Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases and Clinical Research Centre, Copenhagen University Hospital, Hvidovre, Kettegaard Alle 30, DK-2650 Hvidovre, Denmark.
J Virol. 2011 Sep;85(17):8913-28. doi: 10.1128/JVI.00049-11. Epub 2011 Jun 22.
To facilitate genotype-specific high-throughput studies of hepatitis C virus (HCV), we have developed reporter viruses using JFH1-based recombinants expressing core-nonstructural protein 2 (NS2) of genotype 1 to 7 prototype isolates. We introduced enhanced green fluorescent protein (EGFP) into NS5A domain III of the genotype 2a virus J6/JFH1 [2a(J6)]. During Huh7.5 cell culture adaptation, 2a(J6)-EGFP acquired a 40-amino-acid (aa) (Δ40) or 25-aa (Δ25) deletion in NS5A domain II, rescuing the impairment of viral assembly caused by the EGFP insertion. Δ40 conferred efficient growth characteristics to 2a(J6) tagged with EGFP, DsRed-Express2, mCherry, or Renilla luciferase (RLuc), yielding peak supernatant infectivity titers of 4 to 5 log(10) focus-forming units (FFU)/ml. 2a(J6) with Δ40 or Δ25 was fully viable in Huh7.5 cells. In human liver chimeric mice, 2a(J6)-EGFPΔ40 acquired various deletions in EGFP, while 2a(J6)Δ40 did not show an impaired viability. We further developed panels of JFH1-based genotype 1 to 7 core-NS2 recombinants expressing EGFP- or RLuc-NS5AΔ40 fusion proteins. In cell culture, the different EGFP recombinants showed growth characteristics comparable to those of the nontagged recombinants, with peak infectivity titers of 4 to 5 log(10) FFU/ml. RLuc recombinants showed slightly less efficient growth characteristics, with peak infectivity titers up to 10-fold lower. Overall, the EGFP and RLuc recombinants were genetically stable after one viral passage. The usefulness of these reporter viruses for high-throughput fluorescence- and luminescence-based studies of HCV-receptor interactions and serum-neutralizing antibodies was demonstrated. Finally, using RLuc viruses, we showed that the genotype-specific core-NS2 sequence did not influence the response to alfa-2b interferon (IFN-alfa-2b) and that genotype 1 to 7 viruses all responded to treatment with p7 ion channel inhibitors.
为了便于针对丙型肝炎病毒 (HCV) 的基因型进行高通量研究，我们使用基于 JFH1 的重组体开发了报告病毒，这些重组体表达了 1 至 7 型原型分离株的核心-非结构蛋白 2 (NS2)。我们将增强型绿色荧光蛋白 (EGFP) 引入基因型 2a 病毒 J6/JFH1 [2a(J6)] 的 NS5A 结构域 III 中。在 Huh7.5 细胞培养适应过程中，2a(J6)-EGFP 在 NS5A 结构域 II 中获得了 40 个氨基酸 (aa) (Δ40) 或 25 个氨基酸 (Δ25) 的缺失，从而挽救了 EGFP 插入引起的病毒装配受损。Δ40 赋予 2a(J6) 标记 EGFP、DsRed-Express2、mCherry 或 Renilla 荧光素酶 (RLuc) 的高效生长特性，上清液感染性滴度峰值达到 4 至 5 log(10) 焦点形成单位 (FFU)/ml。2a(J6) 中的 Δ40 或 Δ25 在 Huh7.5 细胞中完全具有活力。在人肝嵌合小鼠中，2a(J6)-EGFPΔ40 在 EGFP 中获得了各种缺失，而 2a(J6)Δ40 并未显示出活力受损。我们进一步开发了基于 JFH1 的基因型 1 至 7 的核心-NS2 重组体，这些重组体表达 EGFP-或 RLuc-NS5AΔ40 融合蛋白。在细胞培养中，不同的 EGFP 重组体表现出与未标记重组体相当的生长特性，感染性滴度峰值达到 4 至 5 log(10) FFU/ml。RLuc 重组体的生长特性略低，但感染性滴度峰值低 10 倍。总体而言，这些报告病毒在经过一次病毒传代后具有遗传稳定性。证明了这些报告病毒在 HCV-受体相互作用和血清中和抗体的高通量荧光和发光研究中的有用性。最后，我们使用 RLuc 病毒表明，基因型特异性核心-NS2 序列不会影响对 alfa-2b 干扰素 (IFN-alfa-2b) 的反应，并且基因型 1 至 7 的病毒均对 p7 离子通道抑制剂的治疗有反应。
