昆虫内吞体分选转运复合物III调控杆状病毒BV出芽释放分子机理的研究

In the life cycle, baculoviruses produce two phenotypes viruses: occlusion-derived virus (ODV) and budded virus (BV). ODV initiate viral infection of insect midgut and obtain the envelope from host cells nuclear membrane, whereas BV spread the secondary infection from cell-to-cell within insect tissues and in vitro cultured cell lines. The nucleocapsids of BV are enveloped while budding from the plasma membrane. Our previous studies revealed that the insect endosomal sorting complex required for transport (ESCRT)-III is involved in the budding and release of BV. However, the detailed mechanism of this involvement is not clear. In this grant proposal, we will investigate the mechanism of ESCRT-III in regulating the budding and release of BV by using protein-protein interaction detection methods and microscopic technologies. Initially, we will determine which BV-related viral proteins interacting with the subunits of ESCRT-III by using bimolecular fluorescent complementation (BiFC) and co-immunoprecipitation (Co-IP) assays. Then, we will determine the colocalization of ESCRT-III subunits, BV-related viral proteins, and the nucleocapsids of BV in infected insect cells using confocal microscopy. Finally, by utilizing transmission electronic microscopy (TEM), we will observe the distribution of the subunits of ESCRT-III at the budding and release regions of the nucleocapsids of BV at nuclear and plasma membranes. We will also evaluate the effect of inhibition of the activity of ESCRT-III on the budding and release efficiency of the nucleocapsids of BV. Together these proposed results we will try to elucidate the mechanism of ESCRT-III in regulating the budding and release of BV. This study will be important for understanding the infection mechanism of budded viruses of baculoviruses, and also be beneficial for improving the application of baculoviruses.

在感染周期中，杆状病毒产生包埋型病毒（ODV）和出芽型病毒（BV）。ODV初始感染昆虫中肠并从核膜获得囊膜。BV在细胞间进行次级感染，由质膜出芽时获得囊膜。前期研究发现，昆虫内吞体分选转运复合物III（ESCRT-III）与BV出芽释放有关，但具体作用机制尚不清楚。本项目采用蛋白质互作检测及显微技术开展ESCRT-III调控BV出芽释放机理研究。首先进行双分子荧光互补及免疫共沉淀分析确定哪些BV相关蛋白与ESCRT-III亚基互作；其次利用激光共聚焦显微镜探索ESCRT-III亚基、BV相关蛋白及BV核衣壳在细胞内的共定位关系；最后利用电子显微镜观察ESCRT-III亚基在BV出芽释放区域的分布及抑制ESCRT-III活性对BV经核膜和质膜出芽释放的影响，初步阐明ESCRT-III调控BV出芽释放机理。该项研究对于揭示杆状病毒BV感染机制有重要意义，为杆状病毒应用改良奠定基础。

在真核细胞内，内吞分选转运复合体（the endosomal sorting complex required for transport，ESCRT）由ESCRT-0，I，II，III及Vps4等五个复合体组成。其中，ESCRT-0-II负责分选，ESCRT-III剪切脂质膜，而Vps4则水解ATP促使ESCRT-III解聚和循环利用。前期的研究发现，过表达ESCRT-I、ESCRT-III或内吞体标志蛋白Rab5或Rab11的显性-负性突变体显著影响杆状病毒代表种苜蓿丫纹夜蛾核多角病毒（Autographa californica multiple nucleopolyhedrovirus, AcMNPV）的入侵或出芽释放。本项目通过RNA干扰进一步证实AcMNPV的入侵依赖于Rab5、Rab11、ESCRT-I以及ESCRT-III，而病毒出芽释放则仅依赖于ESCRT-III。蛋白质互作解析发现，65个AcMNPV蛋白与9个ESCRT-III亚基之间形成复杂的互作网络，而ESCRT-III核心亚基与病毒核心蛋白及核衣壳共定位于核膜区域，揭示病毒核心蛋白可能负责募集ESCRT-III亚基促进核衣壳释放。深入分析发现，病毒核心蛋白Ac93 的C-末端存在一个MIM1-like模序。该模序中保守的亮氨酸残基介导Ac93与ESCRT-III亚基互作，揭示Ac93在ESCRT-III募集过程中扮演重要角色。显微观察发现，ESCRT-III核心亚基Vps24与Snf7分布在病毒诱导的核膜和质膜特定区域，而抑制其功能显著降低子代病毒核衣壳经核膜或质膜的出芽释放。上述结果揭示ESCRT-III在杆状病毒BVs入侵和出芽释放过程中具有重要调控作用。