HCV NS5A is a dimeric phosphoprotein involved in HCV replication. NS5A inhibitors are among direct-acting antivirals (DAA) for HCV therapy. The Y93H mutant of NS5A is resistant to NS5A inhibitors, but the precise mechanism remains unclear. In this report, we proposed a Ser38-His93-Asn91 triad to dissect the mechanism. Using pymol 1.3 software, the homology structure of JFH1 NS5A was determined based on the dimer structure of genotype 1b extracted from the database Protein DataBank (www.ebi.ac.uk/pdbsum) with codes 1ZH1 and 3FQM/3FQQ. FLAG-NS5A-WT failed to form dimer in the absence of nonstructural proteins from subgenomic replicon (NS3-5A); however, FLAG-NS5A-Y93H was able to form dimer without the aid of NS3-5A. The Ser38-His93-Asn91 triad in the dimer of the Y93H variant predicts a structural crash of the cleft receiving the NS5A inhibitor daclatasvir. The dimerization assay revealed that the existence of JFH1-NS5A-1ZH1 and -3FQM homology dimers depended on each other for existence and that both NS5A-WT 1ZH1 and 3FQM dimers cooperated to facilitate RNA replication. However, NS5A-Y93H 1ZH1 alone could form dimer and conduct RNA replication in the absence of the 3FQM structure. In conclusion, this study provides novel insight into the functional significance of the Ser38-His93-Asn91 triad in resistance of the Y93H variant to NS5A inhibitors.

中文翻译：

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Ser38-His93-Asn91 三联体赋予 JFH1 HCV NS5A-Y93H 变异体对 NS5A 抑制剂的抗性

HCV NS5A 是一种参与 HCV 复制的二聚体磷蛋白。NS5A 抑制剂属于用于 HCV 治疗的直接作用抗病毒药物 (DAA)。NS5A的Y93H突变体对NS5A抑制剂具有耐药性，但确切机制仍不清楚。在本报告中，我们提出了 Ser38-His93-Asn91 三联体来剖析该机制。使用pymol 1.3软件，根据从数据库Protein DataBank(www.ebi.ac.uk/pdbsum)中提取的基因型1b二聚体结构，确定JFH1 NS5A的同源结构，代码为1ZH1和3FQM/3FQQ。在缺乏亚基因组复制子 (NS3-5A) 的非结构蛋白的情况下，FLAG-NS5A-WT 无法形成二聚体；然而，FLAG-NS5A-Y93H 无需 NS3-5A 的帮助即可形成二聚体。Y93H 变体二聚体中的 Ser38-His93-Asn91 三联体预测接受 NS5A 抑制剂达拉他韦治疗后裂隙会发生结构性崩溃。二聚化测定表明，JFH1-NS5A-1ZH1 和 -3FQM 同源二聚体的存在相互依赖，并且 NS5A-WT 1ZH1 和 3FQM 二聚体协同促进 RNA 复制。然而，在缺乏 3FQM 结构的情况下，NS5A-Y93H 1ZH1 单独可以形成二聚体并进行 RNA 复制。总之，这项研究为 Ser38-His93-Asn91 三联体在 Y93H 变体对 NS5A 抑制剂的耐药性中的功能意义提供了新的见解。