Using fluorescent proteins to study poxvirus morphogenesis.
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.
Ward
Brian M
BM
Department of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA.
D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism*; D011212:Poxviridae / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019065:Virus Assembly
D008164:Luminescent Proteins