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Open AccessArticle

A Designed “Nested” Dimer of Cyanovirin-N Increases Antiviral Activity

School of Molecular Sciences, Arizona State University, Tempe, AZ 85287-1604, USA
Molecular Targets Laboratory, Center for Cancer Research, National Cancer Institute at Frederick, Frederick, MD 21702, USA
Basic Science Program, Leidos Biomedical Research, Inc., Frederick National Laboratory, Frederick, MD 21702, USA
Sanford Burnham Prebys Medical Discovery Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA
Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI 53226, USA
School of Life Sciences and The Biodesign Institute, Arizona State University, Tempe, AZ 85287, USA
Author to whom correspondence should be addressed.
Academic Editor: Eric O. Freed
Viruses 2016, 8(6), 158;
Received: 15 January 2016 / Revised: 26 May 2016 / Accepted: 30 May 2016 / Published: 6 June 2016
(This article belongs to the Special Issue Lectins as Antiviral)
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. View Full-Text
Keywords: Cyanovirin-N; antiviral lectins; glycan-binding proteins; oligomannose; gp120 Cyanovirin-N; antiviral lectins; glycan-binding proteins; oligomannose; gp120
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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.

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