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Michael R Boyd - One of the best experts on this subject based on the ideXlab platform.
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influenza a h1n1 virus resistance to cyanovirin n arises naturally during adaptation to mice and by passage in cell culture in the presence of the inhibitor
Antiviral Chemistry & Chemotherapy, 2007Co-Authors: Donald F Smee, Carrie J. Saucedo, Barry R Okeefe, Michael R Boyd, Vasiliy P Mishin, Larisa V Gubareva, Miles K Wandersee, Melissa B CheckettsAbstract:Influenza A/New Caledonia/20/99 (H1N1) virus was studied for development of resistance to Cyanovirin-N (CVN). CVN neutralizes virus infectivity by binding to specific high-mannose oligosaccharides on the viral haemagglutinin 1 (HA1) subunit. During virus adaptation to mice in the absence of CVN treatment the virus became resistant to CVN (CVN-MR virus), as did virus passaged in cell culture in the presence of CVN (CVN-R virus). The CVN-R virus possessed a single amino acid change at position 94a (Asn94aAsp) of HA1 that eliminated this glycosylation site. The CVN-MR virus at mouse passage 7 was a mixture of clones, consisting of a single mutation (Asp225Gly) and double mutations (Asn63Ser+Asp225Gly or Asn94a+Asp225Gly), eliminating glycosylation sites. CVN did not bind well to the CVN-R and CVN-MR viruses. Propagating these viruses in cells treated with 1 mM deoxymannojirimycin (dMJ, mannosidase inhibitor) increased sensitivity to CVN, suggesting that glycans attached at other sites on HA1 that typically a...
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development of a fluorescent microplate assay for determining cyanovirin n levels in plasma
Analytical and Bioanalytical Chemistry, 2004Co-Authors: Scott D Bringans, Barry R Okeefe, Michael Bray, Chris A Whitehouse, Michael R BoydAbstract:A sensitive immunosorbent competition assay was developed for quantitation of the anti-HIV protein Cyanovirin-N (CV-N) in plasma using a 96-well plate format and a fluorescent endpoint. The assay is based on the binding of CV-N in plasma to plate-bound anti-CV-N antibodies, followed by removal of the plasma and addition of europium-labeled CV-N (Eu3+-CV-N) to compete for the remaining antibody sites. Detection by addition of a dissociative fluorescence enhancement solution and time-resolved fluorescence measurements allowed correlation to the concentration of the native CV-N in plasma. A linear detection range of 1–100 nM (r2>0.99) was obtained for CV-N in mouse plasma. This assay was then utilized for analysis of plasma levels of CV-N samples following subcutaneous injection of CV-N into mice. The results of these studies confirmed the reliability and sensitivity of this assay and the feasibility of its use for pharmacokinetic studies in a variety of species.
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cyanovirin n inhibits aids virus infections in vaginal transmission models
AIDS Research and Human Retroviruses, 2004Co-Authors: Che Chung Tsai, Kirk R Gustafson, Peter Emau, Yonghou Jiang, William R Morton, Robin J Shattock, Ann Schmidt, Michael R BoydAbstract:The cyanobacterial protein Cyanovirin-N (CV-N) potently inactivates diverse strains of HIV-1 and other lentiviruses due to irreversible binding of CV-N to the viral envelope glycoprotein gp120. In this study, we show that recombinant CV-N effectively blocks HIV-1Ba-L infection of human ectocervical explants. Furthermore, we demonstrate the in vivo efficacy of CV-N gel in a vaginal challenge model by exposing CV-N-treated female macaques (Macaca fascicularis) to a pathogenic chimeric SIV/HIV-1 virus, SHIV89.6P. All of the placebo-treated and untreated control macaques (8 of 8) became infected. In contrast, 15 of 18 CV-N-treated macaques showed no evidence of SHIV infection. Further, CV-N produced no cytotoxic or clinical adverse effects in either the in vitro or in vivo model systems. Together these studies suggest that CV-N is a good candidate for testing in humans as an anti-HIV topical microbicide.
