A Designed “Nested” Dimer of Cyanovirin-N Increases Antiviral Activity
AbstractCyanovirin-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. View Full-Text
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Woodrum, B.W.; Maxwell, J.; Allen, D.M.; Wilson, J.; Krumpe, L.R.; Bobkov, A.A.; Hill, R.B.; Kibler, K.V.; O’Keefe, B.R.; Ghirlanda, G. A Designed “Nested” Dimer of Cyanovirin-N Increases Antiviral Activity. Viruses 2016, 8, 158.
Woodrum BW, Maxwell J, Allen DM, Wilson J, Krumpe LR, Bobkov AA, Hill RB, Kibler KV, O’Keefe BR, Ghirlanda G. A Designed “Nested” Dimer of Cyanovirin-N Increases Antiviral Activity. Viruses. 2016; 8(6):158.Chicago/Turabian Style
Woodrum, Brian W.; Maxwell, Jason; Allen, Denysia M.; Wilson, Jennifer; Krumpe, Lauren R.; Bobkov, Andrey A.; Hill, R. B.; Kibler, Karen V.; O’Keefe, Barry R.; Ghirlanda, Giovanna. 2016. "A Designed “Nested” Dimer of Cyanovirin-N Increases Antiviral Activity." Viruses 8, no. 6: 158.
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