol. In addition, this study reinforces that CV-N acts uniquely to prevent essential interactions between the envelope glycoprotein and target cell receptors and further supports the potential broad utility of CV-N as a microbicide to prevent the transmission of HIV and AIDS.
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Cyanovirin-N Binds to gp120 To Interfere with CD4-Dependent Human Immunodeficiency Virus Type 1 Virion Binding, Fusion, and Infectivity but Does Not Affect the CD4 Binding Site on gp120 or Soluble CD4-Induced Conformational Changes in gp120
Journal of virology, 1999Co-Authors: Mark T. Esser, Michael R Boyd, Toshiyuki Mori, Isabelle Mondor, Quentin J. Sattentau, Barna Dey, Edward A. Berger, Jeffrey D. LifsonAbstract:Cyanovirin-N (CV-N), an 11-kDa protein isolated from the cyanobacterium Nostoc Ellipsosporum, potently inactivates diverse strains of human immunodeficiency virus type 1 (HIV-1), HIV-2, and simian immunodeficiency virus. While it has been well established that the viral surface envelope glycoprotein gp120 is a molecular target of CV-N, the detailed mechanism of action is of further interest. We compared matched native and CV-N-treated virus preparations in a panel of assays that measure viral replication, assessing successive stages of the viral life cycle. CV-N-treated virions failed to infect cells as detected by p24 production and quantitative PCR for HIV-1 reverse transcription products, whereas treatment of the target cells did not block infection, confirming that CV-N acts at the level of the virus, not the target cell, to abort the initial infection process. Compared to native HIV-1 preparations, CV-N-treated HIV-1 virions showed impaired CD4-dependent binding to CD4(+) T cells and did not mediate "fusion from without" of CD4(+) target cells. CV-N also blocked HIV envelope glycoprotein Env-induced, CD4-dependent cell-cell fusion. Mapping studies with monoclonal antibodies (MAbs) to defined epitopes on the HIV-1 envelope glycoprotein indicated that CV-N binds to gp120 in a manner that does not occlude or alter the CD4 binding site or V3 loop or other domains on gp120 recognized by defined MAbs and does not interfere with soluble CD4-induced conformational changes in gp120. Binding of CV-N to soluble gp120 or virions inhibited subsequent binding of the unique neutralizing MAb 2G12, which recognizes a glycosylation-dependent epitope. However, prior binding of 2G12 MAb to gp120 did not block subsequent binding by CV-N. These results help clarify the mechanism of action of CV-N and suggest that the compound may act in part by preventing essential interactions between the envelope glycoprotein and target cell receptors. This proposed mechanism is consistent with the extensive activity profile of CV-N against numerous isolates of HIV-1 and other lentiviruses and supports the potential broad utility of this protein as a microbicide to prevent the sexual transmission of HIV.
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isolation primary sequence determination and disulfide bond structure of cyanovirin n an anti hiv human immunodeficiency virus protein from the cyanobacteriumNostoc Ellipsosporum
Biochemical and Biophysical Research Communications, 1997Co-Authors: Kirk R. Gustafson, Umamaheswari Rajamani, Louis E. Henderson, John H. Cardellina, Lewis K. Pannell, Raymond C Sowder, James B Mcmahon, Michael R BoydAbstract:Abstract A novel anti-HIV protein, cyanovirin-N (CV-N), was isolated from an aqueous cellular extract of the cultured cyanobacterium (blue-green alga)Nostoc Ellipsosporum,purified by reverse-phase HPLC, and sequenced by N-terminal Edman degradation of the intact protein and peptide fragments produced by endoproteinase digestions. CV-N consists of a single 101 amino acid chain which exhibits significant internal sequence duplication, but no significant homology to previously described proteins or to the transcription products of known nucleotide sequences. Alignment of residues 1-50 with residues 51-101 reveals 13 conservative amino acid changes as well as direct homology between 16 amino acid residues. CV-N contains four cysteines which form two intrachain disulfide bonds. The positions of the disulfide linkages were established by fast atom bombardment mass spectral studies of peptide fragments generated by a tryptic digestion of the native protein. Reductive cleavage of these crosslinks resulted in loss of anti-HIV activity.
