Autographa californica Multiple Nucleopolyhedrovirus ac75 Is Required for the Nuclear Egress of Nucleocapsids and Intranuclear Microvesicle Formation
Autographa californica Multiple Nucleopolyhedrovirus ac75 Is Required for the Nuclear Egress of Nucleocapsids and Intranuclear Microvesicle Formation
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苜蓿银纹夜蛾多重核多角体病毒 ac75 是核衣壳核出口和核内微泡形成所必需的
DOI：
10.1128/jvi.01509-17
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发表时间：
2017-12
影响因子：
5.4
通讯作者：
Yang Kai
中科院分区：
文献类型：
--
作者：
Shi Anqi;Hu Zhaoyang;Zuo Yachao;Wang Yan;Wu Wenbi;Yuan Meijin;Yang Kai
苜蓿银纹夜蛾核型多角体病毒（Autographa californica multiple nucleopolyhedrovirus，AcMNPV）orf 75（ac 75）基因是一个高度保守的基因，其功能尚不清楚。在这项研究中，我们构建了一个ac 75敲除AcMNPV杆粒和ac 75在杆状病毒的生活周期中的作用进行了研究。Ac 75蛋白的表达和分布进行了表征，并分析了其与另一种病毒蛋白的相互作用，以进一步了解其功能。我们的数据表明，ac 75所需的核衣壳，核内微泡的形成，和随后的出芽病毒体（BV）的形成，以及闭塞衍生的病毒体（ODV）的装配和嵌入到多面体的ODV的核出口。Western印迹分析表明，两种形式，18和15 kDa的FLAG标记的Ac 75蛋白被检测到。Ac 75与BV的核衣壳和包膜组分相关，但仅与ODV的核衣壳组分相关; 18-kDa形式仅与BV相关，而15-kDa形式与两种类型的病毒体相关。Ac 75在感染过程中主要位于核内环区，并在感染早期表现出核边缘分布。相分离分析表明，Ac 75不是一个完整的膜蛋白。免疫共沉淀试验揭示了Ac 75和膜蛋白Ac 76之间的相互作用，双分子荧光互补试验确定了AcMNPV感染细胞的细胞质内和核膜和环区的相互作用的网站。我们的研究结果已经确定ac 75作为第二个基因，这是所需的核出口的核衣壳和核内微泡的形成。重要性在杆状病毒的生命周期中，芽殖病毒体（BV）和闭合衍生病毒体（ODV）的形态发生都涉及核膜上的芽殖过程，该过程发生在核衣壳从核中排出或产生核内微泡时。然而，病毒粒子形态发生的确切机制仍然未知。在这项研究中，我们确定ac 75作为第二个基因，除了ac 93，这是必不可少的核衣壳，核内微泡的形成，和随后的BV形成，以及ODV的表达和嵌入到多面体的ODV的核出口。Ac 75不是一个完整的膜蛋白。然而，它与一个完整的膜蛋白（Ac 76）相互作用，并与核膜相关。这些数据增强了我们对核衣壳的核出口和核内微泡形成之间的共性的理解，并可能有助于揭示杆状病毒病毒粒子形态发生机制的见解。
ABSTRACT Autographa californica multiple nucleopolyhedrovirus (AcMNPV) orf75 (ac75) is a highly conserved gene of unknown function. In this study, we constructed an ac75 knockout AcMNPV bacmid and investigated the role of ac75 in the baculovirus life cycle. The expression and distribution of the Ac75 protein were characterized, and its interaction with another viral protein was analyzed to further understand its function. Our data indicated that ac75 was required for the nuclear egress of nucleocapsids, intranuclear microvesicle formation, and subsequent budded virion (BV) formation, as well as occlusion-derived virion (ODV) envelopment and embedding of ODVs into polyhedra. Western blot analyses showed that two forms, of 18 and 15 kDa, of FLAG-tagged Ac75 protein were detected. Ac75 was associated with both nucleocapsid and envelope fractions of BVs but with only the nucleocapsid fraction of ODVs; the 18-kDa form was associated with only BVs, whereas the 15-kDa form was associated with both types of virion. Ac75 was localized predominantly in the intranuclear ring zone during infection and exhibited a nuclear rim distribution during the early phase of infection. A phase separation assay suggested that Ac75 was not an integral membrane protein. A coimmunoprecipitation assay revealed an interaction between Ac75 and the integral membrane protein Ac76, and bimolecular fluorescence complementation assays identified the sites of the interaction within the cytoplasm and at the nuclear membrane and ring zone in AcMNPV-infected cells. Our results have identified ac75 as a second gene that is required for both the nuclear egress of nucleocapsids and the formation of intranuclear microvesicles. IMPORTANCE During the baculovirus life cycle, the morphogenesis of both budded virions (BVs) and occlusion-derived virions (ODVs) is proposed to involve a budding process at the nuclear membrane, which occurs while nucleocapsids egress from the nucleus or when intranuclear microvesicles are produced. However, the exact mechanism of virion morphogenesis remains unknown. In this study, we identified ac75 as a second gene, in addition to ac93, that is essential for the nuclear egress of nucleocapsids, intranuclear microvesicle formation, and subsequent BV formation, as well as ODV envelopment and embedding of ODVs into polyhedra. Ac75 is not an integral membrane protein. However, it interacts with an integral membrane protein (Ac76) and is associated with the nuclear membrane. These data enhance our understanding of the commonalities between nuclear egress of nucleocapsids and intranuclear microvesicle formation and may help to reveal insights into the mechanism of baculovirus virion morphogenesis.
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