Bimolecular Fluorescence Complementation analysis to reveal protein interactions in herpes virus infected cells

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.

► Bimolecular Fluorescence Complementation allows visualization of protein interactions.
► BiFC can reveal direct protein–protein interactions and subcellular localization in living cells.
► BiFC is simple to perform and only requires a fluorescence microscope.
► BiFC complexes are very stable and thus likely nonreversible once formed.