Replication efficiency of chimeric replicon containing NS5A-5B genes derived from HCV-infected patient sera.
Replication efficiency of chimeric replicon containing NS5A-5B genes derived from HCV-infected patient sera.
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DOI：
10.1016/j.antiviral.2006.07.005
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发表时间：
2007
影响因子：
7.6
通讯作者：
R. Tripathi;Preethi Krishnan;Yupeng He;T. Middleton;T. Pilot-matias;C. Chen;D. Lau;S. Lemon;H. Mo;W. Kati;A. Molla
中科院分区：
文献类型：
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作者：
R. Tripathi;Preethi Krishnan;Yupeng He;T. Middleton;T. Pilot-matias;C. Chen;D. Lau;S. Lemon;H. Mo;W. Kati;A. Molla
建立了一种基于亚基因组复制子的穿梭载体系统，用于研究基因异质性对HCV复制效率的影响。使用RT-PCR从来自HCV基因型1a或1b患者血清的“准种”池扩增单个NS 5A或NS 5 B基因或含有NS 5A和NS 5 B基因的盒，并克隆到它们各自的穿梭载体中。所有含有NS 5A或NS 5A-5 B基因的穿梭载体都用S2204 I“适应性”突变构建，因为缺乏S2204 I突变的复制子复制较差。基因型1a或1b患者样本内准种池的基因序列的同一性范围为94%至95%。相对于实验室优化的1b复制子，含有患者来源的NS 5A或NS 5 B基因的1b穿梭载体的复制能力分别平均为67%和75%。相比之下，相对于各自的实验室优化的复制子，含有患者来源的NS 5A-5 B基因盒的1a和1b穿梭载体的复制效率平均约为2%。在瞬时试验中检测所有患者来源的复制子对干扰素-α（IFN-α）或聚合酶抑制剂A-782759的敏感性。尽管复制效率存在差异，但对大多数患者来源的复制子测量的IC 50值与对照实验室菌株复制子中测量的相应值相当。这些结果表明，无论何时将患者来源的NS 5A-5 B基因插入实验室优化的复制子中，患者序列异质性都会影响复制效率。这些发现也证明了穿梭载体系统用于测试患者来源的基因序列对IFN-α和小分子抑制剂的敏感性的实用性。
A transient subgenomic replicon-based shuttle vector system has been developed to investigate how genetic heterogeneity affects HCV replication efficiency. Individual NS5A or NS5B genes or cassettes containing both NS5A and NS5B genes were amplified from “quasispecies” pools derived from HCV genotype 1a or 1b patient sera using RT-PCR and cloned into their respective shuttle vectors. All shuttle vectors containing NS5A or NS5A–5B genes were constructed with the S2204I “adaptive” mutation because replicons lacking the S2204I mutation replicated poorly. Gene sequences of the quasispecies pools within either genotype 1a or 1b patient samples ranged from 94 to 95% in identity. The replication capacity of 1b shuttle vectors containing patient-derived NS5A or NS5B genes averaged 67 and 75%, respectively, relative to the laboratory-optimized 1b replicon. In contrast, the replication efficiencies of both 1a and 1b shuttle vectors containing patient-derived NS5A–5B gene cassettes averaged around 2% relative to the respective laboratory-optimized replicon. All patient-derived replicons were tested in a transient assay for their sensitivity to either interferon-α (IFN-α) or to the polymerase inhibitor A-782759. Despite the differences in replication efficiency, IC50values measured for most of the patient-derived replicons were equivalent to the respective values measured in the control laboratory strain replicons. These results demonstrate that patient sequence heterogeneity affects replication efficiency whenever patient-derived NS5A–5B genes are inserted into the laboratory-optimized replicon. The findings also demonstrate the utility of the shuttle vector system to test patient-derived gene sequences for sensitivity to IFN-α and to small molecule inhibitors.