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discovery of cyanovirin n a novel human immunodeficiency virus inactivating protein that binds viral surface envelope glycoprotein gp120 potential applications to microbicide development
Antimicrobial Agents and Chemotherapy, 1997Co-Authors: Michael R Boyd, Robert J Gulakowski, M I Rivera, Robert H Shoemaker, Kirk R. Gustafson, Toshiyuki Mori, Barry R Okeefe, James B Mcmahon, Lin Wu, C M LaurencotAbstract: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.
Tran Hai - One of the best experts on this subject based on the ideXlab platform.
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The role of the C-terminal region of cyanophycin synthetase from Nostoc Ellipsosporum NE1 in its enzymatic activity and thermostability: A key
2009Co-Authors: Tran Hai, Jin-sook Lee, Tae-jong Kim, Joo-won SuhAbstract:article i nfo The biosynthesis of cyanophycin granule polypeptides is catalyzed by cyanophycin synthetase, CphA. In this study, the role of the C-terminal region of CphA from Nostoc Ellipsosporum NE1, CphANE1, was analyzed using a tailor-made C-terminus truncated library. The expression level of truncated CphANE1 in E. coli depended on the stop codons that were used. The expression vector that had the amber stop codon TAG produced more than twice amount of CphANE1 as a vector that contained the ochre codon TAA. CphANE1ΔC45, which was truncated up to 45 amino acids at its C-terminus, retained full enzymatic activity and produced polymers. However, the removal of one additional amino acid, Glu 856 , resulted in complete inactivation of CphANE1ΔC46. 856 by valine or alanine confirmed the importance of this residue for the activity of CphANE1, as it resulted in the complete inactivation of the enzyme. In addition, thermostability analysis revealed a dramatic decrease in the thermostability of CphANE1 after removal of the region from Leu 867 to Leu 870 . The gel filtration analysis showed that CphANE1Δ46C still formed a dimer form even its enzyme activity was lost completely. These results suggest that Glu 856 is critical for CphANE1 catalytic activity and that the
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the role of the c terminal region of cyanophycin synthetase from Nostoc Ellipsosporum ne1 in its enzymatic activity and thermostability a key function of glu856
Biochimica et Biophysica Acta, 2009Co-Authors: Tran Hai, Jin-sook Lee, Tae-jong Kim, Joo-won SuhAbstract:The biosynthesis of cyanophycin granule polypeptides is catalyzed by cyanophycin synthetase, CphA. In this study, the role of the C-terminal region of CphA from Nostoc Ellipsosporum NE1, CphANE1, was analyzed using a tailor-made C-terminus truncated library. The expression level of truncated CphANE1 in E. coli depended on the stop codons that were used. The expression vector that had the amber stop codon TAG produced more than twice amount of CphANE1 as a vector that contained the ochre codon TAA. CphANE1ΔC45, which was truncated up to 45 amino acids at its C-terminus, retained full enzymatic activity and produced polymers. However, the removal of one additional amino acid, Glu856, resulted in complete inactivation of CphANE1ΔC46. Replacement of Glu856 by valine or alanine confirmed the importance of this residue for the activity of CphANE1, as it resulted in the complete inactivation of the enzyme. In addition, thermostability analysis revealed a dramatic decrease in the thermostability of CphANE1 after removal of the region from Leu867 to Leu870. The gel filtration analysis showed that CphANE1Δ46C still formed a dimer form even its enzyme activity was lost completely. These results suggest that Glu856 is critical for CphANE1 catalytic activity and that the predicted α-helical region that ranges from Val858 to Leu870 is important for the thermostability of the enzyme.
