新型猪冠状病毒SADS-CoV入侵过程中S蛋白与受体识别机制的结构与功能研究

Since the beginning of the 21st century, coronaviruses have emerged as a serious threat to human health and economy. In recent years, a new coronavirus found in bats, named swine acute diarrhea coronavirus (SADS-CoV), has devastated pig farming industry in Southern China. The mechanisms underlying interaction between SADS-CoV (i.e. S protein) and its receptor, as well as subsequent viral entry, are not well understood. In terms of elucidating these processes, we have already solved a trimeric cryo-EM structure of extracellular domain of the viral S protein at 3.55 Å and carried out a preliminary screening of its receptor. Moreover, we observed that SADS-CoV can replicate in the human 293T cell line, implying that the virus may potentially infect humans. Based on these findings, we will use a CRISPR-Cas9 library to perform further screening and identify the S protein membrane receptor. The results of the screening will be verified by using SADS-CoV to infect different cell lines in which the putative receptor will be knocked out or overexpressed. We will also solve the structure of a complex between the S protein trimer or its receptor binding domain and the receptor. This structure will then be analyzed to identify key residues involved in the protein-protein interaction; the importance of these residues will be confirmed in vitro by measuring protein binding affinities with SPR and other methods. Our project will contribute to understanding of mechanisms underlying viral entry and difference/similarities between homologous receptors of different species, thus providing valuable information for determining zoonotic potential of SADS-CoV, as well as possible strategies for antiviral therapies and prevention.

21世纪以来冠状病毒给人类社会带来了巨大威胁，最近新发蝙蝠源猪冠状病毒（SADS-CoV）给华南养殖业造成重大损失，其入侵机制尚不清楚。本项目前期利用冷冻电镜解析了其S蛋白胞外域三聚体3.55Å的结构，并对其受体进行了初步筛选，申请人还发现该病毒可以在人293T细胞中复制，意味着它可能对人类具有潜在威胁。鉴于此，本项目拟继续通过基于CRISPR-Cas9文库的功能基因筛选技术鉴定该病毒的受体，并通过病毒感染受体敲除和过表达的细胞等方法进行验证；同时解析病毒S蛋白三聚体或受体识别区（RBD）与受体复合物结构，确定二者相互识别的关键位点，通过定点突变和SPR等蛋白互作测定手段进行体外验证，最终结合病毒抑制实验等技术从体内水平阐明二者的识别机制。本项目的开展不仅能为比较不同种属间该受体的异同点和深入理解病毒入侵机制提供帮助，还能为制订针对性防控措施和治疗手段及评估其跨种传播风险提供重要参考信息。

21世纪以来冠状病毒给人类社会带来了巨大威胁，2017年新发的蝙蝠源猪冠状病毒（SADS-CoV）给华南养殖业造成重大损失，同时还有研究发现该病毒可以在多种物种的细胞中复制，包括人293细胞，表明它可能对人类具有潜在威胁。冠状病毒依赖于其表面的S蛋白和宿主细胞表面受体完成入侵过程，然而该病毒S蛋白的结构还尚未解析，其入侵宿主细胞的受体及详细机制亦不清楚。本项目获得资助后，首先利用冷冻电镜解析了SADS-CoV S蛋白胞外域三聚体3.55Å的结构，并与其它冠状病毒S蛋白结构进行比对，阐明了其S蛋白结构特点和进化过程。然后通过基于CRISPR-Cas9文库的功能基因筛选技术对该病毒感染宿主细胞过程中入侵与复制相关的蛋白进行了筛选鉴定，获得了一些候选蛋白。因此，我们的研究不仅为深入理解冠状病毒不同亚型间S蛋白进化特点提供帮助，并为基于S蛋白的疫苗设计提供精确的结构信息，还为了解该病毒入侵过程中需要的宿主蛋白提供参考，也为评估其跨种传播风险提供参考信息。

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