Centre of Immunobiology, Blizard Institute, Queen Mary University of London, London.
Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom.
Cell Mol Gastroenterol Hepatol. 2021;11(4):1163-1175. doi: 10.1016/j.jcmgh.2020.11.012. Epub 2020 Nov 26.
BACKGROUND & AIMS: Despite recent advances in antiviral therapy for hepatitis C virus (HCV), a proportion of patients with genotype 3 (G3) HCV infection do not respond to current all oral treatment regimens. Genomic analyses have identified key polymorphisms correlating with increased resistance to direct-acting antivirals. We previously reported that amino the acid polymorphism, A150V, in the polymerase (NS5B) of G3 HCV reduces response to sofosbuvir. We now demonstrate that this polymorphism alters the response to interferon alpha.
Quantitative polymerase chain reaction, immunofluorescence, luciferase activity assay, immunoblotting, and flow cytometry were used to study the antiviral effect of interferon (IFN) on DBN G3 HCV-infected cells and G3 HCV replicons.
We show the presence of the A150V polymorphism markedly reduces the response to IFN alpha (IC of S52_WT = 1.162 IU/mL and IC of S52_A150V = 14.45 IU/mL, 12.4-fold difference). The induction of IFN-stimulated genes in A150V replicon cells is unaffected, but nuclear localization of active protein kinase R (PKR) is reduced. Blockade of PKR activity reduced the antiviral effect of IFN on wild-type replicons, whereas augmented PKR activation promoted the antiviral effect of IFN on A150V replicons. Furthermore, we show that impaired activation of PKR in A150V replicon cells diminishes cellular apoptosis.
These results demonstrate that polymorphisms reducing response rates to direct-acting antivirals may function beyond conferring drug resistance by modulating the intrinsic cellular antiviral response.
尽管丙型肝炎病毒（HCV）的抗病毒治疗在最近取得了进展，但一部分基因型 3（G3）HCV 感染患者对目前所有口服治疗方案没有反应。基因组分析已经确定了与直接作用抗病毒药物耐药性增加相关的关键多态性。我们之前报道过 G3 HCV 聚合酶（NS5B）中的氨基酸多态性 A150V 降低了对索非布韦的反应。我们现在证明这种多态性改变了对干扰素α的反应。
采用定量聚合酶链反应、免疫荧光、荧光素酶活性测定、免疫印迹和流式细胞术研究干扰素（IFN）对 DBN G3 HCV 感染细胞和 G3 HCV 复制子的抗病毒作用。
我们发现 A150V 多态性的存在显著降低了对 IFNα的反应（S52_WT 的 IC = 1.162 IU/mL，S52_A150V 的 IC = 14.45 IU/mL，相差 12.4 倍）。A150V 复制子细胞中 IFN 刺激基因的诱导不受影响，但活性蛋白激酶 R（PKR）的核定位减少。PKR 活性阻断降低了 IFN 对野生型复制子的抗病毒作用，而增强的 PKR 激活促进了 IFN 对 A150V 复制子的抗病毒作用。此外，我们还表明，A150V 复制子细胞中 PKR 激活受损会减少细胞凋亡。
这些结果表明，降低直接作用抗病毒药物反应率的多态性可能通过调节细胞内在的抗病毒反应来发挥作用，而不仅仅是赋予耐药性。
