Anti-HIV Activity of Defective Cyanovirin-N Mutants Is Restored by Dimerization

Matei, Elena; Zheng, Andrew; Furey, W.; Rose, Jeremy J.; Aiken, Christopher; Gronenborn, Angela M.

doi:10.1074/jbc.m109.094938

Cited by 40 publications

(61 citation statements)

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“…Variants with three binding sites knocked out were unable to neutralize HIV at the concentrations tested (data not shown). These data are consistent with previously published accounts, which showed that at least two functional binding sites are required for activity (16,19), and are also consistent with the hypothesis that avidity is an important factor for viral neutralization by CV-N. Interestingly, CVN 2 L0 variants that contain one functional A and one functional B binding site did not neutralize with the same potency as WT CV-N. CVN 2 L0 ΔA∥B and CVN 2 L0 ΔA×B in which the active A and B binding sites are on the same pseudomonomer or opposite pseudomonomer (Fig. 4C), respectively, were 15-to 75-fold less potent than monomeric WT CV-N, which also contains only a single A and single B binding site.…”

Section: Cv-n Dimers Exhibit Broad Cross-clade Reactivity and Are Comsupporting

confidence: 83%

“…These linked dimers show enhanced HIV neutralization compared to WT CV-N against 33 strains from 3 clades. In addition, we show that although two carbohydrate binding sites are sufficient for activity as previously reported (18,19), variants with more binding sites (three or four) have increased neutralization activity.…”

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confidence: 47%

“…Numerous studies have shown, however, that both sites are necessary for viral neutralization and that destruction of either site renders the CV-N variant inactive (16,17). However, a recent study showed that in the context of a CV-N dimer that was covalently crosslinked using disulfide bonds, two out of the four possible binding sites are sufficient to maintain neutralization activity, indicating that it is the number and not the identity of sites that is important for neutralization (18,19). These results point toward a key role for avidity in the viral neutralization activity of CV-N.…”

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confidence: 99%

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Designed oligomers of cyanovirin-N show enhanced HIV neutralization

Keeffe¹,

Gnanapragasam²,

Gillespie³

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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Cyanovirin-N (CV-N) is a small, cyanobacterial lectin that neutralizes many enveloped viruses, including human immunodeficiency virus type I (HIV-1). This antiviral activity is attributed to two homologous carbohydrate binding sites that specifically bind high mannose glycosylation present on envelope glycoproteins such as HIV-1 gp120. We created obligate CV-N oligomers to determine whether increasing the number of binding sites has an effect on viral neutralization. A tandem repeat of two CV-N molecules (CVN 2 ) increased HIV-1 neutralization activity by up to 18-fold compared to wild-type CV-N. In addition, the CVN 2 variants showed extensive cross-clade reactivity and were often more potent than broadly neutralizing anti-HIV antibodies. The improvement in activity and broad cross-strain HIV neutralization exhibited by these molecules holds promise for the future therapeutic utility of these and other engineered CV-N variants.

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Section: Cv-n Dimers Exhibit Broad Cross-clade Reactivity and Are Comsupporting

confidence: 83%

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confidence: 47%

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confidence: 99%

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Designed oligomers of cyanovirin-N show enhanced HIV neutralization

Keeffe¹,

Gnanapragasam²,

Gillespie³

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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“…Likewise, superposition of free Cyt-CVNH (black contours) and protein in the presence of 6 molar equivalents of Man-3 or 2 molar equivalents of Man-9 (magenta) are provided in Fig. 2 In the past, attempts to structurally monitor Man-9 binding to CV-N by NMR were hampered by extreme line broadening and ultimately disappearance of resonances in the 1 H-15 N HSQC spectra, accompanied by precipitation of the sugar⅐ protein caused by multisite/multivalent cross-linking (7,19,20). In contrast to the findings with CV-N, no aggregation or precipitation was observed for Man-9 binding to Cyt-CVNH.…”

Section: Resultsmentioning

confidence: 99%

“…For CV-N, the presence of two binding sites and the multivalency of the carbohydrate have been implicated as important factors for its anti-HIV activity (6). Unfortunately, cross-linking-mediated aggregation and precipitation have hampered studies of CV-N/Man-9 interactions at the atomic level in vitro (7,19,20) and have precluded unambiguous determination of whether multivalent and multisite sugar/protein interactions are a necessary prerequisite for the antiviral activity of cyanovirin-N homolog (CVNH) proteins. However, in contrast, considerable atomic level information is available on CV-N binding to substructures of Man-8 and Man-9.…”

