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.
Department of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA.
Brian M
BM
Ward
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