Differential Sensitivity of 5′UTR-NS5A Recombinants of Hepatitis C Virus Genotypes 1-6 to Protease and NS5A Inhibitors
Differential Sensitivity of 5′UTR-NS5A Recombinants of Hepatitis C Virus Genotypes 1-6 to Protease and NS5A Inhibitors
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DOI：
10.1053/j.gastro.2013.11.009
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发表时间：
2014-03-01
期刊：
影响因子：
29.4
通讯作者：
Bukh, Jens
中科院分区：
文献类型：
--
作者：
Li, Yi-Ping;Ramirez, Santseharay;Bukh, Jens
背景与目的：丙型肝炎病毒(HCV)的治疗将受益于直接作用的抗病毒药物在感染培养系统中的临床前评估，以测试对不同病毒基因型的影响。我们构建了包含1-6和2a(JFH1)NS5B-3‘非翻译区的5’非翻译区-NS5A(5-5A)的重组体，并检测了NS3蛋白酶和NS5A抑制剂对这些重组体的影响。方法：对先前发现NS3解旋酶(F1464L)、NS4A(A1672S)和NS5B(D2979G)突变的丙型肝炎病毒5-5A重组体进行改造和改进，加入额外的恢复突变以增加其在Huh7.5细胞中的繁殖。在复制感染的Huh7.5细胞中测定每种DAA试剂的浓度-反应曲线。结果：所构建的1a(H77)、1a(TN)、3a(S52)、4a(ED43)、5a(SA13)和6a(HK6a)5-5A重组体在病毒传代后不需要在药物靶向的NS3蛋白酶或NS5A结构域-I区发生突变。NS3蛋白水解酶抑制剂telaprevir、bocprevir、asunaprevir、simeprevir、vaniprevir、fordaprevir和MK-5172以及NS5A抑制剂达拉塔韦均以浓度依赖的方式抑制它们的生长。1a(TN)5-5A和JFH1非依赖的全长病毒对DAA试剂的敏感性相似，证实了5-5A重组体在DAA检测中作为全长病毒的替代品。与1a(TN)全长病毒相比，3a(S52)5-5A重组体对所有酶抑制剂高度耐药，4a(ED43)重组体对替拉维韦和博西普韦高度抗性，但对其他酶抑制剂最敏感。与其他蛋白水解酶抑制剂相比，MK-5172对所有的丙型肝炎病毒都有很强的抑制作用。NS5A抑制剂达拉塔韦的效力最高，但其活性与基因型有关。结论：突变F1464L、A1672S和D2979G可使高效的丙型肝炎病毒重组体的形成，该重组体包含了基因特异性的50个非翻译区-NS5A(5-5A)，其中包括天然的NS3蛋白酶和NS5A结构域-I药物靶点。适配的5-5A重组体的强劲复制允许将NS3蛋白酶和NS5A抑制剂与1-6型丙型肝炎病毒株进行直接比较。
BACKGROUND & AIMS: Hepatitis C virus (HCV) therapy will benefit from the preclinical evaluation of direct-acting antiviral (DAA) agents in infectious culture systems that test the effects on different virus genotypes. We developed HCV recombinants comprising the 5' untranslated region-NS5A (5-5A) from genotypes 1-6 and 2a(JFH1) NS5B-3' untranslated region, and tested the effects of NS3 protease and NS5A inhibitors on these recombinants. METHODS: The HCV 5-5A recombinants with previously identified mutations in the NS3-helicase (F1464L), NS4A (A1672S), and NS5B (D2979G) were adapted and improved, by incorporating additional recovered mutations that increased their propagation in Huh7.5 cells. Concentration-response profiles were determined for each DAA agent in replicate infected Huh7.5 cells. RESULTS: Developed efficient 1a(H77), 1a(TN), 3a(S52), 4a(ED43), 5a(SA13), and 6a(HK6a) 5-5A recombinants did not require mutations after viral passage in the NS3 protease or NS5A domain-I regions targeted by the drugs. They were inhibited in a concentration-dependent manner by the NS3 protease inhibitors telaprevir, boceprevir, asunaprevir, simeprevir, vaniprevir, faldaprevir, and MK-5172 and by the NS5A inhibitor daclatasvir. The 1a(TN) 5-5A and JFH1-independent full-length viruses had similar levels of sensitivity to the DAA agents, validating the 5-5A recombinants as surrogates for full-length viruses in DAA testing. Compared with the 1a(TN) full-length virus, the 3a(S52) 5-5A recombinant was highly resistant to all protease inhibitors, and the 4a(ED43) recombinant was highly resistant to telaprevir and boceprevir, but most sensitive to other protease inhibitors. Compared with other protease inhibitors, MK-5172 had exceptional potency against all HCV genotypes. The NS5A inhibitor daclatasvir had the highest potency observed, but with genotype-dependent activity. CONCLUSIONS: The mutations F1464L, A1672S, and D2979G permitted the development of efficient HCV recombinants comprising genotype-specific 50 untranslated region-NS5A (5-5A), which include the natural NS3 protease and NS5A domain-I drug targets. The robust replication of adapted 5-5A recombinants allowed for direct comparison of NS3 protease and NS5A inhibitors against HCV strains of genotypes 1- 6.