GSF: Fluorescence technique visualises proteins interacting in cells

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Scientists from the GSF National Research Center for Environment and Health in Germany have developed a method for the visualisation of individual proteins and their interactions in living cells, by engineering the proteins to constantly emit red or blue fluorescent signals and to produce an additional yellow fluorescent signals upon interaction.

In previous assays, signals were generated only when proteins interacted and it was not possible to visualise individual proteins, says Ruth Brack-Werner, Director of the GSF Institute of Molecular Virology. “However, the absence of signals could not be used to rule out protein interactions since the absence of one or both interaction partners would have the same effect.”

To overcome this problem Brack-Werner and her colleagues developed the extended bimolecular fluorescence complementation (exBiFC) technique to allow simultaneous monitoring of individual proteins and their interactions.

The team is using exBiFC to study mechanisms that control replication of the HIV virus. HIV replication is based on the interaction of cellular proteins with viral proteins. Interactions involving viral regulatory factors have a direct impact on the amount of virus produced by the HIV host cell.

“Preventing HIV proteins from interacting with their counterparts is a promising approach to developing novel therapies,” says Brack-Werner. The exBiFC fluorescence technology has been validated with the HIV Rev protein, which is an accelerator of HIV production.

The activity of Rev depends on the interaction of Rev molecules with each other and with cellular proteins, including exportin 1, which transports proteins from the nucleus to the cytoplasm and RISP, a modulator of HIV gene expression. exBIFC allows visualisation of interactions of Rev with itself, and with exportin1 and RISP in living cells. In addition, it is possible to compare the strengths of the interactions of Rev with its partners by analysing the intensities of the signals in cell images.

Beyond HIV the researchers believe ExBiFC has a wide range of potential applications, representing an important tool for the elucidation of protein interaction networks and the discovery of novel antiviral factors.