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A Comparative Approach to Characterize the Landscape of Host-Pathogen Protein-Protein Interactions
Published on: July 18, 2013
Virus-derived platforms for visualizing protein associations inside cells
Cathy L Miller1, Michelle M Arnold, Teresa J Broering
1Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA. clm@iastate.edu
Insights
A new method uses orthoreovirus protein muNS to visualize protein-protein interactions within cells, identifying novel protein associations and complexes. This technique is simple, adaptable, and aids in understanding cellular functions.
Area of Science:
- Cell Biology
- Virology
- Molecular Biology
Background:
- Protein-protein interactions are fundamental to cellular processes.
- Visualizing these interactions in living cells is crucial for understanding cell function.
- Existing methods can be complex or lack throughput.
Purpose of the Study:
- To develop a novel, simplified method for visualizing protein-protein associations within cells.
- To demonstrate the utility of this method in identifying new protein interactions and complexes.
- To leverage orthoreovirus protein muNS for creating cellular inclusions that recruit interacting proteins.
Main Methods:
- Utilized orthoreovirus protein muNS to form cytoplasmic inclusions for protein recruitment.
- Employed fluorescently tagged muNS fusion proteins to present other proteins (e.g., p53).
- Applied light microscopy and three-color microscopy to visualize and analyze protein associations and complexes.
Main Results:
- Identified a sixth orthoreovirus protein, RNA-dependent RNA polymerase lambda3, recruited by muNS.
- Demonstrated recruitment of simian virus 40 large T antigen by a muNS-p53 fusion, revealing key interaction regions.
- Confirmed p53 oligomerization and association with CREB-binding proteins within inclusions.
- Identified a ternary complex of p53, simian virus 40 large T antigen, and retinoblastoma protein.
Conclusions:
- The developed method provides a simple and adaptable platform for studying protein-protein interactions in cells.
- This technique facilitates the identification of novel protein associations and the characterization of protein complexes.
- The findings highlight the potential of using viral proteins as tools in cell biology research.
Abstract:
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 muNS 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 lambda3, that was recruited to the structures through an association with muNS. We then established the broader utility of this technology by using a truncated, fluorescently tagged form of muNS 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 muNS-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 muNS 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.
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