Abstract
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.
Highlights
Cyanovirin-N (CV-N) is a small (11 kDa) cyanobacterial protein with potent antiviral activity towards HIV, Ebola, influenza, hepatitis C, and other enveloped viruses [1,2,3,4,5]
We found that Nested CV-N interacts with both glycoproteins with apparent affinity comparable to WT CV-N
Nested CV-N is stable to thermal denaturation, and contains four functional dimannose binding domains, two with high affinity and two with low affinity as assessed by Isothermal titration calorimetry (ITC), consistent with the presence of two sets of domains as observed in WT CV-N
Summary
Cyanovirin-N (CV-N) is a small (11 kDa) cyanobacterial protein with potent antiviral activity towards HIV, Ebola, influenza, hepatitis C, and other enveloped viruses [1,2,3,4,5]. Its mechanism of action involves high affinity binding to the oligomannose glycans tethered to the viral envelope glycoproteins, gp120 in the case of HIV. Viruses 2016, 8, 158 comprises two quasi-symmetric glycan binding sites, A (residues 1–38/90–101) and B (residues 39–89), connected on each side by a short helical linker. Each carbohydrate binding site binds Man (1 Ñ 2)Man termini in oligomannose selectively, domain B binds dimannose more tightly than domain A, and domain A shows selectivity for linear trimannose [1,2,3,4,5,7]. Site B consists of a contiguous stretch of amino acids, whereas N- and C-terminal residues fold to form glycan binding site
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