Department of Internal Medicine 1, Goethe University Hospital Frankfurt, D-60590 Frankfurt a.M., Germany; Max-Planck-Institute for Informatics, Computational Biology & Applied Algorithmics, D-66123 Saarbrücken, Germany.
Department of Internal Medicine 1, Goethe University Hospital Frankfurt, D-60590 Frankfurt a.M., Germany.
J Hepatol. 2015 Apr;62(4):779-84. doi: 10.1016/j.jhep.2014.11.009. Epub 2014 Nov 21.
BACKGROUND & AIMS: The hepatitis C virus (HCV) NS3-4A protease is essential for the HCV life cycle and a prime target of antiviral treatment strategies. Protease inhibitors, however, are limited by emergence of resistance-associated amino acid variants (RAVs). The capacity to cleave and inactivate mitochondrial antiviral-signaling protein (MAVS) in the RIG-I-signaling pathway is a cardinal feature of NS3-4A, by which HCV blocks induction of interferon-(IFN)-β, thereby promoting viral persistence. Here, we aimed to investigate the impact of NS3-4A RAVs on MAVS cleavage.
The impact of NS3-4A RAVs on MAVS cleavage was assessed using immunoblot analyses, luciferase reporter assays and molecular dynamics simulations to study the underlying molecular principles. IFN-β was quantified in serum from patients with different NS3-4A RAVs.
We show that macrocyclic NS3-4A RAVS with substitutions at residue D168 of the protease result in an increased capacity of NS3-4A to cleave MAVS and suppress IFN-β induction compared with a comprehensive panel of RAVs and wild type HCV. Mechanistically, we show the reconstitution of a tight network of electrostatic interactions between protease and the peptide substrate that allows much stronger binding of MAVS to D168 RAVs than to the wild-type protease. Accordingly, we could show IFN-β serum levels to be lower in patients with treatment failure due to the selection of D168 variants compared to R155 RAVs.
Our data constitutes a proof of concept that the selection of RAVs against specific classes of direct antivirals can lead to the predominance of viral variants with possibly adverse pathogenic characteristics.
丙型肝炎病毒（HCV）NS3-4A 蛋白酶对于 HCV 生命周期至关重要，也是抗病毒治疗策略的主要靶点。然而，蛋白酶抑制剂受到耐药相关氨基酸变异（RAV）的限制。HCV 通过 NS3-4A 切割和失活 RIG-I 信号通路中的抗病毒信号蛋白（MAVS），阻断干扰素-（IFN）-β的诱导，从而促进病毒持续存在，这是 NS3-4A 的一个主要特征。在此，我们旨在研究 NS3-4A RAV 对 MAVS 切割的影响。
使用免疫印迹分析、荧光素酶报告基因测定和分子动力学模拟来研究潜在的分子原理，评估 NS3-4A RAV 对 MAVS 切割的影响。从具有不同 NS3-4A RAV 的患者血清中定量 IFN-β。
我们表明，与广泛的 RAV 和野生型 HCV 相比，具有蛋白酶残基 D168 取代的大环 NS3-4A RAV 导致 NS3-4A 切割 MAVS 和抑制 IFN-β诱导的能力增强。从机制上讲，我们表明蛋白酶和肽底物之间重新形成了静电相互作用的紧密网络，这使得 MAVS 与 D168 RAV 的结合比与野生型蛋白酶的结合更强。因此，与 R155 RAV 相比，由于选择 D168 变异导致治疗失败的患者血清中 IFN-β 水平较低。
我们的数据构成了一个概念验证，即针对特定类别直接抗病毒药物的 RAV 选择可能导致具有潜在不良致病特征的病毒变异的优势。
