Virus-derived platforms for visualizing protein associations inside cells.
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.
Miller
Cathy L
CL
Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA. clm@iastate.edu
Arnold
Michelle M
MM
Broering
Teresa J
TJ
Eichwald
Catherine
C
Kim
Jonghwa
J
Dinoso
Jason B
JB
Nibert
Max L
ML
D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000378:metabolism; D019556:COS Cells; D002522:Chlorocebus aethiops; D022083:Cytoplasmic Structures / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D018165:Orthoreovirus / Q000737:chemistry*; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016160:Retinoblastoma Protein / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry / Q000378:metabolism; D014764:Viral Proteins / Q000378:metabolism*
D000952:Antigens, Polyomavirus Transforming; D011993:Recombinant Fusion Proteins; D016160:Retinoblastoma Protein; D016159:Tumor Suppressor Protein p53; D014764:Viral Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins