Brandariz Núñez, AlbertoMenaya Vargas, RebecaBenavente Martínez, Francisco JavierMartínez Costas, José Manuel2020-06-062020-06-062010Brandariz-Nuñez A, Menaya-Vargas R, Benavente J, Martinez-Costas J (2010) IC-Tagging and Protein Relocation to ARV muNS Inclusions: A Method to Study Protein-Protein Interactions in the Cytoplasm or Nucleus of Living Cells. PLoS ONE 5(11): e13785. https://doi.org/10.1371/journal.pone.0013785http://hdl.handle.net/10347/22843Background: Characterization of protein-protein interactions is essential for understanding cellular functions. Although there are many published methods to analyze protein-protein interactions, most of them present serious limitations. In a different study we have characterized a novel avian reovirus muNS-based protein tagging and inclusion targeting method, and demonstrated its validity to purify free an immobilized protein. Methodology/Principal Findings: Here we present a method to identify protein-protein interactions inside living eukaryotic cells (tested in primate and avian cells). When p53 was tagged with Intercoil (IC; muNS residues 477–542), it not only got integrated into muNS cytoplasmic inclusions, but also attracted its known ligand SV40 large T antigen (TAg) to these structures. We have also adapted this system to work within the cell nucleus, by creating muNS-related protein chimeras that form nuclear inclusions. We show that nuclear muNS-derived inclusions are as efficient as cytoplasmic ones in capturing IC-tagged proteins, and that the proteins targeted to nuclear inclusions are able to interact with their known ligands. Conclusions/Significance: Our protein redistribution method does not present the architectural requirement of reconstructing a transcription factor as any of the two-hybrid systems do. The method is simple and requires only cell transfection and a fluorescence microscope. Our tagging method can be used either in the cytoplasm or the nucleus of living cells to test protein-protein interactions or to perform functional studies by protein ligand sequestration.eng© 2010 Brandariz-Nuñez et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are creditedhttp://creativecommons.org/licenses/by/2.0Cytoplasmic inclusionsImmunostainingProtein expressionProtein interactionsBirdsPlasmid constructionSV40Protein-protein interactionsjournal article10.1371/journal.pone.00137851932-6203open access