Barrientos Laura G, Matei Elena, Lasala Fátima, Delgado Rafael, Gronenborn Angela M
Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases National Institutes of Health, Bethesda, MD 20892, USA.
Protein Eng Des Sel. 2006 Dec;19(12):525-35. doi: 10.1093/protein/gzl040. Epub 2006 Sep 29.
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-N(mutDA) and CV-N(mutDB), 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-N(mutDA) and CV-N(mutDB) comprise changes in amino acids that alter the trimannose specificity of domain A(M) and abolish the sugar binding site on domain B(M), respectively. We demonstrate that carbohydrate binding via domain B(M) is essential for antiviral activity, whereas alterations in sugar binding specificity on domain A(M) have little effect on envelope glycoprotein recognition and antiviral activity. Changes in A(M), 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.
HIV 灭活蛋白氰病毒素 -N（CV-N）是一种蓝细菌凝集素，通过与病毒糖蛋白上的高甘露糖碳水化合物相互作用，在纳摩尔浓度下表现出强大的抗病毒活性。鉴于实验测得的对小糖的微摩尔亲和力以及高甘露糖/CV-N 复合物聚集和沉淀所遇到的问题，迄今为止，对于这种强大的杀病毒活性尚无分子层面的解释。在此，我们展示了两种 CV-N 变体 CV-N(mutDA) 和 CV-N(mutDB) 的结果，比较了它们与单体 [P51G]CV-N（野生型 CV-N 的稳定版本）的结合特性，并测试了它们的体外活性。CV-N(mutDA) 和 CV-N(mutDB) 中的突变分别包括氨基酸变化，这些变化改变了结构域 A(M) 的三甘露糖特异性并消除了结构域 B(M) 上的糖结合位点。我们证明，通过结构域 B(M) 的碳水化合物结合对于抗病毒活性至关重要，而结构域 A(M) 上糖结合特异性的改变对包膜糖蛋白识别和抗病毒活性影响很小。然而，A(M) 的变化会影响 CV-N 的交联活性。我们的研究结果补充并阐明了 CV-N 与病毒糖蛋白上 N 连接聚糖结合的现有模型，并证明 CV-N 的纳摩尔抗病毒效力与病毒糖蛋白聚糖簇中甘露糖的紧密且空间拥挤的排列有关，而不是由于 CV-N 诱导的病毒颗粒凝集，这使得 CV-N 成为一种真正的病毒进入抑制剂。
