Resistance to cyclosporin A derives from mutations in hepatitis C virus nonstructural proteins.
Resistance to cyclosporin A derives from mutations in hepatitis C virus nonstructural proteins.
复制标题
对环孢菌素 A 的耐药性源自丙型肝炎病毒非结构蛋白的突变。
DOI：
10.1016/j.bbrc.2014.04.053
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发表时间：
2014
期刊：
影响因子：
--
通讯作者：
Kohara M
中科院分区：
文献类型：
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作者：
Arai M;Tsukiyama-Kohara K;Takagi A;Tobita Y;Inoue K;Kohara M
环孢素A（CsA）是一种免疫抑制药物，靶向亲环素，调节免疫系统的细胞辅因子。丙型肝炎病毒（HCV）的复制被CsA抑制，但这种抑制的分子基础仍然没有完全理解。为了研究这种抑制，我们培养的HCV复制子细胞（Con 1，HCV基因型1b，FLR-N细胞）在CsA的存在下，并获得了9个CsA耐药FLR-N细胞系。我们确定了所有九个克隆的全长HCV序列，并选择了九个克隆中在CsA存在下具有高复制活性的两个（克隆#6和#7）用于进一步分析。两个克隆均显示两个共有突变，一个在NS 3（T1280 V）中，另一个在NS 5A（D2292 E）中。各种突变体的表征表明，D2292 E突变赋予了对高浓度CsA（高达2 μM）的抗性。此外，错义突变T1280 V有助于集落形成活性的恢复。这些突变的影响在两个已建立的HCV复制子细胞系-HCV-RMT（[1]，基因型1a）和JFH 1（基因型2a）中也是明显的。此外，NS 5A中的其他三个错义突变-D2303 H、S2362 G和E2414 K-增强了D2292 E赋予的对CsA的抗性;这些双重或全部四重突变体可以抵抗比野生型Con 1高约8至25倍的CsA浓度。这四个突变，无论是单一或组合，也使Con 1菌株耐两个其他亲环素抑制剂，N-甲基-4-异亮氨酸-环孢菌素（NIM 811）或Debio-025。有趣的是，由这些突变中的每一种引起的IC 50值的变化在Debio-025处理的细胞中最低，表明其对适应性突变的抗性活性最高。
Cyclosporine A (CsA) is an immunosuppressive drug that targets cyclophilins, cellular cofactors that regulate the immune system. Replication of hepatitis C virus (HCV) is suppressed by CsA, but the molecular basis of this suppression is still not fully understood. To investigate this suppression, we cultured HCV replicon cells (Con1, HCV genotype 1b, FLR-N cell) in the presence of CsA and obtained nine CsA-resistant FLR-N cell lines. We determined full-length HCV sequences for all nine clones, and chose two (clones #6 and #7) of the nine clones that have high replication activity in the presence of CsA for further analysis. Both clones showed two consensus mutations, one in NS3 (T1280V) and the other in NS5A (D2292E). Characterization of various mutants indicated that the D2292E mutation conferred resistance to high concentrations of CsA (up to 2 μM). In addition, the missense mutation T1280V contributed to the recovery of colony formation activity. The effects of these mutations are also evident in two established HCV replicon cell lines—HCV-RMT ([1], genotype 1a) and JFH1 (genotype 2a). Moreover, three other missense mutations in NS5A—D2303H, S2362G, and E2414K—enhanced the resistance to CsA conferred by D2292E; these double or all quadruple mutants could resist approximately 8- to 25-fold higher concentrations of CsA than could wild-type Con1. These four mutations, either as single or combinations, also made Con1 strain resistant to two other cyclophilin inhibitors, N-methyl-4-isoleucine-cyclosporin (NIM811) or Debio-025. Interestingly, the changes in IC50values that resulted from each of these mutations were the lowest in the Debio-025-treated cells, indicating its highest resistant activity against the adaptive mutation.