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Figure 2 | Genome Medicine

Figure 2

From: Virus-host interactomics: new insights and opportunities for antiviral drug discovery

Figure 2

Methods used for high-throughput screening of virus-host protein-protein interactions. (a) The yeast two-hybrid approach. The generic principle of a Y2H system is based on the reconstitution of a functional transcription factor following interaction between a bait protein and a prey protein. One construct comprises the DNA-binding domain of the transcription factor (BD) in fusion with a bait protein, whereas the prey protein is fused with the transcription activation domain (AD). Upon interaction of the bait with the prey in the nucleus of the yeast, the transcription factor activity is reconstituted, leading to the transcription of a reporter gene. In general, reporter genes are selected for their ability to allow the growth of yeast on selective medium or the use of a colorimetric assay so that their active transcription can be easily monitored. Bait and prey interactions can be tested pairwise in an array when both baits and preys have been individually cloned or upon the screening of fusion proteins expressed from cDNA libraries followed by sequencing of selected preys. (b) The co-affinity purification/MS technique. This approach is typically divided into two technical steps consisting of the capture of cellular proteins with the bait protein and identification of affinity-purified proteins by mass spectrometry (MS; method reviewed in [86]). (c) The protein array. Functional protein arrays, also called ‘protein chips’, can comprise a thousand different proteins attached at high density on a solid surface [30]. Following binding of a protein of interest with its target, the interaction can be detected with fluorescent, radioisotope or photochemical tags. (d) Protein-complementation assays. These assays employ a split Gaussia princeps luciferase (Gluc) assay together with bait and prey proteins that are expressed in mammalian cells in fusion with two inactive fragments of the luciferase. Interaction between bait and prey brings the two fragments into close proximity, restoring the enzymatic activity.

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