期刊
JOURNAL OF STRUCTURAL BIOLOGY
卷 161, 期 3, 页码 439-446出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.jsb.2007.09.009
关键词
electron microscope tomography; correlated fluorescence and electron microscopy; in vivo virus assembly
资金
- NCRR NIH HHS [P41 RR004050, RR004050] Funding Source: Medline
- NIGMS NIH HHS [R37 GM034220-21, F32 GM074536-02, R37 GM034220-23, F32 GM074536, R01 GM034220, R37 GM034220-25, GM034220, F32 GM074536-01, R37 GM034220, GM074536, R01 GM072881, R01 GM053491, R37 GM034220-22, R01 GM065937, GM072881, R37 GM034220-24, GM53491] Funding Source: Medline
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.
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