title: Bimolecular Fluorescence Complementation analysis to reveal protein interactions in herpes virus infected cells.
abstract: Protein interactions are at the basis of all processes in living organisms. In particular, regulatory proteins do not act alone but participate in multifaceted sets of interactions that are organized into complex networks. In herpes simplex virus (HSV-1) infected cells, viral proteins interact with cellular proteins and with other viral proteins to form the protein complexes required for virus production, including transcription complexes, replication complexes and virion assembly complexes. While a number of methods have been developed to investigate protein-protein interactions such as coimmunoprecipitation, GST-binding assays and yeast 2-hybrid analyses, these approaches require removal of the proteins from the cellular environment and do not provide information on the spatial localization of the protein-protein interaction in living cells. The fluorescence based approach Bimolecular Fluorescence Complementation (BiFC) allows direct visualization of the subcellular localization of the protein complex in living cells. In BiFC, two halves of a fluorescent protein are fused to each of two interacting proteins of interest, resulting in nonfluorescent fusion proteins. Interaction of the protein partners tethers the fused fluorescent fragments in close proximity, which facilitates their association and restoration of fluorescence. Two limitations of BiFC are that there is a delay between the time that the interacting proteins associate and fluorescence complex formation and thus complex formation cannot be measured in real-time, and fluorescence complex formation is irreversible in vivo. Despite these limitations, BiFC is a powerful and sensitive approach that can be performed using standard molecular biology and cell culture protocols and a fluorescence microscope.
authors.affiliation: Department of Microbiology and Molecular Genetics, University of California, Irvine, CA 92697, USA.
authors.forename: Felicia P
authors.initials: FP
authors.lastname: Hernandez
authors.forename: Rozanne M
authors.initials: RM
authors.lastname: Sandri-Goldin
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism*; D018929:Cell Culture Techniques; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D006367:HeLa Cells; D006561:Herpes Simplex / Q000821:virology*; D018259:Herpesvirus 1, Human; D006801:Humans; D017874:Immediate-Early Proteins / Q000378:metabolism; D008164:Luminescent Proteins / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism*; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods*; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D014162:Transfection / Q000379:methods; D014709:Vero Cells
chemical_list: D001426:Bacterial Proteins; C057120:ICP27 protein, human herpesvirus 1; D017874:Immediate-Early Proteins; D008164:Luminescent Proteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; C054562:yellow fluorescent protein, Bacteria