Autographa californica Nucleopolyhedrovirus Ac76: a Dimeric Type II Integral Membrane Protein That Contains an Inner Nuclear Membrane-Sorting Motif
Autographa californica Nucleopolyhedrovirus Ac76: a Dimeric Type II Integral Membrane Protein That Contains an Inner Nuclear Membrane-Sorting Motif
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DOI：
10.1128/jvi.02392-13
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发表时间：
2014-01-01
影响因子：
5.4
通讯作者：
Yang, Kai
中科院分区：
文献类型：
--
作者：
Wei, Denghui;Wang, Yan;Yang, Kai
我们的前期研究表明苜蓿银纹夜蛾核型多角体病毒（Autographa californica nucleopolyhedrovirus，AcMNPV）的ac 76基因对出芽病毒体（Budded virion，BV）和闭合源病毒体（occlusion derived virion，ODV）的发育都是必需的。更重要的是，ac 76的缺失影响核内微泡的形成。然而，ac 76影响病毒粒子形态发生的确切作用仍然未知。在这份报告中，我们的特点Ac 76的表达，分布和拓扑结构，以进一步了解Ac 76在病毒形态发生中的功能作用。Ac 76含有一个α-螺旋跨膜结构域，相分离表明它是一个完整的膜蛋白。在AcMNPV感染的细胞中，检测到Ac 76作为稳定的二聚体，其对SDS和热变性具有抗性，并且仅检测到痕量的单体。免疫共沉淀试验表明，Ac 76的二聚化的高亲和力的自我协会。纯化的病毒粒子和它们的核衣壳和包膜组分的蛋白质印迹分析表明，Ac 76与BV和ODV的包膜组分相关。免疫电镜显示Ac 76定位于质膜、内质网、核膜、核内微泡和ODV包膜。Ac 76的氨基酸15至48被鉴定为非典型的内核膜分选基序，因为它足以在不存在病毒感染的情况下将融合蛋白靶向ER和核膜，并在感染期间靶向核内微泡和ODV包膜。Ac 76的拓扑结构分析表明，Ac 76是一个II型的膜整合蛋白与N端暴露于细胞质和C端隐藏在内质网腔。
Our previous study showed that the Autographa californica nucleopolyhedrovirus (AcMNPV) ac76 gene is essential for both budded virion (BV) and occlusion-derived virion (ODV) development. More importantly, deletion of ac76 affects intranuclear microvesicle formation. However, the exact role by which ac76 affects virion morphogenesis remains unknown. In this report, we characterized the expression, distribution, and topology of Ac76 to further understand the functional role of Ac76 in virion morphogenesis. Ac76 contains an alpha-helical transmembrane domain, and phase separation showed that it was an integral membrane protein. In AcMNPV-infected cells, Ac76 was detected as a stable dimer that was resistant to SDS and thermal denaturation, and only a trace amount of monomer was detected. A coimmunoprecipitation assay demonstrated the dimerization of Ac76 by high-affinity self-association. Western blot analyses of purified virions and their nucleocapsid and envelope fractions showed that Ac76 was associated with the envelope fractions of both BVs and ODVs. Immunoelectron microscopy revealed that Ac76 was localized to the plasma membrane, endoplasmic reticulum (ER), nuclear membrane, intranuclear microvesicles, and ODV envelope. Amino acids 15 to 48 of Ac76 were identified as an atypical inner nuclear membrane-sorting motif because it was sufficient to target fusion proteins to the ER and nuclear membrane in the absence of viral infection and to the intranuclear microvesicles and ODV envelope during infection. Topology analysis of Ac76 by selective permeabilization showed that Ac76 was a type II integral membrane protein with an N terminus exposed to the cytosol and a C terminus hidden in the ER lumen.