Aloia Amanda L, Eyre Nicholas S, Black Stuart, Bent Stephen J, Gaeguta Adriana, Guo Zhuyan, Narayana Sumudu K, Chase Robert, Locarnini Stephen, Carr Jill M, Howe John A, Beard Michael R
School of Molecular and Biomedical Science, Adelaide, The University of Adelaide and Centre for Cancer Biology, SA Pathology, Adelaide, South Australia, Australia.
Antivir Ther. 2015;20(3):271-80. doi: 10.3851/IMP2850. Epub 2014 Sep 15.
Genotype (gt)6 HCV is common amongst HCV-positive populations of the Asia-Pacific region but cell culture models for this gt have only recently been developed. Boceprevir (SCH503034) is a clinically available inhibitor of the HCV NS3 protein. We investigated the efficacy of boceprevir for inhibiting replication of a chimeric gt1b replicon encoding a gt6a NS3 protease and defined the development of mutations in the protease when boceprevir treatment was applied.
We constructed a chimeric gt1b subgenomic replicon encoding a gt6 NS3 protease (NS3p) sequence (gt6NS3p_gt1b). The boceprevir EC50 value against replication of this replicon was determined using quantitative reverse transcriptase PCR. Next-generation sequencing was used to identify nucleotide changes associated with boceprevir resistance. The replication capacities of chimeric replicons containing mutations associated with boceprevir resistance were determined by colony formation efficiency assays.
The boceprevir EC50 value for the gt6NS3p_gt1b replicon was 535 ±79 nM. Boceprevir-resistant gt6NS3p_gt1b replicon cell lines could be selected and they demonstrated drug-associated amino acid changes that have previously been reported in other HCV gts. Interestingly, no mutations were observed at A156, a position defined for boceprevir resistance in gt1 NS3p, while mutation at N122, which is rarely reported in boceprevir-resistant gt1 proteases, was frequently observed. Re-introduction of these mutations into the chimeric replicon altered their replication capacity, ranging from complete abolishment of replication (A156T) to increasing replication capacity (V36A, N122S). This report provides the first characterization of gt6 HCV resistance to boceprevir.
A chimeric HCV replicon encoding gt6 NS3 protease is sensitive to boceprevir and develops drug-resistant mutations at amino acid sites previously reported for other gts. Mutation at N122 also appears to be associated with boceprevir resistance in the gt6 NS3 protease.
基因1型（gt）6丙型肝炎病毒在亚太地区丙型肝炎病毒阳性人群中较为常见，但针对该基因1型的细胞培养模型直到最近才得以开发。波西普韦（SCH503034）是一种临床上可用的丙型肝炎病毒NS3蛋白抑制剂。我们研究了波西普韦抑制编码基因1型6a NS3蛋白酶的嵌合基因1型b复制子复制的效果，并确定了应用波西普韦治疗时蛋白酶中突变的发生情况。
我们构建了一个编码基因1型6 NS3蛋白酶（NS3p）序列（基因1型6 NS3p_基因1型b）的嵌合基因1型b亚基因组复制子。使用定量逆转录聚合酶链反应测定波西普韦针对该复制子复制的半数有效浓度（EC50）值。利用下一代测序技术鉴定与波西普韦耐药相关的核苷酸变化。通过集落形成效率试验确定含有与波西普韦耐药相关突变的嵌合复制子的复制能力。
基因1型6 NS3p_基因1型b复制子的波西普韦EC50值为535±79 nM。可以筛选出对波西普韦耐药的基因1型6 NS3p_基因1型b复制子细胞系，并且它们表现出先前在其他丙型肝炎病毒基因1型中报道过的与药物相关的氨基酸变化。有趣的是，在基因1型NS3p中被定义为波西普韦耐药的A156位点未观察到突变，而在波西普韦耐药的基因1型蛋白酶中很少报道的N122位点的突变却经常被观察到。将这些突变重新引入嵌合复制子会改变它们的复制能力，范围从完全抑制复制（A156T）到增加复制能力（V36A、N122S）。本报告首次描述了基因1型6丙型肝炎病毒对波西普韦的耐药性。
编码基因1型6 NS3蛋白酶的嵌合丙型肝炎病毒复制子对波西普韦敏感，并在先前报道的其他基因1型的氨基酸位点发生耐药性突变。N122位点的突变似乎也与基因1型6 NS3蛋白酶对波西普韦的耐药性有关。
