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Methods Mol Biol. 2009;515:1-11. doi: 10.1007/978-1-59745-559-6_1.

Using fluorescent proteins to study poxvirus morphogenesis.

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  • 1Department of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA.

Abstract

Fluorescent protein (FP) fusions not only allow for the convenient visualization of a protein of -interest's subcellular localization but also permit the real-time monitoring of their subcellular trafficking. The subcellular fluorescent pattern of FP-fusions can also serve as a visual marker for various subcellular processes using either live or static microscopy. We have employed FP-fusions for the study of poxvirus morphogenesis. Fusion of FP with either a viral core protein or an extracellular virion-specific protein can serve as a visual read-out for normal poxvirus morphogenesis at the subcellular level. Recombinant viruses expressing a FP-fusion, in conjunction with the deletion of a gene involved in either morphogenesis or egress, usually display an aberrant FP pattern. Functional domains in the missing protein are then mapped by complementation in-trans followed by fluorescent microscopy for analysis of the FP pattern. The methods presented here describe how to infect and transfect cells for trans-complementation for the purpose of functional domain mapping. The imaging and analysis of these cells is described.

PMID:
19378136
[PubMed - indexed for MEDLINE]

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