Visualizing flock house virus infection in Drosophila cells with correlated fluorescence and electron microscopy
Visualizing flock house virus infection in Drosophila cells with correlated fluorescence and electron microscopy
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DOI：
10.1016/j.jsb.2007.09.009
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发表时间：
2008-03-01
影响因子：
3
通讯作者：
Johnson, John E.
中科院分区：
文献类型：
--
作者：
Lanman, Jason;Crum, John;Johnson, John E.
病毒组装发生在一个复杂的环境中，依赖于病毒和细胞组件在时间和空间上的适当关联。鸡舍病毒(FHV)衣壳的简单性和广泛的结构、生化和基因特征使其成为研究体内病毒组装的极佳系统。四半胱氨酸基序(CCPGCC)可以诱导结合的双砷化合物(Flash和ReAsH)中的荧光，它被基因插入到外壳蛋白中，以显示病毒感染时的基因产物。与传统的荧光蛋白相比，这种修饰的尺寸很小，最大限度地减少了对外壳蛋白众多功能的破坏。ReAsH不仅在与四半胱氨酸基序结合时发出荧光，而且还允许在光转化和Os染色后对同一细胞进行相关电子显微镜(EM)观察。这些研究表明，外壳蛋白集中在细胞内的离散斑块中。感染细胞的高压冷冻(HPF)和冷冻置换(FS)表明，这些斑块是由结晶阵列中的病毒颗粒形成的。对制备的HPF/FS样品的电子显微镜断层扫描(EMT)显示，这些阵列位于高度修饰的线粒体的近端，先前被确定为RNA复制的位置。线粒体修饰的两个特征类似于排列在外膜上的60 nm球体和由整个细胞器中诱导的卷曲产生的大腔。由爱思唯尔公司出版。
Virus assembly occurs in a complex environment and is dependent upon viral and cellular components being properly correlated in time and space. The simplicity of the flock house virus (FHV) capsid and the extensive structural, biochemical and genetic characterization of the virus make it an excellent system for studying in vivo virus assembly. The tetracysteine motif (CCPGCC), that induces fluorescence in bound biarsenical compounds (FlAsH and ReAsH), was genetically inserted in the coat protein, to visualize this gene product during virus infection. The small size of this modification when compared to those made by traditional fluorescent proteins minimizes disruption of the coat proteins numerous functions. ReAsH not only fluoresces when bound to the tetracysteine motif but also allows correlated electron microscopy (EM) of the same cell following photoconversion and osmium staining. These studies demonstrated that the coat protein was concentrated in discrete patches in the cell. High pressure freezing (HPF) followed by freeze substitution (FS) of infected cells showed that these patches were formed by virus particles in crystalline arrays. EM tomography (EMT) of the HPF/FS prepared samples showed that these arrays were proximal to highly modified mitochondria previously established to be the site of RNA replication. Two features of the mitochondrial modification are similar to 60 nm spherules that line the outer membrane and the large chamber created by the convolution induced in the entire organelle. Published by Elsevier Inc.