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potent anti influenza activity of cyanovirin n and interactions with viral hemagglutinin
Antimicrobial Agents and Chemotherapy, 2003Co-Authors: Barry R Okeefe, Carrie J. Saucedo, Kirk R Gustafson, Toshiyuki Mori, Jim A Turpin, Robert W Buckheit, Donald F Smee, Dennis Blakeslee, Michael R BoydAbstract:The novel antiviral protein Cyanovirin-N (CV-N) was initially discovered based on its potent activity against the human immunodeficiency virus (HIV). Subsequent studies identified the HIV envelope glycoproteins gp120 and gp41 as molecular targets of CV-N. More recently, mechanistic studies have shown that certain high-mannose oligosaccharides (oligomannose-8 and oligomannose-9) found on the HIV envelope glycoproteins comprise the specific sites to which CV-N binds. Such selective, carbohydrate-dependent interactions may account, at least in part, for the unusual and unexpected spectrum of antiviral activity of CV-N described herein. We screened CV-N against a broad range of respiratory and enteric viruses, as well as flaviviruses and herpesviruses. CV-N was inactive against rhinoviruses, human parainfluenza virus, respiratory syncytial virus, and enteric viruses but was moderately active against some herpesvirus and hepatitis virus (bovine viral diarrhea virus) strains (50% effective concentration [EC 50 ] = ∼1 μg/ml) while inactive against others. Remarkably, however, CV-N and related homologs showed highly potent antiviral activity against almost all strains of influenza A and B virus, including clinical isolates and a neuraminidase inhibitor-resistant strain (EC 50 = 0.004 to 0.04 μg/ml). When influenza virus particles were pretreated with CV-N, viral titers were lowered significantly (>1,000-fold). Further studies identified influenza virus hemagglutinin as a target for CV-N, showed that antiviral activity and hemagglutinin binding were correlated, and indicated that CV-N′s interactions with hemagglutinin involved oligosaccharides. These results further reveal new potential avenues for antiviral therapeutics and prophylaxis targeting specific oligosaccharide-comprised sites on certain enveloped viruses, including HIV, influenza virus, and possibly others.
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cyanovirin n gel as a topical microbicide prevents rectal transmission of shiv89 6p in macaques
AIDS Research and Human Retroviruses, 2003Co-Authors: Che Chung Tsai, Kirk R Gustafson, Peter Emau, Yonghou Jiang, Baoping Tian, William R Morton, Michael R BoydAbstract:Cyanovirin-N (CV-N), an 11-kDa cyanobacterial protein, potently inactivates diverse strains of HIV-1, HIV-2, and simian immunodeficiency virus (SIV) and also prevents virus-to-cell fusion, virus en...
Barry R Okeefe - One of the best experts on this subject based on the ideXlab platform.
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a designed nested dimer of cyanovirin n increases antiviral activity
Viruses, 2016Co-Authors: Brian Woodrum, Barry R Okeefe, Jason D Maxwell, L. R. H. Krumpe, Jennifer A Wilson, Denysia Allen, Andrey A Bobkov, R Hill, Karen V Kibler, Giovanna GhirlandaAbstract:Cyanovirin-N (CV-N) is an antiviral lectin with potent activity against enveloped viruses, including HIV. The mechanism of action involves high affinity binding to mannose-rich glycans that decorate the surface of enveloped viruses. In the case of HIV, antiviral activity of CV-N is postulated to require multivalent interactions with envelope protein gp120, achieved through a pseudo-repeat of sequence that adopts two near-identical glycan-binding sites, and possibly involves a 3D-domain-swapped dimeric form of CV-N. Here, we present a covalent dimer of CV-N that increases the number of active glycan-binding sites, and we characterize its ability to recognize four glycans in solution. A CV-N variant was designed in which two native repeats were separated by the “nested” covalent insertion of two additional repeats of CV-N, resulting in four possible glycan-binding sites. The resulting Nested CV-N folds into a wild-type-like structure as assessed by circular dichroism and NMR spectroscopy, and displays high thermal stability with a Tm of 59 °C, identical to WT. All four glycan-binding domains encompassed by the sequence are functional as demonstrated by isothermal titration calorimetry, which revealed two sets of binding events to dimannose with dissociation constants Kd of 25 μM and 900 μM, assigned to domains B and B’ and domains A and A’ respectively. Nested CV-N displays a slight increase in activity when compared to WT CV-N in both an anti-HIV cellular assay and a fusion assay. This construct conserves the original binding specifityies of domain A and B, thus indicating correct fold of the two CV-N repeats. Thus, rational design can be used to increase multivalency in antiviral lectins in a controlled manner.
