Université Lille Nord de France, F-59000 Lille, France.
J Biol Chem. 2011 Jun 10;286(23):20441-54. doi: 10.1074/jbc.M110.182436. Epub 2011 Apr 13.
Nonstructural protein 5A (NS5A) is essential for hepatitis C virus (HCV) replication and constitutes an attractive target for antiviral drug development. Although structural data for its in-plane membrane anchor and domain D1 are available, the structure of domains 2 (D2) and 3 (D3) remain poorly defined. We report here a comparative molecular characterization of the NS5A-D3 domains of the HCV JFH-1 (genotype 2a) and Con1 (genotype 1b) strains. Combining gel filtration, CD, and NMR spectroscopy analyses, we show that NS5A-D3 is natively unfolded. However, NS5A-D3 domains from both JFH-1 and Con1 strains exhibit a propensity to partially fold into an α-helix. NMR analysis identifies two putative α-helices, for which a molecular model could be obtained. The amphipathic nature of the first helix and its conservation in all genotypes suggest that it might correspond to a molecular recognition element and, as such, promote the interaction with relevant biological partner(s). Because mutations conferring resistance to cyclophilin inhibitors have been mapped into NS5A-D3, we also investigated the functional interaction between NS5A-D3 and cyclophilin A (CypA). CypA indeed interacts with NS5A-D3, and this interaction is completely abolished by cyclosporin A. NMR heteronuclear exchange experiments demonstrate that CypA has in vitro peptidyl-prolyl cis/trans-isomerase activity toward some, but not all, of the peptidyl-prolyl bonds in NS5A-D3. These studies lead to novel insights into the structural features of NS5A-D3 and its relationships with CypA.
非结构蛋白 5A（NS5A）是丙型肝炎病毒（HCV）复制所必需的，是抗病毒药物开发的有吸引力的靶标。尽管其平面膜锚和结构域 D1 的结构数据已经可用，但结构域 2（D2）和 3（D3）的结构仍然定义不明确。我们在这里报告 HCV JFH-1（基因型 2a）和 Con1（基因型 1b）株的 NS5A-D3 结构域的比较分子特征。通过凝胶过滤、CD 和 NMR 光谱分析，我们表明 NS5A-D3 是天然无规卷曲的。然而，来自 JFH-1 和 Con1 株的 NS5A-D3 结构域都表现出部分折叠成α-螺旋的倾向。NMR 分析确定了两个可能的α-螺旋，对于这两个螺旋，我们可以获得分子模型。第一个螺旋的两亲性及其在所有基因型中的保守性表明，它可能对应于一个分子识别元件，并因此促进与相关生物伴侣的相互作用。由于已经将赋予环孢菌素抑制剂抗性的突变映射到 NS5A-D3 中，我们还研究了 NS5A-D3 与亲环蛋白 A（CypA）之间的功能相互作用。CypA 确实与 NS5A-D3 相互作用，并且这种相互作用完全被环孢菌素 A 消除。NMR 异核交换实验表明，CypA 在体外对 NS5A-D3 中的一些，但不是所有的肽脯氨酰顺反异构酶活性。这些研究为 NS5A-D3 的结构特征及其与 CypA 的关系提供了新的见解。
