杆状病毒核衣壳装配相关顺式作用元件的鉴定和功能研究

Baculovirus transfer vectors have unique advantages in numerous aspects, such as biosafety, transduction efficacy, and gene capacity. However, the ambiguous mechanism of baculovirus nucleocapsid assembly limits the improvement and further application of the baculovirus vectors. Previous studies generally focus on proteins that are essential for the nucleocapsid assembly. For better understanding of the relevant molecular mechanism, the current project aims to identify the cis-acting elements that mediate the packaging of baculovirus genomes into the capsids. During serial passage, viruses usually produce defective genomes in which packaging elements are always retained. Thus, by constructing recombinant plasmids, we plan to determine which segment of baculovirus defective genome has the ability to mediate the packaging of a reporter gene into capsids. Subsequently, the sufficiency and necessity of the identified packaging elements will be investigated by using gene knockout, electronic microscopy, and so on. Furthermore, electrophoretic mobility shift assay, DNA pull-down, and chromatin immunoprecipitation-sequencing will be used to investigate the interaction between the packaging elements and related proteins. The aim of this project is to identify packaging elements in the baculovirus genome, to determine the sufficiency and necessity of these elements, and to identify protein factors that interact with the elements. Our research will be a valuable and important reference for studies on the replication mechanism of baculovirus and other large circular dsDNA viruses, and may be an inspiring clue for revealing the evolutionary relationship of these DNA viruses. The research on baculovirus packaging element will also provide novel ideas and theoretical basis for improving baculovirus transfer vectors.

杆状病毒基因转导载体安全、高效、容量大，但其核衣壳装配机制的不明确限制了该类载体的改良和应用。与过去只针对相关蛋白因子的研究不同，本课题的目标是鉴定和分析杆状病毒基因组中参与核衣壳装配的顺式作用元件。病毒经连续传代后产生的缺损型基因组中常保留其装配元件。本课题将通过构建重组质粒，研究杆状病毒缺损型基因组中各片段介导报告基因装配至病毒核衣壳的能力。以基因敲除和电子显微镜观察等手段验证鉴定到的元件在病毒核衣壳装配的作用，确定顺式作用元件在功能上的充分性和必需性。再通过电泳迁移率实验、DNA pull-down和ChIP-seq等检测核酸-蛋白互作的技术，鉴定与该元件相互作用的蛋白因子以及研究病毒装配元件与相关蛋白的相互作用。本研究对揭示杆状病毒及其他大型环状双链DNA病毒的复制机制和进化关系有重要参考意义，也可为改良杆状病毒基因转导载体提供新思路和和理论基础。

杆状病毒衣壳呈杆状，其长短可以随着病毒基因组的大小而变化。目前一般病毒载体的容量仅为5-8 kb，而杆状病毒基因组大小在130 kbp左右，并可容纳38 kbp的外源片段插入，所以杆状病毒是具有相当应用潜力的基因转导载体。虽然参与杆状病毒基因组装配的蛋白元件大都已被鉴定，但装配信号，即位于病毒基因组上的装配相关顺式作用元件一直未被发现。本研究以杆状病毒的模式种苜蓿丫纹夜蛾核多角体病毒（Autographa californica multiple nucleopolyhedrovirus, AcMNPV）为材料，主要研究内容包括：检验杆状病毒基因组中是否存在特定的核衣壳装配元件；对杆状病毒的装配元件进行特征描述；鉴定与杆状病毒装配元件相互作用的蛋白；以及杆状病毒装配元件和相关蛋白相互作用机制的初探。我们发现：AcMNPV基因组中存在特定的核衣壳装配元件。该元件位于病毒基因ac83的nt 1651-1850区域，是一段大小为200 bp的富含A/T的序列。由于该序列是AcMNPV核衣壳正常装配所必需的，但不是充分的，故命名为病毒核衣壳装配必需元件(nucleocapsid assembly-essential element, NAE)。病毒蛋白VLF-1可特异性地与NAE结合。上述部分结果已发表在Journal of Virology杂志（影响因子=4.32，中科院一区）【2017, Volume 91 Issue 5 e02110-16】。本研究所报道得DNA元件是在大型环状DNA病毒中鉴定到的首个核衣壳装配元件。其结果有助于阐明DNA病毒的复制机制和进化关系，揭示杆状病毒核衣壳的装配机制，简化杆状病毒基因转导载体的构建，增加杆状病毒载体的外源片段容量。