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cyanovirin n produced in rice endosperm offers effective pre exposure prophylaxis against hiv 1bal infection in vitro
Plant Cell Reports, 2016Co-Authors: E. Vamvaka, Barry R Okeefe, Robin J Shattock, A. Evans, K. Ramessar, L. R. H. Krumpe, P. Christou, T. CapellAbstract:Cyanovirin-N produced in rice endosperm provides efficient pre-exposure prophylaxis against HIV-1 BaL infection in vitro. Cyanovirin-N (CV-N) is a lectin with potent antiviral activity that has been proposed as a component of microbicides for the prevention of infection with Human immunodeficiency virus (HIV). The production of protein-based microbicide components requires a platform that is sufficiently economical and scalable to meet the demands of the large at-risk population, particularly in resource poor developing countries. We, therefore, expressed CV-N in rice endosperm, because the dried seed is ideal for storage and transport and crude extracts could be prepared locally and used as a microbicide component without further purification. We found that crude extracts from rice seeds expressing up to 10 µg CV-N per gram dry seed weight showed dose-dependent gp120 binding activity, confirming that the protein was soluble, correctly folded and active. The recombinant lectin (OSCV-N) reduced the infectivity of HIV-1BaL (an R5 virus strain representing the majority of transmitted infections) by ~90 % but showed only weak neutralization activity against HIV-1RF (representative of X4 virus, rarely associated with transmission), suggesting it would be highly effective for pre-exposure prophylaxis against the vast majority of transmitted strains. Crude extracts expressing OSCV-N showed no toxicity towards human cells at working dilutions indicating that microbicide components produced in rice endosperm are safe for direct application as topical microbicides in humans.
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engineering soya bean seeds as a scalable platform to produce cyanovirin n a non arv microbicide against hiv
Plant Biotechnology Journal, 2015Co-Authors: Barry R Okeefe, Carrie J. Saucedo, K. Ramessar, Jennifer A Wilson, Andre M Murad, G R Vianna, Karen W Buckheit, Nicolau B Da Cunha, Ana Claudia Guerra Araujo, Cristiano LacorteAbstract:Summary There is an urgent need to provide effective anti-HIV microbicides to resource-poor areas worldwide. Some of the most promising microbicide candidates are biotherapeutics targeting viral entry. To provide biotherapeutics to poorer areas, it is vital to reduce the cost. Here, we report the production of biologically active recombinant Cyanovirin-N (rCV-N), an antiviral protein, in genetically engineered soya bean seeds. Pure, biologically active rCV-N was isolated with a yield of 350 μg/g of dry seed weight. The observed amino acid sequence of rCV-N matched the expected sequence of native CV-N, as did the mass of rCV-N (11 009 Da). Purified rCV-N from soya is active in anti-HIV assays with an EC50 of 0.82–2.7 nM (compared to 0.45–1.8 nM for E. coli-produced CV-N). Standard industrial processing of soya bean seeds to harvest soya bean oil does not diminish the antiviral activity of recovered rCV-N, allowing the use of industrial soya bean processing to generate both soya bean oil and a recombinant protein for anti-HIV microbicide development.
