Department of Medicine, University of Wisconsin-Madison, Madison, WI 53706, USA.
GlaxoSmithKline, 5 Moore Drive, 3.3204 RTP, NC 27709, USA.
J Gen Virol. 2014 Feb;95(Pt 2):363-372. doi: 10.1099/vir.0.054569-0. Epub 2013 Aug 30.
Non-structural 5A protein (NS5A) has emerged as an important pharmacological target for hepatitis C virus (HCV). However, little is known about the conformation of NS5A intracellularly or how NS5A inhibitors achieve the picomolar (pM) inhibition of virus replication. Here, we have presented two structurally related small molecules, one that potently inhibits HCV replication and selects for resistance in NS5A, and another that is inactive. Resistance to this antiviral was greater in genotype 1a than in genotype 1b replicons and mapped to domain 1 of NS5A. Using a novel cell-based assay that measures the intracellular proximity of fluorescent tags covalently attached to NS5A, we showed that only the active antiviral specifically disrupted the close proximity of inter- and intramolecular positions of NS5A. The active antiviral, termed compound 1, caused a repositioning of both the N and C termini of NS5A, including disruption of the close approximation of the N termini of two different NS5A molecules in a multimolecular complex. These data provide the first study of how antivirals that select resistance in domain 1 of NS5A alter the cellular conformation of NS5A. This class of antiviral disrupts the close proximity of the N termini of domain 1 in a NS5A complex but also alters the conformation of domain 3, and leads to large aggregates of NS5A. Current models predict that a multicomponent cocktail of antivirals is needed to treat HCV infection, so a mechanistic understanding of what each component does to the viral machinery will be important.
非结构蛋白 5A（NS5A）已成为丙型肝炎病毒（HCV）的重要药理学靶点。然而，人们对 NS5A 的细胞内构象以及 NS5A 抑制剂如何达到皮摩尔（pM）抑制病毒复制的了解甚少。在这里，我们提出了两种结构相关的小分子，一种能有效抑制 HCV 复制并在 NS5A 中选择耐药性，另一种则没有活性。对这种抗病毒药物的耐药性在基因型 1a 中比在基因型 1b 复制子中更强，并且定位于 NS5A 的结构域 1。使用一种新的基于细胞的测定法，该测定法测量共价连接到 NS5A 的荧光标记物的细胞内接近度，我们表明只有活性抗病毒药物才能特异性破坏 NS5A 的分子间和分子内位置的紧密接近。这种活性抗病毒药物称为化合物 1，它导致 NS5A 的 N 和 C 末端重新定位，包括破坏多分子复合物中两个不同 NS5A 分子的 N 末端的紧密接近。这些数据首次研究了在 NS5A 的结构域 1 中选择耐药性的抗病毒药物如何改变 NS5A 的细胞构象。这类抗病毒药物会破坏 NS5A 复合物中结构域 1 的 N 末端的紧密接近，但也会改变结构域 3 的构象，并导致 NS5A 的大聚集体。目前的模型预测需要一种多组分抗病毒药物鸡尾酒来治疗 HCV 感染，因此了解每种成分对病毒机制的作用将非常重要。
