寄主亚细胞膜及脂类组分参与烟草坏死病毒TNV-A粒子装配的机制研究

Plant plus-strand RNA virus particle assembly is coupled with replication. It was observed under electron microscope that sub-cellular membranes participate in plant plus-strand RNA viruses particle assembly by enclosing many viral particles inside membrane pockets, which were often found in vacuole or cytoplasm. However the molecular and cellular mechanism of such observation did not receive enough attention by scientists. In this project, we aim to understand this observation in cellular and biochemical level by using Tobacco necrosis virus-A (TNV-A) as a model virus. First, bioinformatics analysis of the coat protein sequence from TNV-A and other close related viruses in Tombusviridae identified a hydrophobic domain and a closely positioned sterol binding motif. Mutagenesis analysis on the sterol binding motif suggested that it is required for efficient viral particle assembly, cytoplasm localization and the ability to infect systemic leaves of plant. With ours and other people's finding, it is likely that sub-cellular membranes play a positive role in plant positive strand RNA virus particle assembly. This scientific question will be studied in several ways: 1) identify the membrane origin of the membrane pockets observed under electron microscope; 2) testing the binding ability of TNV-A CP with membranes and various lipids; 3) find important host factors that involve in making the membrane pockets that enclose lots of viral particles; 4) establish yeast system to assemble TNV-A particles from which the cellular pathways involved in viral particle assembly will be identified. Our study promises to answer an unsolved question in plant virology study and will contribute to understand the complex virus-host interaction process.

植物正义RNA病毒粒子装配和基因组复制是偶联的。早期对植物正义RNA病毒侵染的植物细胞的电镜研究发现，一部分病毒粒子往往被亚细胞膜包裹，这类膜结构存在于液泡或者细胞质中。但对亚细胞膜及脂类参与病毒粒子装配的机制并不清楚。我们通过对烟草坏死病毒A (TNV-A)和同科不同病毒的生物信息学分析发现，此类病毒的外壳蛋白（coat protein, CP）均含有一段位置保守的疏水区域以及一个脂类/固醇（sterol）结合序列。功能实验证明此固醇结合序列决定病毒粒子的正确包装与细胞质定位。结合此前的研究以及我们的初步结果，我们认为亚细胞膜在番茄丛矮病毒科病毒粒子的装配过程中起重要的作用。本项目利用番茄丛矮病毒科（Tombusviridae）的TNV-A为研究材料，来研究亚细胞膜及脂类组分参与植物病毒粒子装配的过程和机理。

植物正义链RNA病毒粒子的装配及成熟需要细胞组分的参与。我们以烟草坏死病毒中国大豆分离物（Tobacco necrosis virus A Chinese isolate, TNV-AC）为研究材料，对其CP蛋白与脂分子互作的研究发现，CP可以特异性地结合磷脂酰肌醇磷酸酯（phosphatidylinositol phosphates ,PIPs），特别是PI45P。利用免疫荧光分析发现病毒粒子在细胞核与细胞质均有分布，并且特异性地富集在VAP27标记的ER与PM接触位点附近。透射电镜观察TNV-AC侵染的本氏烟叶片发现病毒侵染早期病毒粒子部分地被包裹在类似ER结构的膜中。同时电镜观察也发现TNV-AC诱导了大量的胞外囊泡产生，通过对叶片胞外洗脱物的分析发现其中有大量病毒粒子，进一步的遗传学发现VPS4调节病毒粒子及胞外囊泡的分泌。我们发现TNV-AC诱导的胞外囊泡中含有病毒双链RNA，将胞外囊泡提纯后注射接种了病毒的本氏烟植株，接种植物的系统性症状大大减轻，提示病毒诱导的胞外囊泡可以诱导植物对病毒的系统性抗性。本研究揭示了一个膜组分参与病毒粒子包装和分泌，及病毒胞外囊泡参与植物系统型抗性的模型，对病毒与植物互作过程有了更深入理解。