Assembly and intracellular localization of the bluetongue virus core protein VP3
Assembly and intracellular localization of the bluetongue virus core protein VP3
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DOI：
10.1128/jvi.79.17.11487-11495.2005
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发表时间：
2005-09-01
影响因子：
5.4
通讯作者：
Roy, P
中科院分区：
文献类型：
--
作者：
Kar, AK;Iwatani, N;Roy, P
蓝舌病病毒（BTV）核心蛋白VP 3在病毒体组装和复制过程中起关键作用。虽然蛋白质的结构是很好的特点，少得多是已知的细胞内加工和定位的蛋白质在感染的宿主细胞。在BTV感染的细胞中，新合成的病毒核心颗粒以称为病毒包涵体（VIB）的结构聚集在宿主细胞内的特定位置，该结构主要由非结构蛋白NS 2组成。然而，核心蛋白的位置，在不存在的VIBs仍然不清楚。在这项研究中，我们研究了VP 3的位置和降解的情况下，任何其他病毒蛋白和NS 2或VP 3的天然相关蛋白，VP 7的存在下。为了能够在宿主细胞内实时跟踪和处理VP 3，合成了全功能增强型绿色荧光蛋白（EGFP）-VP 3嵌合体，并使用特异性标记物和抑制剂监测融合蛋白在不同细胞类型中的分布。在没有其他BTV蛋白的情况下，EGFP-VP 3表现出明显的细胞质焦点形成。进一步的证据表明，EGFP-VP 3靶向宿主细胞的蛋白酶体，但当加入特异性蛋白酶体抑制剂MG 132时，EGFP-VP 3分散在整个细胞质中。然而，嵌合EGFP-VP 3蛋白的分布发生了显着变化时，蛋白质表达的BTV核心蛋白VP 7的存在下，在BTV组装过程中的VP 3的正常伴侣。EGFP-VP 3和VP 7的相互作用以及随后的核心样颗粒的组装通过可视化荧光颗粒进一步检查，并通过生物化学分析和电子显微镜证实。这些数据表明EGFP-VP 3亚核心的正确组装，表明核心形成可以被真实的实时监测。当EGFP-VP 3在BTV感染的BSR细胞中表达时，该蛋白不与蛋白酶体相关，而是分布在BTV包涵体中，在那里它与NS 2共定位。这些发现扩展了我们对VP 3定位及其在宿主细胞内命运的了解，并说明了具有大氨基末端延伸的VP 3分子的组装能力。这也打开了作为一个交付系统的应用的可能性。
The bluetongue virus (BTV) core protein VP3 plays a crucial role in the virion assembly and replication process. Although the structure of the protein is well characterized, much less is known about the intracellular processing and localization of the protein in the infected host cell. In BTV-infected cells, newly synthesized viral core particles accumulate in specific locations within the host cell in structures known as virus inclusion bodies (VIBs), which are composed predominantly of the nonstructural protein NS2. However, core protein location in the absence of VIBs remains unclear. In this study, we examined VP3 location and degradation both in the absence of any other viral protein and in the presence of NS2 or the VP3 natural associate protein, VP7. To enable real-time tracking and processing of VP3 within the host cell, a fully functional enhanced green fluorescent protein (EGFP)-VP3 chimera was synthesized, and distribution of the fusion protein was monitored in different cell types using specific markers and inhibitors. In the absence of other BTV proteins, EGFP-VP3 exhibited distinct cytoplasmic focus formation. Further evidence suggested that EGFP-VP3 was targeted to the proteasome of the host cells but was dispersed throughout the cytoplasm when MG132, a specific proteasome inhibitor, was added. However, the distribution of the chimeric EGFP-VP3 protein was altered dramatically when the protein was expressed in the presence of the BTV core protein VP7, a normal partner of VP3 during BTV assembly. Interaction of EGFP-VP3 and VP7 and subsequent assembly of core-like particles was further examined by visualizing fluorescent particles and was confirmed by biochemical analysis and by electron microscopy. These data indicated the correct assembly of EGFP-VP3 subcores, suggesting that core formation could be monitored in real time. When EGFP-VP3 was expressed in BTV-infected BSR cells, the protein was not associated with proteasomes but instead was distributed within the BTV inclusion bodies, where it colocalized with NS2. These findings expand our knowledge about VP3 localization and its fate within the host cell and illustrate the assembly capability of a VP3 molecule with a large amino-terminal extension. This also opens up the possibility of application as a delivery system.