杆状病毒衣壳蛋白VP39保守半胱氨酸残基在病毒复制包装中的作用机制

The long-term irrational use of chemical pesticides has caused a lot of problems such as ecological imbalance and environmental contamination. Baculoviruses are insect pathogens and are amongst the safest insecticides in bio-control with no effect on non-target organisms and environment. However, the use of baculovirus as insecticide has not reached its full potentials due to several unsolved problems such as undetermined mechanism of virus nucleocapsid assembly. The solving of how nucleocapsid assembles will contribute to the understanding of the speed-limit of virion packaging and the stability of virion structure, which is the foundation for the improvement of baculovirus insecticide. VP39 is the capsid protein that plays a key role during baculovirus nucleocapsid assembly but how it assembles the capsid structure is unclear. VP39 contains eight conserved cysteines and our preliminary data showed that the VP39 polymers in the capsid structure are formed by disulfide bonds. The aims of this proposal are to elucidate the functions of the conserved cysteines in VP39 by constructing recombinant viruses; to determine the disulfide bonds between VP39 polymers by non-reducing gels and Mass spectrometry; and to investigate the molecular mechanism of the effect on baculovirus replication cycle resulting from the mutations of these cysteines by means of yeast two hybrid, Co-immunoprecipitation, qPCR and RNA-Seq. The results of these studies will contribute important new information to the understanding of the mechanism of the nucleocapsid assembly and potentially lead to new strategies to improve baculovirus as bio-insecticide.

昆虫杆状病毒是安全有效的生物杀虫剂，但存在杀虫速度慢，杀虫谱窄等不足，其病毒核衣壳装配机制的不明确限制了进一步的改良和应用。为揭示核衣壳装配机制，阐释衣壳结构蛋白VP39组装衣壳的机理是关键基础。申请人初步研究表明VP39通过半胱氨酸残基之间的二硫键形成多聚体并组装成衣壳结构。为研究VP39在此过程的作用机制，本课题首先通过点突变技术构建重组病毒检测VP39保守半胱氨酸残基对病毒装配和杀虫效力的影响，然后结合非还原胶和蛋白质谱技术鉴定衣壳结构VP39多聚体中形成二硫键的半胱氨酸残基位点并分析二硫键在衣壳组装过程中的功能，最后通过酵母双杂交、免疫共沉淀、qPCR和RNA-Seq高通量测序等手段探究VP39半胱氨酸残基对病毒复制包装所产生影响的分子机理。本研究将解析VP39如何组装成衣壳结构，有助于理解病毒包装的限速步骤和病毒粒子结构稳定性因素，为杆状病毒杀虫剂的改良和完善提供新的思路和理论。

杆状病毒是一种广泛分布于自然界且专一性感染昆虫的病原微生物，在害虫的生物防治中具有广阔的应用前景，但也存在杀虫速度慢以及杀虫谱窄等不足之处，因此对杆状病毒进行改造可提高其在生物防治上的应用价值。由于杆状病毒复制包装的分子机制未明确，因此限制了其进一步的改良。本课题以杆状病毒主要衣壳蛋白VP39为出发点，对其组装核衣壳的机制进行研究。首先通过点突变技术将VP39的8个保守半胱氨酸分别突变，发现Cys18，Cys36和Cys49突变后VP39无法入核，也无法形成由二硫键搭建而成的VP39多聚体复合物，因此这三个点对病毒核衣壳的组装至关重要。此外，发现Cys132和Cys169这一对二硫键的缺失导致VP39只能形成二聚体复合物，而无法形成更高级别的多聚体复合物。虽然这对二硫键的缺失不影响病毒BV的产量，但是病毒核衣壳的组装出现了异常，核内形成大量长度异常的核衣壳结构，暗示VP39需要更高级别的多聚体复合物才能更有效的组装正常的核衣壳结构。后续通过质谱技术鉴定了VP39分子间的二硫键位点。为了更好的解析VP39入核组装衣壳的机制，对VP39蛋白的互作网络进行了探讨。通过pull-down实验和后续的质谱技术筛选出可能与VP39存在互作的蛋白并进行了CoIP验证，新发现了VP39的互作蛋白Pp78/83和C42，结合前人的研究结果，暗示VP39存在“VP39-38K-VP1054-C42-Pp78/83”的通路模式。最后通过原核表达纯化出可溶性VP39蛋白，在高压冷冻电镜下可观察到所纯化的VP39蛋白的颗粒，为进一步分析VP39的晶体结构提供了材料。为更好的解析杆状病毒核衣壳装配机制，还对Alpha杆状病毒特有基因pkip和ac34进行了功能研究，发现PKIP能促进核衣壳的装配，从而使得病毒粒子BV和OB的产量最大化；而Ac34能利用宿主蛋白SfMEF转运入核行使功能，从而有效的促进了病毒在核内的复制包装。本课题的研究结果为杆状病毒载体的改良提供了新的理论基础。