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The role of the C-terminal region of cyanophycin synthetase from Nostoc Ellipsosporum NE1 in its enzymatic activity and thermostability: A key function of Glu856
Biochimica et biophysica acta, 2008Co-Authors: Tran Hai, Jin-sook Lee, Tae-jong Kim, Joo-won SuhAbstract:The biosynthesis of cyanophycin granule polypeptides is catalyzed by cyanophycin synthetase, CphA. In this study, the role of the C-terminal region of CphA from Nostoc Ellipsosporum NE1, CphA(NE1), was analyzed using a tailor-made C-terminus truncated library. The expression level of truncated CphA(NE1) in E. coli depended on the stop codons that were used. The expression vector that had the amber stop codon TAG produced more than twice amount of CphA(NE1) as a vector that contained the ochre codon TAA. CphA(NE1DeltaC45), which was truncated up to 45 amino acids at its C-terminus, retained full enzymatic activity and produced polymers. However, the removal of one additional amino acid, Glu(856), resulted in complete inactivation of CphA(NE1DeltaC46). Replacement of Glu(856) by valine or alanine confirmed the importance of this residue for the activity of CphA(NE1), as it resulted in the complete inactivation of the enzyme. In addition, thermostability analysis revealed a dramatic decrease in the thermostability of CphA(NE1) after removal of the region from Leu(867) to Leu(870). The gel filtration analysis showed that CphA(NE1Delta46C) still formed a dimer form even its enzyme activity was lost completely. These results suggest that Glu(856) is critical for CphA(NE1) catalytic activity and that the predicted alpha-helical region that ranges from Val(858) to Leu(870) is important for the thermostability of the enzyme.
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engineered cyanophycin synthetase cpha from Nostoc Ellipsosporum confers enhanced cpha activity and cyanophycin accumulation to escherichia coli
Applied and Environmental Microbiology, 2006Co-Authors: Tran Hai, Kay M. Frey, Alexander SteinbüchelAbstract:The cyanophycin (CGP) synthetase gene (cphANE1) of the transposon-induced argL mutant NE1 of the cyanobacterium Nostoc Ellipsosporum, which exhibits a CGP-leaky phenotype during diazotrophical growth, was cloned and expressed in Escherichia coli strain TOP10. Its amino acid sequence exhibited high similarities to CphAs of other cyanobacteria. Recombinant cells of E. coli, which harbored a fragment comprising the complete cphANE1 gene plus 400 bp of its downstream region in colinear orientation to the lacZ promoter, accumulated CGP up to 17 and 8.5% (wt/wt) of cellular dry matter (CDM) if cultivated in complex medium in the presence or absence of isopropyl-β-d-thiogalactopyranoside, respectively. Two truncated CphAs, lacking 31 (CphANE1del96) or 59 (CphANE1del180) amino acids of the C-terminal region, were derived from cphANE1 by deleting 96 or 180 bp from its 3′ region through the introduction of stop codons. In comparison to the wild-type gene, cphANE1del96 conferred about 2.1- to 2.2-fold-higher enzyme activity (up to 5.75 U/mg protein) on E. coli. Furthermore, these cells accumulated about twofold more CGP (up to 34.5% [wt/wt] of CDM) than cells expressing the wild-type gene. An engineered CphA possessing significantly enhanced activity and conferring the highest CGP content on E. coli is demonstrated. In contrast, CphANE1del180 was inactive and did not confer CGP accumulation on E. coli. Interestingly, a short conserved stretch of 4 to 5 hydrophobic amino acids is located in the protein region present in CphANE1del96 but absent in CphANE1del180. In addition, CphANE1 and CphANE1del96 are, besides CphA from Acinetobacter baylyi, the only CphAs exhibiting rigid substrate specificities that do not enable the incorporation of lysine instead of arginine into CGP.
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Engineered cyanophycin synthetase (CphA) from Nostoc Ellipsosporum confers enhanced CphA activity and cyanophycin accumulation to Escherichia coli.