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confidence: 99%

Structure and Glycan Binding of a New Cyanovirin-N Homolog

Matei

Basu

Furey

et al. 2016

Journal of Biological Chemistry

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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 Å x-ray structure of Cyt-CVNH, a recently identified lectin from the cyanobacterium Cyanothece 7424 , 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 Man␣(1-2)Man␣ units on the D1 and D3 arms of high mannose glycans. However, in contrast to CV-N, no cross-linking and precipitation of the crosslinked 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 ϳ4-fold higher potency than CV-N P51G , a stabilized version of wild type CV-N. Therefore, Cyt-CVNH may qualify as a valuable lectin for potential microbicidal use.Despite the effective use of anti-retroviral therapies as a means to treat HIV infection and prolong the lifespan of those affected by AIDS, the number of HIV infections worldwide continues to grow. Unfortunately, at present, no vaccine is available to protect against HIV, creating the need to develop safe, effective, and acceptable prevention strategies that will help halt the spread of HIV infection globally. Promising candidates for inclusion into microbicides are lectins, which are carbohydrate-binding proteins that are present in a variety of plant, fungal, and cyanobacterial species. Over the last two decades, several lectins have been identified that potently block viral infection, being active against HIV and influenza virus (1-4). One such example is the extensively explored cyanobacterial lectin cyanovirin-N (CV-N), 2 which possesses virucidal properties against HIV types I and II, simian immunodeficiency virus, and other enveloped viruses like Ebola and influenza at nanomolar concentrations (5-7). Its anti-HIV activity is mediated by recognizing and interacting with high mannose glycans (Man-8, Man-9) that are present on the envelope glycoprotein gp120. Indeed, HIV-1 gp120 is highly glycosylated, and N-linked glycans account for approximately half of its molecular mass (8, 9). CV-N specifically binds the terminal Man␣(1-2)Man␣ epitopes on the D1 and D3 arms of Man-8 and Man-9 glycans (10 -14). The mechanism of action for mannose-binding lectins is assumed to involve the inability of the lectinbound gp120 to productively engage the host cell CD4 and CCR5 receptors, effectively preventing the necessary conformational changes required for membrane fusion and viral entry into the host cell. At present, efforts to develop lectins such as CV-N for therapeutic use are focused on topical applications in microbicides. All known anti-HIV lectins vary in their degrees of potency and some were found to ...

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¹⁹F Paramagnetic Relaxation Enhancement: A Valuable Tool for Distance Measurements in Proteins

Matei

Gronenborn

2015

Angewandte Chemie

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Fluorine NMR paramagnetic relaxation enhancement was evaluated as a versatile approach for extracting distance information in selectively F-labeled proteins. Proof of concept and initial applications are presented for the HIV-inactivating lectin Cyanovirin-N. Single F atoms were introduced at the 4-, 5-, 6-or 7 positions of Trp49 and the 4 position of Phe4, Phe54 and Phe80. The paramagnetic MTSL label was attached to Cys residues that were placed into the protein at positions 50 or 52. 19 F-T 2 NMR spectra with different relaxation delays were recorded and the transverse 19 F-PRE rate, 19 F-Γ 2 , was used to determine the average distance between the F nucleus and the paramagnetic center. Our data show that experimental 19 F PRE based distances correspond to ~0.93 of the 1 H N -PRE distances, in perfect agreement with the gyromagnetic γ 19 F/γ 1 H ratio, thereby demonstrating that 19 F PREs are excellent alternative parameters for quantitative distance measurements in selectively F-labeled proteins. Keywords fluorine; paramagnetic; NMR; distance; protein In Paramagnetic Relaxation Enhancement (PRE) NMR experiments the increase in longitudinal or transverse relaxation rates, induced by the presence of a paramagnetic center, is measured. The effect is distance dependent, and, as a result, information on the distance between the paramagnetic moiety to the measured nuclei is obtained.In biomolecular NMR, PREs are increasingly used for providing long-range distance information that complements nuclear Overhauser effect (NOE)-derived, short (≤ 5 Å) interproton distance restraints. In cases where NOE data were found limited, distances derived from PREs provided valuable supplemental restraints and permitted either characterization of the global folds for some proteins, [1,2] or delineate structural properties of disorder proteins. [3,4] Correspondence to: Angela M. Gronenborn, amg100@pitt.edu. Supporting information for this article is given via a link at the end of the document. HHS Public AccessAuthor manuscript Angew Chem Int Ed Engl. Author manuscript; available in PMC 2017 January 04. Author Manuscript Author ManuscriptAuthor Manuscript Author ManuscriptThe most widely used spin label, originally developed for EPR studies, [5,6] is the nitroxide radical MTSL (1-oxyl-2,2,5,5-tetramethyl-Δ 3 -pyrroline-3-methyl)methanethiosulfonate. [7,8] It enhances the transverse relaxation rate (R 2 ) of protons and is introduced into proteins via site specific labeling of cysteine residues. [9][10][11] PRE measurements in proteins commonly monitor 1 H line broadening or intensity attenuation of proton resonances, caused by R 2 relaxation rate enhancements, and these are used to derive distances, with an accessible distance range for the MTSL spin label to protons of 13-25 Å. Resonances of residue in close proximity to the spin label are broadened beyond detection, while for long distances, the effect becomes undetectable.

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Anti-HIV Activity of Defective Cyanovirin-N Mutants Is Restored by Dimerization

Cited by 40 publications

References 39 publications

Designed oligomers of cyanovirin-N show enhanced HIV neutralization

Designed oligomers of cyanovirin-N show enhanced HIV neutralization

Structure and Glycan Binding of a New Cyanovirin-N Homolog

¹⁹F Paramagnetic Relaxation Enhancement: A Valuable Tool for Distance Measurements in Proteins

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Anti-HIV Activity of Defective Cyanovirin-N Mutants Is Restored by Dimerization

Cited by 40 publications

References 39 publications

Designed oligomers of cyanovirin-N show enhanced HIV neutralization

Designed oligomers of cyanovirin-N show enhanced HIV neutralization

Structure and Glycan Binding of a New Cyanovirin-N Homolog

19F Paramagnetic Relaxation Enhancement: A Valuable Tool for Distance Measurements in Proteins

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Product

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¹⁹F Paramagnetic Relaxation Enhancement: A Valuable Tool for Distance Measurements in Proteins