Protease Inhibitor-Resistant Hepatitis C Virus Mutants With Reduced Fitness From Impaired Production of Infectious Virus
Protease Inhibitor-Resistant Hepatitis C Virus Mutants With Reduced Fitness From Impaired Production of Infectious Virus
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DOI：
10.1053/j.gastro.2010.10.056
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发表时间：
2011-02-01
期刊：
影响因子：
29.4
通讯作者：
Lemon, Stanley M.
中科院分区：
文献类型：
--
作者：
Shimakami, Tetsuro;Welsch, Christoph;Lemon, Stanley M.
背景与目的：几种丙型肝炎病毒非结构蛋白3/4A蛋白水解酶的小分子抑制剂已成功进入临床试验。然而，耐药突变体的选择是蛋白酶抑制剂(PI)的一个重要问题。NS3蛋白结构域上的多种氨基酸替换可导致对PI的抗性。其中许多都严重损害了丙型肝炎病毒RNA复制子的复制适合性。然而，目前尚不清楚这些突变是否也对传染性病毒的组装和释放产生不利影响，NS3也参与了这一过程。方法：我们研究了先前发现的25个PI抗性突变对1a型H77S RNA在细胞培养中复制和产生感染性病毒能力的影响。结果：虽然V36A/L/M、R109K和D168E的复制能力与野生型相当，但大多数PI抗性突变导致复制能力的中度丧失，而另一些突变(S138T和A156V)在RNA复制和感染性病毒产生方面都严重受损。虽然对于大多数突变，RNA复制能力的降低与感染性病毒产量的下降相关，但突变的子集(Q41R、F43S、R155T、A156S和I170A/T)在其产生病毒的能力方面的损害比预期的RNA复制能力降低更严重。对I170A突变体的详细检查显示，病毒从细胞中释放没有缺陷，细胞外病毒颗粒的特异性感染性也没有明显差异。结论：基于复制子的分析可能低估了PI耐药突变造成的适合性损失，因为NS3蛋白酶域的一些突变特别地损害了病毒生命周期中涉及感染性病毒细胞内组装的后期步骤。
BACKGROUND & AIMS: Several small molecule inhibitors of the hepatitis C virus (HCV) nonstructural protein (NS) 3/4A protease have advanced successfully to clinical trials. However, the selection of drug-resistant mutants is a significant issue with protease inhibitors (PIs). A variety of amino acid substitutions in the protease domain of NS3 can lead to PI resistance. Many of these significantly impair the replication fitness of HCV RNA replicons. However, it is not known whether these mutations also adversely affect infectious virus assembly and release, processes in which NS3 also participates. METHODS: We studied the impact of 25 previously identified PI-resistance mutations on the capacity of genotype 1a H77S RNA to replicate in cell culture and produce infectious virus. RESULTS: Most PI-resistance mutations resulted in moderate loss of replication competence, although several (V36A/L/M, R109K, and D168E) showed fitness comparable to wild type, whereas others (S138T and A156V) were severely impaired both in RNA replication and infectious virus production. Although reductions in RNA replication capacity correlated with decreased yields of infectious virus for most mutations, a subset of mutants (Q41R, F43S, R155T, A156S, and I170A/T) showed greater impairment in their ability to produce virus than predicted from reductions in RNA replication capacity. Detailed examination of the I170A mutant showed no defect in release of virus from cells and no significant difference in specific infectivity of extracellular virus particles. CONCLUSIONS: Replicon-based assays might underestimate the loss of fitness caused by PI-resistance mutations, because some mutations in the NS3 protease domain specifically impair late steps in the viral life cycle that involve intracellular assembly of infectious virus.