Applied and environmental microbiology, 2006Co-Authors: Tran Hai, Kay M. Frey, Alexander SteinbüchelAbstract:The cyanophycin (CGP) synthetase gene (cphANE1) of the transposon-induced argL mutant NE1 of the cyanobacterium Nostoc Ellipsosporum, which exhibits a CGP-leaky phenotype during diazotrophical growth, was cloned and expressed in Escherichia coli strain TOP10. Its amino acid sequence exhibited high similarities to CphAs of other cyanobacteria. Recombinant cells of E. coli, which harbored a fragment comprising the complete cphANE1 gene plus 400 bp of its downstream region in colinear orientation to the lacZ promoter, accumulated CGP up to 17 and 8.5% (wt/wt) of cellular dry matter (CDM) if cultivated in complex medium in the presence or absence of isopropyl-beta-D-thiogalactopyranoside, respectively. Two truncated CphAs, lacking 31 (CphANE1del96) or 59 (CphANE1del180) amino acids of the C-terminal region, were derived from cphANE1 by deleting 96 or 180 bp from its 3' region through the introduction of stop codons. In comparison to the wild-type gene, cphANE1del96 conferred about 2.1- to 2.2-fold-higher enzyme activity (up to 5.75 U/mg protein) on E. coli. Furthermore, these cells accumulated about twofold more CGP (up to 34.5% [wt/wt] of CDM) than cells expressing the wild-type gene. An engineered CphA possessing significantly enhanced activity and conferring the highest CGP content on E. coli is demonstrated. In contrast, CphANE1del180 was inactive and did not confer CGP accumulation on E. coli. Interestingly, a short conserved stretch of 4 to 5 hydrophobic amino acids is located in the protein region present in CphANE1del96 but absent in CphANE1del180. In addition, CphANE1 and CphANE1del96 are, besides CphA from Acinetobacter baylyi, the only CphAs exhibiting rigid substrate specificities that do not enable the incorporation of lysine instead of arginine into CGP.
Joo-won Suh - One of the best experts on this subject based on the ideXlab platform.
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The role of the C-terminal region of cyanophycin synthetase from Nostoc Ellipsosporum NE1 in its enzymatic activity and thermostability: A key
2009Co-Authors: Tran Hai, Jin-sook Lee, Tae-jong Kim, Joo-won SuhAbstract:article i nfo The biosynthesis of cyanophycin granule polypeptides is catalyzed by cyanophycin synthetase, CphA. In this study, the role of the C-terminal region of CphA from Nostoc Ellipsosporum NE1, CphANE1, was analyzed using a tailor-made C-terminus truncated library. The expression level of truncated CphANE1 in E. coli depended on the stop codons that were used. The expression vector that had the amber stop codon TAG produced more than twice amount of CphANE1 as a vector that contained the ochre codon TAA. CphANE1ΔC45, which was truncated up to 45 amino acids at its C-terminus, retained full enzymatic activity and produced polymers. However, the removal of one additional amino acid, Glu 856 , resulted in complete inactivation of CphANE1ΔC46. 856 by valine or alanine confirmed the importance of this residue for the activity of CphANE1, as it resulted in the complete inactivation of the enzyme. In addition, thermostability analysis revealed a dramatic decrease in the thermostability of CphANE1 after removal of the region from Leu 867 to Leu 870 . The gel filtration analysis showed that CphANE1Δ46C still formed a dimer form even its enzyme activity was lost completely. These results suggest that Glu 856 is critical for CphANE1 catalytic activity and that the
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the role of the c terminal region of cyanophycin synthetase from Nostoc Ellipsosporum ne1 in its enzymatic activity and thermostability a key function of glu856
Biochimica et Biophysica Acta, 2009Co-Authors: Tran Hai, Jin-sook Lee, Tae-jong Kim, Joo-won SuhAbstract:The biosynthesis of cyanophycin granule polypeptides is catalyzed by cyanophycin synthetase, CphA. In this study, the role of the C-terminal region of CphA from Nostoc Ellipsosporum NE1, CphANE1, was analyzed using a tailor-made C-terminus truncated library. The expression level of truncated CphANE1 in E. coli depended on the stop codons that were used. The expression vector that had the amber stop codon TAG produced more than twice amount of CphANE1 as a vector that contained the ochre codon TAA. CphANE1ΔC45, which was truncated up to 45 amino acids at its C-terminus, retained full enzymatic activity and produced polymers. However, the removal of one additional amino acid, Glu856, resulted in complete inactivation of CphANE1ΔC46. Replacement of Glu856 by valine or alanine confirmed the importance of this residue for the activity of CphANE1, as it resulted in the complete inactivation of the enzyme. In addition, thermostability analysis revealed a dramatic decrease in the thermostability of CphANE1 after removal of the region from Leu867 to Leu870. The gel filtration analysis showed that CphANE1Δ46C still formed a dimer form even its enzyme activity was lost completely. These results suggest that Glu856 is critical for CphANE1 catalytic activity and that the predicted α-helical region that ranges from Val858 to Leu870 is important for the thermostability of the enzyme.
