鸡β-半乳糖凝集素蛋白-1在新城疫病毒感染中的作用及其分子机制

Newcastle disease virus (NDV) is an important pathogen which causes significant economic losses to the poultry industry, the contributions of host factors during the course of NDV infection remain largely unknown. Galectin-1 has been extensively reported as an important immune regulation factor, in recent years, it has become clear that galectin-1 could also bind the envelope glycoprotein of viruses to influence the process of virus infection. However, the roles of chicken Galectin-1 in the course of virus infection have not been reported. In this study, we explore the potential roles of chicken Galectin-1B（CG-1B）during NDV infection, which was up-regulated in NDV- infected chickens. The CG-1B recombinant protein will be expressed in vitro and purified, the impact of CG-1B on NDV hemagglutination activity will be further detected. An ELISA based method will be developed to demonstrate the direct binding between CG-1B and NDV virion. Furthermore, protein interaction methods would be applied to identify the NDV proteins which interact with CG-1B, mutant NDVs will be constructed through reverse genetics to determine the specific sites on NDV proteins that bind to CG-1B. The impacts of extracellular and intracellular CG-1B on NDV replication will be determined, and the mechanism of extracellular CG-1B in the course of NDV adsorption and intracellular CG-1B in the course of process and transport of NDV proteins would be further revealed, and then elucidates the biological roles of CG-1B during NDV infection and its molecular mechanisms.

新城疫病毒（NDV）是严重危害家禽养殖业的病原体，而宿主因子在NDV感染中的作用知之甚少。β-半乳糖凝集素蛋白-1（Galectin-1）是一种免疫调控分子，并能与病毒糖蛋白结合从而影响其感染细胞的进程，禽Galectin-1在病毒感染中的作用尚未有报道。本项目选择NDV感染后上调表达的chicken Galectin-1B（CG-1B）为研究对象，探索CG-1B在NDV感染中的作用机制。通过体外表达获得CG-1B，检测其对NDV血凝活性的影响。建立ELISA检测CG-1B与NDV之间的直接结合。通过蛋白相互作用筛选与CG-1B相互作用的NDV蛋白，利用反向遗传构建突变NDV，确定与CG-1B相互作用的位点。检测CG-1B对NDV复制的影响，揭示细胞外CG-1B在NDV吸附细胞进程中、细胞内CG-1B在NDV蛋白成熟运输进程中的作用，从而阐明CG-1B在NDV感染中的作用及其分子机制。

本团队前期研究发现，NDV弱毒株La Sota感染鸡后能够诱导靶器官气管中Chicken galectin-1B（CG-1B）的表达水平升高。由于NDV表面含有糖基化修饰的囊膜蛋白，而β-半乳糖凝集素蛋白（galectin）具有能够与糖蛋白结合的特性，因此我们推测鸡源的β-半乳糖凝集素蛋白CG-1B有与NDV的糖蛋白结合从而参与NDV感染过程的潜在可能。本研究中，我们发现NDV强毒株F48E9感染同样能够引起CG-1B在靶器官中的上调表达。此外，ELISA检测结果显示CG-1B能够与NDV强毒株F48E9、弱毒株La Sota直接结合，并且能够在体外抑制二者的红细胞凝集活性。蛋白相互作用检测结果表明CG-1B是与F48E9和La Sota中的Hemagglutinin-neuraminidase（HN）糖蛋白相结合的。通过反向遗传技术构建获得HN糖蛋白糖基化位点缺失的突变NDV La Sota-G1、-G2、-G3、-G4和-G12，与CG-1B进行相互作用检测，结果表明HN糖蛋白中的G4 N-glycans在CG-1B与HN糖蛋白之间的相互作用中起着至关重要的作用。体外细胞感染试验结果表明细胞上清中加入CG-1B后能够与HN糖蛋白结合抑制NDV的吸附过程，而对吸附之后的进入过程没有影响。并且CG-1B可能通过结合细胞表面的HN糖蛋白从而抑制子代病毒的释放。而细胞内表达的CG-1B能够与HN糖蛋白相互作用，导致HN糖蛋白在细胞表面的表达水平减少，从而抑制子代病毒的产生。此外，为分析CG-1B表达水平与NDV复制的相关性，分别测定了亲本病毒和HN蛋白糖基化位点缺失的突变病毒在SPF鸡胚、鸡靶器官、CG-1B过表达和敲低表达的LMH细胞中的生长曲线，结果表明NDV的复制水平与CG-1B的表达水平是负相关的。上述结果表明CG-1B通过结合NDV HN糖蛋白中的N-glycans发挥抗NDV活性。本研究首次表明在NDV的感染过程中，鸡的galectin-1B通过调节病毒-宿主之间的相互作用从而参与宿主的抗病毒防御反应。