Protease inhibitors (PIs) are important components of treatment regimens for patients with chronic hepatitis C virus (HCV) infection. However, emergence and persistence of antiviral resistance could reduce their efficacy. Thus, defining resistance determinants is highly relevant for efforts to control HCV. Here, we investigated patterns of PI resistance–associated substitutions (RASs) for the major HCV genotypes and viral determinants for persistence of key RASs. We identified protease position 156 as a RAS hotspot for genotype 1‐4, but not 5 and 6, escape variants by resistance profiling using PIs grazoprevir and paritaprevir in infectious cell culture systems. However, except for genotype 3, engineered 156‐RASs were not maintained. For genotypes 1 and 2, persistence of 156‐RASs depended on genome‐wide substitution networks, co‐selected under continued PI treatment and identified by next‐generation sequencing with substitution linkage and haplotype reconstruction. Persistence of A156T for genotype 1 relied on compensatory substitutions increasing replication and assembly. For genotype 2, initial selection of A156V facilitated transition to 156L, persisting without compensatory substitutions. The developed genotype 1, 2, and 3 variants with persistent 156‐RASs had exceptionally high fitness and resistance to grazoprevir, paritaprevir, glecaprevir, and voxilaprevir. A156T dominated in genotype 1 glecaprevir and voxilaprevir escape variants, and pre‐existing A156T facilitated genotype 1 escape from clinically relevant combination treatments with grazoprevir/elbasvir and glecaprevir/pibrentasvir. In genotype 1 infected patients with treatment failure and 156‐RASs, we observed genome‐wide selection of substitutions under treatment. Conclusion: Comprehensive PI resistance profiling for HCV genotypes 1‐6 revealed 156‐RASs as key determinants of high‐level resistance across clinically relevant PIs. We obtained in vitro proof of concept for persistence of highly fit genotype 1‐3 156‐variants, which might pose a threat to clinically relevant combination treatments.

中文翻译：

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高度适应性和耐药性丙型肝炎病毒蛋白酶抑制剂逃逸变体持续存在的进化途径

蛋白酶抑制剂（PI）是慢性丙型肝炎病毒（HCV）感染患者治疗方案的重要组成部分。然而，抗病毒耐药性的出现和持续可能会降低其疗效。因此，确定耐药性决定因素与控制 HCV 的努力高度相关。在这里，我们研究了主要 HCV 基因型的 PI 耐药相关替代 (RAS) 模式以及关键 RAS 持续存在的病毒决定因素。我们在感染性细胞培养系统中使用 PI grazoprevir 和 paritaprevir 进行耐药性分析，将蛋白酶位置 156 确定为基因型 1-4 的 RAS 热点，但不是基因型 5 和 6 的 RAS 热点。然而，除了基因型 3 之外，工程化的 156-RAS 并未得到维持。对于基因型 1 和 2，156-RAS 的持久性取决于全基因组替换网络，在持续 PI 处理下共同选择，并通过具有替换连锁和单倍型重建的下一代测序进行鉴定。基因型 1 的 A156T 的持续存在依赖于增加复制和组装的补偿性替代。对于基因型 2，A156V 的初始选择促进了向 156L 的转变，并且在没有补偿性取代的情况下持续存在。开发的具有持久 156-RAS 的基因型 1、2 和 3 变体对 Grazoprevir、paritaprevir、glecaprevir 和 voxilaprevir 具有极高的适应性和耐药性。A156T 在基因型 1 glecaprevir 和 voxilaprevir 逃逸变异中占主导地位，预先存在的 A156T 促进基因型 1 从 Grazoprevir/elbasvir 和 glecaprevir/pibrentasvir 的临床相关联合治疗中逃逸。在治疗失败且患有 156-RAS 的基因 1 型感染患者中，我们观察到治疗下的全基因组替换选择。结论：HCV 基因型 1-6 的综合 PI 耐药性分析表明，156-RAS 是临床相关 PI 中高水平耐药性的关键决定因素。我们获得了高度拟合基因型 1-3 156 变异持续存在的体外概念证明，这可能对临床相关的联合治疗构成威胁。