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The role of the C-terminal region of cyanophycin synthetase from Nostoc Ellipsosporum NE1 in its enzymatic activity and thermostability: A key function of Glu856
Biochimica et biophysica acta, 2008Co-Authors: Tran Hai, Jin-sook Lee, Tae-jong Kim, Joo-won SuhAbstract:The biosynthesis of cyanophycin granule polypeptides is catalyzed by cyanophycin synthetase, CphA. In this study, the role of the C-terminal region of CphA from Nostoc Ellipsosporum NE1, CphA(NE1), was analyzed using a tailor-made C-terminus truncated library. The expression level of truncated CphA(NE1) in E. coli depended on the stop codons that were used. The expression vector that had the amber stop codon TAG produced more than twice amount of CphA(NE1) as a vector that contained the ochre codon TAA. CphA(NE1DeltaC45), which was truncated up to 45 amino acids at its C-terminus, retained full enzymatic activity and produced polymers. However, the removal of one additional amino acid, Glu(856), resulted in complete inactivation of CphA(NE1DeltaC46). Replacement of Glu(856) by valine or alanine confirmed the importance of this residue for the activity of CphA(NE1), as it resulted in the complete inactivation of the enzyme. In addition, thermostability analysis revealed a dramatic decrease in the thermostability of CphA(NE1) after removal of the region from Leu(867) to Leu(870). The gel filtration analysis showed that CphA(NE1Delta46C) still formed a dimer form even its enzyme activity was lost completely. These results suggest that Glu(856) is critical for CphA(NE1) catalytic activity and that the predicted alpha-helical region that ranges from Val(858) to Leu(870) is important for the thermostability of the enzyme.
Kirk R. Gustafson - One of the best experts on this subject based on the ideXlab platform.
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treatment of influenza a h1n1 virus infections in mice and ferrets with cyanovirin n
Antiviral Research, 2008Co-Authors: Donald F Smee, Vasiliy P. Mishin, Kirk R. Gustafson, Minhui Wong, Kevin W Bailey, Barry R Okeefe, Larisa V GubarevaAbstract:Abstract Cyanovirin-N (CV-N), a protein derived from Nostoc Ellipsosporum , neutralizes influenza virus infectivity by binding to specific high-mannose oligosaccharides (oligomannose-8 and -9) at glycosylation sites on the viral hemagglutinin HA1 subunit. Mouse-adapted viruses lose sensitivity to CV-N due to HA1 mutations that eliminate these glycosylation sites. Recently we created a hybrid (reassortant) influenza A/WSN/33 (H1N1) virus containing the HA gene of A/New Caledonia/20/99 (H1N1) with an Asp225Gly mutation in the HA1, that was lethal to mice yet retained sensitivity to CV-N. We then utilized this model system to test the efficacy of CV-N against influenza. CV-N efficacy was dose-responsive from 0.0625 to 1 mg/kg/day when administered intranasally (i.n.) twice daily for 4 days starting 4 h prior to virus exposure. In a second study, survival benefit was seen with CV-N treatments (0.5 mg/kg/day for 4 days) beginning at −4 or +6 h, but was significantly reduced at +12 h. The early treatment resulted in up to 100% survival and 1000-fold reduction in lung virus titer on day 3 of the infection. In contrast, ribavirin (a positive control—75 mg/kg/day) treatment resulted in 30% survival and 30-fold decrease in lung virus titers. Lung consolidation scores and lung weights were significantly reduced by CV-N and ribavirin treatment on day 6 of the infection. Ferrets infected with a non-animal adapted influenza A/Charlottesville/31/95 (H1N1) virus were treated intranasally with CV-N (50 μg twice daily for 5 days starting 24 h before virus challenge). They exhibited 100-fold lower viral titers in nasal washes than placebos 1 day after treatment, but virus titers were equivalent on days 2–7. CV-N has the potential for prophylaxis and early initiation of treatment of influenza virus infections.
