灰飞虱肠蛋白在水稻条纹病毒穿越肠屏障传播中的作用机理

Most plant viruses are transmitted by insect vector. During horizontal transmission of persistent-transmitted virus, virus always initials its infection by invading the insect midgut epithelium and overcomes the gut barrier, which is the first barrier of vectors, to spread into the heamocoel. Various gut molecules facilitate the virus infection and dissemination via specific interactions with the virus particle or its capsid protein (CP). This proposal takes Rice stripe virus (RSV) - Laodelphax striatellus interaction as research model, investigating the function of the L. striatellus gut proteins in RSV infecting and escaping the gut barrier. In specific, we will first identify L. striatellus midgut proteins that interact with RSV, and then address the protein function in RSV infection and dissemination. The previous work, by co-immunoprecipitation of L. striatellus total proteins and RSV particles, has identified interaction proteins and established the protein database. Those that were confirmed induction by RSV infection and preferential expression in midgut were selected. For each of the selected L. striatellus gut protein, we will perform RNAi experiment to address the gene function and alter proteins that have function in RSV transmission for the further study. We will then confirm the relationship of gut protein and RSV, by expressing the corresponding recombinant proteins to retest the interaction with RSV, and by determining the in vivo localization of gut protein and RSV. Based on the detailed phenotype of each protein and the predicted function domain of each protein, further physiological and biochemical experiments would be performed to clarify the working mechanism for RSV transmission through gut barrier. The conclusion of this study would hopefully contribute to the mechanisms for RSV transmission within the vector.

大多数植物病毒依赖昆虫介体进行传播。持久型传播的病毒在水平传播时，昆虫的肠壁是其传播的第一道屏障，病毒初始感染及克服肠屏障进入血腔的过程涉及病毒和介体之间复杂的分子互作。项目拟以水稻条纹病毒（Rice stripe virus, RSV）-介体昆虫灰飞虱为模型，探索肠蛋白在RSV穿越肠壁运输中的作用机制。前期工作通过免疫共沉淀鉴定了灰飞虱中与RSV互作的蛋白，选定了9个受RSV诱导，并在肠中特异性表达的蛋白为本课题研究对象。项目拟通过dsRNA介导的基因沉默实验确定蛋白在RSV建立感染及跨肠传播中的功能，并筛选功能蛋白做机制研究；通过体外表达的肠蛋白与RSV之间的特异性蛋白互作实验和对两者在组织及细胞水平的定位研究，验证两者之间存在的互作关系。根据肠蛋白发挥作用的具体过程及功能预测，进一步解析肠蛋白在RSV穿越肠屏障运输时发挥作用的分子机制。研究结果将部分填补RSV水平传播研究的空白。

植物病毒侵染昆虫介体细胞时，往往需要借助内吞过程完成，而介导昆虫体内病毒的侵染细胞的受体知之甚少。本课题对水稻条纹病毒（Rice stripe virus，RSV）侵染其昆虫介体灰飞虱的细胞过程进行了研究。课题主要对侵染肠细胞及卵巢细胞的过程进行了研究，鉴定了介导RSV侵染细胞的重要的蛋白，并且发现了卵黄蛋白受体（vitellogenin receptor, VgR）参与了病毒的侵染过程。卵黄蛋白（vitellogenin , Vg）是昆虫卵巢发育的重要营养蛋白，由VgR介导的内吞作用从血淋巴向卵内运输。我们前期研究发现RSV和Vg互作，利用Vg的入卵过程侵染卵巢，从而实现卵传。本项目工作通过电镜实验发现，RSV侵染卵巢细胞时，病毒在细胞膜表面聚集，形成内吞小泡，从细胞表面向细胞质中的卵黄体运输的各个运输阶段，都和VgR定位在一起。干扰了VgR表达时，病毒的入卵传播水平显著下降。工作确定了VgR受体介导的Vg运输过程，协助了RSV的入卵传播过程，完善了对RSV卵传机制的研究，建立了完整的RSV垂直传播的分子模型。RSV借助已有的物质运输途径及其受体入细胞传播，是一种新型的病毒运输途径，在载体生物学研究中有突破性进展。以建立的RSV-Vg-VgR互作运输途径为靶标，设计干扰机制，对于有效抑制病毒传播有重要的指导意义。本项工作2019年发表在了Phil. Trans. R. Soc. B上。