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Molecular & Cellular Proteomics 6:1027-1038, 2007.
© 2007 by The American Society for Biochemistry and Molecular Biology, Inc.

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Research

Virus-derived Platforms for Visualizing Protein Associations inside Cells^*

Cathy L. Miller^,,¶, Michelle M. Arnold^,||,**, Teresa J. Broering^,, Catherine Eichwald, Jonghwa Kim^,, Jason B. Dinoso^,¶¶ and Max L. Nibert^,||,||||

From the Department of Microbiology and Molecular Genetics and ^|| Program in Virology, Harvard Medical School, Boston, Massachusetts 02115 and the Department of Veterinary Microbiology and Preventive Medicine, College of Veterinary Medicine, Iowa State University, Ames, Iowa 50011

Protein-protein associations are vital to cellular functions. Here we describe a helpful new method to demonstrate protein-protein associations inside cells based on the capacity of orthoreovirus protein µNS to form large cytoplasmic inclusions, easily visualized by light microscopy, and to recruit other proteins to these structures in a specific manner. We introduce this technology by the identification of a sixth orthoreovirus protein, RNA-dependent RNA polymerase 3, that was recruited to the structures through an association with µNS. We then established the broader utility of this technology by using a truncated, fluorescently tagged form of µNS as a fusion platform to present the mammalian tumor suppressor p53, which strongly recruited its known interactor simian virus 40 large T antigen to the µNS-derived structures. In both examples, we further localized a region of the recruited protein that is key to its recruitment. Using either endogenous p53 or a second fluorescently tagged fusion of p53 with the rotavirus NSP5 protein, we demonstrated p53 oligomerization as well as p53 association with another of its cellular interaction partners, the CREB-binding proteins, within the inclusions. Furthermore using the p53-fused fluorescent µNS platform in conjunction with three-color microscopy, we identified a ternary complex comprising p53, simian virus 40 large T antigen, and retinoblastoma protein. The new method is technically simple, uses commonly available resources, and is adaptable to high throughput formats.

^|||| Supported by a junior faculty grant from the Giovanni Armenise-Harvard Foundation. To whom correspondence may be addressed: Dept. of Microbiology and Molecular Genetics, Harvard Medical School, 200 Longwood Ave., Boston, MA 02115. Tel.: 617-432-4829; Fax: 617-738-7664; E-mail: mnibert{at}hms.harvard.edu

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