Gastrointestinal Unit, Department of Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA.
J Virol. 2012 Aug;86(16):8581-91. doi: 10.1128/JVI.00533-12. Epub 2012 Jun 6.
Responses to alpha interferon (IFN-α)-based treatment are dependent on both host and viral factors and vary markedly among patients infected with different hepatitis C virus (HCV) genotypes (GTs). Patients infected with GT3 viruses consistently respond better to IFN treatment than do patients infected with GT1 viruses. The mechanisms underlying this difference are not well understood. In this study, we sought to determine the effects of HCV NS5A proteins from different genotypes on IFN signaling. We found that the overexpression of either GT1 or GT3 NS5A proteins significantly inhibited IFN-induced IFN-stimulated response element (ISRE) signaling, phosphorylated STAT1 (P-STAT1) levels, and IFN-stimulated gene (ISG) expression compared to controls. GT1 NS5A protein expression exhibited stronger inhibitory effects on IFN signaling than did GT3 NS5A protein expression. Furthermore, GT1 NS5A bound to STAT1 with a higher affinity than did GT3 NS5A. Domain mapping revealed that the C-terminal region of NS5A conferred these inhibitory effects on IFN signaling. The overexpression of HCV NS5A increased HCV replication levels in JFH1-infected cells through the further reduction of levels of P-STAT1, ISRE signaling, and downstream ISG responses. We demonstrated that the overexpression of GT1 NS5A proteins resulted in less IFN responsiveness than did the expression of GT3 NS5A proteins through stronger binding to STAT1. We confirmed that GT1 NS5A proteins exerted stronger IFN signaling inhibition than did GT3 NS5A proteins in an infectious recombinant JFH1 virus. The potent antiviral NS5A inhibitor BMS-790052 did not block NS5A-mediated IFN signaling suppression in an overexpression model, suggesting that NS5A's contributions to replication are independent of its subversive action on IFN. We propose a model in which the binding of the C-terminal region of NS5A to STAT1 leads to decreased levels of P-STAT1, ISRE signaling, and ISG transcription and, ultimately, to preferential GT1 resistance to IFN treatment.
对α干扰素（IFN-α）治疗的反应取决于宿主和病毒因素，并且在感染不同丙型肝炎病毒（HCV）基因型（GT）的患者中差异很大。感染 GT3 病毒的患者对 IFN 治疗的反应始终优于感染 GT1 病毒的患者。这种差异的机制尚不清楚。在这项研究中，我们试图确定来自不同基因型的 HCV NS5A 蛋白对 IFN 信号的影响。我们发现，与对照相比，GT1 或 GT3 NS5A 蛋白的过表达均显著抑制 IFN 诱导的干扰素刺激反应元件（ISRE）信号、磷酸化 STAT1（P-STAT1）水平和 IFN 刺激基因（ISG）表达。GT1 NS5A 蛋白表达对 IFN 信号的抑制作用强于 GT3 NS5A 蛋白表达。此外，GT1 NS5A 与 STAT1 的结合亲和力高于 GT3 NS5A。结构域映射表明 NS5A 的 C 末端区域赋予了这些对 IFN 信号的抑制作用。HCV NS5A 的过表达通过进一步降低 P-STAT1、ISRE 信号和下游 ISG 反应，增加了 JFH1 感染细胞中的 HCV 复制水平。我们证明，通过与 STAT1 的更强结合，GT1 NS5A 蛋白的过表达导致比 GT3 NS5A 蛋白的表达更低的 IFN 反应性。我们证实，在感染性重组 JFH1 病毒中，GT1 NS5A 蛋白比 GT3 NS5A 蛋白发挥更强的 IFN 信号抑制作用。强效抗病毒 NS5A 抑制剂 BMS-790052 并未在过表达模型中阻断 NS5A 介导的 IFN 信号抑制，这表明 NS5A 对复制的贡献与其对 IFN 的颠覆性作用无关。我们提出了一种模型，其中 NS5A 的 C 末端区域与 STAT1 的结合导致 P-STAT1、ISRE 信号和 ISG 转录水平降低，最终导致 GT1 对 IFN 治疗的耐药性增加。