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isolation primary sequence determination and disulfide bond structure of cyanovirin n an anti hiv human immunodeficiency virus protein from the cyanobacteriumNostoc Ellipsosporum
Biochemical and Biophysical Research Communications, 1997Co-Authors: Kirk R. Gustafson, Umamaheswari Rajamani, Louis E. Henderson, John H. Cardellina, Lewis K. Pannell, Raymond C Sowder, James B Mcmahon, Michael R BoydAbstract:Abstract A novel anti-HIV protein, cyanovirin-N (CV-N), was isolated from an aqueous cellular extract of the cultured cyanobacterium (blue-green alga)Nostoc Ellipsosporum,purified by reverse-phase HPLC, and sequenced by N-terminal Edman degradation of the intact protein and peptide fragments produced by endoproteinase digestions. CV-N consists of a single 101 amino acid chain which exhibits significant internal sequence duplication, but no significant homology to previously described proteins or to the transcription products of known nucleotide sequences. Alignment of residues 1-50 with residues 51-101 reveals 13 conservative amino acid changes as well as direct homology between 16 amino acid residues. CV-N contains four cysteines which form two intrachain disulfide bonds. The positions of the disulfide linkages were established by fast atom bombardment mass spectral studies of peptide fragments generated by a tryptic digestion of the native protein. Reductive cleavage of these crosslinks resulted in loss of anti-HIV activity.
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discovery of cyanovirin n a novel human immunodeficiency virus inactivating protein that binds viral surface envelope glycoprotein gp120 potential applications to microbicide development
Antimicrobial Agents and Chemotherapy, 1997Co-Authors: Michael R Boyd, Robert J Gulakowski, M I Rivera, Robert H Shoemaker, Kirk R. Gustafson, Toshiyuki Mori, Barry R Okeefe, James B Mcmahon, Lin Wu, C M LaurencotAbstract: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.
Alexander Wlodawer - One of the best experts on this subject based on the ideXlab platform.
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cyanovirin n a sugar binding antiviral protein with a new twist
Cellular and Molecular Life Sciences, 2003Co-Authors: Istvan Botos, Alexander WlodawerAbstract:Cyanovirin-N (CV-N), an 11-kDa protein from the cyanobacterium Nostoc Ellipsosporum, is a highly potent virucidal agent that has generated interest as a lead natural product for the prevention and chemotherapy of human immunodeficiency virus infection. The antiviral activity of CV-N is mediated through specific, high-affinity interactions with the viral surface envelope glycoproteins. A number of structures of wild-type, mutant and sequence-shuffled CV-N have been solved by nuclear magnetic resonance and crystallography, showing that the protein exists as either a quasi-symmetric two-domain monomer or a domain-swapped dimer. Structures of several complexes of CV-N with oligosaccharides help in explaining the unique mode of high-affinity binding of these molecules to both forms of CV-N.
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structures of the complexes of a potent anti hiv protein cyanovirin n and high mannose oligosaccharides
Journal of Biological Chemistry, 2002Co-Authors: Istvan Botos, Laura K Cartner, Daniel M. Ratner, Michael R Boyd, Peter H. Seeberger, Shilpa R Shenoy, Barry R Okeefe, Alexander WlodawerAbstract:Abstract The development of anti-human immunodeficiency virus (HIV) microbicides for either topical or ex vivo use is of considerable interest, mainly due to the difficulties in creating a vaccine that would be active against multiple clades of HIV. Cyanovirin-N (CV-N), an 11-kDa protein from the cyanobacterium (blue-green algae) Nostoc Ellipsosporum with potent virucidal activity, was identified in the search for such antiviral agents. The binding of CV-N to the heavily glycosylated HIV envelope protein gp120 is carbohydrate-dependent. Since previous CV-N-dimannose structures could not fully explain CV-N-oligomannose binding, we determined the crystal structures of recombinant CV-N complexed to Man-9 and a synthetic hexamannoside, at 2.5- and 2.4-A resolution, respectively. CV-N is a three-dimensional domain-swapped dimer in the crystal structures with two primary sites near the hinge region and two secondary sites on the opposite ends of the dimer. The binding interface is constituted of three stacked α1→2-linked mannose rings for Man-9 and two stacked mannose rings for hexamannoside with the rest of the saccharide molecules pointing to the solution. These structures show unequivocally the binding geometry of high mannose sugars to CV-N, permitting a better understanding of carbohydrate binding to this potential new lead for the design of drugs against AIDS.
