PEDS Advance Access originally published online on September 29, 2006
Protein Engineering Design and Selection 2006 19(12):525-535; doi:10.1093/protein/gzl040
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Published by Oxford University Press.
Dissecting carbohydrate–Cyanovirin-N binding by structure-guided mutagenesis: functional implications for viral entry inhibition
1 Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases National Institutes of Health, Bethesda, MD 20892, USA 2 Special Pathogens Branch, Division of Viral and Rickettsial Diseases National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, GA 30333, USA 3 Laboratory of Molecular Microbiology, Hospital Universitario 12 de Octubre Madrid, Spain 4 Department of Structural Biology, University of Pittsburgh Medical School Pittsburgh, PA 15260, USA
5To whom correspondence should be addressed. E-mail: amg100{at}pitt.edu; lbarrientos1{at}cdc.gov
The HIV-inactivating protein Cyanovirin-N (CV-N) is a cyanobacterial lectin that exhibits potent antiviral activity at nanomolar concentrations by interacting with high-mannose carbohydrates on viral glycoproteins. To date there is no molecular explanation for this potent virucidal activity, given the experimentally measured micromolar affinities for small sugars and the problems encountered with aggregation and precipitation of high-mannose/CV-N complexes. Here, we present results for two CV-N variants, CV-NmutDA and CV-NmutDB, compare their binding properties with monomeric [P51G]CV-N (a stabilized version of wtCV-N) and test their in vitro activities. The mutations in CV-NmutDA and CV-NmutDB comprise changes in amino acids that alter the trimannose specificity of domain AM and abolish the sugar binding site on domain BM, respectively. We demonstrate that carbohydrate binding via domain BM is essential for antiviral activity, whereas alterations in sugar binding specificity on domain AM have little effect on envelope glycoprotein recognition and antiviral activity. Changes in AM, however, affect the cross-linking activity of CV-N. Our findings augment and clarify the existing models of CV-N binding to N-linked glycans on viral glycoproteins, and demonstrate that the nanomolar antiviral potency of CV-N is related to the constricted and spatially crowded arrangement of the mannoses in the glycan clusters on viral glycoproteins and not due to CV-N induced virus particle agglutination, making CV-N a true viral entry inhibitor.
Keywords: Cyanovirin-N/high-mannose oligosaccharides/mutant design/viral env glycoprotein/virucidal agent
Received July 3, 2006; revised August 11, 2006; accepted August 29, 2006.