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anti retroviral lectins have modest effects on adherence of trichomonas vaginalis to epithelial cells in vitro and on recovery of tritrichomonas foetus in a mouse vaginal model
PLOS ONE, 2015Co-Authors: A. Chatterjee, Barry R Okeefe, Daniel M Ratner, Christopher M. Ryan, Evan W Secor, Phillips W. Robbins, Patricia J Johnson, Deborah J. Anderson, John SamuelsonAbstract:Trichomonas vaginalis causes vaginitis and increases the risk of HIV transmission by heterosexual sex, while Tritrichomonas foetus causes premature abortion in cattle. Our goals were to determine the effects, if any, of anti-retroviral lectins, which are designed to prevent heterosexual transmission of HIV, on adherence of Trichomonas to ectocervical cells and on Tritrichomonas infections in a mouse model. We show that Trichomonas Asn-linked glycans (N-glycans), like those of HIV, bind the mannose-binding lectin (MBL) that is part of the innate immune system. N-glycans of Trichomonas and Tritrichomonas bind anti-retroviral lectins (Cyanovirin-N and griffithsin) and the 2G12 monoclonal antibody, each of which binds HIV N-glycans. Binding of Cyanovirin-N appears to be independent of susceptibility to metronidazole, the major drug used to treat Trichomonas. Anti-retroviral lectins, MBL, and galectin-1 cause Trichomonas to self-aggregate and precipitate. The anti-retroviral lectins also increase adherence of ricin-resistant mutants, which are less adherent than parent cells, to ectocervical cell monolayers and to organotypic EpiVaginal tissue cells. Topical application of either anti-retroviral lectins or yeast N-glycans decreases by 40 to 70% the recovery of Tritrichomonas from the mouse vagina. These results, which are explained by a few simple models, suggest that the anti-retroviral lectins have a modest potential for preventing or treating human infections with Trichomonas.
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the lectins griffithsin cyanovirin n and scytovirin inhibit hiv 1 binding to the dc sign receptor and transfer to cd4 cells
Virology, 2012Co-Authors: Kabamba B. Alexandre, Ereck Chakauya, Hazel Mufhandu, Elin S. Gray, Barry R Okeefe, James B. Mcmahon, Rachel Chikwamba, Lynn MorrisAbstract:It is generally believed that during the sexual transmission of HIV-1, the glycan-specific DC-SIGN receptor binds the virus and mediates its transfer to CD4+ cells. The lectins griffithsin (GRFT), Cyanovirin-N (CV-N) and scytovirin (SVN) inhibit HIV-1 infection by binding to mannose-rich glycans on gp120. We measured the ability of these lectins to inhibit both the HIV-1 binding to DC-SIGN and the DC-SIGN-mediated HIV-1 infection of CD4+ cells. While GRFT, CV-N and SVN were moderately inhibitory to DC-SIGN binding, they potently inhibited DC-SIGN-transfer of HIV-1. The introduction of the 234 glycosylation site abolished HIV-1 sensitivity to lectin inhibition of binding to DC-SIGN and virus transfer to susceptible cells. However, the addition of the 295 glycosylation site increased the inhibition of transfer. Our data suggest that GRFT, CV-N and SVN can block two important stages of the sexual transmission of HIV-1, DC-SIGN binding and transfer, supporting their further development as microbicides.
James B. Mcmahon - One of the best experts on this subject based on the ideXlab platform.
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Mechanisms of HIV-1 subtype C resistance to GRFT, CV-N and SVN
Virology, 2013Co-Authors: Kabamba B. Alexandre, Penny L. Moore, Molati Nonyane, Nthabeleng Ranchobe, Ereck Chakauya, Barry R. O'keefe, Elin S. Gray, James B. Mcmahon, Rachel Chikwamba, Lynn MorrisAbstract:We examined the ability of HIV-1 subtype C to develop resistance to the inhibitory lectins, griffithsin (GRFT), Cyanovirin-N (CV-N) and scytovirin (SVN), which bind multiple mannose-rich glycans on gp120. Four primary HIV-1 strains cultured under escalating concentrations of these lectins became increasingly resistant tolerating 2 to 12 times their 50% inhibitory concentrations. Sequence analysis of gp120 showed that most had deletions of 1 to 5 mannose-rich glycans. Glycosylation sites at positions 230, 234, 241, 289 located in the C2 region and 339, 392 and 448 in the C3-C4 region were affected. Furthermore, deletions and insertions of up to 5 amino acids in the V4 region were observed in 3 of the 4 isolates. These data suggest that loss of glycosylation sites on gp120 as well as rearrangement of glycans in V4 are mechanisms involved in HIV-1 subtype C escape from GRFT, CV-N and SVN. © 2013 Elsevier Inc.
