HCV核心蛋白变异致干扰素应答抵抗的位点定位及机制研究

In consideration of the critical role of IFN in treatment for chronic hepatitis C (CHC) and the fact that mutations of HCV core protein relate to IFN resistance. The project aims to focus on the mechanisms of HCV core protein site/struction mutation induced IFN resistance in three groups (SVR group,no-response group,relapse group). The analysis of HCV core protein quasispecies and frameshift coding phenomenon in CHC patients will be carryied out by clone sequencing and software prediction. The locating suspicious sites in core protein that lead to IFN resistance by generating mutations and chimeric replicon though gene directional modification and Parallel displacement, variant-core-protein transfer into cells, use technologies of expression profile chip, mass spectroscopy and electron microscopy to investigate the molecular mechanism of core protein induced IFN resistance. We hope to identify the hypothesis that functional sites which affect IFN response existing in HCV core protein, and further validate mutation of these sites resist to IFN response by the following three ways: generating quasispecies of F protein and core protein by spontaneous mutation, which two proteins compete with each other; reversible posttranslational modification happened in lipid microenvironment; change of space structure of HCV core protein. The common effect pathways are response reactions that influence the interac groups(SVR group,no-response group, relapse group), through clone sequencing and software prediction analysis of HCV core protein quasispecies and drift coding phenomenon in CHC patients, and locating suspicious sites in core protein that lead to IFN resistance by generating mutations and chimeric replicon though gene directional modification and parallel displacement, then investigate the molecular mechanism of core protein induced IFN resistance. we hope to test the hypothesis that functional sites that affect IFN response exist in HCV core protein, and further validate these sites affect IFN response by the following three ways: generating quasispecies that producing F protein and core protein(two proteins that compete with each other) by spontaneous mutation; reversible posttranslational modification happened in lipid microenvironment; change of space structure of HCV core protein. Then they influent the interaction between core functional sites and anti-viral effector molecules in IFN response pathway, leading to various response to IFN between individuals or before and after treatment in one case. Finally, a prediction model for the sensitivity of IFN response will be set up, and it will be applied as an individual assessment before therapy for CHC patients.

鉴于IFN在抗HCV治疗中的核心地位及HCV核心蛋白变异致干扰素应答抵抗的事实，本项目拟重点探讨核心蛋白位点/结构变异致IFN应答抵抗的机制，在SVR、无应答、复发的三个CHC患者队列中，通过克隆测序及软件预测分析CHC患者HCV核心蛋白准种和移码编码现象；通过基因定向修饰或平行置换的方法制造突变体及嵌合复制子定位核心蛋白致IFN应答抵抗的位点；通过芯片、质谱和电镜技术探讨核心蛋白变异致IFN应答抵抗的分子机制。验证假设：HCV核心蛋白存在一组影响IFN应答的功能位点，通过①自发突变产生相互竞争的F蛋白与核心蛋白准种；或②在脂质微环境中发生可逆的翻译后修饰及③空间结构改变，共同效应通路是影响其与IFN通路中抗病毒效应分子的相互作用，使个体之间、同一个体治疗前后出现有差别的应答反应。为开发IFN应答敏感性的预测模型并最终用于CHC患者治疗前的个体化测评、指导再治疗决策提供依据。

鉴于IFN 在抗HCV 治疗中的核心地位及HCV 核心蛋白变异致干扰素应答抵抗的事实，本项目计划重点探讨核心蛋白位点/结构变异致IFN 应答抵抗的机制，在SVR、无应答、复发的三个CHC 患者队列中。. 本课题2014年启动，按照课题任务进行了大量的实验研究工作，也取得了一些进展和成果。我们建立了HCV 1型，2/3型，6型患者的临床资料、辅助检查结果、样本血清的数据库，进行了干扰素应答抵抗情况在HCV 2/3、6型患者间的回顾性分析研究，并发表了文章；在HCV核心蛋白的实验研究中，我们发现在hcv-1b型感染患者中，血清mir-93-5p水平升高，在hcv-1b清除后降至正常水平。但是，与健康的受试者相比，产生干扰素抵抗的患者，mir-93-5p水平持续增加。在Huh 7细胞中研究发现，HCV 1b核心蛋白增加了mir-93-5p的表达从而诱导失活了IFN的信号通路。进一步研究显示，IFNAR1被证实是miR-93-5p调控的靶基因，表明了miR-93-5p-IFNAR1信号轴调节了干扰素信号通路,这个信号轴可能是HCV 1b感染中的一个潜在治疗靶点。该部分的研究已发表文章；由于医学的进步和发展，在课题执行中期，临床上针对HCV的治疗出现了新的手段，即直接抗病毒药物，该类药物相比IFN对HCV患者具有更高的应答率和治愈率, 直接取代了IFN在HCV治疗中的核心地位，随着这类药物的上市和应用，目前针对HCV的治疗方案发生了巨大变化，所以本课题针对IFN治疗HCV产生应答抵抗机制的研究将变得无实用价值。鉴于此，课题组讨论后进行了研究方向的调整，即HCV广泛中和抗体的研究,淘选出2株与HCV E2高保守线性表位高度特异性结合的抗体。目前文章已撰写完毕，正在投稿过程中；. 本课题培养硕士研究生2名，博士研究生2名。