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HCV 基因型 1-6 NS3 残基 80 位取代影响蛋白酶抑制剂活性并促进病毒逃逸。

HCV genotype 1-6 NS3 residue 80 substitutions impact protease inhibitor activity and promote viral escape.

机构信息

Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases, Copenhagen University Hospital, Hvidovre and Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark.

Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases, Copenhagen University Hospital, Hvidovre and Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark; Department of Clinical Microbiology, Copenhagen University Hospital, Hvidovre, Denmark.

出版信息

J Hepatol. 2019 Mar;70(3):388-397. doi: 10.1016/j.jhep.2018.10.031. Epub 2018 Nov 3.

Abstract

BACKGROUND & AIMS: Protease inhibitors (PIs) are of central importance in the treatment of patients with chronic hepatitis C virus (HCV) infection. HCV NS3 protease (NS3P) position 80 displays polymorphisms associated with resistance to the PI simeprevir for HCV genotype 1a. We investigated the effects of position-80-substitutions on fitness and PI-resistance for HCV genotypes 1-6, and analyzed evolutionary mechanisms underlying viral escape mediated by pre-existing Q80K.

METHODS

The fitness of infectious NS3P recombinants of HCV genotypes 1-6, with engineered position-80-substitutions, was studied by comparison of viral spread kinetics in Huh-7.5 cells in culture. Median effective concentration (EC50) and fold resistance for PIs simeprevir, asunaprevir, paritaprevir, grazoprevir, glecaprevir and voxilaprevir were determined in short-term treatment assays. Viral escape was studied by long-term treatment of genotype 1a recombinants with simeprevir, grazoprevir, glecaprevir and voxilaprevir and of genotype 3a recombinants with glecaprevir and voxilaprevir, next generation sequencing, NS3P substitution linkage and haplotype analysis.

RESULTS

Among tested PIs, only glecaprevir and voxilaprevir showed pan-genotypic activity against the original genotype 1-6 culture viruses. Variants with position-80-substitutions were all viable, but fitness depended on the specific substitution and the HCV isolate. Q80K conferred resistance to simeprevir across genotypes but had only minor effects on the activity of the remaining PIs. For genotype 1a, pre-existing Q80K mediated accelerated escape from simeprevir, grazoprevir and to a lesser extent glecaprevir, but not voxilaprevir. For genotype 3a, Q80K mediated accelerated escape from glecaprevir and voxilaprevir. Escape was mediated by rapid and genotype-, PI- and PI-concentration-dependent co-selection of clinically relevant resistance associated substitutions.

CONCLUSIONS

Position-80-substitutions had relatively low fitness cost and the potential to promote HCV escape from clinically relevant PIs in vitro, despite having a minor impact on results in classical short-term resistance assays.

LAY SUMMARY

Among all clinically relevant hepatitis C virus protease inhibitors, voxilaprevir and glecaprevir showed the highest and most uniform activity against cell culture infectious hepatitis C virus with genotype 1-6 proteases. Naturally occurring amino acid changes at protease position 80 had low fitness cost and influenced sensitivity to simeprevir, but not to other protease inhibitors in short-term treatment assays. Nevertheless, the pre-existing change Q80K had the potential to promote viral escape from protease inhibitors during long-term treatment by rapid co-selection of additional resistance changes, detected by next generation sequencing.

摘要

背景与目的

蛋白酶抑制剂(PI)在慢性丙型肝炎病毒(HCV)感染患者的治疗中具有重要意义。HCV NS3 蛋白酶(NS3P)位置 80 存在与对 HCV 基因型 1a 的 PI 西咪普韦耐药相关的多态性。我们研究了位置 80 取代对 HCV 基因型 1-6 的适应性和 PI 耐药性的影响,并分析了由预先存在的 Q80K 介导的病毒逃逸的进化机制。

方法

通过比较培养的 Huh-7.5 细胞中病毒传播动力学,研究了具有工程化位置 80 取代的 HCV 基因型 1-6 的传染性 NS3P 重组体的适应性。通过短期治疗试验确定了西咪普韦、阿那普韦、帕立瑞韦、格拉瑞韦、格卡瑞韦和 voxilaprevir 的半数有效浓度(EC50)和倍耐药性。通过对基因型 1a 重组体用西咪普韦、格拉瑞韦、格卡瑞韦和 voxilaprevir 以及基因型 3a 重组体用格卡瑞韦和 voxilaprevir 进行长期治疗、下一代测序、NS3P 取代连锁和单倍型分析来研究病毒逃逸。

结果

在所测试的 PI 中,只有格卡瑞韦和 voxilaprevir 对原始基因型 1-6 培养病毒具有泛基因型活性。具有位置 80 取代的变体均具有生存能力,但适应性取决于特定的取代和 HCV 分离株。Q80K 赋予了对所有基因型的 simeprevir 耐药性,但对其余 PI 的活性只有很小的影响。对于基因型 1a,预先存在的 Q80K 介导了对 simeprevir、格拉瑞韦和程度较小的格卡瑞韦的快速逃逸,但不能介导对 voxilaprevir 的逃逸。对于基因型 3a,Q80K 介导了对格卡瑞韦和 voxilaprevir 的快速逃逸。逃逸是由快速、与基因型、PI 和 PI 浓度相关的临床相关耐药相关取代的共同选择介导的。

结论

位置 80 取代具有相对较低的适应性成本,并有潜力在体外促进对临床相关 PI 的 HCV 逃逸,尽管它们对经典短期耐药试验的结果影响较小。

临床意义

在所有临床相关的丙型肝炎病毒蛋白酶抑制剂中,voxilaprevir 和格卡瑞韦对携带基因型 1-6 蛋白酶的细胞培养传染性丙型肝炎病毒表现出最高和最一致的活性。蛋白酶位置 80 处的天然氨基酸变化具有较低的适应性成本,并且在短期治疗试验中影响对 simeprevir 的敏感性,但不影响其他蛋白酶抑制剂。然而,预先存在的 Q80K 有可能通过快速共同选择其他耐药性变化,通过下一代测序检测到,从而促进蛋白酶抑制剂治疗期间的病毒逃逸。

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