猪瘟病毒新型细胞受体的鉴定及其介导病毒入侵的分子机制

Classical swine fever virus (CSFV) is the causative agent of classical swine fever, a devastating disease of pigs worldwide. Previously, we identified several cellular proteins (including poly(C)-binding protein 1, thioredoxin 2, guanylate-binding protein 1, etc.) that interact with CSFV proteins and regulate viral replication, and demonstrated that the laminin receptor (LamR) acts as an attachment receptor alternative to heparan sulfate (HS). To date, however, the cellular receptor(s) that mediate CSFV entry into susceptible cells and the underlying mechanisms are poorly elucidated. The aim of this proposal is to: 1) high-throughput screen cellular receptor(s) for CSFV using siRNA library along with luciferase-expressing recombinant CSFV, lentivirus-packaged cDNA library of swine peripheral blood mononuclear cells in combination with neomycin-expressing recombinant CSFV, virus overlay protein blot assay, and glycan chip; 2) investigate the interplay between the identified receptor(s) and the known attachment receptors (HS and LamR) interacting with the envelope glycoproteins of CSFV using the Biacore platform; 3) map the domain(s) of the screened receptor(s) interacting with the viral envelope proteins using receptor reconstruction assay, blocking assay with antisera against receptor(s) or soluble receptor proteins, RNA interference, co-immunoprecipitation, GST pulldown, in situ proximity ligation assay, and bimolecular fluorescence complementation assay; 4) resolve the three-dimensional structure of the receptor-ligand complexes using X-ray diffraction, nuclear magnetic resonance spectroscopy, and cryo-electron microscopy; 5) identify the steps of virus attachment and entry, and the contributory roles of the receptor(s) using attachment, fusion, and internalization assays; and 6) generate a highly productive cell line stably expressing the receptor(s). This project is expected to provide scientific data for better understanding of CSFV infection and entry, to present targets for novel antivirals, and to establish a robust cell culture system for production of high-titer CSFV vaccines.

猪瘟是严重危害养猪业的一种烈性传染病，其病原为猪瘟病毒（CSFV）。此前我们发现了多个参与调控CSFV复制的宿主细胞蛋白，并证实层粘连蛋白受体是继硫酸乙酰肝素之后的另一吸附受体，但介导CSFV入侵细胞的受体及其分子机制尚不清楚。本项目拟通过siRNA文库结合荧光素酶标记CSFV、cDNA表达文库结合新霉素抗性基因标记CSFV、病毒铺覆蛋白印迹及糖芯片技术，高通量筛选CSFV新型受体；借助Biacore平台分析新型受体与已知受体的关系；通过X射线晶体衍射、核磁共振和冷冻电镜技术解析CSFV囊膜蛋白与受体分子相互作用的结构基础；借助动态激光共聚焦技术示踪CSFV感染过程；利用吸附、膜融合和内化试验分析受体介导CSFV入侵细胞的具体通路；构建高表达受体细胞系提高CSFV的增殖效率。本项目将揭示CSFV的入侵机制，为设计抗病毒策略提供新靶标，并为猪瘟疫苗提供高效生产平台。

猪瘟是严重危害养猪业的一种烈性传染病，其病原为猪瘟病毒（CSFV）。此前我们发现层粘连蛋白受体是CSFV的另一吸附受体，但介导CSFV入侵细胞的受体及其分子机制尚不清楚。本项目利用猪源细胞膜蛋白siRNA文库结合表达荧光素酶报告基因的CSFV（rCSFV-Rluc）进行高通量筛选，发现原癌基因酪氨酸激酶（MERTK）下调后可明显抑制rCSFV-Rluc的增殖，进一步应用CSFV石门株进行了验证。PK-15细胞中过表达MERTK后，明显促进了CSFV的感染。可溶性MERTK蛋白以及抗MERTK抗体处理细胞后，能明显地抑制CSFV的感染。进一步研究发现，MERTK与CSFV囊膜蛋白E2相互作用并促进CSFV的内化，表明MERTK是CSFV入侵的关键宿主因子。本研究还发现CSFV经由网格蛋白介导的内吞途径侵入宿主细胞，此过程需要胆固醇的参与。CSFV侵入宿主细胞后，利用宿主分子OASL、MEK2拮抗I型IFN和JAK-STAT信号通路促进病毒复制；宿主分子RNF114通过泛素化NS4B进而拮抗CSFV复制，阐明了CSFV细胞内复制的分子调控机制。此外，我们发现，E2蛋白既参与CSFV的吸附，又介导了CSFV的内化过程；利用纯化的重组E2蛋白进行了蛋白结晶，获得分辨率为4 Å的晶体；发现CSFV强弱毒株E2蛋白的第986位氨基酸糖基化修饰存在差异，将CSFV强毒株中引入该糖基化修饰可导致其体外复制水平和体内致病力下降，深入研究表明，该糖基化修饰改变后影响了病毒蛋白二聚体的形成和病毒扩散。总之，本项目鉴定了CSFV入侵的关键宿主因子，明确了CSFV的入侵通路，解析了CSFV入侵以及细胞内复制的分子调控机制，揭示了E2蛋白关键糖基化位点修饰在病毒感染和致病过程中的关键作用，为研发抗病育种靶标和快速开发减毒疫苗提供科学依据。