E基因变异的MERS-CoV亚基因组形成机制及其功能研究

Recently, WHO published that MERS-CoV was one of the top emerging diseases likely to cause major epidemics. We previously found that two deletion subgenomes of MERS-CoV were detected in the first imported patient in China in additional to the wild type (WT) genome. The deletion region of subgenomes located at ORF5 and E gene, which could result in truncation or/and fusion of ORF5 and E protein. According to the references and lab results, the viruses of deletion variants might be defective because the propagation was deficient due to lack of E gene. Currently, the mechanism of subgenomes generation and their effect on the capacity of replication and propagation and the pathogenicity of wild type virus are unclear. In this program, we will take the advantage of technologies, such as infectious clone, virus rescue and deep sequencing, to investigate the mechanism of subgenomes generation from extrinsic factors (host cell types, infection titration of virus and passage numbers ) and intrinsic factor (RNA secondary structure of virus); to reveal the effect on the capacity of replication and propagation and the pathogenicity of wild type virus. Solving these problems will contribute to understand the mechanism of replication and subgenomes generation of MERS-CoV; to determine the interfering effect of defective virus on pathogenicity of the wild type virus; to learn about the action of the defective virus on the process of MERS disease; to explore the novel measure to prevent and cure MERS.

MERS是近期WHO指出的8种最危险的传染病之一。本课题组前期测定中国第一例输入性MERS-CoV序列时发现了两个亚基因组序列（ORF5和E基因之间分别存在419bp和414bp的缺失，致ORF5和E蛋白的截短或/和融合）。研究表明E基因缺失的MERS-CoV不能传代。至今尚未见到缺陷MERS-CoV的报道。目前这种序列缺失的机制、缺陷病毒对野生型病毒的作用不明。本项目拟采用感染性克隆构建、病毒拯救、二代测序等技术从外在因素（病毒接种滴度、细胞类型、传代次数）和内在因素（病毒RNA的二级结构）研究该亚基因组形成的影响因素，探讨E基因缺陷的MERS-CoV在复制、传代能力和致病性方面对野生型病毒的影响。本项目的研究结果，将有助于：揭示该亚基因组产生的分子机制；确定E基因缺陷的MERS-CoV对野生型病毒的作用；了解其产生对疾病过程可能的影响；为MERS-CoV的预防和治疗提供新思路。

通过本项目研究，我们发现了MERS-CoV的变异体，在ORF5基因与E基因之间存在缺失，导致ORF5基因截短和ORF5基因与部分E基因融合变异，通过生物信息学分析，这种变化可能由于自身的二级结构引发的复制跳越产生。通过基因重组技术，我们成功构建了MERS CoV基因组全长感染性克隆和变异体的克隆；为后续病毒致病性研究打下基础。运用探针和基因转技术、免疫共沉淀、质谱技术等发现MERS CoV病毒E蛋白主要定位于内质网和高尔基体内，与细胞中的TIMM50蛋白相互作用，具有诱导细胞自噬的作用。克隆MERS CoV的ORF5野生型和两种变异体，转染至细胞，发现ORF5基因在细胞内表达较为复杂，在细胞内表达量少；其变异体在细胞内具有多个翻译起始点。通过双荧光素酶分析发现ORF5具有一定的IRES功能启动后续基因的表达。运用免疫共沉淀、质谱技术，发现ORF5蛋白与细胞内PHB2相互作用，最终定位于溶酶体，诱导细胞的自噬，抑制干扰素系统的激活。这些功能可能与MERS CoV的致病性密切相关。