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the lectins griffithsin cyanovirin n and scytovirin inhibit hiv 1 binding to the dc sign receptor and transfer to cd4 cells
Virology, 2012Co-Authors: Kabamba B. Alexandre, Ereck Chakauya, Hazel Mufhandu, Elin S. Gray, Barry R Okeefe, James B. Mcmahon, Rachel Chikwamba, Lynn MorrisAbstract:It is generally believed that during the sexual transmission of HIV-1, the glycan-specific DC-SIGN receptor binds the virus and mediates its transfer to CD4+ cells. The lectins griffithsin (GRFT), Cyanovirin-N (CV-N) and scytovirin (SVN) inhibit HIV-1 infection by binding to mannose-rich glycans on gp120. We measured the ability of these lectins to inhibit both the HIV-1 binding to DC-SIGN and the DC-SIGN-mediated HIV-1 infection of CD4+ cells. While GRFT, CV-N and SVN were moderately inhibitory to DC-SIGN binding, they potently inhibited DC-SIGN-transfer of HIV-1. The introduction of the 234 glycosylation site abolished HIV-1 sensitivity to lectin inhibition of binding to DC-SIGN and virus transfer to susceptible cells. However, the addition of the 295 glycosylation site increased the inhibition of transfer. Our data suggest that GRFT, CV-N and SVN can block two important stages of the sexual transmission of HIV-1, DC-SIGN binding and transfer, supporting their further development as microbicides.
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The lectins griffithsin, Cyanovirin-N and scytovirin inhibit HIV-1 binding to the DC-SIGN receptor and transfer to CD4+cells
Virology, 2012Co-Authors: Kabamba B. Alexandre, Ereck Chakauya, Barry R. O'keefe, Hazel Mufhandu, Elin S. Gray, James B. Mcmahon, Rachel Chikwamba, Lynn MorrisAbstract:It is generally believed that during the sexual transmission of HIV-1, the glycan-specific DC-SIGN receptor binds the virus and mediates its transfer to CD4+cells. The lectins griffithsin (GRFT), Cyanovirin-N (CV-N) and scytovirin (SVN) inhibit HIV-1 infection by binding to mannose-rich glycans on gp120. We measured the ability of these lectins to inhibit both the HIV-1 binding to DC-SIGN and the DC-SIGN-mediated HIV-1 infection of CD4+cells. While GRFT, CV-N and SVN were moderately inhibitory to DC-SIGN binding, they potently inhibited DC-SIGN-transfer of HIV-1. The introduction of the 234 glycosylation site abolished HIV-1 sensitivity to lectin inhibition of binding to DC-SIGN and virus transfer to susceptible cells. However, the addition of the 295 glycosylation site increased the inhibition of transfer. Our data suggest that GRFT, CV-N and SVN can block two important stages of the sexual transmission of HIV-1, DC-SIGN binding and transfer, supporting their further development as microbicides. © 2011 Elsevier Inc.
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the microbicide cyanovirin n expressed on the surface of commensal bacterium streptococcus gordonii captures hiv 1
AIDS, 2002Co-Authors: Barbara Giomarelli, James B. Mcmahon, Toshiyuki Mori, Roberta S Gardella, Roberta Provvedi, Francesca Meacci, Tiziana Maggi, Donata Medaglini, Gianni Pozzi, Michael R BoydAbstract:Objective: To explore the feasibility of expressing the potent HIV-inactivating protein, Cyanovirin-N (CV-N), in the human commensal bacterium Streptococcus gordonii, as a possible approach for local delivery of CV-N to prevent sexual transmission of HIV-1. Design and methods: To express CV-N in S. gordonii, we used the host-vector system we had previously developed. CV-N was expressed as a fusion protein both attached to the bacterial surface and secreted in soluble form in the supernatant of liquid cultures. The soluble form of recombinant CV-N was tested for gp120-binding activity in an enzyme-linked immunosorbent assay, whereas S. gordonii strain expressing CV-N on the surface was analyzed in an in vitro HIV capturing assay. Results: Two recombinant S. gordonii strains secreting or displaying CV-N on the bacterial surface were constructed and the expression of CV-N was confirmed by immunoblot and flow-cytometric analysis. The secreted form of recombinant CV-N exhibited a concentration-dependent binding to the envelope glycoprotein gp 120 of HIV-1, whereas CV-N displayed on the bacterial surface was able to capture HIV virions efficiently. Conclusion: The anti-HIV protein CV-N in S. gordonii was expressed in a biologically active form. This represents a first step in the development of a system to deliver and maintain an effective concentration of a microbicide in the vaginal mucosa.
