Biswas Siddhartha, Willis Leslie G, Fang Minggang, Nie Yingchao, Theilmann David A
Plant Science, Faculty of Land Food Systems, University of British Columbia, Vancouver, BC, Canada.
Summerland Research and Development Center, Agriculture and Agri-Food Canada, Summerland, BC, Canada.
J Virol. 2018 Jan 17;92(3). doi: 10.1128/JVI.01713-17. Print 2018 Feb 1.
During the infection cycle of Autographa californica multiple nucleopolyhedrovirus (AcMNPV), two forms of virions are produced, budded virus (BV) and occlusion-derived virus (ODV). Nucleocapsids that form BV have to egress from the nucleus, whereas nucleocapsids that form ODV remain inside the nucleus. The molecular mechanism that determines whether nucleocapsids remain inside or egress from the nucleus is unknown. AC141 (a predicted E3 ubiquitin ligase) and viral ubiquitin (vUbi) have both been shown to be required for efficient BV production. In this study, it was hypothesized that vUbi interacts with AC141, and in addition, that this interaction was required for BV production. Deletion of both and restricted viral infection to a single cell, and BV production was completely eliminated. AC141 was ubiquitinated by either vUbi or cellular Ubi, and this interaction was required for optimal BV production. Nucleocapsids in BV, but not ODV, were shown to be specifically ubiquitinated by vUbi, including a 100-kDa protein, as well as high-molecular-weight conjugates. The viral ubiquitinated 100-kDa BV-specific nucleocapsid protein was identified as AC66, which is known to be required for BV production and was shown by coimmunoprecipitation and mass spectrometry to interact with AC141. Confocal microscopy also showed that AC141, AC66, and vUbi interact at the nuclear periphery. These results suggest that ubiquitination of nucleocapsid proteins by vUbi functions as a signal to determine if a nucleocapsid will egress from the nucleus and form BV or remain in the nucleus to form ODV. Baculoviruses produce two types of virions called occlusion-derived virus (ODV) and budded virus (BV). ODVs are required for oral infection, whereas BV enables the systemic spread of virus to all host tissues, which is critical for killing insects. One of the important steps for BV production is the export of nucleocapsids out of the nucleus. This study investigated the molecular mechanisms that enable the selection of nucleocapsids for nuclear export instead of being retained within the nucleus, where they would become ODV. Our data show that ubiquitination, a universal cellular process, specifically tags nucleocapsids of BV, but not those found in ODV, using a virus-encoded ubiquitin (vUbi). Therefore, ubiquitination may be the molecular signal that determines if a nucleocapsid is destined to form a BV, thus ensuring lethal infection of the host.
在苜蓿银纹夜蛾多核多角体病毒（AcMNPV）的感染周期中，会产生两种形式的病毒粒子，即出芽型病毒（BV）和包涵体衍生病毒（ODV）。形成BV的核衣壳必须从细胞核中释放出来，而形成ODV的核衣壳则保留在细胞核内。决定核衣壳是留在细胞核内还是从细胞核中释放出来的分子机制尚不清楚。AC141（一种预测的E3泛素连接酶）和病毒泛素（vUbi）已被证明对高效产生BV都是必需的。在本研究中，推测vUbi与AC141相互作用，此外，这种相互作用是产生BV所必需的。删除两者会将病毒感染限制在单个细胞内，并且完全消除了BV的产生。AC141可被vUbi或细胞泛素泛素化，这种相互作用是最佳产生BV所必需的。结果表明，BV中的核衣壳而非ODV中的核衣壳被vUbi特异性泛素化，包括一种100 kDa的蛋白质以及高分子量缀合物。病毒泛素化的100 kDa BV特异性核衣壳蛋白被鉴定为AC66，已知它是产生BV所必需的，并且通过免疫共沉淀和质谱分析表明它与AC141相互作用。共聚焦显微镜还显示AC141、AC66和vUbi在核周边相互作用。这些结果表明，vUbi对核衣壳蛋白的泛素化作用作为一种信号，来决定核衣壳是从细胞核中释放出来形成BV还是留在细胞核中形成ODV。杆状病毒产生两种类型的病毒粒子，称为包涵体衍生病毒（ODV）和出芽型病毒（BV）。ODV用于经口感染，而BV使病毒能够全身扩散到所有宿主组织，这对于杀死昆虫至关重要。产生BV的一个重要步骤是核衣壳从细胞核中输出。本研究调查了使得选择核衣壳进行核输出而不是保留在细胞核内（在细胞核内它们会变成ODV）的分子机制。我们的数据表明，泛素化这一普遍的细胞过程，使用病毒编码的泛素（vUbi）特异性标记BV的核衣壳，而不标记ODV中的核衣壳。因此，泛素化可能是决定核衣壳是否注定形成BV的分子信号，从而确保对宿主的致命感染。
