Ketoamide Resistance and Hepatitis C Virus Fitness in Val55 Variants of the NS3 Serine Protease
Ketoamide Resistance and Hepatitis C Virus Fitness in Val55 Variants of the NS3 Serine Protease
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DOI：
10.1128/aac.05184-11
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发表时间：
2012-04-01
影响因子：
4.9
通讯作者：
Antes, Iris
中科院分区：
文献类型：
--
作者：
Welsch, Christoph;Schweizer, Sabine;Antes, Iris
耐药病毒变异是慢性丙型肝炎直接作用抗病毒药物使用中的主要问题。酮酰胺是NS 3蛋白酶的有效抑制剂，其中V55 A被鉴定为与对博赛泼维的耐药性相关的突变。基本的分子机制仅被部分理解。我们应用了一种全面的序列分析来表征全球主要患者菌株中Val 55处的自然变异性。应用残基相互作用网络和分子动力学模拟来确定Val 55变体中酮酰胺抗性和病毒适应性的机制。感染性H77S.3细胞培养系统用于变体表型表征。我们测量了抗病毒的50%有效浓度（EC 50）和倍数变化，以及RNA复制和感染性病毒产量从病毒RNA含有变异体。Val 55在所有丙型肝炎病毒（HCV）基因型中高度保守。从HCV基因型Ia株中鉴定出保守的V55 A和V55 I变体，而在基因型Ib中没有变体。从蛋白酶结构的残基相互作用网络的拓扑测量表明，潜在的Val 55的关键作用，调制的蛋白酶配体结合位点的分子变化。分子动力学表明，变体与收缩的结合口袋和损失的H-键合的相互作用后boceprevir结合的变体蛋白酶。这些效应可能解释了V55 A变体以及Val 55变体中低水平的boceprevir抗性，降低了RNA复制能力。在野生型蛋白酶中发现了较高的结构灵活性，而变体显示出较低的灵活性。降低的结构灵活性可能影响Val 55变体适应NS 3结构域-结构域相互作用的能力，并可能解释在变体菌株中观察到的病毒产量下降。
Drug-resistant viral variants are a major issue in the use of direct-acting antiviral agents in chronic hepatitis C. Ketoamides are potent inhibitors of the NS3 protease, with V55A identified as mutation associated with resistance to boceprevir. Underlying molecular mechanisms are only partially understood. We applied a comprehensive sequence analysis to characterize the natural variability at Val55 within dominant worldwide patient strains. A residue-interaction network and molecular dynamics simulation were applied to identify mechanisms for ketoamide resistance and viral fitness in Val55 variants. An infectious H77S.3 cell culture system was used for variant phenotype characterization. We measured antiviral 50% effective concentration (EC50) and fold changes, as well as RNA replication and infectious virus yields from viral RNAs containing variants. Val55 was found highly conserved throughout all hepatitis C virus (HCV) genotypes. The conservative V55A and V55I variants were identified from HCV genotype la strains with no variants in genotype 1b. Topology measures from a residue-interaction network of the protease structure suggest a potential Val55 key role for modulation of molecular changes in the protease ligand-binding site. Molecular dynamics showed variants with constricted binding pockets and a loss of H-bonded interactions upon boceprevir binding to the variant proteases. These effects might explain low-level boceprevir resistance in the V55A variant, as well as the Val55 variant, reduced RNA replication capacity. Higher structural flexibility was found in the wild-type protease, whereas variants showed lower flexibility. Reduced structural flexibility could impact the Val55 variant's ability to adapt for NS3 domain-domain interaction and might explain the virus yield drop observed in variant strains.