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high throughput screening for cyanovirin n mimetics binding to hiv 1 gp41
Journal of Biomolecular Screening, 2002Co-Authors: John A Beutler, Barry R Okeefe, James B. Mcmahon, Roberta S Gardella, Tanya R Johnson, Randy A Buzzell, Danielle Robbins, Jennifer A Wilson, Michael R BoydAbstract:The human immunodeficiency virus type-1 (HIV-1) envelope glycoprotein gp4l is an important mediator of viral entry into host cells. Previous studies showed that the virucidal protein Cyanovirin-N (CV-N) bound to both gpl20 and gp4l, and that this binding was associated with its antiviral activity. We constructed an HTS assay based on the interaction of europium-labeled CV-N with recombinant glycosylated gp4l ectodomain to support identification of small-molecule mimetics of CV-N that might be developed as antiviral drug leads. Primary screening of over 107,000 natural product extracts in the assay yielded 347 confirmed hits. Secondary assays eliminated extracts that bound directly to labeled CV-N or for which the simple sugars mannose and N-acetylglucosamine blocked the interaction with gp4l (lectin activity). Extracts were further prioritized based on anti-HIV activity and other biological, biochemical, and chemical criteria. The distribution of source organism taxonomy of active extracts was analyzed, a...
Angela M Gronenborn - One of the best experts on this subject based on the ideXlab platform.
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structure and glycan binding of a new cyanovirin n homolog
Journal of Biological Chemistry, 2016Co-Authors: Elena Matei, William Furey, Christopher Aiken, Rohan Basu, Conor Calnan, Angela M GronenbornAbstract:Abstract The HIV-1 envelope glycoprotein gp120 is heavily glycosylated and bears numerous high mannose sugars. These sugars can serve as targets for HIV-inactivating compounds, such as antibodies and lectins, which bind to the glycans and interfere with viral entry into the target cell. We determined the 1.6 A x-ray structure of Cyt-CVNH, a recently identified lectin from the cyanobacterium Cyanothece7424, and elucidated its glycan specificity by NMR. The Cyt-CVNH structure and glycan recognition profile are similar to those of other CVNH proteins, with each domain specifically binding to Manα(1–2)Manα units on the D1 and D3 arms of high mannose glycans. However, in contrast to CV-N, no cross-linking and precipitation of the cross-linked species in solution was observed upon Man-9 binding, allowing, for the first time, investigation of the interaction of Man-9 with a member of the CVNH family by NMR. HIV assays showed that Cyt-CVNH is able to inhibit HIV-1 with ∼4-fold higher potency than CV-NP51G, a stabilized version of wild type CV-N. Therefore, Cyt-CVNH may qualify as a valuable lectin for potential microbicidal use.
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different 3d domain swapped oligomeric cyanovirin n structures suggest trapped folding intermediates
Proceedings of the National Academy of Sciences of the United States of America, 2013Co-Authors: Leonardus M I Koharudin, Angela M GronenbornAbstract:Although it has long been established that the amino acid sequence encodes the fold of a protein, how individual proteins arrive at their final conformation is still difficult to predict, especially for oligomeric structures. Here, we present a comprehensive characterization of oligomeric species of Cyanovirin-N that all are formed by a polypeptide chain with the identical amino acid sequence. Structures of the oligomers were determined by X-ray crystallography, and each one exhibits 3D domain swapping. One unique 3D domain-swapped structure is observed for the trimer, while for both dimer and tetramer, two different 3D domain-swapped structures were obtained. In addition to the previously identified hinge-loop region of the 3D domain-swapped dimer, which resides between strands β5 and β6 in the middle of the polypeptide sequence, another hinge-loop region is observed between strands β7 and β8 in the structures. Plasticity in these two regions allows for variability in dihedral angles and concomitant differences in chain conformation that results in the differently 3D domain-swapped multimers. Based on all of the different structures, we propose possible folding pathways for this protein. Altogether, our results illuminate the amazing ability of Cyanovirin-N to proceed down different folding paths and provide general insights into oligomer formation via 3D domain swapping.
