Forum 02/19/2009

“Visualizing Flock House Virus assembly in Drosophila cells with electron microscope tomography”

Jason Lanman – Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA

irus assembly occurs in a complex cellular environment and is dependent upon both 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. Fluorescent microscopy and electron microscope tomography (EMT) were used to investigate the spatial organization of FHV assembly in the cell. The virus particles consist of a coat protein and two viral RNA segments. RNA 1 encodes for the RNA dependent RNA polymerase (RdRp), which forms spherules on the outer mitochondrial membrane. These spherules are the location for RNA replication. Additionally, the mitochondria are convoluted in on themselves to form large chambers lined with these spherules. RNA2 encodes the coat protein, which forms the T=3 icosahedral viral capsid. Following assembly, the virus particles form paracrystaline arrays in the cytoplasmic space. Using new sample preparation approaches and tomographic imaging technologies we determined the three-dimensional structure of the RdRp protein complex inside these spherules. In these tomograms, the RdRp complex was observed associated with small filaments, and the paracrystaline arrays were associated with large filament bundles (~200 to 300 nm). To our knowledge this is the first time such filaments have been observed associated with viral factories. Our current model is that the virus coat protein is synthesized in close vicinity to the modified mitochondria, binds to the filaments (potentially facilitating assembly) and is then trafficked on the filament bundles to a central location to form viral arrays.

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