It is widely accepted that significant differences between the in-vivo virus cycle and its in-vitro representations exist. In-vitro assays lack the cellular regulatory mechanisms that influence the pathways of viral protein synthesis and self-assembly. We propose to implement a demonstration of a new in-vivo imaging technology, based on virus-like particles (VLPs) and able to bridge the existing gap between in-vitro and in-vivo experiments. Virus-like particles are here hybrid biological/inorganic complexes composed of a viral capsid encapsulating a functionalized nanoparticle instead of nucleic acid. The proposed in vivo imaging technology is of potential high impact since it is not limited to virus assembly but it can be extended to many other self-organizing macromolecular complexes occurring in the confines of a cell. An illustration of the new capabilities is provided through the following proof-of-concept experiments: a) determine real-time disassembly trajectories of individual VLPs in vitro and in vivo with 10 nm spatial resolution and 10 ms time resolution; b) locate individual self-assembled VLPs in cells and their spatial relation with the sites for viral replication. The anticipated results from this proposal will provide a new non-intrusive imaging technology coupled to a means of measuring in-vivo sub-cellular dynamics and will teach us about the rules for virus particle assembly, disassembly, and intracellular transit. We will learn unprecedented basic insight into virus biology. The technology will also broaden the area of future VLP applications and yield a new class of high-output probes that can be adapted to a wide range of targets. ? ? ?
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