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domain swapping proceeds via complete unfolding a 19f and 1h nmr study of the cyanovirin n protein
Journal of the American Chemical Society, 2012Co-Authors: Inja L Byeon, Ivet Bahar, Angela M GronenbornAbstract:Domain swapping creates protein oligomers by exchange of structural units between identical monomers. At present, no unifying molecular mechanism of domain swapping has emerged. Here we used the protein Cyanovirin-N (CV–N) and 19F-NMR to investigate the process of domain swapping. CV–N is an HIV inactivating protein that can exist as a monomer or a domain-swapped dimer. We measured thermodynamic and kinetic parameters of the conversion process and determined the size of the energy barrier between the two species. The barrier is very large and of similar magnitude to that for equilibrium unfolding of the protein. Therefore, for CV–N, overall unfolding of the polypeptide is required for domain swapping.
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multivalent glyconanoparticles with enhanced affinity to the anti viral lectin cyanovirin n
Chemical Communications, 2011Co-Authors: Xin Wang, Elena Matei, Lingquan Deng, Olof Ramstrom, Angela M GronenbornAbstract:Low-mannose (LM) structures were coupled to gold nanoparticles (Au NPs) to amplify the affinity of LMs with Cyanovirin-N (CV-N) lectins and to study the structures of CV-N variants CVNQ50C and CVNMutDB.
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anti hiv activity of defective cyanovirin n mutants is restored by dimerization
Journal of Biological Chemistry, 2010Co-Authors: Elena Matei, Andrew Zheng, William Furey, Jeremy J Rose, Christopher Aiken, Angela M GronenbornAbstract:Cyanovirin-N (CV-N) is a two-domain, cyanobacterial protein that inhibits human immunodeficiency virus (HIV) at nanomolar concentrations by binding to high mannose sugars on the HIV envelope glycoprotein gp120. The wild type protein can exist as a monomer or a domain-swapped dimer with the monomer and dimer containing two or four sugar binding sites, respectively, one on each domain. Here we demonstrate that monomeric, single binding site mutants are completely inactive and that a single site, whether located on domain A or B, is insufficient to impart the antiviral activity. Linking inactive, monomeric proteins in a head-to-head fashion by an intermolecular disulfide bond or by creating an exclusively domain-swapped dimer via a hinge residue deletion restored antiviral activity to levels similar to that of wild type CV-N. These findings demonstrate unequivocally that multisite binding by CV-N type lectins is necessary for viral inhibition.
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, Barry R Okeefe, Kirk R Gustafson, Kevin W Bailey, Minhui Wong, Vasiliy P Mishin, 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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cyanovirin n inhibits hepatitis c virus entry by binding to envelope protein glycans
Journal of Biological Chemistry, 2006Co-Authors: Francois Helle, Kirk R Gustafson, Czeslaw Wychowski, Ngoc Vudac, Cecile Voisset, Jean DubuissonAbstract:Inhibition of viruses at the stage of viral entry provides a route for therapeutic intervention. Because of difficulties in propagating hepatitis C virus (HCV) in cell culture, entry inhibitors have not yet been reported for this virus. However, with the development of retroviral particles pseudotyped with HCV envelope glycoproteins (HCVpp) and the recent progress in amplification of HCV in cell culture (HCVcc), studying HCV entry is now possible. In addition, these systems are essential for the identification and the characterization of molecules that block HCV entry. The lectin Cyanovirin-N (CV-N) has initially been discovered based on its potent activity against human immunodeficiency virus. Because HCV envelope glycoproteins are highly glycosylated, we sought to determine whether CV-N has an antiviral activity against this virus. CV-N inhibited the infectivity of HCVcc and HCVpp at low nanomolar concentrations. This inhibition is attributed to the interaction of CV-N with HCV envelope glycoproteins. In addition, we showed that the carbohydrate binding property of CV-N is involved in the anti-HCV activity. Finally, CV-N bound to HCV envelope glycoproteins and blocked the interaction between the envelope protein E2 and CD81, a cell surface molecule involved in HCV entry. These data demonstrate that targeting the glycans of HCV envelope proteins is a promising approach in the development of antiviral therapies to combat a virus that is a major cause of chronic liver diseases. Furthermore, CV-N is a new invaluable tool to further dissect the early steps of HCV entry into host cells.
