III型干扰素与自噬蛋白作为丙型肝炎病毒复制和表达的宿主应答因子的研究

Hepatitis C virus (HCV）is a main causative factor for virus hepatitis, cirrhosis and hepatocellular carcinomas in the world. Over ten years, the therapy has been aiming at HCV oneself in clinic. However, HCV often escaped the anti-HCV drug attack by frequent variation of itself gene sequences and led to failure of the drug treatment ? drug-tolerance emergence, and also followed the serious toxicity. Thus, a new attractive strategy for new drug research and development become a hot focus, which is taking host factors as the targets of anti-HCV drugs, including to find new host factors that can respond to HCV infection and to reveal the interaction mechanism between the host factors and HCV. This project is planning to study action mechanisms of two kinds of new candidate host factors ?Interferon lambda subfamily (ATG5, mainly) and autophagy related proteins (ATG5 mainly) on HCV RNA sub-replicon and HCV-IRES mediated protein production. In both systems of cell level and zebrafish, we will perform experiments on correlation of HCV, autophagy and IFN?s in interfering of gene expression and their functions, and also on their co-localization on the membrane web induced by HCV, involving in how the host gene promoters, control on HCV-IRES, gene knockdown and overexpression, and deletion or mutation of gene coding sequence interfere to HCV RNA replication and gene expression. The better understanding for the both host factors as HCV-specific responsive factors will facilitate confirmation of the new anti-HCV drug targets. Also we are interested in IFN?s' medicinal potential and will provided substantial data for the research and development of the new types of anti-HCV drugs.

HCV是导致全球病毒性肝炎、肝硬化和肝癌的重要病因之一。数十年来，临床抗HCV感染的治疗手段是针对病毒本身靶位；而病毒可通过其自身基因频繁的变异来逃避药物的攻击，使基于这类靶位的药物失效而产生耐药性，且存在严重毒性等问题。为此，基于宿主因子靶位的新药研发策略受到重视，发现具有成药靶性的宿主因子及与病原因子相互作用机制，成为研究热点。本项目对两类新的潜在的HCV感染相关的宿主因子-IFN?s和自噬蛋白的作用机制进行研究，在细胞水平和斑马鱼个体两个层面，通过HCV-自噬-IFN?三者之间的基因表达干扰与功能相关性和HCV诱导膜网的共定位研究，包括宿主基因启动子、HVC-IRES调控、基因干扰及编码区突变等对HCV 复制和表达的影响，了解这两类宿主因子是否是HCV基因组复制和基因表达的应答因子、是否具有成药靶性，以及IFN?s是否具有潜在的成药性，为新型抗HCV药的研发提供基础性和关键性依据。

HCV是导致全球病毒性肝炎、肝硬化和肝癌的重要病因之一。数十年来，临床抗HCV感染的治疗手段是针对病毒本身靶位；而病毒可通过其自身基因频繁的变异来逃避药物的攻击，使基于这类靶位的药物失效而产生耐药性，且存在严重毒性等问题。为此，基于宿主因子靶位的新药研发策略受到重视，发现具有成药靶性的宿主因子及与病原因子相互作用机制，成为研究热点。本项目对两类潜在的HCV感染相关的宿主因子—IFNs和自噬蛋白的作用机制进行研究，在细胞水平和斑马鱼个体两个层面，通过HCV-自噬-IFN三者之间的基因表达干扰与功能相关性研究，以及HCV蛋白与自噬蛋白和IFN2的共定位研究，发现了自噬蛋白ATG10和ATG10S具有差异性的抗HCV作用；ATG10，一个主流的ATG10自噬蛋白可通过促进自噬体形成、并使得自噬体停留于细胞质外周，导致自噬流受阻，HCV得以在自噬膜网上大量复制。而ATG10S，一个非主流的较短的ATG10蛋白，可活化III型干扰素和与病毒双链RNA相关的先天免疫基因如ddx-58, tlr-3, tlr-7, irf-3 和 irf-7的表达，并通过直接结合并驱动自噬体接近围绕细胞核的溶酶体，促进自噬溶酶体的形成，导致HCV基因组复制子降解。这两种亚型的ATG10蛋白对HCV亚复制子斑马鱼模型具有与细胞HCV模型相同的差异性作用。另一重要发现是III型干扰素IFNλ2可直接与ATG10S结合，促进自噬体与溶酶体的融合。IFNλ2不论是细胞内表达的蛋白还是作用于细胞膜受体的外源性蛋白均可有效抑制HCV的复制。HCV NS5A基因的过表达可促使细胞提高IL28A和两个自噬泛素化酶基因ATG3、ATG5、ATG7、ATG10的表达；但NS5A可通过与IL28A结合，阻止自噬溶酶体的形成。NS5A缺失干扰素敏感区序列(ISDR)后，则失去与IL28A结合的能力，解除了NS5A对自噬流的阻止。采用基因序列系列缺失和点突变方法，探索ATG10两种亚型差异性功能的分子机制。发现位于ATG10第44位和135位的半胱氨酸是两个关键氨基酸。这两氨基酸的缺失或替代，均可导致ATG10S样的功能转变，包括IL28A与溶酶体的共定位。ATG10S可作为新的抗HCV 复制的宿主因子并可作为具有新机制的抗HCV新药筛选的靶标。