Role of VP30 Phosphorylation in Ebola Virus Nucleocapsid Assembly and Transport
Role of VP30 Phosphorylation in Ebola Virus Nucleocapsid Assembly and Transport
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VP30 磷酸化在埃博拉病毒核衣壳组装和运输中的作用
DOI：
10.1128/jvi.01083-22
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发表时间：
2022
影响因子：
5.4
通讯作者：
Noda Takeshi
中科院分区：
文献类型：
--
作者：
Takamatsu Yuki;Yoshikawa Tomoki;Kurosu Takeshi;Fukushi Shuetsu;Nagata Noriyo;Shimojima Masayuki;Ebihara Hideki;Saijo Masayuki;Noda Takeshi
埃博拉病毒（EBOV）VP30通过改变其磷酸化状态来调节病毒基因组的转录和复制。然而，VP30磷酸化和去磷酸化在其他病毒复制过程如核衣壳和病毒体组装中的重要性尚不清楚。有趣的是，VP30主要被病毒包涵体中的细胞磷酸酶去磷酸化，而它在释放的病毒粒子中被磷酸化。因此，关于核衣壳中VP 30磷酸化如何实现以及VP 30磷酸化是否在病毒复制的后续步骤中提供任何优势的不确定性已经出现。在本研究中，为了表征VP30磷酸化在核衣壳形成中的作用，我们使用了电子显微镜分析和活细胞成像系统。我们发现VP30定位于核蛋白（NP）周围的核衣壳形成螺旋结构的突起的表面，这表明参与核衣壳的组装和运输。有趣的是，VP30磷酸化促进了其与核衣壳样结构（NCLS）的结合。相反，VP30磷酸化不影响NCLS离开和返回病毒包涵体的转运特性和数量，表明VP30的磷酸化状态不是NCLS离开的先决条件。此外，VP30的磷酸化状态并没有引起真正的EBOV感染细胞中的核衣壳转运的重大差异。在随后的出芽步骤中，VP30的结合及其磷酸化状态不影响病毒样颗粒的出芽效率。综上所述，EBOV可能利用VP30的磷酸化作用与核衣壳选择性结合，而不影响核衣壳转运和病毒体出芽过程。核衣壳为病毒基因组转录和复制提供模板。因此，了解其形成背后的调控机制对于开发新的治疗方法非常重要。在此之前，我们建立了一个活细胞成像系统的基础上异位表达的病毒荧光融合蛋白，允许可视化和表征胞质内转运的核帽样结构。EBOV VP30是病毒基因组合成的必需转录因子，尽管其在病毒基因组转录和复制中的作用已被充分理解，但VP30磷酸化在核衣壳组装中的功能重要性仍不清楚。我们的工作确定了VP30在皱褶核衣壳表面的定位，这与EBOV感染细胞中聚合酶的定位不同。这项研究揭示了VP30磷酸化在核衣壳组装中的新作用，这是病毒体形成的重要先决条件。
Ebola virus (EBOV) VP30 regulates viral genome transcription and replication by switching its phosphorylation status. However, the importance of VP30 phosphorylation and dephosphorylation in other viral replication processes such as nucleocapsid and virion assembly is unclear. Interestingly, VP30 is predominantly dephosphorylated by cellular phosphatases in viral inclusions, while it is phosphorylated in the released virions. Thus, uncertainties regarding how VP30 phosphorylation in nucleocapsids is achieved and whether VP30 phosphorylation provides any advantages in later steps in viral replication have arisen. In the present study, to characterize the roles of VP30 phosphorylation in nucleocapsid formation, we used electron microscopic analyses and live cell imaging systems. We identified VP30 localized to the surface of protrusions surrounding nucleoprotein (NP)-forming helical structures in the nucleocapsid, suggesting the involvement in assembly and transport of nucleocapsids. Interestingly, VP30 phosphorylation facilitated its association with nucleocapsid-like structures (NCLSs). On the contrary, VP30 phosphorylation does not influence the transport characteristics and NCLS number leaving from and coming back into viral inclusions, indicating that the phosphorylation status of VP30 is not a prerequisite for NCLS departure. Moreover, the phosphorylation status of VP30 did not cause major differences in nucleocapsid transport in authentic EBOV-infected cells. In the following budding step, the association of VP30 and its phosphorylation status did not influence the budding efficiency of virus-like particles. Taken together, it is plausible that EBOV may utilize the phosphorylation of VP30 for its selective association with nucleocapsids, without affecting nucleocapsid transport and virion budding processes.IMPORTANCEEbola virus (EBOV) causes severe fevers with unusually high case fatality rates. The nucleocapsid provides the template for viral genome transcription and replication. Thus, understanding the regulatory mechanism behind its formation is important for the development of novel therapeutic approaches. Previously, we established a live-cell imaging system based on the ectopic expression of viral fluorescent fusion proteins, allowing the visualization and characterization of intracytoplasmic transport of nucleocapsid-like structures. EBOV VP30 is an essential transcriptional factor for viral genome synthesis, and, although its role in viral genome transcription and replication is well understood, the functional importance of VP30 phosphorylation in assembly of nucleocapsids is still unclear. Our work determines the localization of VP30 at the surface of ruffled nucleocapsids, which differs from the localization of polymerase in EBOV-infected cells. This study sheds light on the novel role of VP30 phosphorylation in nucleocapsid assembly, which is an important prerequisite for virion formation.
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