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cyanovirin n inhibits aids virus infections in vaginal transmission models
AIDS Research and Human Retroviruses, 2004Co-Authors: Che Chung Tsai, Kirk R Gustafson, Peter Emau, Yonghou Jiang, William R Morton, Robin J Shattock, Ann Schmidt, Michael R BoydAbstract:The cyanobacterial protein Cyanovirin-N (CV-N) potently inactivates diverse strains of HIV-1 and other lentiviruses due to irreversible binding of CV-N to the viral envelope glycoprotein gp120. In this study, we show that recombinant CV-N effectively blocks HIV-1Ba-L infection of human ectocervical explants. Furthermore, we demonstrate the in vivo efficacy of CV-N gel in a vaginal challenge model by exposing CV-N-treated female macaques (Macaca fascicularis) to a pathogenic chimeric SIV/HIV-1 virus, SHIV89.6P. All of the placebo-treated and untreated control macaques (8 of 8) became infected. In contrast, 15 of 18 CV-N-treated macaques showed no evidence of SHIV infection. Further, CV-N produced no cytotoxic or clinical adverse effects in either the in vitro or in vivo model systems. Together these studies suggest that CV-N is a good candidate for testing in humans as an anti-HIV topical microbicide.
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potent anti influenza activity of cyanovirin n and interactions with viral hemagglutinin
Antimicrobial Agents and Chemotherapy, 2003Co-Authors: Barry R Okeefe, Carrie J. Saucedo, Kirk R Gustafson, Toshiyuki Mori, Jim A Turpin, Robert W Buckheit, Donald F Smee, Dennis Blakeslee, Michael R BoydAbstract:The novel antiviral protein Cyanovirin-N (CV-N) was initially discovered based on its potent activity against the human immunodeficiency virus (HIV). Subsequent studies identified the HIV envelope glycoproteins gp120 and gp41 as molecular targets of CV-N. More recently, mechanistic studies have shown that certain high-mannose oligosaccharides (oligomannose-8 and oligomannose-9) found on the HIV envelope glycoproteins comprise the specific sites to which CV-N binds. Such selective, carbohydrate-dependent interactions may account, at least in part, for the unusual and unexpected spectrum of antiviral activity of CV-N described herein. We screened CV-N against a broad range of respiratory and enteric viruses, as well as flaviviruses and herpesviruses. CV-N was inactive against rhinoviruses, human parainfluenza virus, respiratory syncytial virus, and enteric viruses but was moderately active against some herpesvirus and hepatitis virus (bovine viral diarrhea virus) strains (50% effective concentration [EC 50 ] = ∼1 μg/ml) while inactive against others. Remarkably, however, CV-N and related homologs showed highly potent antiviral activity against almost all strains of influenza A and B virus, including clinical isolates and a neuraminidase inhibitor-resistant strain (EC 50 = 0.004 to 0.04 μg/ml). When influenza virus particles were pretreated with CV-N, viral titers were lowered significantly (>1,000-fold). Further studies identified influenza virus hemagglutinin as a target for CV-N, showed that antiviral activity and hemagglutinin binding were correlated, and indicated that CV-N′s interactions with hemagglutinin involved oligosaccharides. These results further reveal new potential avenues for antiviral therapeutics and prophylaxis targeting specific oligosaccharide-comprised sites on certain enveloped viruses, including HIV, influenza virus, and possibly others.
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cyanovirin n gel as a topical microbicide prevents rectal transmission of shiv89 6p in macaques
AIDS Research and Human Retroviruses, 2003Co-Authors: Che Chung Tsai, Kirk R Gustafson, Peter Emau, Yonghou Jiang, Baoping Tian, William R Morton, Michael R BoydAbstract:Cyanovirin-N (CV-N), an 11-kDa cyanobacterial protein, potently inactivates diverse strains of HIV-1, HIV-2, and simian immunodeficiency virus (SIV) and also prevents virus-to-cell fusion, virus en...
